InteractiveFly: GeneBrief

shibire: Biological Overview | References

BIOLOGICAL OVERVIEW

Plasma membrane clathrin-coated vesicles form after the directed assembly of clathrin and the adaptor complex, AP2, from the cytosol onto the membrane. In addition to these structural components, several other proteins have been implicated in clathrin-coated vesicle formation. These include the large molecular weight GTPase, dynamin, and several Src homology 3 (SH3) domain-containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (see model of the role of dynamin and some of its binding partners in clathrin-coated vesicle formation). To understand the mechanism of coated vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in coated pit assembly, invagination, and scission (Hill, 2001).

To address the role of dynamin and its binding partners in the early stages of endocytosis in mammalian cells, this study used well-established in vitro assays for the late stages of coated pit invagination and coated vesicle scission. Dynamin has been shown to have a role in scission of coated vesicles. This study also shows that dynamin is also required for the late stages of invagination of clathrin-coated pits. Furthermore, dynamin must bind and hydrolyze GTP for its role in sequestering ligand [in the case of this study, the SH3 domain of amphiphysin-2 (Amph2)] into deeply invaginated coated pits (Hill, 2001).

This study also demonstrates that the SH3 domain of endophilin, which binds both synaptojanin and dynamin, inhibits both late stages of invagination and also scission in vitro. This inhibition results from a reduction in phosphoinositide 4,5-bisphosphate levels which causes dissociation of AP2, clathrin, and dynamin from the plasma membrane. The dramatic effects of the SH3 domain of endophilin led to the proposal of a model for the temporal order of addition of endophilin and its binding partner synaptojanin in the coated vesicle cycle (Hill, 2001).

The directed assembly of clathrin and the AP2 adaptor complex from the cytosol onto the membrane leads to the formation of clathrin-coated pits (Smythe 1996; Schmid 1997). In addition to these structural components, many other proteins have been implicated in clathrin-coated vesicle formation. In particular, the large molecular weight GTPase, dynamin, has been implicated in the late stages of coated vesicle budding. Several Src homology (SH) 3 domain-containing proteins which bind to dynamin via interactions with its COOH-terminal proline/arginine-rich domain (PRD) (Schmid, 1998) have also been shown to have a role in endocytosis. These include amphiphysin (Shupliakov, 1997; Wigge, 1997; see Drosophila Amphiphysin) and endophilin (Ringstad, 1997; Schmidt, 1999; Hill, 2001 and references therein).

Dynamin was first identified as having a role in endocytosis when it was shown to be the mammalian homologue of the shibire protein in Drosophila (van der Bliek, 1991). Temperature-sensitive mutations in shibire give rise to a paralytic phenotype at the nonpermissive temperature. Morphological analysis revealed an accumulation of membrane invaginations and coated pits at the neuromuscular junction suggestive of a defect in the scission of coated pits to form coated vesicles (Kosaka, 1983a). Electron-dense collars believed to be composed of dynamin were visible around the necks of these pits. Dynamin was later shown to self-assemble into rings and spirals when incubated in buffers of low ionic strength (Hinshaw, 1995). These rings and spirals had the same dimensions as the collars seen in shibire mutant flies and spirals that formed around invaginations on permeabilized rat brain synaptosomes in the presence of GTP{gamma}S (Takei, 1995). The self-assembly of dynamin stimulates its GTPase activity (Warnock, 1996) and a model was proposed in which dynamin would assemble around the neck of an invaginated pit. The energy released by GTP hydrolysis would allow it to act as a mechanochemical enzyme in the final stage of scission (Warnock, 1996; McNiven 1998; Hill, 2001 and references therein).

The role of dynamin as a 'pinchase' has been somewhat controversial, however (Kelly, 1999; Yang, 1999; Sever, 2000b), and recent studies have suggested that rather than requiring GTP hydrolysis to perform its function, dynamin functions as a regulator in its GTP conformation, recruiting other binding partners which are then required for scission (Sever, 1999, Sever, 2000a; Hill, 2001 and references therein).

Several proteins have been demonstrated to bind to the COOH-terminal PRD of dynamin via their SH3 domains (Schmid, 1998). These include amphiphysin and endophilin. Amphiphysin has been implicated in the endocytic process by studies where an inhibition of endocytosis was observed when its SH3 domain was microinjected into the synapse of the giant lamprey (Shupliakov, 1997) or transfected into fibroblasts (Wigge, 1997). In addition to binding dynamin, amphiphysin can also bind to the appendage domain of α-adaptin (see Drosophila α-adaptin), suggesting that it acts to recruit dynamin to coated pits (Wang, 1995; Wigge, 1998). This model has been supported by observations showing that amphiphysin can enhance the association of clathrin buds with dynamin on purified liposomes. Amphiphysin can also coassemble with dynamin into rings and spirals (Takei, 1999; Hill, 2001 and references therein).

Endophilin is another SH3 domain-containing protein whose major binding partner is the inositol 5-phosphatase, synaptojanin, although it also binds dynamin (Micheva, 1997; Ringstad, 1997). Recent studies have shown that endophilin is required for the formation of synaptic-like microvesicles at the plasma membrane (Schmidt, 1999). Endophilin has lysophosphatidic acid acyl transferase activity which is essential for its function in synaptic-like microvesicle formation. Synaptojanin knockout mice have elevated levels of phosphatidylinositol 4,5-bisphosphate (PtdIns_P2) and show an accumulation of clathrin-coated vesicles indicative of a role for synaptojanin in uncoating (Hill, 2001 and references therein).

To understand the mechanism of coated vesicle formation, it is essential to determine the hierarchy by which individual components are targeted to and act in coated pit assembly. To address the role of dynamin and its binding partners in the early stages of endocytosis, well-established in vitro assays were used for the late stages of coated pit invagination and coated vesicle scission (Smythe, 1989; Schmid, 1991; Smythe, 1992). Using these functional assays, this study demonstrated that dynamin, in addition to its role in scission, is also required for the late stages of coated pit invagination. Furthermore, both GTP binding and hydrolysis are required for this step. It was also demonstrated that the SH3 domain of endophilin causes a reduction in PtdIns_P2 levels, a loss of AP2 from the permeabilized cell membranes, and a concomitant inhibition of clathrin-coated pit invagination and scission. This leads to the suggestion of a model for the sequential action of endophilin and synaptojanin in the coated vesicle cycle (Hill, 2001).

This paper used the SH3 domains of two proteins implicated in endocytosis, amphiphysin and endophilin, as tools to investigate the nature of interactions involved in edocytosis. Using the SH3 domain of amphiphysin, the requirement for dynamin during the late stages of clathrin-coated pit invagination and coated vesicle scission in permeabilized A431 cells was analyzed. Dynamin is required not only for scission of coated vesicles but also for the late stages of clathrin-coated pit invagination. Using the SH3 domain of endophilin, it was demonstrated that this domain is sufficient to cause a dramatic reduction in PtdIns_P2 levels and a concomitant inhibition of ligand sequestration and internalization (Hill, 2001).

The SH3 domain of amphiphysin inhibits both ligand sequestration and internalization. Ligand sequestration was defined as the loss of accessibility of biotinylated transferrin (B-SS-Tfn) to exogenously added avidin. The deeply invaginated structures which sequester B-SS-Tfn have not yet pinched off because the biotinylated ligand is still sensitive to the small membrane-impermeant reducing agent MesNa. By contrast, B-SS-Tfn which is internalized into coated vesicles is inaccessible to avidin and also resistant to MesNa reduction. The SH3 domain of amphiphysin interacts strongly with dynamin and the results show that the inhibitory effects result from interactions with membrane-associated dynamin. The inhibitory effects of the SH3 domain of amphiphysin are consistent with results in intact cells where transfection of the SH3 domain into fibroblasts (Wigge, 1997). or injection into lamprey synapses (Shupliakov, 1997), resulted in an inhibition of receptor-mediated endocytosis via clathrin-coated pits. Injection of the SH3 domain of amphiphysin into lamprey synapses resulted in an accumulation of invaginated coated pits many of which still have a clearly discernible 'neck.' Given that this SH3 domain in the permeabilized A431 cell system causes an increase in the accessibility of avidin to B-SS-Tfn, it would be interesting to know if these coated pits might represent a stage of invagination in which receptor-bound ligand is still accessible to avidin. In permeabilized A431 cells which have been preincubated with GST-amph2 SH3^D36R, Western blotting reveals that the bulk of dynamin remains associated with the membranes, indicating that GST-amph2 SH3^D36R acts by binding to dynamin on the membrane and preventing it from performing its biological function (Hill, 2001).

The major binding partners of the SH3 domain of amphiphysin are dynamin and synaptojanin, but the inhibitory effects of GST-amph2 SH3SH3^D36R on the avidin inaccessibility assay were only rescued by purified dynamin and not by synaptojanin. Rescue is not apparently due to displacement of the inhibitory fusion protein. A more likely possibility is that dynamin is being recruited directly to deeply invaginating coated pits and so compensates for nonfunctional dynamin already on the membrane. It has been proposed (Shupliakov, 1997) that the effect of the SH3 domain is to block the recruitment of dynamin since after injection of this domain there was no electron-dense collar as is observed in the shibire mutant of Drosophila at the nonpermissive temperature. This electron-dense collar is thought to be composed of dynamin although this has not yet been formally demonstrated. Therefore, it is likely that recruitment of dynamin to the neck of pits is blocked when the permeabilized cells are preincubated with the SH3 domain of amphiphysin. Another possibility is that it interferes with the assembly/disassembly of dynamin as was demonstrated for the SH3 domain in vitro, thus preventing the formation of a dynamin collar (Owen, 1998). The ability of dynamin rather than synaptojanin to rescue coated pit invagination indicates that the inhibitory effect of the SH3 domain of amphiphysin is specific for dynamin (Hill, 2001).

Purified dynamin, however, is unable to rescue the inhibitory effects of GST-amph2 SH3SH3^D36R in the MesNa resistance assay. This suggests that for coated vesicle scission, the SH3 domain of amphiphysin has a dominant negative effect. This is consistent with previous studies which indicated that the population of coated pits that are capable of pinching off in A431 cells is 'primed' for scission and has many of the components required for the late stages of coated vesicle budding already assembled (Schmid, 1991; Smythe, 1992). The dominant negative effect of the SH3 domain on the MesNa resistance assay may operate in one of two ways. It is possible that GST-amph2 SH3SH3^D36R acts directly to interfere with dynamin function. Alternatively, it may exert its inhibitory effects by blocking access of other binding partners of dynamin, e.g., full length endophilin, required for the final scission step. However, because it was not possible to rescue the inhibitory effects of GST-amph2 SH3SH3^D36R with concentrated cytosol, dynamin, or synaptojanin, the possibility cannot be ruled out that there is another target of this SH3 domain in addition to dynamin that is essential for the final scission step (Hill, 2001).

The requirement for dynamin to execute both the late stages of invagination and scission is consistent with experiments in cells transfected with GTP binding domain mutants of dynamin where endocytosis was blocked after coat assembly and preceding the sequestration of ligand into deeply invaginated coated pits (van der Bliek, 1993). Similarly the GED (GTPase effector domain) of dynamin inhibits B-SS-Tfn as measured by the avidin inaccessibility assay in permeabilized A431 cells (Sever, 1999). The results are also consistent with studies on the assembly of dynamin onto liposomes which indicate that dynamin can tubulate liposomes and cause them to evaginate (Sweitzer, 1998; Takei, 1998; Hill, 2001 and references therein).

Consistent with rescue experiments, the addition of purified dynamin to untreated permeabilized cells resulted in a stimulation of the avidin inaccessibility assay by 10%-15%. This increase in the avidin inaccessibility signal likely corresponds to the increased invagination of a population of coated pits where dynamin is limiting and which, in the presence of exogenously added dynamin, can become sufficiently invaginated to confer avidin inaccessibility. Although the addition of exogenous dynamin can increase the extent of invagination of these pits, it is insufficient to cause them to pinch off and therefore become MesNa resistant. This is again consistent with the idea that those pits which are capable of pinching off have already assembled all of the essential components. However, the inhibitory effect of the SH3 domain of amphiphysin indicates that, as expected, dynamin function appears still to be required for the late stages of coated scission. Binding of the SH3 domain interferes with this function (Hill, 2001).

The rescue by dynamin is dependent on the ability of dynamin to bind GTP. In contrast to the ability of wild-type dynamin to rescue the inhibitory effects of the SH3 domain of amphiphysin, neither S45N nor K44A dynamin is capable of rescue. The S45N mutant is deficient in the ability to bind GTP. The K44A mutant, although considered as an hydrolysis mutant, also has reduced binding of GTP (van der Bliek, 1993). However, even in the presence of high concentrations of GTP (500 microM), the K44A mutant failed to rescue the inhibitory effects of the amphiphysin SH3 domain. Surprisingly, mutant dynamin deficient in the ability to hydrolyze GTP (T65A) is also unable to rescue the inhibitory effects of the SH3 domain of amphiphysin. This implies that dynamin must bind and hydrolyze GTP in order to promote the formation of deeply invaginated coated pits in vitro which are inaccessible to avidin. In vitro results were confirmed by studies in HEK293 cells overexpressing wild-type and mutant forms of dynamin. Overexpression of both dynamin K44A and dynamin T65A resulted in an inhibition of B-SS-Tfn uptake as measured by both the avidin inaccessibility and MesNa resistance assays. It has previously been demonstrated that these assays can distinguish between the formation of deeply invaginated coated pits and pinched off coated vesicles in intact cells (Schmid, 1990). These results are in contrast to recent results reporting an increase in the uptake of transferrin on overexpression of mutant dynamin deficient in GTP hydrolysis and assembly (Sever, 1999; Sever, 2000a). Using dynamin that has point mutations in the GED (GTPase effector domain), these workers concluded that dynamin functions as a classical GTPase, recruiting effectors when in the GTP conformation. The current results indicate, however, that GTP hydrolysis by dynamin is required for the late stages of invagination. The data are therefore most consistent with a ratchet model of dynamin action (Smirnova, 1999), whereby dynamin molecules would interact with those molecules on an adjacent rung of the dynamin collar. The energy of GTP hydrolysis would then be harnessed to the movement of the dynamin molecules resulting in an increase in constriction which would result in an increase in avidin inaccessibility in experimental system (see Model of the role of dynamin and some of its binding partners in clathrin-coated vesicle formation). The action of dynamin would thus be akin to that of kinesin and myosin (Vale 1996; Smirnova, 1999; Hill, 2001 and references therein).

Despite the stimulatory effect of dynamin in the avidin inaccessibility assay, depletion of dynamin from bovine brain cytosol has no significant effect on the extent of avidin inaccessibility. This suggests either that the concentration of dynamin (0.1 microM) present at the highest concentration of cytosol used was too little to have a significant effect or, alternatively, that in the preparation of bovine brain cytosol used the dynamin is somehow inactivated. It has recently been reported that several SH3 domains inhibit clathrin-mediated endocytosis in permeabilized 3T3L1 cells (Simpson, 1999). However, in contrast to the current results, the SH3 domains of amphiphysin and endophilin only inhibited scission in this cell line. This study also showed that depletion of dynamin from cytosol inhibited scission as measured by the MesNa resistance assay in permeabilized 3T3L1 cells. The reasons for these differences are unknown, but one possible explanation for the discrepancies may be that the cytosol preparation used by Simpson and colleagues contains an active pool of dynamin. If this were the case then this cytosol might be capable of overcoming the inhibitory effects of the amphiphysin and endophilin SH3 domains on invagination. This would also explain why the inhibitory effects of these SH3 domains in 3T3L1 cells were only observed after preincubation of the permeabilized cell membranes and with concentrations of SH3 domains that are 10-fold higher than those used in this study (Hill, 2001).

The current study does not allow drawing of any conclusions about the mechanism of action of dynamin in scission. As discussed above, the specificity of the inhibitory effects of GST-amph2 SH3^D36R on invagination and its similar concentration-dependent inhibition of scission led to the conclusion that it is required for scission, as has been shown by many other laboratories (Takei, 1995; Sweitzer, 1998; Stowell, 1999). Although the A431 permeabilized cell system has several limitations for the study of scission, these limitations actually provide an advantage for the study of the formation of deeply invaginated coated pits since they allow dissociation of the two events (Hill, 2001).

The SH3 domain of endophilin also inhibited both deep invagination and scission in permeabilized A431 cells. The major physiological binding partner for endophilin is synaptojanin although it also binds dynamin to a lesser extent (Micheva, 1997; Ringstad, 1997). Synaptojanin was unable to rescue the inhibitory effects of GST-endoSH3 in either the avidin inaccessibiltiy or MesNa resistance assays. In contrast to the inhibitory effect of the SH3 domain of amphiphysin, dynamin (at concentrations up to twofold higher than that required for full rescue of GST-amph2 SH3^D36R) was also unable to rescue the inhibitory effects of the SH3 domain of endophilin (Hill, 2001).

Synaptojanin is an inositol 5-phosphatase whose principal substrates are PtdIns_P2 and PtdInsP3. There is no significant effect on the levels of PtdInsP3 in permeabilized cells treated with GST-endoSH3. However, the presence of GST-endoSH3 resulted in a dramatic reduction in PtdIns_P2 levels. The reduction in PtdIns_P2 levels was specific for GST-endoSH3 since GST-amph2 SH3^D36R had no effect. Phosphoinositides have been implicated in the coated vesicle cycle in the binding of both AP2 and dynamin to the plasma membrane. AP2 can bind a variety of phospholipids and its biological activity is modulated by these interactions. For example, PtdIns_P2 and PtdInsP3 enhance the binding of AP2 to internalization motifs, and the enhancement is comparable to the binding of AP2 to receptor tails within assembled coats although the latter is independent of phosphoinositides. A phosphoinositide binding site on the α-subunit of AP2 has been identified which is essential for correct targeting of AP2 to clathrin-coated pits at the plasma membrane. The PH domain of PLCδ1, which specifically binds PtdIns_P2, has been shown to interfere with ligand sequestration and internalization in permeabilized A431 cells. This study demonstrated that GST-endoSH3 reduced PtdIns_P2 levels by >60%, leading to the loss of AP2 from the permeabilized cell membranes and a concomitant inhibition of ligand sequestration and internalization. Similarly, the pleckstrin homology domain of dynamin which interacts with membrane phospholipids has been shown to be essential for endocytosis (Achiriloaie, 1999; Lee, 1999; Vallis, 1999), and interestingly this interaction is dependent on its oligomeric state (Lee, 1999). GST-endoSH3-induced reduction of PtdIns_P2 levels also leads to a loss of dynamin from the permeabilized cell membranes, providing further evidence of the requirement for PtdIns_P2 in the association of dynamin with membranes. Treatment of the permeabilized cells with GST-endoSH3 also caused a loss in membrane-associated clathrin, measured both by Western blotting and by immunofluorescence. This presumably is an indirect effect of the loss of AP2 from the plasma membrane and further confirms the requirement for PtdIns_P2 in coated pit assembly. By contrast, there is no significant loss of membrane-associated synaptojanin or transferrin receptor after treatment with either of the SH3 domains, indicating that the reduction in PtdIns_P2 levels has a specific effect on clathrin-coated pit components and is not due to gross disruption of membranes. GST-amph2 SH3^D36R caused a small reduction in the amount of clathrin and dynamin associated with the membranes, but the reduction was much less than that observed in the presence of GST-endoSH3. It has been estimated that the amount lost is no more than 5-10% of the total (Hill, 2001).

Synaptojanin was unable to rescue the inhibitory effects of either of the SH3 domains. The inability of synaptojanin to rescue GST-amph2 SH3^D36R treated membranes further demonstrates the specificity of the rescue of these membranes by dynamin, indicating that dynamin is the true target for this SH3 domain. The lack of rescue of GST-endoSH3 treated membranes is consistent with the results, showing that these membranes have reduced PtdIns_P2 levels. Since synaptojanin, if recruited to the membrane, would be predicted to itself cause a reduction in PtdIns_P2 levels, the most likely mechanism by which it could rescue the inhibitory effects of the SH3 domain would be by competition of the SH3 domain from the membrane. Western blots using anti-GST antibodies revealed that there was no significant reduction in the amount of GST-endoSH3 associated with the membrane in the presence of either dynamin or synaptojanin. It is worth noting that it is estimated that a molar excess of at least 100-fold of dynamin and synaptojanin was added over the amount of SH3 domain associated with the membrane. This suggests that the fusion proteins may either form a tighter association with membrane rather than cytosolic components or, alternatively, be incorporated into a relatively stable complex (Hill, 2001).

Recent data using a mouse knockout for synaptojanin have strongly implicated synaptojanin as having a role in uncoating of clathrin-coated vesicles (Cremona, 1999). Cortical neurons from the knockout mouse exhibited increased levels of clathrin-coated vesicles and also of PtdIns_P2, providing a link between the requirement to hydrolyze PtdIns_P2 and uncoating. In C. elegans, mutations in synaptojanin lead to pleiotropic defects in vesicle trafficking including synaptic vesicle budding and uncoating. The current data are most consistent with a model where it appears that the SH3 domain of endophilin causes an untimely reduction in PtdIns_P2 levels, most likely by mislocalization and/or activation of synaptojanin. Western blotting of membranes separated from assay mixtures which had been incubated in the presence or absence of GST-endoSH3 showed no significant recruitment of recombinant synaptojanin by GST-endoSH3. Similarly, addition of recombinant synaptojanin had only a very modest inhibitory effect on coated pit invagination and scission and caused a similar modest reduction in PtdIns_P2 levels (data not shown). This strongly suggests that the functional pool of synaptojanin in permeabilized cells is associated with the membrane, and its untimely activation prevents coat assembly and invagination and probably scission. However, it would be predicted that normally synaptojanin works later in the endocytic cycle, in the uncoating of coated vesicles (Hill, 2001).

This hypothesis is supported by recent studies carried out in parallel (Gad, 2000). The Gad study showed that microinjection of antisynaptojanin antibodies and also of a peptide which specifically interferes with the interaction of endophilin with synaptojanin and dynamin caused an accumulation of coated vesicles in the lamprey synapse. Microinjection of the peptide and also GST-endoSH3 caused an accumulation of coated pits, but interestingly, synapses microinjected with GST-endoSH3 showed no uncoating defect. These results suggest that the interaction between endophilin and synaptojanin is important for uncoating while SH3-mediated effects of endophilin (perhaps by interaction with dynamin) are necessary for scission (Schmidt, 1999). In the permeabilized cell system, GST-endoSH3 has inhibitory effects on both invagination and scission. The inhibition appears to result principally from a loss of coat components from the membrane as a result of the reduction in PtdIns_P2 levels. However, the possibility that the inhibition of scission is a direct effect on this process as is observed in the lamprey synapse cannot be ruled out. The differential effects of GST-endoSH3 seen in the lamprey synapse versus permeabilized A431 cells are most likely due to compensatory mechanisms within the synapse which are lost in permeabilized cells and perhaps allow a replenishment of PtdIns_P2 levels. Such mechanisms appear to exist in vivo to maintain the levels of essential phosphoinositides because, for example, the increase in PtdIns_P2 in the synaptojanin knockout mouse, although significant, is only 1.6-fold (Cremona, 1999; Hill, 2001 and references therein).

In summary, this study has shown that dynamin, in addition to its well-established role in scission, is also necessary for the late stages of coated pit invagination. Its function in invagination is dependent on GTP binding and hydrolysis, pointing, at the very least, to a mechanochemical role. However, the current findings do not exclude the possibility of additional regulatory components in this process. The results with the SH3 domain of endophilin indicate the importance of PtdIns_P2 in the recruitment of AP2 and dynamin to the membrane to initiate coated vesicle formation (Hill, 2001).

Drosophila motor neuron retraction during metamorphosis is mediated by inputs from TGF-beta/BMP signaling and orphan nuclear receptors; Disruption of shi function specifically in glial cells results in an unpruned mushroom body γ neuron phenotype and prevents glial cell infiltration into the mushroom body

Larval motor neurons remodel during Drosophila neuro-muscular junction dismantling at metamorphosis. This study describes the motor neuron retraction as opposed to degeneration based on the early disappearance of β-Spectrin and the continuing presence of Tubulin. By blocking cell dynamics with a dominant-negative form of Dynamin, this study shows that phagocytes have a key role in this process. Importantly, the presence of peripheral glial cells is shown close to the neuro-muscular junction that retracts before the motor neuron. In muscle, expression of EcR-B1 encoding the steroid hormone receptor required for postsynaptic dismantling, is under the control of the ftz-f1/Hr39 orphan nuclear receptor pathway but not the TGF-β signaling pathway. In the motor neuron, activation of EcR-B1 expression by the two parallel pathways (TGF-β signaling and nuclear receptor) triggers axon retraction. This study interrupted TGF-β signaling in motor neurons using expression of dominate negative Wishful thinking. It is proposed that a signal from a TGF-β family ligand is produced by the dismantling muscle (postsynapse compartment) and received by the motor neuron (presynaptic compartment) resulting in motor neuron retraction. The requirement of the two pathways in the motor neuron provides a molecular explanation for the instructive role of the postsynapse degradation on motor neuron retraction. This mechanism insures the temporality of the two processes and prevents motor neuron pruning before postsynaptic degradation (Boulanger, 2012).

It is a general feature of maturing brains, both in vertebrates and in invertebrates, that neural circuits are remodeled as the brain acquires new functions. In holometabolous insects, the difference in lifestyle is particularly apparent between the larval and the adult stages. These insects possess two distinct nervous systems at the larval and adult stages. A class of neurons is likely to function in both the larval and the adult nervous systems. The neuronal remodeling occurring during this developmental period is expected to be necessary for the normal functioning of the new circuits (Boulanger, 2012).

The pruning of an axon can involve a retraction of the axonal process, its degeneration or both a retraction and degeneration. The MB γ axon is pruned through a local degeneration mechanism. In contrast, axons may retract their cellular processes from distal to proximal in the absence of fragmentation and this mechanism is called retraction. Interestingly, the two mechanisms can occur sequentially in the same neuron, as in the case of the dendrites of the da neurons, where branches degenerate and the remnant distal tips retract (Boulanger, 2012).

This study provides evidence that the motor neuron innervating larval muscle 4 (NMJ 4) is pruned predominantly through a retraction mechanism. The first morphological indication of motor neuron retraction is the absence of fragmentation observed with anti-HRP staining at the level of the presynapse in all the developmental stages analyzed, together with a decrease in perimeter size observed after 2 h APF. The continuity of this HRP staining is in contrast to the pronounced interruptions between blebs observed with an antibody against mCD8 in γ axons. A molecular indication of motor neuron retraction in these studies is the fact that β-Spectrin disappears at the synapse 5 h APF, before motor neuron pruning takes place. Indeed, it has been shown using an RNA interference approach that loss of presynaptic β-Spectrin leads to presynaptic retraction and synapse elimination at the NMJ during larval stages. The modifications of the microtubule morphology that were observed, such as an increase in microtubule thickness and withdrawal, provide additional evidence of axonal retraction during NMJ remodeling. Finally, a strong argument in favor of a motor neuron retraction mechanism is the fact that Tubulin is present at the NMJ throughout all stages of axonal pruning at the start of metamorphosis (0-7 h APF). This stands in clear contrast to the abolition of Tubulin expression observed before the first signs of γ axon degeneration. It is also interesting to note that the motor neuron retraction observed in this study at metamorphosis and at larval stages are morphologically different. During metamorphosis, retraction bulbs or postsynaptic footprints, which have been reported at larval stages, were never visualized. The fact that the postsynapse dismantles at metamorphosis before motor neuron retraction might explain these discrepancies. Worth noting is the mechanistic correlation between accelerated debris shedding observed here for NMJ pruning at the start of metamorphosis and axosome shedding occurring during vertebrate motor neuron retraction (Boulanger, 2012).

In vertebrates, glia play an essential role in the developmental elimination of motor neurons. In Drosophila, the role of glia in sculpting the developing nervous system is becoming more apparent. Clear examples of a role for engulfing glial cells in axon pruning are well documented during the MB γ axon degeneration at metamorphosis. Also, glia are required for clearance of severed axons of the adult brain. A distinct protective role of glia has been recently discovered during the patterning of dorsal longitudinal muscles by motor neurobranches. This study describes the presence of glia processes close to the end of the pupal NMJ. The observations suggest that the glial extensions retract at 5 h APF, just before motor neuron retraction is observed. When the glial dynamic is blocked, the NMJ dismantling might be also blocked. It is hypothesized that during development in larvae and early pupae, glial processes have a protective role and aid in the maintenance of the NMJ. Then, between 2 and 5 h APF, glial retraction would be a necessary initial step that allows NMJ dismantling. In accordance with this hypothesis, glia play a protective role in the maintenance of NMJ during pruning of second order motor neuron branches 31 h APF (Boulanger, 2012).

Disruption of shi function specifically in glial cells results in an unpruned mushroom body γ neuron phenotype and prevents glial cell infiltration into the mushroom body (Awasaki, 2004). One can note that at the NMJ the role of the glia is proposed to be essentially opposite from its role in MB γ axons pruning but in both cases blocking the glia dynamics results in a similar blocking of the pruning process (Boulanger, 2012).

In vertebrates, phagocytes are recruited to the injured nerve where they clear, by engulfment, degenerating axons. In Drosophila, phagocytic blood cells engulf neuronal debris during elimination of da sensory neurons. This study shows that blocking phagocyte dynamics with shi produces a strong blockade of the NMJ dismantling process. One possibility is that phagocytes attack and phagocytose the postsynaptic material, a process blocked by compromising shi function resulting in postsynaptic protection. In accordance, it has been shown that phagocytes attack not only the da dendrites to be pruned, but also the epidermal cells that are the substrate of these dendrites (Boulanger, 2012).

During NMJ dismantling, the muscle has an instructive role for motor neuron retraction. In all the situations where postsynapse dismantling is blocked, the corresponding presynaptic motor neuron retraction is also blocked. Therefore, it is sufficient to propose that both glial cells and phagocytes affect only the postsynaptic compartment. Nevertheless, one cannot rule out that these two cell types both act directly at the pre and at the postsynapse (Boulanger, 2012).

ECR-B1 is highly expressed and/or required for pruning in remodeling neurons of the CNS. MB γ neurons and antennal lobe projection neurons remodeling require both the same TGF-β signaling to upregulate EcR-B1. In the MBs only neurons destined to remodel show an upregulation of EcR-B1. At least two independent pathways insure EcR-B1 differential expression. The TGF-β pathway and the nuclear receptor pathway are thought to provide the necessary cell specificity of EcR-B1 transcriptional activation. This study shows that in the motor neuron pruning these two pathways are also necessary to activate EcR-B1. Noteworthy, showing an analogous requirement of ftz-f1/Hr39 pathway in two different remodeling neuronal systems unravels the fundamental importance of this newly described pathway (Boulanger, 2012).

The following model is proposed for the sequential events that are occurring during NMJ dismantling at early metamorphosis. First, EcR-B1 is expressed in the muscle under the control of FTZ-F1. FTZ-F1 activates EcR-B1 and represses Hr39. This repression is compulsory for EcR-B1 activation. Importantly, TGF-β/BMP signaling does not appear to be required for EcR-B1 activation in this tissue, however, a result of EcR-B1 activation in the muscle would be the production of a secreted TGF-β family ligand. Then, this secreted TGF-β family ligand reaches the appropriate receptors and activates the TGF-β signaling in the motor neuron. Finally, TGF-β signaling in association with the nuclear receptor pathway activates EcR-B1 expression resulting in motor neuron retraction. Since glial cells and phagocytes are required for the dismantling process, it is possible that a TGF-β/BMP family ligand(s) be produced by one or both of these cell types and not by the postsynaptic compartment. Noteworthy, a recent study shows that glia secrete myoglianin, a TGF-β ligand, to instruct developmental neural remodeling in Drosophila MBs (Awasaki, 2011). Nevertheless, one can note that the requirement of the two pathways in the motor neuron provides a simple molecular explanation of the instructive role of postsynapse degradation on motor neuron retraction. This mechanism insures the temporality of the two processes and prevents motor neuron pruning before postsynaptic degradation. It was proposed that in the MBs, the association of these two pathways provides the cell (spatial) specificity of pruning. In this paper, this association is proposed to provide the temporal specificity of the events. Future studies will be necessary to understand how EcR-B1 controls the production of a TGF-β/BMP ligand(s) in the muscle, the reception of this signal by the motor neuron and the ultimate response by the motor neuron to initiate retraction. These steps will be necessary to unravel the molecular mechanisms underlying the NMJ dismantling process and related phenomenon in vertebrate NMJ development and disease. Interestingly, it appears that TGF-β ligands on the one hand are positive regulators of synaptic growth during larval development and on the other hand, they are positive regulators of synaptic retraction, at the onset of metamorphosis. In both situations signaling provides a permissive role, sending a signal from the target tissue to the neuron. The consequence of this signal would be dependent on developmental timing thus, on a change in context (Boulanger, 2012).

The BAR domain of amphiphysin is required for cleavage furrow tip-tubule formation during cellularization in Drosophila embryos

De novo formation of cells in the Drosophila embryo is achieved when each nucleus is surrounded by a furrow of plasma membrane. Remodeling of the plasma membrane during cleavage furrow ingression involves the exocytic and endocytic pathways, including endocytic tubules that form at cleavage furrow tips (CFT-tubules). The tubules are marked by amphiphysin but are otherwise poorly understood. This study identified the septin family of GTPases as new tubule markers. Septins do not decorate CFT-tubules homogeneously: instead, novel septin complexes decorate different CFT-tubules or different domains of the same CFT-tubule. Using these new tubule markers, it was determined that all CFT-tubule formation requires the BAR domain of amphiphysin. In contrast, dynamin activity is preferentially required for the formation of the subset of CFT-tubules containing the septin Peanut. The absence of tubules in amphiphysin-null embryos correlates with faster cleavage furrow ingression rates. In contrast, upon inhibition of dynamin, longer tubules formed, which correlated with slower cleavage furrow ingression rates. These data suggest that regulating the recycling of membrane within the embryo is important in supporting timely furrow ingression (Su, 2013).

Cellularization in the Drosophila embryo involves de novo generation of 6000 columnar epithelial cells, which are generated by the ingression of plasma membrane furrows (cleavage furrows) that enclose each nucleus. At the tip of ingressing cleavage furrows, CFT-tubules form. This study demonstrated the existence of three populations of CFT-tubules, which can de defined by different septin family members. The different populations of CFT-tubules are differentially regulated, and their presence or absence correlates with changes in cleavage furrow ingression kinetics (Su, 2013).

Septins were identified as additional factors localizing to the CFT-tubules. Of interest, not all septins localize to the same CFT-tubules or the same domain within a single CFT-tubule. This suggests that although the CFT-tubules are formed by an endocytic pathway (Sokac, 2008), the tubules are not homogeneous. Instead, tubules can contain different domains that may have different functions. Three distinct types of tubules were identified: those that contain only amphiphysin and the septins Sep1 and Sep2, those that contain only the septins Peanut, Sep4, and Sep5, and those that possess heterogeneous subdomains each defined by a distinct composition of these various components. Of importance, localization studies suggest that distinct septin complexes localize to different structures. Because Peanut, Sep4, and Sep5 do not colocalize with Sep1 and Sep2 on CFT-tubules, it is predicted that Peanut, Sep4, and Sep5 form a novel septin complex. This new septin complex may resemble the previously isolated complex of Peanut, Sep1, and Sep2, as Sep2 is most closely related to Sep5 (72% identity) and Sep1 is most closely related to Sep4 (47% identity). It was not possible to isolate individual septin complexes by immunoprecipitation, as all septins coimmunoprecipitated. This finding is consistent with studies in mammalian cells and reflects either the heterogeneous nature of septin complexes within the entire embryo or that, in part, partial septin filaments were being immunoprecipitated. Unexpectedly, Peanut did not colocalize with Sep1 and Sep2 on CFT-tubules. This observation raises the possibility that Sep1 and Sep2 alone form a complex. Septin filaments in yeast and mammalian systems are generated from octamers containing two copies of four different septins arranged in an inverted repeat; however, this may not be true for all systems. In the case of Drosophila a hexamer of Peanut, Sep1, and Sep2 has been isolated, and in Caenorhabditis elegans there are only two septin genes (Su, 2013).

Septins have predominantly been implicated in modulating events at the plasma membrane in conjunction with the actin cytoskeleton. In mammalian cells, septins have also been linked to potential roles in membrane trafficking, especially in the exocytic pathway, possibly by regulating vesicle fusion. It seems unlikely that the septins on CFT-tubules are regulating exocytosis, as all evidence suggests that exocytosis occurs at distinct apical sites in the syncytial embryo). In contrast, one study suggests a role for septins in the endocytic pathway by regulating recruitment of the coat protein complex AP-3 to lysosomal membranes (Baust, 2008). The precise roles for septins in this process are unclear. In CFT-tubules, it is possible that septins exert an effect directly on the membrane. Septins can tubulate membranes containing phosphatidylinositol (4,5)-bisphosphate, a lipid that has a key role in cytokinesis. However, the current data demonstrate that CFT-tubule formation is dependent on amphiphysin. Septins have been proposed to stabilize membranes. Therefore septins could stabilize the CFT-tubules once formed. Indeed, reduced recruitment of septins to cleavage furrows destabilizes the entire cleavage cleavage furrow. Furthermore, embryos depleted of Peanut form unstable yolk channels at the end of cellularization, further supporting the model that septins can stabilize membrane structures to which they localize. These findings also suggest that mutations that deplete septins will not allow examination of the role of septins in CFT-tubule organization and function (Su, 2013).

This study found that CFT-tubule formation requires the BAR domain of amphiphysin. The N-BAR subfamily, to which amphiphysin belongs, can bind to membranes and promote their curvature. Amphiphysin is also involved in t-tubule formation in Drosophila indirect flight muscles and mouse heart muscle. These findings suggest a conserved role for amphiphysin in promoting tubule formation and organization (Su, 2013).

Loss of amphiphysin and the prevention of CFT-tubule formation did not inhibit furrow ingression, suggesting that amphiphysin is not required for remodeling of the membrane to drive furrow ingression. Instead, loss of amphiphysin increased the rate of furrow ingression. Because amphiphysin localizes to the tip of the furrow, it is possible that amphiphysin acts as a negative regulator of furrow ingression. Alternatively, by preventing CFT-tubule formation, amphiphysin may render more plasma membrane accessible for furrow ingression, and therefore the rate of furrow ingression increases. Consistent with this model, when CFT-tubules become longer upon disruption of dynamin, the rate of cleavage furrow ingression is reduced. One potential consequence of inhibiting endocytosis at the furrow tip would be to reduce the amount of membrane available for the expansion of the plasma membrane and the ingression of the furrow. In such a scenario membrane derived from endocytosis at the tip of the furrow would be recycled back to the plasma membrane through the exocytic pathway, thereby providing sufficient membrane for the expansion and ingression of the furrow. This reduced availability of membrane could manifest itself as a reduced rate of furrow ingression seen in shibire^ts embryos at the nonpermissive temperature, where CFT-tubules elongate due to a failure to pinch off. The additional membrane may be especially important for the rapid increase in furrow ingression that is seen once the furrow has ingressed ∼10 μm, a depth of ingression where CFT-tubules normally become shorter and disappear (Su, 2013).

Changes in tubule parameters correlate with changes in cleavage furrow ingression kinetics, especially in the fast phase of ingression; longer, more persistent tubules correlate with slower ingression kinetics, and the absence of tubules correlates with faster ingression kinetics. If the fast phase of cleavage furrow ingression were dependent upon new membrane being inserted into the plasma membrane, then restricting membrane insertion would suppress the fast phase. If membrane was recycled by endocytosis at the cleavage furrow tips through an endocytic compartment back to the plasma membrane, then changes in CFT-tubule parameters might be expected to affect cleavage furrow ingression kinetics (Su, 2013).

In the models outlined in this study CFT-tubules would function to buffer the amount of available membrane that is accessible for efficient cleavage furrow ingression. However, no comparable measurements have been made with respect to t-tubules in muscles. Therefore it remains unclear whether the tubules in these different systems have a common function, whether they are examples of specialized endocytosis, or whether the creation of extra membrane surface area facilitates specialized functions in these different systems (Su, 2013).

Principles of E-Cadherin supramolecular organization in vivo

E-cadherin plays a pivotal role in tissue morphogenesis by forming clusters that support intercellular adhesion and transmit tension. What controls E-cadherin mesoscopic organization in clusters is unclear. This study used 3D superresolution quantitative microscopy in Drosophila embryos to characterize the size distribution of E-cadherin nanometric clusters. The cluster size follows power-law distributions over three orders of magnitude with exponential decay at large cluster sizes. By exploring the predictions of a general theoretical framework including cluster fusion and fission events and recycling of E-cadherin, two distinct active mechanisms setting the cluster-size distribution were identified. Dynamin-dependent endocytosis targets large clusters only, thereby imposing a cutoff size. Moreover, interactions between E-cadherin clusters and actin filaments control the fission in a size-dependent manner. It is concluded that E-cadherin clustering depends on key cortical regulators, which provide tunable and local control over E-cadherin organization. The data provide the foundation for a quantitative understanding of how E-cadherin distribution affects adhesion and might regulate force transmission in vivo (Truong Quang, 2013).

Perturbing dynamin reveals potent effects on the Drosophila circadian clock

Transcriptional feedback loops are central to circadian clock function. However, the role of neural activity and membrane events in molecular rhythms in the fruit fly Drosophila is unclear. To address this question, a temperature-sensitive, dominant negative allele was expressed of the fly homolog of dynamin called shibire^ts1 (shi^ts1), an active component in membrane vesicle scission. Broad expression in clock cells resulted in unexpectedly long, robust periods (>28 hours) comparable to perturbation of core clock components, suggesting an unappreciated role of membrane dynamics in setting period. Expression in the pacemaker lateral ventral neurons (LNv) was necessary and sufficient for this effect. Manipulation of other endocytic components exacerbated shi^ts1's behavioral effects, suggesting its mechanism is specific to endocytic regulation. PKA overexpression rescued period effects suggesting shi^ts1 may downregulate PKA pathways. Levels of the clock component Period were reduced in the shi^ts1-expressing pacemaker small LNv of flies held at a fully restrictive temperature (29°C). Less restrictive conditions (25 degrees C) delayed cycling proportional to observed behavioral changes. Levels of the neuropeptide Pigment-dispersing factor (PDF), the only known LNv neurotransmitter, were also reduced, but Period cycling was still delayed in flies lacking PDF, implicating a PDF-independent process. Further, shi^ts1 expression in the eye also results in reduced Per protein and per and vri transcript levels, suggesting that shibire-dependent signaling extends to peripheral clocks. The level of nuclear Clk, transcriptional activator of many core clock genes, is also reduced in shi^ts1 flies, and Clk overexpression suppresses the period-altering effects of shi^ts1. It is proposed that membrane protein turnover through endocytic regulation of PKA pathways modulates the core clock by altering Clk levels and/or activity. These results suggest an important role for membrane scission in setting circadian period (Kilman, 2009).

Daily rhythms of sleep and wake are driven by transcriptional feedback loops in pacemaker neurons. In Drosophila, the transcription factor Clock (Clk) heterodimerizes with cycle (cyc) to directly activate key components of a principal feedback loop, period (per) and timeless (tim), and of a secondary feedback loop, par domain protein 1 (pdp-1) and vrille (vri). Per and perhaps Tim feed back and repress Clk/Cyc DNA binding leading to molecular oscillations in clock components. Vri feeds back to repress transcription of Clk, while Ppd may regulate clock output. Clk also activates clockwork orange (cwo), which represses Clk-activated transcription of its targets. These molecular feedback loops are thought to operate in a cell-autonomous manner. Several components of these feedback loops are conserved with mammals (Kilman, 2009).

Molecular clocks are evident in many peripheral tissues, such as the eye, as well as the central brain. Brain clocks are divided into 7 anatomical clusters: small and large ventral lateral neurons (sLNv, lLNv), dorsal lateral neurons (LNd), three groups of dorsal neurons (DN1, DN2, DN3), and the lateral posterior neurons (LPN). The neuropeptide Pigment Dispersing Factor (PDF) is expressed uniquely by and is the only known transmitter of the LNv. Mutants of PDF or its receptor display short period damping rhythms. pdf⁰¹ pacemaker molecular oscillations are eventually low amplitude or phase-dispersed, indicating PDF feeds back to maintain synchrony. Mammalian rhythms are also lost in mutants of the Vasoactive Intestinal Peptide (VIP) system, indicating a conserved role for neuropeptidergic signaling in clocks. Under light-dark conditions (LD), the PDF+ sLNv mediate behavioral anticipation of the transition from dark to light ('morning') while 'evening' anticipation is mediated by PDF- clocks: the DN1, LNd, and one sLNv. Under constant darkness (DD), the LNv dominate behavioral period determination and reset non-PDF clocks. PDF neurons may also receive a number of other neurotransmitter inputs. In addition, electrical silencing of PDF neurons suppresses core clock function. A number of intracellular signaling pathways have been identified as contributing to core circadian function. However, the mechanisms of feedback between receptor and/or ion channel signaling and transcriptional feedback rhythms remain unclear (Kilman, 2009).

To explore the role of the network in circadian function, vesicle traffic was perturbed as a way of disrupting intercellular communication. shibire (shi), the Drosophila homolog of dynamin, is a GTPase necessary for vesicle scission (Kosaka, 1983a and Kosaka, 1983b). The dominant negative shi^ts1 allele (van der Bliek, 1991;; Kitamoto, 2001) has been used at the restrictive temperature (29^oC) to inhibit synaptic transmission (Kitamoto, 2001). However shi is also involved in other endocytic pathways (Guha, 2003) that may affect intercellular signaling including receptor-mediated endocytosis and recycling of membrane proteins, such as ion channels. This study shows shi^ts1 expression in clock cells at 25^oC results in robust long behavioral rhythms. Period effects are exacerbated by perturbing endocytic/endosomal pathways and suppressed by overexpressing arrestin2 or a catalytic subunit of Protein Kinase A (PKA-C1). Long period results from PDF-independent delays in the molecular clock of the sLNv. With further impairment at 29^oC, shi^ts1 expression in either the LNv or in peripheral eye clocks also drastically reduces Clk target gene levels. Clk itself is reduced in the sLNv and the long period is suppressed by Clk overexpression. These results suggest that modulation of cell membrane processes such as receptor signaling pathways may powerfully affect the molecular clock (Kilman, 2009).

These data suggest an important function for membrane events, specifically endocytosis, in circadian timing. While previous studies have demonstrated roles for neural activity in circadian output, in sustaining molecular rhythms, and in synchrony, this work strongly suggests a substantial role in circadian timing. Expression of shi^ts1 in pacemaker neurons results in strikingly long periods, suggesting potent effects on circadian timing through perturbing vesicle scission. These effects are enhanced by co-expression of other components of endocytic pathways leading to early endosomes, consistent with shi function in traffic, recycling, and turnover of cell membrane components. PKA expression rescues period defects induced by shi^ts1, suggesting a functional link between the membrane, PKA, and behavioral period. The LNv-expressed shi^ts1 results in delays in the phase of Per molecular rhythms in the sLNv sufficient to account for the delay in behavior. While shi^ts1 effects on behavior require Pdf, those on the molecular clock of the sLNv are Pdf-independent, implicating a novel pathway. In fact these perturbations of the molecular clock are not specific to locomotor pacemakers, but appear in peripheral clocks as well, suggesting membrane-clock interactions are a general property of clock cells. Reductions in the levels of Clk and Clk-activated transcripts are consistent with the hypothesis that membrane events regulate the molecular clock through Clk (Kilman, 2009).

Several lines of evidence indicate that shi^ts1 effects are not operating principally by blocking pacemaker neural output. Expression of tetanus toxin in PDF neurons blocks responses to arousing effects of cocaine, indicating that PDF neurons use a classical neurotransmitter and that tetanus toxin is expressed at functional levels capable of blocking this process. Yet tetanus toxin expression in PDF+ cells does not significantly alter period or rhythmicity. In shi^ts1 expressing flies, delayed PDF neuronal clocks still delay the offset of evening behavior, implying PDF cells can still reset evening clocks. In addition, no desynchronization was observed of molecular rhythms among the sLNv as might be expected if communication were disrupted. Period altered shi^ts1-expressing flies also largely preserve rhythmicity at 25^oC suggesting a primary clock effect rather than an output effect. Likewise PDF, the only known sLNv output, is also not necessary for shi^ts1 molecular effects. In pdf⁰¹ mutants, shi^ts1 expression blocks the effects on behavioral period but does not block the effect of shi^ts1 on PER LNv rhythms. The uncoupling of sLNv molecular rhythms from behavioral rhythms clearly demonstrates an output function for PDF in pacemaker neuron function. This also implies that other neural clusters drive behavior in pdf⁰¹. Moreover, these results demonstrate that shi^ts1's effects on sLNv PER do not operate through PDF. Taken together these data suggest the period differences that were seen do not result primarily from alterations of sLNv transmitter output. Instead it seems likely shi^ts perturbs another target or pathway regulating sLNv activity (Kilman, 2009).

While effects of shi^ts1 are typically tested at 29°C or above, shi^ts1 effects noted in this study have been observed at just 25°C, below the reported paralytic temperature for shi^ts1 (Masur, 1991). However, ultrastructural shi^ts1 effects have been observed even at the nominal permissive temperature (18°C) for behavioral paralysis (Masur, 1991). Thus, shi^ts1 is likely modestly defective at 18°C and this impairment grows with increasing temperature until a threshold is reached at which paralysis is evident when driven in motorneurons. However under conditions of overexpression, the temperature threshold for various phenotypes may differ from paralysis. The finding of slight period lengthening relative to controls even at 18°C is consistent with a modest defect, with core clock effects getting stronger gradually as the temperature increases. The evidence that shi^ts1 is not perturbing outputs (at least at 25°C) raises that possibility that other membrane scission-sensitive processes, such as receptor endocytosis, may have a lower threshold for disruption than synaptic transmission (Kilman, 2009).

What might be the nature of the membrane perturbation evoked by shi^ts1? More broadly, endocytosis regulates membrane protein turnover, and a variety of targets could influence neuronal activity, including ion channels, pumps, and transporters, which in turn could feedback to regulate the core clock. Endocytosis has a well-established role in down-regulation of metabotropic or ionotropic receptors. In the sLNv, potential receptors include (but are not limited to) acetylcholine, GABA, serotonin, dopamine, histamine, and neuropeptides such as IPNamide. Ion channel density may also be modulated by endocytosis and could influence core clock rhythms. In contrast, the finding that PKA overexpression can suppress shi^ts1 effects on period provide evidence that down regulation of G-protein coupled receptors that stimulate cAMP and PKA may be a mechanism for shi action. The identification and functional analysis of the relevant membrane targets of shi^ts1 will be critical to understanding the role of the membrane in circadian function (Kilman, 2009).

Awd, the homolog of metastasis suppressor gene Nm23, regulates Drosophila epithelial cell invasion

Border cell migration during Drosophila oogenesis is a highly pliable model for studying epithelial to mesenchymal transition and directional cell migration. The process involves delamination of a group of 6 to 10 follicle cells from the epithelium followed by guided migration and invasion through the nurse cell complex toward the oocyte. The guidance cue is mainly provided by the homolog of platelet-derived growth factor/vascular endothelial growth factor family of growth factor, or Pvf, emanating from the oocyte, although Drosophila epidermal growth factor receptor signaling also plays an auxiliary role. Earlier studies implicated a stringent control of the strength of Pvf-mediated signaling since both down-regulation of Pvf and overexpression of active Pvf receptor (Pvr) resulted in stalled border cell migration. This study shows that the metastasis suppressor gene homolog Nm23/awd (abnormal wing discs) is a negative regulator of border cell migration. Its down-regulation allows for optimal spatial signaling from two crucial pathways, Pvr and JAK/STAT. Its overexpression in the border cells results in stalled migration and can revert the phenotype of overexpressing constitutive Pvr or dominant-negative dynamin (Shibire or Shi). The functional relationship between awd and shi is highly specific and almost exclusive in the endocytic pathway. The functional relationship between Nm23/Awd and dynamin has prompted the suggestion that Nm23/Awd is a GTP supplier for dynamin, a GTPase. Nonetheless, the putative antimetastasis activity of Nm23/Awd has never been demonstrated in a physiologically relevant metastasis or epithelial to mesenchymal transition model. This is a rare example demonstrating the relevance of a metastasis suppressor gene function utilized in a developmental process involving cell invasion (Nallamothu, 2008).

This report describes a novel role of a negative regulator of directional migration in border cells. Specifically, the significance of developmentally regulated loss of Awd expression in border cells during their active migratory phase was studied. Ectopic expression of Awd effectively blocks border cell movement, suggesting that Awd is involved in modulating the directional movement of the border cell complex. Conversely, a high level of Awd expression was observed in the nonmigrating follicle cells, possibly promoting rapid turnover of surface receptors to prevent ectopic cell migration. Indeed, loss of awd in these cells and in premigratory border cells resulted in up-regulation of Pvr and stalling of border cells, consistent with the phenotype of overactive Pvr signaling reported previously observation of overexpression of wild-type Pvr. The results show that the function of Awd is to promote down-regulation of the surface receptors such as Pvr and Dome, in collaboration with Shi/dynamin, thereby regulating the chemotactic signal strength. Although the function of Awd has been linked to dynamin (Dammai, 2003; Krishnan, 2001), this report is the first on the relevance of the Nm23/Awd antimetastasis function in an analogous developmental model. This study has demonstrated that border cell migration is stalled by both ectopic expression of Awd in the migrating cells and knockdown of Awd in premigrating cells, although through opposite consequences of reducing and increasing Pvr expression, respectively. This is consistent with the published finding that both loss of function and gain of function in Pvr signaling can disrupt border cell migration, due to loss of chemotactic signal or overwhelming signal, respectively. It was proposed and subsequently demonstrated by time-lapse microscopy that border cells that are oversaturated with Pvf signaling spin around without moving forward, consistent with the overborne, nondirectional chemotactic signaling response; while pvr loss-of-function border cells do not move. That is, although the phenotypic outcomes are the same, the cellular behaviors of the two genetic conditions are just the opposite (Nallamothu, 2008).

At this time, the cellular events that precisely down-regulate Awd expression in migrating border cells remain unknown. However, the observations suggest that the regulatory mechanism, besides potential transcriptional regulation, could at least in part be posttranscriptional. For example, the slbo-GAL4 driver can usually induce very high levels of ectopic expression, as evidenced by the expression of UAS-{lambda}pvr in this study. However, with UAS-awd (without the endogenous 3' untranslated region), it was possible to achieve at best a level equal to the endogenous one in nearby follicle cells and very often much lower (Nallamothu, 2008).

The histidine-dependent phosphotransferase activity of Nm23/Awd has functional correlation with the production and usage of GTP and the Awd-GTP link is worth noting since dynamin is a GTPase. In a classic study to identify components of eye color pathway, one peculiar, otherwise healthy mutant caused dominant lethality in the viable eye color prune null mutant background (Sturtevant, 1956). This dominant conditional lethal allele was named Killer-of-prune (K-pn) and turned out to be a missense mutant allele of awd. This is highly interesting because the Drosophila eye pigmentation is determined by pteridines that is also a precursor of essential enzyme cofactors. The rate-limiting enzyme in pteridine biosynthesis is GTP cyclohydrolase, which uses substrate GTP to generate dihydroneopterin triphosphate. It was suggested that the Prune protein, which contains pyrophosphatase activity, stabilizes or promotes Nm23/Awd multimeric protein activity by channeling the phosphate. It is possible that Awd and Prune proteins together form a relay system for generating GTP. Therefore, the K-pn mutation of awd in the prune mutant background renders the phosphate transfer function of the Prune-Awd protein complex even less stable. Indeed, among the myriad of interacting proteins of Nm23 in mammalian cells, many are related directly or indirectly to the GTPases, such as Arf6, TIAM1 (a guanine exchange factor for Rac), Lbc (a guanine exchange factor for Rho), and Rad. Whether or not these GTPase-related functions hold true requires further in vivo investigation. Recently, the lysophosphatidic acid receptor EDG2 was found to be overexpressed in Nm23-H1 mutant metastatic breast cancer cells, which can account for the metastatic activity of this cell line. However, whether the up-regulation is a direct or downstream effect of Nm23 loss of function is not clear. It therefore remained to be determined whether the similar receptor down-regulation mechanism by Awd observed in this report is applicable to EDG2 regulation (Nallamothu, 2008).

It should be noted, however, that although the observed genetic interaction between awd and shi suggests that Awd may promote the endocytic activity of Shi/dynamin, it is formally possible that Awd may promote protein turnover that is downstream of the initial endocytic event. On this note, it is also worth considering other activities of Nm23/Awd. The results showed that substitution of the active-site histidine residue that is critical for the nucleoside diphosphate kinase activity could not stall border cell migration. This is consistent with previous finding that this residue is required for rescuing the enhancer of shi phenotype (Krishnan, 2001). Curiously, this residue is not required for suppressing the in vitro motility (assayed by Boyden chamber) of the metastatic breast cancer cells. However, the histidine substitutions employed in the two systems are different (phenylalanine in human versus alanine in fly). It is therefore difficult at this time to draw a direct comparison. In contrast, human mutants that affect the histidine-dependent protein kinase activity failed to suppress the in vitro motility of the cancer cells. So far, very few Nm23 protein kinase targets have been identified and none verified in physiological settings. Nonetheless, the protein kinase activity may be of specific functional significance since the range of targets is likely limited, so that specific pathways that contribute to metastasis may be identified more readily. The border cell migration model describe here should be used in future studies to test the functions of Nm23/Awd based on the above-mentioned human mutations (Nallamothu, 2008).

Drosophila Cip4 and WASp define a branch of the Cdc42-Par6-aPKC pathway regulating E-Cadherin endocytosis

Integral to the function and morphology of the epithelium is the lattice of cell-cell junctions known as adherens junctions (AJs). AJ stability and plasticity relies on E-Cadherin exocytosis and endocytosis. A mechanism regulating E-Cadherin (E-Cad) exocytosis to the AJs has implicated proteins of the exocyst complex, but mechanisms regulating E-Cad endocytosis from the AJs remain less well understood. This study shows that Cdc42, Par6, or aPKC loss of function is accompanied by the accumulation of apical E-Cad intracellular punctate structures and the disruption of AJs in Drosophila epithelial cells. These punctate structures derive from large and malformed endocytic vesicles that emanate from the AJs; a phenotype that is also observed upon blocking vesicle scission in dynamin mutant cells. The Drosophila Cdc42-interacting protein 4 (Cip4) is a Cdc42 effector that interacts with Dynamin and the Arp2/3 activator WASp in Drosophila. Accordingly, Cip4, WASp, or Arp2/3 loss of function also results in defective E-Cadherin endocytosis. Altogether These results show that Cdc42 functions with Par6 and aPKC to regulate E-Cad endocytosis and define Cip4 and WASp as regulators of the early E-Cad endocytic events in epithelial tissue (Leibfried, 2008).

Cdc42 has been implicated in the regulation of polarity establishment in the early Drosophila embryo. The function was shown to be dependent upon the interaction of Cdc42 with the Baz-Par6-aPKC complex that promotes the exclusion of Lgl through Lgl phosphorylation by aPKC. However, the role of Cdc42 in epithelial tissue is unlikely to depend only on its regulation of aPKC because aPKC was shown to be dispensable for apico-basal polarity establishment in the Drosophila embryo. The role of Cdc42 in mammalian epithelial cells has so far been examined by the expression of constitutively active and dominant-negative forms of Cdc42, and such an examination has led to conflicting results in establishing the exact role of Cdc42 in apico-basal polarity maintenance. Nonetheless, they point toward an important role of Cdc42 in the regulation of polarized trafficking. The possible role of Cdc42 in polarized trafficking in epithelial cells was further strengthened by the identification of Cdc42 and the Par complex as regulators of endocytosis in both mammalian cells and C. elegans. Nevertheless, the precise role of Cdc42 and the Par complex in the regulation of endocytosis has remained poorly understood except in migrating cells in which the Par complex was shown to inhibit integrin endocytosis via Numb (Leibfried, 2008).

Cdc42 and its effector Drosophila Cip4 have been found to regulate E-Cad endocytosis and that their loss of function is associated with the formation of long tubular endocytic structures similar to what is observed upon blocking Dynamin function. It is therefore proposed that in Drosophila epithelial cells, Cdc42 controls the early steps of E-Cad endocytosis via Cip4. Because Cdc42, aPKC, and Par6 loss of function are associated with similar defects in E-Cad and Cip4 localization, a simple model is favored, in which the loss of aPKC or Par6 activity disrupts Cdc42 localization or activity and in turn prevents Cip4 function (Leibfried, 2008).

The identified role of PCH family of protein stems in part from the biochemical analysis of Toca-1 as a regulator of actin polymerization. Toca-1 is necessary to activate actin polymerization and actin comet formation downstream of PIP2 and Cdc42 in a WASp-dependent manner (Ho, 2004). On the basis of elegant biochemical assays, Toca-1 was further shown to be necessary to alleviate the WIP inhibitory activity on WASp, in order to allow efficient Arp2/3 activation by WASp (Ho, 2004). Toca-1 was proposed to play an essential role in the fine spatial and temporal regulation of actin polymerization in both cell migration and vesicle movement. Cip4 has been implicated in microtubule organizing center (MTOC) polarization in immune natural killer cells (Banerjee, 2007), a process in which Cdc42 and the Par complex are also involved. Importantly, because Cip4 was shown to bind microtubules, the interaction between Cdc42 and Cip4 might indicate that Cip4 might also be an effector of Cdc42-Par complex in the regulation of MTOC polarization (Leibfried, 2008).

In mammalian cells, regulation of endocytic-vesicle formation has been proposed to be dependent upon both branched actin-filament formation and Dynamin. The role of WASp and Arp2/3 in the regulation of E-Cad endocytosis may therefore indicate that Cip4, which is also known to form dimers, can promote vesicle scission by recruiting Dynamin and promoting actin polymerization via WASp. Therefore, it is proposed that Cip4 and WASp act as a link between Cdc42-Par6-aPKC and the early endocytic machinery to regulate E-Cadherin endocytosis in epithelial cells (Leibfried, 2008).

Dopamine-mushroom body circuit regulates saliency-based decision-making in Drosophila

Fruit flies can make appropriate choices among alternative flight options on the basis of the relative salience (prominence) of competing visual cues. This choice behavior consists of early and late phases; the former requires activation of the dopaminergic system and mushroom bodies, whereas the latter is independent of these activities. Immunohistological analysis showed that mushroom bodies are densely innervated by dopaminergic axons. Thus, the circuit from the dopamine system to mushroom bodies is crucial for choice behavior in Drosophila (Zhang, 2007).

Value-based decision-making is a complex behavior controlled, in part, by the dopamine system. Primates make choices among many available options to produce an advantageous response. The complexity of the mammalian brain has made it difficult to fully understand the neural circuits underlying value-based decision-making. To discern these circuits, this phenomenon was studied in Drosophila, because the functions of dopamine neurons are largely conserved evolutionarily. For example, forming aversive olfactory memories in Drosophila requires dopamine, allows punishment prediction, and involves neural activities that are similar to primates and rodents during conditioning (Zhang, 2007 and references therein).

To explore the circuitry mediating value-based choice behavior of fruit flies, a novel paradigm was developed involving relative saliency evaluation of contradictory cues. Flies were trained in a flight simulator to associate heat punishment with one of two bars with compound cues, position (upper and lower) and color (blue and green). After training with one bar (e.g., upper and blue), flies were confronted with conflicting cues (e.g. upper-green and lower-blue) and had to decide whether to follow the position or color cue depending on their relative saliency. Position and color saliency were quantified by vertical separation between the bar center of gravity (DeltaCOG) and color intensity (CI), respectively. Amount of time spent in the conditioned quadrants was quantified as a preference index (PI) over 2-min blocks. Wild-type Berlin (WTB), Canton-S (CS), and mutant mushroom body miniature¹ (mbm¹) flies were trained with an upper-blue bar (CI = 1.0 and DeltaCOG = 60°). They were then tested for choice behavior by changing both the color (blue to green; CI unchanged) and position cue saliency (DeltaCOG from 60° to 40°). Wild types preferentially chose the position cue and followed the upper-green bar, whereas mutants could not decide which bar to follow, as evidenced by substantially reduced PIs (Zhang, 2007).

To further characterize choice behavior, WTB, CS and mbm¹ flies were tested by using a wide range of position cue saliencies (DeltaCOG: 0° to 60° in 5° or 10° increments) without changing the color cue CI. The choice curve of wild types (WTB and CS) exhibited a distinct transition in the preference for position cues, as a function of relative salience (position versus color), at DeltaCOG = 30° and could be fit by a sigmoid function (Boltzmann fit, r² = 0.97 for WTB and CS). In contrast, position cue preference in mbm¹ flies climbed up progressively (linear fit, r² = 0.92). Mushroom bodies (MBs) are essential for olfactory but not visual reinforcement learning, and, in the visual choice paradigm, mbm¹ flies could not distinguish pertinent position or color cues when their saliencies varied. This is consistent with previous findings that MBs participate in decision-making when Drosophila confronts a shape-color dilemma (Tang, 2001). Flies could interpret cue saliency as a representation of punishment probability and alter their choice strategy accordingly. Along these lines, without prior training wild types randomly chose all saliency cues (Zhang, 2007).

Primate studies suggest two general categories of decision-making: simple perceptual and value-based. The former is based on simple linear subtraction of alternative sensory inputs, and the latter on nonlinear calculation of the relative values of stimuli. This stud investigated which decision-making type fruit flies used when faced with conflicting visual cues. For this purpose, flies were trained with both color and position cues (CI = 1.0 and DeltaCOG = 60°), and then their preference for a single cue (each tested separately) was assessed during the posttraining session. When position cue saliency was varied (DeltaCOG from 0° to 60°), a sigmoid retrieval curve was not evident. Wild-type and mbm¹ flies performed similarly under these conditions indicating that retrieval of single visual cues is not MB dependent. It was then asked how visual perception of separated cues after compound training contributes to decision-making. The choice curve predicted by subtracting the PI at CI = 1.0 from the PIs of position cues (DeltaCOG from 0° to 60°) was linear and similar to the performance of mbm¹ flies in the position-color dilemma. Thus, mbm¹ flies make perceptual decisions in conflict situations by a simple subtraction mechanism, which is thought a general mechanism for perceptual decision-making in the human brain. In contrast to mbm¹, wild-type flies performed according to a sigmoid choice curve, and the mechanism underlying should be beyond simple comparison of the different cues perception (Zhang, 2007).

How and when MBs contribute to the decision-making process was investigated by selectively disrupting their function at different stages of choice behavior with shibire^ts1 (shi^ts1), a temperature-sensitive mutant form of dynamin. In shi^ts1 mutants, synaptic transmission is normal at permissive temperature (PT, below 30°C) and blocked at restrictive temperature (RT, above 30°C). Transgenic 247/upstream activation sequence (UAS)-shi^ts1 flies, with restricted shi^ts1 expression in MBs, were trained to follow bars with compound cues (CI = 1.0 and DeltaCOG = 60°) at PT (24°C) then tested at RT (30°C) for 6 min of choice performance with conflicting cues. They showed a sigmoid choice curve at PT, but a linear one at RT, which is similar to mbm¹ flies; wild types were unaffected by the temperature shift (Zhang, 2007).

Dopamine plays a crucial role in the motivation to acquire a reward or avoid a punishment. In Drosophila, dopaminergic transmission also mediates punishment prediction and associates punishment with a conditioned stimulus (Riemensperger, 2005). Expression of shi^ts1 in dopaminergic neurons is triggered by tyrosinse hydroxylase (TH)-Gal4 and dopa decarboxylase (Ddc)-Gal4. Ddc/UAS-shi^ts1 flies express shi^ts1 in both dopaminergic and serotoninergic neurons, whereas TH-Gal4/UAS-shi^ts1 flies express it only in the former. Both types of transgenic flies were tested for choice behavior and exhibited a sigmoid choice curve at PT, similar to wild types. However, their choice behavior was severely impaired at RT, as evidenced by a linear choice curve, indicating that dopamine deprivation was sufficient to disturb decision-making based on relative cue saliency (Zhang, 2007).

Because dopaminergic synaptic activity is necessary for memory acquisition in aversive olfactory conditioning, blocking it could impair visual memory required for decision-making rather than the process itself. To address this issue, flies were trained at PT and their preference tested for conditioned cues at RT, which required memory retrieval. Flies of all genotypes (CS, 247/UAS-shi^ts1, Ddc/UAS-shi^ts1, and TH/UAS-shi^ts1) performed similarly at both temperatures. Therefore, reduced dopaminergic transmission specifically disrupts saliency-based decision-making (Zhang, 2007).

In addition to MBs, the ellipsoid body (EB) in the Drosophila central complex was examined for its potential contribution to decision-making. Transgenic flies c507/UAS-shi^ts1 expressing shi^ts1 specifically in the EB showed normal sigmoid choice behavior at both temperatures, indicating that the EB is not critical for this behavior (Zhang, 2007).

Both dopamine and MBs are involved in saliency-based decision-making, and D1-type dopamine receptors are densely distributed in MB lobes. To determine how dopamine and MBs interact, the anatomical relation between them was examined by simultaneously expressing a red fluorescent protein (RFP), driven by 247-Gal4, specifically in MB neurons and visualizing dopaminergic neurons with immunostaining for TH, an enzyme specifically used in dopamine synthesis. Dopaminergic fibers were broadly distributed in Drosophila brain, with the highest density around MBs. Higher magnification showed that TH staining was concentrated in MB lobes rather than calyces or peduncles; thus, dopaminergic processes occupy MB lobes containing Kenyon cell axons, as confirmed by labeling dopaminergic neurons with green fluorescent protein (GFP)–tagged synaptic vesicle protein Synaptotagmin I (Syt I). Furthermore, dopaminergic axons, not dendrites, invade MB lobes, because the dendrite-specific Drosophila Down Syndrome Cell Adhesion Molecule conjugated to GFP (Dscam[17.1]-GFP) in dopaminergic neurons did not colocalize with immunostaining for the MB marker Fasciclin II (Fas II). Dopaminergic axons specifically innervate MB lobes, because the prominent lobe-like profile of dopaminergic fibers was largely abolished in flies treated with hydroxyurea (HU) to ablate MBs. Their absence in calyces suggests that dopamine regulates MBs by acting on Kenyon cell output (Zhang, 2007).

To determine whether choice behavior is time dependent, decision-making was examined at different times after flies encountered conflicting visual cues. During the first 30 s of conflict cues presentation, wild types (WTB and CS) showed linear choice performance according to position cue saliency; however, sigmoid choice behavior was evident at 90 to 120 and 330 to 360 s. These results suggest that decisive choices are time dependent and that the early test phase likely involved simple perceptual decision-making. To explore the circuits involved, MBs and dopaminergic function were selectively disrupted at varying times after choice behavior testing began with temperature-sensitive 247/UAS-shi^ts1 and TH/UAS-shi^ts1 flies. Flies were given a choice test using a DeltaCOG shift of 60° to 40° with CI = 1.0 because these parameters caused the largest difference in choice behavior between mutants and wild types. After testing started, flies were kept at PT for 1, 2, or 4 min before exposure to RT. Both 247/UAS-shi^ts1 and TH/UAS-shi^ts1 flies executed clear choices at PT; however, those kept at PT for 1 or 2 min, but not 4 min, performed worse at RT. These findings indicate that MB dopaminergic activity is only required during the first 4 min after encountering conflicting cues and not after stable choice behavior is established (Zhang, 2007).

The above results suggest that choice behavior of flies requires two phases: an initial involving dopaminergic and MBs activities and a later executing phase that is independent of these activities. Accordingly, it was hypothesized that, if flies were presented with a second set of conflicting cues, then dopamine system and MB would be reactivated. This hypothesis was tested by first determining whether wild types correctly discern the salient cue after sequential transition of cue positions (DeltaCOG shift from 60° to 40° then to 20°, at CI = 1.0); their choice was not significantly different from that seen after a direct transition (DeltaCOG shift from 60° to 20°). Next, TH/UAS-shi^ts1 and 247/UAS-shi^ts1 flies were exposed to two sequential sets of conflicting position-color cues and exhibited normal choice behavior with notable PIs for the first choice test at PT (DeltaCOG = 40°, upper-green bar, and CI = 1.0). However, when these flies were tested for the second cue set (DeltaCOG = 20°, upper-green bar, and CI = 1.0), they followed the color rather than the position cue, resulting in negative PIs. Both transgenic fly strains performed correctly at PT but incorrectly (PIs near zero) when the second choice test was performed at RT, whereas their visual perception to DeltaCOG = 20° was still normal. Flies were also tested with a shape-color dilemma as the second choice and acted similarly to the performance in position-color dilemma. Thus, the dopamine- and MB-independent execution of a decision is specific for an established choice condition; a new conflicting set again requires dopamine and MB activities for decision-making (Zhang, 2007).

This study demonstrated two distinct decision-making processes in Drosophila: one that is nonlinear and saliency-based and the other that is linear, simple perceptual. The latter process could be performed in the absence of dopaminergic-MB circuits by subtracting the saliency of conflicting cues, but the ability to amplify the difference at crucial points was compromised. Thus, linear choice performance was displayed instead of the sigmoid pattern of wild types. It is proposed that changing from linear to nonlinear decision-making depends on a gating mechanism of the dopaminergic-MB circuit whereby only the stronger 'winner' signal is transmitted to the MB while other weaker inputs are inhibited. Thus, flies implementing the gating function in MBs and the amplification effects of dopamine can accomplish a winner-takes-all decision. Two different phases, namely formation and execution, are involved in saliency-based decision-making in Drosophila, and a dynamic balance must be established between maintaining an existing choice and switching to a new decision (Zhang, 2007).

Roles for Drosophila mushroom body neurons in olfactory learning and memory

Olfactory learning assays in Drosophila have revealed that distinct brain structures known as mushroom bodies (MBs) are critical for the associative learning and memory of olfactory stimuli. However, the precise roles of the different neurons comprising the MBs are still under debate. The confusion surrounding the roles of the different neurons may be due, in part, to the use of different odors as conditioned stimuli in previous studies. This study investigated the requirements for the different MB neurons, specifically the α/ß versus the γ neurons, and whether olfactory learning is supported by different subsets of MB neurons irrespective of the odors used as conditioned stimuli. The rutabaga (rut)-encoded adenylyl cyclase was expressed in either the γ or α/ß neurons and the effects were examined on restoring olfactory associative learning and memory of rut mutant flies. A temperature-sensitive shibire (shi) transgene was expressed in these neuron sets and the effects of disrupting synaptic vesicle recycling on Drosophila olfactory learning was examined. These results indicate that although odor-pair-specific learning was not detected using GAL4 drivers that primarily express in γ neurons, expression of the transgenes in a subset of α/ß neurons resulted in both odor-pair-specific rescue of the rut defect as well as odor-pair-specific disruption of learning using shi^ts1 (Akalal, 2006).

Drosophila olfactory learning is typically assayed using olfactory classical conditioning. Using this assay, several memory mutants and the genes involved in olfactory memory formation have been identified. This assay has also been used to investigate the roles of the different MB lobes. In each case, GAL4 drivers that express in distinct lobes of the MBs were used. When G-protein signaling was disrupted using pan-MB GAL4 drivers (238y, c747, and c309) to express a constitutively activated stimulatory heterotrimeric GTP-binding protein α-subunit (Gα_s*), associative olfactory learning was reported to be completely abolished. Using 201y-GAL4, a line that expresses extensively in the γ lobes but only in the narrow core elements of the α/ß lobes, learning was only reduced by ~50%. In a study that investigated the effects of expressing shi^ts1 using c739-GAL4, an α/ß driver, and 201y-GAL4, a significant impairment of performance was observed when neurotransmission was transiently inactivated through the α/ß lobes with only a slight, but nonsignificant, decrease in memory performance observed using 201y-GAL4, suggesting a greater role for MB α/ß lobes in olfactory memory. Other experiments involving rescue of the rut mutant defect by expressing a wild-type rut cDNA showed that memory was restored to wild-type levels using broad-MB GAL4 drivers (247, c772, and 30y) and the γ lobe driver H24-GAL4. However, memory was only partially rescued using 201y-GAL4, and no rescue was observed using the GAL4 drivers 189y and 17d, which both express primarily in the MB α/ß lobes. This, therefore, suggested a greater role of MB γ lobes in olfactory learning. The apparent contradictions among each of these studies could be due to the fact that in each of these experiments different combinations of odors were used for the olfactory learning assay: MCH-OCT, MCH-BEN, and OCT-BEN. To resolve the apparent discrepancies and inconsistencies among the different studies, this study used three commonly used odor combinations (MCH-OCT, MCH-BEN, and OCT-BEN) and two different assays, shi^ts1 inactivation of neurotransmission as well as rescue of the rut memory defect, to examine the roles of the different neurons comprising the MB lobes (Akalal, 2006).

Expressing transgenes using the two γ lobe GAL4 drivers NP1131 and H24 did not produce any odor-pair-specific learning effects. Using these γ drivers to express a rut cDNA in a rut mutant background results in a partial rescue of the learning defect for each of the odor combinations tested. The γ driver of choice has typically been H24-GAL4, but the expression pattern of this driver is not limited to the γ neurons. In fact, it expresses very robustly in the ALs, and one concern is that this might affect performance scores since the learning assay is based on olfactory cues. The use of NP1131-GAL4 that expresses primarily in the γ neurons and a small subset of α'/ß' neurons is important to validate the results for H24-GAL4. A prior study using H24-GAL4 to rescue the rut learning defect resulted in performance scores that were statistically indistinguishable from wild-type flies using MCH and BEN. However, among the different lines that were observed to rescue the rut defect, H24-GAL4 yielded the lowest scores, and another γ driver, 201y-GAL4, used in the same study failed to rescue the defect. Yet another study that used H24-GAL4 to restore a rut cDNA in a rut mutant background failed to produce rescue of the defect using MCH and OCT as odorants. The current data suggest that driving a rut cDNA in γ neurons results in rut levels that partially rescue the defect. Although no odor-pair-specific learning was detected using the two γ drivers tested, this does not preclude the possibility that when γ drivers that express in other subsets of γ neurons are discovered, this would remain the case. It is likewise important to note that the three odorants used in in the current study do not represent the whole repertoire of odors that a fly responds and learns to. Thus, it remains possible that the γ neurons labeled by NP1131-GAL4 and H24-GAL4 may still be involved in the odor-pair-specific associative learning of other smells (Akalal, 2006).

Rut rescue using GAL4 drivers that express in the α/ß neurons reveals some odor-pair-specific effects. Performance scores for flies carrying c739-GAL4 together with the UAS-rut transgene in a rut mutant background show no rescue of the rut defect for all odor combinations tested. In contrast, driving a rut cDNA using 17d-GAL4 shows partial rescue of the rut defect when MCH-BEN and OCT-BEN are used for the assay but not for MCH-OCT. To address the observed contrast in the rescue using the different odor combinations for these two lines, the expression patterns for the two α/ß drivers was compared. The expression level for c739-GAL4 is greater than 17d-GAL4, and it appears that only a subset of neurons, a thin core, is labeled for 17d-GAL4. Using antibodies raised against glutamate, a further subdivision of the lobes has been described as a slender core of glutamatergic neurons, the α and ß core neurons (αc and ßc), that lie posteriorly and are partly enclosed by the α and ß neurons. Whether the core neurons that are marked by 17d-GAL4 correspond to the glutamatergic subdivision of the α/ß lobes remains to be determined. One explanation for the observed odor-pair-specific effects is that 17d-GAL4 and c739-GAL4 express in nonoverlapping regions of the MB α/ß neurons. Behavioral experiments were performed on flies that were 2–5 d old, and although the neuron counts make clear that the expression of c739-GAL4 is broader than 17d-GAL4, it was not possible to confirm whether there is nonoverlapping expression at the α/ß core. Preliminary data suggest, however, that during this time period, the spatial expression of 17d-GAL4 is in the core region, while c739-GAL4 expression is more peripheral with a slight overlap of expression as a ring around this core. Additional experiments are needed to extend these observations (Akalal, 2006).

What is the significance of organizing the Drosophila MBs into different lobes, and is there differential representation of odors in the different lobes? These findings indicate that to answer this question one must take into account the choice of odor pairs used in the olfactory assay. The differential effects seen when using different odor combinations for 17d-GAL4 suggest that even though this driver expresses in a subset of α/ß neurons, some of these neurons must be important in MCH-BEN and OCT-BEN learning since driving rut using c739-GAL4, a line that expresses in a greater number of α/ß neurons, is insufficient to see a partial rescue of the rut learning defect. Clearly, this partial rescue is more than a mass effect of rut-expressing MB neurons. This raises an important point: It may not be accurate to describe GAL4 driver patterns based solely on lobe-specificity, since different GAL4 drivers may highlight subsets of the neurons comprising individual lobes. The significance of achieving complete rescue when the double GAL4 c739;H24 was used to express UAS-rut in a rut mutant background is still unclear. Although it is tempting to speculate that this complete rescue occurs by completing a spatial circuit that involves odorant representation in both α/ß and γ neurons, more experiments need to be performed to investigate whether it may just represent the massed effect of expression in more MB neurons (Akalal, 2006).

Examination of the odor-evoked activity in Drosophila MB neurons by expressing a green fluorescent protein-based Ca²⁺ indicator, G-CaMP, have revealed remarkable spatial stereotypy. In fact, several studies have shown that stereotypical anatomical and functional organization can be found at the different levels of the insect olfactory pathway. Each olfactory receptor neuron (ORN) likely expresses a single olfactory receptor (OR) gene, and ORNs that express the same OR genes converge on a common glomerulus in the AL, resulting in a stereotyped projection pattern. A near complete map of ORN connectivity constructed through a systematic survey of Drosophila OR expression has validated the principles of 'one neuron-one receptor' and 'one glomerulus-one receptor.' Different odors activate different combinations of ORs, and individual receptors can mediate both excitatory and inhibitory responses to different odors in the same cell. In addition, a topographic organization of the AL has been described wherein ORNs in distinct sensilla types project into distinct regions of the AL. At the level of the MB neuron cell bodies and the calyx, different odors evoked distribution patterns of fluorescence that were odor-specific and conserved across flies, resulting in stereotyped responses for BEN-, MCH-, and OCT-evoked fluorescence activity at both the wide-field and single-neuronal level. The current results demonstrate an odor-pair-specific effect with 17d-GAL4 using two odor combinations that have BEN as one of the odors. Several studies have indicated that Drosophila processes BEN differently from other odors. In fact, although surgical removal of the antennae and palps of wild-type flies results in the abolishment of MCH and OCT avoidance, BEN avoidance is only partially affected in both T-maze and arena paradigms, suggesting that BEN is sensed through other nonolfactory pathways. To eliminate naive odor bias, experiments are usually performed in a counterbalanced design, with half of the flies used in the calculation of the performance index being trained to the first odor and the other half to the second odor. An examination of the half P.I. scores for these experiments does not reveal obvious asymmetries between BEN and the two counter-odors. Moreover, the fact that the odor-pair-specific effects are seen in both rescue as well as disruption of memory experiments suggests that it is the expression of transgenes in the subset of neurons defined by 17d-GAL4 that confers the odor-pair-specific behavioral phenotypes observed in this study (Akalal, 2006).

The existence of such stereotypical anatomical and functional organization at the various levels of the Drosophila olfactory pathway may explain the odor-pair-specific rut rescue and shi^ts1-mediated disruption of learning that was observed in this study. The spatial pattern of odor-evoked fluorescence activity for BEN has been reported to occur mostly in the center of the calyx, OCT-evoked activities distribute more laterally and medially, while MB neurons that displayed fluorescence transients in response to MCH occur primarily in the top and middle portions of the soma layer. To investigate whether different lobes of the MBs receive olfactory information from different subsets of AL glomeruli, the spatial correlation between MB neurons and projection neurons (PNs) have been examined. The MB dendrites of γ neurons were found to preferentially occupy the center of the calyx, and although the dendrites of the α'/ß' and α/ß core and surface neurons were more widespread across the calyx, their distribution was slightly nuanced. Based on these observations, it is speculated that olfactory learning using different odors is, in part, a function of the relationships between the expression pattern of the GAL4 driver and the degree and pattern of overlap and nonoverlap in the populations of MB neurons that respond to the odor combinations chosen. This is a more complex picture than just having the different odors mapping to distinct lobes of the MBs, and, in fact, a shift toward describing GAL4 drivers based on actual patterns of expression may be more useful than describing them solely on MB lobe-specificity. Ultimately, determining the precise manner by which odors are encoded in the Drosophila brain and how this links to specific behavioral outputs will require careful analyses of the expression patterns of the GAL4 drivers and the representation of the odors in the different MB neurons (Akalal, 2006).

Activity-dependent retrograde laminin A signaling regulates synapse growth at Drosophila neuromuscular junctions

Retrograde signals induced by synaptic activities are derived from postsynaptic cells to potentiate presynaptic properties, such as cytoskeletal dynamics, gene expression, and synaptic growth. However, it is not known whether activity-dependent retrograde signals can also depotentiate synaptic properties. This study shows that laminin A (LanA) functions as a retrograde signal to suppress synapse growth at Drosophila neuromuscular junctions (NMJs). The presynaptic integrin pathway consists of the integrin subunit βν and focal adhesion kinase 56 (Fak56), both of which are required to suppress crawling activity-dependent NMJ growth. LanA protein is localized in the synaptic cleft and only muscle-derived LanA is functional in modulating NMJ growth. The LanA level at NMJs is inversely correlated with NMJ size and regulated by larval crawling activity, synapse excitability, postsynaptic response, and anterograde Wnt/Wingless signaling, all of which modulate NMJ growth through LanA and βν. These data indicate that synaptic activities down-regulate levels of the retrograde signal LanA to promote NMJ growth (Tsai, 2012).

This study proposes a plasticity mechanism by which the synapse growth (or size) can be modulated by larva crawling and synaptic activities. These activities modulate LanA-integrin signaling that functions to constrain NMJ growth. This trans-synaptic signaling functions in a retrograde manner, which requires postsynaptic muscle-derived LanA and presynaptic integrin. The model suggests various activities modulate NMJ growth by regulating the LanA level and integrin signaling (Tsai, 2012).

Regulation of LanA levels at NMJs is the major mechanism underlying this synaptic structural plasticity. The LanA levels at NMJs are tightly coupled to several synaptic activities that are involved in synaptic structural plasticity at NMJs. Wg signaling in both pre- and postsynaptic compartments are shown to modify synaptic structure at Drosophila NMJs. The channel mutations para and eag Sh alter both synaptic potential and NMJ size. Finally, manipulation of postsynaptic responses by altering the GluRIIA and GluRIIB compositions also fine tunes synapse size and pFAK levels. Activities that promote NMJ growth also down-regulated LanA levels at NMJs. In contrast, NMJ growth suppression was accompanied with LanA accumulation, establishing an inverse correlation between the LanA level and the NMJ size. Importantly, manipulation of the gene dosage of LanA (or βν) could override these synaptic activities in NMJ growth regulation. This study also showed that LanA down-regulation at NMJs preceded synaptic structural remodeling induced by larval crawling, further supporting that LanA is a major mediator of these activities to modulate NMJ growth (Tsai, 2012).

Integrin signaling activities play important roles in synapse development and plasticity. In mammalian central synapses, various integrin subunits are important to transmit postsynaptic signaling in various plasticity models may function redundantly with βν to mediate integrin signaling. This study indicates a distinct presynaptic integrin pathway that is likely composed of βν and αPS3 (encoded by Volado), as suggested by their strong genetic interaction in NMJ growth. In response to postsynapse-secreted LanA signals, activation of the presynaptic integrin is transmitted through Fak56 activation. Interestingly, the signaling activity is rather local, limited by the range of LanA distribution, and shown by muscle 6-specific rescue, although this does not exclude the involvement of signaling to the nuclei of motor neurons. The presynaptic integrin/Fak56 signaling is in turn mediated by two downstream signaling activities. The activation of NF1/cAMP signaling, which suppressed NMJ overgrowth induced by crawling activity or βν mutation. The integrin/Fak56 pathway also suppresses Ras/MAPK signaling Tsai, 2008), as shown by diphospho-ERK (dpERK) accumulation and Fas2 reduction at NMJs in high crawling condition. These pathways have been shown to regulate cell adhesion and cytoskeletal organization, leading to the stabilization of synapses. The activity-dependent depletion of the LanA laminins in the synaptic cleft would allow the remodeling of synapses and further growth of NMJs (Tsai, 2012).

The activity-dependent structural plasticity is specific to the presynaptic integrin pathway. hiw mutants that show large NMJ size still retained the structural plasticity and constant pFAK levels at NMJs. Interestingly, LanA levels were increased in hiw mutants, in contrast to other NMJ overgrown mutants. Two nonmutually exclusive mechanisms can regulate activity-dependent LanA expressions at NMJs. First, within hours of activity induction, the LanA levels can be regulated at NMJs by putative ECM regulators such as matrix metalloproteinases. Second, transcription regulation of LanA can provide long-term changes of LanA levels at NMJs. Activity-triggered presynaptic Wg secretion promotes Wg receptor DFz2 activation on both post- and presynaptic compartments. The LanA level is regulated by the anterograde Wg signaling that is transduced through nuclear entry of the DFz2 intracellular domain and its transcription activity. However, LanA is unlikely to mediate all aspects of Wg signaling activity as overexpression of LanA in postsynapses suppressed ghost bouton formation, a hallmark in disrupting Wg signaling. Postsynaptic BMP/Gbb functions as a retrograde signal to activate presynaptic BMP type II receptor Wit in response to synaptic activity. With the lack of genetic interaction between BMP/Gbb and integrin signaling components, and constant levels of phosphorylated Mothers against dpp (pMad) in different crawling activities, it is proposed that both BMP/Gbb and LanA pathways can function in parallel by retrograde mechanisms to regulate NMJ growth (Tsai, 2012).

Drosophila dopamine synthesis pathway genes regulate tracheal morphogenesis

While studying the developmental functions of the Drosophila dopamine synthesis pathway genes, interesting and unexpected mutant phenotypes were noticed in the developing trachea, a tubule network that has been studied as a model for branching morphogenesis. Specifically, Punch (Pu) and pale (ple) mutants with reduced dopamine synthesis show ectopic/aberrant migration, while Catecholamines up (Catsup) mutants that over-express dopamine show a characteristic loss of migration phenotype. Expression of Punch, Ple, Catsup and dopamine was seen in tracheal cells. The dopamine pathway mutant phenotypes can be reproduced by pharmacological treatments of dopamine and a pathway inhibitor 3-iodotyrosine (3-IT), implicating dopamine as a direct mediator of the regulatory function. Furthermore, these mutants genetically interact with components of the endocytic pathway, namely shibire/dynamin and awd/nm23, that promote endocytosis of the chemotactic signaling receptor Btl/FGFR. Consistent with the genetic results, the surface and total cellular levels of a Btl-GFP fusion protein in the tracheal cells and in cultured S2 cells are reduced upon dopamine treatment, and increased in the presence of 3-IT. Moreover, the transducer of Btl signaling, MAP kinase, is hyper-activated throughout the tracheal tube in the Pu mutant. Finally it was shown that dopamine regulates endocytosis via controlling the dynamin protein level (Hsouna, 2007).

This report demonstrates that genes involved in DA biosynthesis also regulate tracheal cell migration, and that this function is mediated by DA. This is unexpected since DA is normally associated with neuronal function. However, this novel developmental function is not fortuitous because of the strong and highly specific expression of Punch in tracheal cells during the migratory phase of tracheal development. In addition, the ectopic migration tracheal phenotypes in Pu/GTPCH mutants can be rescued by expressing a Pu/GTPCH transgene in developing trachea using a btl promoter, demonstrating the trachea-specific function of the DA pathway. Moreover, the expression of this transgene shows a dosage-dependent progression of phenotypic outcomes. That is, mild expression rescues ectopic migration, while over-expression tips the balance to blocked migration (Hsouna, 2007).

The product of GTPCH enzymatic activity, BH₄, is also a stabilizing cofactor of nitric oxide synthase (NOS), which is needed for hypoxia-induced outgrowth of terminal tracheal branches. However, the Pu mutant phenotypes reported in this study are most likely unrelated to this developmental process because of the following reasons: (1) Terminal branching occurs near the end of embryogenesis and during larval development whereas the defects in primary and secondary branch migration occur during mid-embryogenesis. (2) Mutations in ple, which has no role in NOS function, also results in ectopic migration phenotypes. (3) DA and the DA pathway inhibitor can phenocopy the genetic mutants. (4) DA treatment can rescue Pu/GTPCH mutant phenotypes, implicating a direct role of DA in primary and secondary branch migration. Interestingly, DA is enriched in the trachea (Hsouna, 2007).

An inhibitory role for DA in the regulation of cell migration has been reported for a number of cell types over recent years. DA is capable of blocking the migration of vascular smooth muscle cells, a factor in the formation of atherosclerotic lesions, in a process mediated through the D1 class of DA receptors. DA also was reported to interfere with activated neutrophil transendothelial migration. Similarly, DA, acting through D1 receptors, is reported to reduce the migration of regulatory T cells in damaged neural regions, thereby providing protection against T cell-mediated neurodegeneration. Finally, DA acting through the D2 class of DA receptors, can inhibit tumor angiogenesis in highly vascularized gastric and ovarian tumors. It would be interesting to determine whether the mammalian GTPCH-TH-DA enzymatic pathways also have a non-neurological function in modulating tubulogenesis during development (Hsouna, 2007).

DA pathway mutants show tracheal abnormalities that are strikingly similar to the perturbations of tracheal cell migration in embryos with abnormalities in FGF signaling. Diminution of DA expression in Drosophila embryos, either by mutations (Pu and ple) or by pharmacological depletion (3-IT treatment), has dramatic effects on the stereotyped migratory behavior of tracheal cells and patterning of the resulting branched structure. Entire branches are misdirected and individual cells move away from the tracheal branches, a phenotype that has been termed 'run-away cells'. Under conditions of excess DA, via mutations (Catsup) or pharmacological modification (DA treatment), the converse phenotype, one of blocked migration, is evident. This phenotype is often associated with clustering of tracheal cells near the stunted ends of tracheal tubes. These opposing phenotypes are correlated with the increased or decreased levels of MAPK activation, the mediator of FGFR signaling (Hsouna, 2007).

The data further demonstrate that DA regulates FGF receptor turnover. Btl;;GFP fusion protein is down-regulated in the presence of DA and up-regulated in the presence of 3-IT, either in trachea or in cultured S2 cells. These results are consistent with the model that DA promotes internalization of Btl/FGFR, leading to its degradation through the endocytic pathway. The role of DA in internalization of FGFR is further suggested by the genetic interaction between the DA synthesis and endocytic pathway genes. Mutations in the human tumor suppressor nucleoside diphosphate kinase (NDK) gene (nm23) are strongly associated with tumor metastatic activity. Its functional homolog in Drosophila, abnormal wing discs (awd), has been shown to genetically interact with a temperature-sensitive allele of the shibire gene (shi^ts), which encodes dynamin, a large GTPase required for the formation of clathrin-coated endocytic vesicles. A function for awd/nm23 in the migratory phase of tracheal development has been demonstrated and functional interactions occur with shi/dynamin that are required for the modulation of FGFR/Btl levels in tracheal cells. This report shows that Pu and ple phenotypes are exacerbated by awd and rescued by btl, while the Catsup phenotypes are rescued by awd but exacerbated by btl. These results indicate that Pu/GTPCH and Ple/TH, and by extension, DA, are positive regulators of endocytosis (Hsouna, 2007).

This analysis was extended by implicating direct involvement of DA in regulating the Shi/dynamin protein itself. Mutations in Pu/GTPCH, which regulates DA pools, result in reduction of the Shi/dynamin levels and, in consequence, the subsequent accumulation of FGFR/Btl in the plasma membranes of tracheal cells, thus accounting for ectopic migratory behavior of these mutant cells. Importantly, down-regulation of Shi/dynamin level in the Pu mutants can be rescued by treatment with DA. A direct correlation between DA and dynamin levels is also demonstrated in cultured S2 cells. Thus, the genetic and pharmacological evidence in this report supports the hypothesis that the diminished DA pools that accompany loss-of-function mutations in the Pu/GTPCH and ple/TH genes result in deficits of DA-mediated signaling necessary for Shi/dynamin accumulation (Hsouna, 2007).

Evidence of a role of DA in receptor endocytosis has emerged recently. For instance, DA can promote VEGFR endocytosis in cultured human endothelial cells. DA D₃ receptor-mediated modulation of GABA_A receptor and DA-regulated endocytosis of the renal cell Na⁺, K⁺-ATPase are similarly dynamin-mediated events. Why is a neurohormone also a mediator of endocytosis in other cell types? It is interesting to consider the possibility that the endocytic activity of DA may in fact be its ancestral function, which was adopted by the neurons and tubular cells later. Indeed, primitive, nerve-less multicellular organisms such as sponge can produce dopamine. The precise mechanism(s) by which DA regulates dynamin assembly is not yet clear. However, DA signaling promotes dynamin stabilization and assembly at the plasma membrane in cultured human kidney cells, and thus endocytosis, by activating protein phosphatase 2A which dephosphorylates dynamin-2. It is possible that a similar mechanism of action occurs in the tracheal cells and future experiments will help to address this possibility (Hsouna, 2007).

Functional analysis of fruitless gene expression by transgenic manipulations of Drosophila courtship: fruitless controls singing behavior in identified neurons

A gal4-containing enhancer-trap called C309, which is expressed broadly in central-brain and VNC regions, has been shown to cause subnormal courtship of Drosophila males toward females and courtship among males when driving a conditional disrupter of synaptic transmission (shi^TS). These manipulations have been extended to analyze all features of male-specific behavior, including courtship song, which was almost eliminated by driving shi^TS at high temperature. In the context of singing defects and homosexual courtship affected by mutations in the fru gene, a tra-regulated component of the sex-determination hierarchy, C309/tra^F combination was also found to induce high levels of courtship between pairs of males and 'chaining' behavior in groups; however, these doubly transgenic males sang normally. Because production of male-specific FRU^M protein is regulated by Tra, it was hypothesized that a fru-derived transgene encoding the male (M) form of an Inhibitory RNA (fru^MIR) would mimic the effects of tra^F; but C309/fru^MIR males exhibited no courtship chaining, although they courted other males in single-pair tests. Double-labeling of neurons in which GFP was driven by C309 revealed that 10 of the 20 CNS clusters containing FRU^M in wild-type males included coexpressing neurons. Histological analysis of the developing CNS could not rationalize the absence of tra^F or fru^MIR effects on courtship song, because C309 was found to be coexpressed with FRU^M within the same 10 neuronal clusters in pupae. Thus, it is hypothesized that elimination of singing behavior by the C309/shi^TS combination involves neurons acting downstream of FRU^M cells (Villella, 2005).

Various portions of the CNS in Drosophila are inferred to control separate elements of normal male courtship, in part by analysis of abnormal behavior. Some such studies have involved brain-behavioral analyses of the fruitless (fru) gene and its mutants. Different fru mutants exhibit courtship subnormalities to varying degrees and at separate stages of the courtship sequence, depending on the mutant allele. Most fru mutants court other males substantially above levels normally exhibited by pairs or groups of wild-type males. The original fruitless mutation leads to spatially nonrandom decreases of fru-product presence within particular subsets of the normal CNS expression pattern, which may be causally connected with the breakdown of recognition that is a salient effect of fru¹ on male behavior. fru-like courtship can be induced by the effects of a transgene that encodes GAL4 (a transcription factor derived from yeast). When this broadly expressed C309 enhancer trap was combined with a GAL4-drivable factor containing a dominant-negative, conditionally expressed variant of the shibire gene (shi^TS), heat treatment of doubly transgenic males caused them to court females subnormally and to court other males vigorously. Although this strain had been termed a mushroom body enhancer trap in terms of the gal4 sequence it contains, being expressed 'predominantly' within that dorsal-brain structure, C309 drives marker expression in a widespread manner. Therefore, attempts were made to correlate various CNS regions in which this transgene is expressed with its effects on male behavior, emphasizing a search for 'C309 neurons' that might overlap with elements of the FRU^M pattern (Villella, 2005).

The possibility was also entertained that the C309/shi^TS combination causes a mere caricature of fruitless-like behavior. Therefore, what would be the courtship effects of C309 driving a transgene that produces the female form of the transformer gene product? This Tra protein participates in posttranscriptional control of fru's primary 'sex transcript,' so that FRU^M protein is not produced in females. If C309 and tra^F are naturally coexpressed in a subset of the to-be-analyzed neurons, feminization of the overlapping cells should eliminate this protein. These transgenic experiments were extended to target fruitless expression specifically by gal4 driving of an inhibitory RNA (IR) construct, which was generated with fru DNA. Their experiments furnish one object lesson as to how 'enhancer–trap mosaics' can delve into the neural substrates of a complex behavioral process, an approach commonly taken to manipulate brain structures and functions in courtship experiments. Because few genetic loci putatively identified by such transposons have been specified, the tactics applied are in the context of CNS regions in which expression of a 'real gene' is hypothesized to underlie well defined behaviors (Villella, 2005).

Mutations at the fruitless locus and the C309/UAS-shi^TS transgene combination each cause similar courtship subnormalities and anomalies. In this context, the C309 enhancer trap is expressed in many CNS neurons that contain male-specific FRU^M protein. One element of the courtship effects of C309/UAS-shi^TS involves subnormal interactions between males and females. From correlating C309/FRU^M coexpression with the fact that fruitless mutations lead to lower-than-normal male–female courtship, it is speculated that FRU^M brain regions 2, 8, 13, and 14 are connected with the deleterious effects of shi^TS. As to why two additional neuronal groups that coexpress FRU^M and C309 are not noted here (groups 5 and 7), see below (Villella, 2005).

With respect to males courting females, special attention might be paid to clusters 13 and 14. These posteriorly located groups of FRU^M neurons are likely to include brain regions within which genetic maleness is required if sex mosaics are to exhibit orientation toward and following of females. A problem with this interpretation is that feminizing substantial proportions of the C309/FRU^M coexpressing cells led to no decrements in male-female interactions, despite the 7- to 10-fold coexpression reductions caused by XY/C309/UAS-tra^F within clusters 13 and 14. Perhaps the relevant 'overlap percentages' would have had to drop from 47 and 31 to 0 for both of these clusters if a tra^F-affected courtship decrement were to be realized. An alternative to viewing this matter in the context of C309/FRU^M coexpression is that certain neurons in which this gal4 driver is active could be anatomically downstream of the fru-expressing brain cells that influence a male's ability to initiate and sustain courtship of a female (Villella, 2005).

This conception is relevant to the striking elimination of courtship song in recordings of C309/UAS-shi^TS males. Once again, UAS-tra^F has no such effect. Neurogenetic findings pertinent to this matter are that C309 is expressed in imaginal thoracic ganglia; fru is 'song-involved'; this gene makes its products within several regions of the ventral nerve cord, coexpressing C309 within most of them; and genetic maleness within Drosophila's VNC has been implicated in song control. Thus, turning off synaptic transmission emanating from one or more subsets of the C309/UAS-shi^TS neurons in the thoracic ganglia could be the etiology of heat-induced songlessness exhibited by these doubly transgenic males. Regarding the absence of a tra^F effect, what if C309 was not expressed in any FRU^M-containing song-relevant neurons during metamorphosis? In other words, C309 expression in VNC neurons underlying song control could be activated late in the life cycle, allowing for the shi^TS effect to take hold after adult males are heated; however, the progenitors of such cells might not express C309 during an earlier 'feminization-relevant' stage, so that post-metamorphic activation of tra^F would occur too late to affect singing ability. However, substantial coexpression of FRU^M and C309 was found within the pupal VNC. In this respect, it is submitted that assessing the C309's expression pattern throughout the life cycle is a valuable object lesson as to what must be done properly to interpret the biological effects of a given enhancer trap (Villella, 2005).

As to the divergent effects C309-driven shi^TS vs. tra^F, recall that the former factor seems broadly to impinge on VNC functioning, in that the fly's general ability to vibrate its wings is shut down by the synaptic disruptor; in contrast, songless fruitless mutants fly normally. Thus, consider a scenario in which C309 neurons would include those that mediate wing vibrations during flight, and that this transgene is expressed in separate VNC cells hypothetically dedicated to such vibrations during courtship. Therefore, it is speculated that the expression domain of C309 includes inter- or motor-neurons functioning within and downstream of a 'command center' for flight as well as neurons located in relatively distal regions of a separate anatomical pathway. The latter would originate where fru-expressing cells exert the gene's crucial regulation of courtship song (Villella, 2005).

Turning to anomalous courtship interactions among males, focus shifts back to the brain: FRU^M clusters 5 and 7, where fru¹ causes an apparent absence of this protein. This mutation minimally affects the gene's expression in other brain regions. It is notable that the C309/UAS-tra^F combination knocked down driver/FRU^M coexpression to ~10% of normal in cluster 5. Cluster 7 was similarly affected, but special attention should be paid to cluster 5. One reason that this group was thought to be the etiology of frantic courtship among fru¹ males is that cluster 5 is located near the antennal lobes; and transgenically mediated feminization of a brain region near these structures induces intermale courtships, although none of the gal4 drivers in that study included C309. Therefore, if proper male-specific structure or function of cluster 5 is involved in normal sex recognition, the mutation's demasculinizing effect on this brain region, or transgene-effected feminization of it, could cause this aspect of courtship to break down (Villella, 2005).

Elements of the current findings suggest that abnormal formation of the brain region in question is not necessary for it to mediate anomalous interactions between males; this is because deactivating synaptic transmission in cluster 5, after CNS development has been completed in a male manner, is sufficient to induce intermale courtship. Perhaps this behavioral effect of driving UAS-shi^TS involves removal of inhibitory neurotransmission relevant to the functioning of this brain region, which in normal males would block their wherewithal to sustain courtship between males. Therefore, the fru¹ effect on cluster 5 and that of driving Tra production in this region might not involve the formation of a sex recognition center (such that a hypothetical circuit involved in inhibiting intermale courtship is not present or miswired), but instead the intracellular quality and function of neurons in the mature brain (Villella, 2005).

Considering further that certain cluster 5 neurons comprise the subset of FRU^M's spatial domain for shi^TS- or tra^F-induced intermale courtship, the relevant cells would be those in which both fruitless and C309 generate their gene products (20% of the 35 neurons within this group). One problem with this supposition is that C309/UAS-tra^F flies elicit fairly high levels of courtship. Thus, groups of C309/UAS-tra^F males may form chains for reasons extending beyond a given fly's inappropriate 'motivation' to court another male: The extent to which a C309/UAS-tra^F fly is feminized could include self-stimulation that might contribute to intermale courting. However, recall the case of C309/UAS-tra^F/Cha-gal80 males, a transgenic type that is similarly feminine externally and elicits courtship. The diminished extent to which C309's gal4 is effective when combined with Cha-gal80 led to weakened homosexual courtship in single-pair tests and dramatically reduced chaining behavior, although there was essentially no effect of Cha-gal80 on the basic courtship ability of these triply transgenic males. Thus, the effects of this 'neurons-only' manipulation suggest that hypothetical self-stimulation, which did not cause C309/UAS-tra^F/Cha-gal80 males vigorously to court other ones, is minimally operating to induce the homosexual courtships performed by XY/C309/UAS-tra^F flies. Males carrying C309 and UAS-fru^MIR are also not feminized externally; however, they courted other males robustly in single-pair tests, an effect that was diminished by adding Cha-gal80. Therefore, it is surmised that flies carrying a given fruitless-affecting transgene exhibit intermale courtship because the relevant CNS neurons are demasculinized (Villella, 2005).

However, what about neural structures not analyzed in the current study that could be involved in the behavioral effects of C309 driving either tra^For fru^MIR? Thus, consider that Tra affects the primary transcript emanating from the doublesex (dsx) gene and that dsx null mutations cause XY flies to exhibit modest levels of intermale courtships. C309 driving of tra^F could lead to the female (F) form of DSX (thus, no DSX^M, as in dsx^–) within brain cells connected to sex recognition other than those analyzed. Indeed, dsx⁺ is expressed in the brain; however, the functional significance of these cells is unknown, let alone whether any of them also express fru⁺. In this regard, it was important to home in on disruption of fruitless's CNS expression alone by combining C309 with the UAS-fru^MIR transgenes; this was sufficient to induce courtship between a given pair of doubly transgenic males but led to no chaining. Thus, anomalously high levels of courtship between two males has been disconnected from courtship chaining. [The same disconnect between these different kinds of intermale courtship occurred when Cha-gal80 was added to the C309/UAS-tra^F combination. It is as if the broad neural effects of a genetic abnormality such as a fruitless mutation, or combining C309 with UAS-tra^F, is necessary to cause sustained courtship among several variant males; however, if the impingement on fru⁺ expression is more limited, only courtship between a pair of males can occur (Villella, 2005).

In this regard, the C309/UAS-fru^MIR flies were substantially less affected in terms of numbers of brain neurons within which FRU^M became undetectable, compared with the effect of the same driver combined with UAS-tra^F. This brings up the matter of additional neurons that are potentially relevant to courtship and should be analyzed in context of the C309 effects. Here, the many PNS cells recently discovered to express fruitless in external sensory structures are referred to. It is unknown whether any of these neurons coexpress C309, such that sensory inputs relevant to courtship may have been impinged upon by combining that transgene with UAS-shi^TS or with the sex-affecting transgenes. However, fru⁺ expression in external appendages is not required for a fly to recognize, follow, and perform wing extension at a female: when these structures are genetically female in certain gynandromorphs, maleness within the brain is sufficient to trigger mosaic-with-female courtship (Villella, 2005).

The current study aimed to delve into various regions of the male CNS in which the fruitless gene is expressed: Do certain subsets of the spatial pattern govern a male's ability to perform a discrete feature of the reproductive sequence? Using the gal4-containing C309 enhancer trap was valuable, because it leads to impersonations of certain fru-mutant behaviors when this driver is combined with a shi^TS-containing factor that broadly disrupts neural functioning. By limiting C309's efficacy to disrupt by causing it to drive sex-related transgenes succeeded in provisionally partitioning fru-related 'sex recognition' neurons to a subset of the normal brain pattern. By subtraction, the partitioning was further delimited by knocking out the driver's efficacy in a subset C309's spatial domain: adding a neurally driven gal80 transgene substantially attenuated anomalous intermale courtships. A pleasant surprise occurred when the C309/UAS-fru^MIR combination was found not to mimic the effects on courtship among males of combining the driver with UAS-tra^F. Thus, the broader pattern of FRU^M expression, unaffected by the IR compared with the substantial decrement caused by tra^F, takes the analysis a further step. For example, the manner by which fru mutations and related factors influence courtship between two males, as opposed to the much more complicated behavioral dynamics that can occur in a group of such Drosophila, are now being teased out (Villella, 2005).

However, inferences about the potentially relevant subsets of a given brain cluster do not approach specifically identifiable neurons. For this, it will be necessary to do more than quantify the cells in which a transgene driver and fruitless are coexpressed. Further brain-behavioral dissections will require assessing the differential connectivity patterns defining a given class of FRU^M neurons, along with variations of cellular content that are likely to discriminate one category of such neurons from another. The relevant object lessons stem from analyses of, so far, only the posterior-most component of fruitless's expression domain in the male CNS: partitioning certain abdominal-ganglion neurons that differentially connect with either a male-specific muscle or with internal reproductive organs, and discovering that the latter type of FRU^M cells uniquely contain serotonin. Neurons containing another neurotransmitter, acetylcholine, are on point; but not all of the C309 effects can be ascribed to neurons affected by Cha-gal80, because certain courtship defects were found to remain when analyzing males that carried this transgene along with C309 and UAS-shi^TS. This finding reinforces that notion that additional neuronal qualities must be uncovered with regard to cells expressing this enhancer-trap, the fruitless gene, or both (Villella, 2005).

Excitatory and inhibitory switches for courtship in the brain of Drosophila melanogaster

Courtship is the best-studied behavior in Drosophila melanogaster, and work on its anatomical basis has concentrated mainly on the functional identification of sexually dimorphic sites in the brain. Much less is known of the more expansive, nondimorphic, but nonetheless essential, neural elements subserving male courtship behavior. Sites in the CNS mediating initiation and early steps of male courtship in Drosophila melanogaster were identified by analyzing the behavior of mosaic flies expressing transgenes designed either to suppress neurotransmission or enhance neuronal excitability. Suppression of neurotransmission was accomplished by means of the dominantly acting, temperature-sensitive dynamin mutation shibire^ts1, whereas enhanced neuronal excitability was produced by means of a novel, dominantly acting, truncated eag potassium channel. By using a new, landmark-based procedure for aligning diverse expression patterns among the various mosaic strains, a comparison of courtship performance and affected brain sites in strains expressing the transgenes identified a cluster of cells in the posterior lateral protocerebrum that exerts reciprocal effects on the initiation of courtship, suppressing it when they are inactivated and enhancing it when they are hyperactivated, indicative of cells that normally play an excitatory, triggering role. A separate group of nearby cells, slightly more anterior in the lateral protocerebrum, was found to inhibit courtship when its activity is enhanced, indicative of an inhibitory role in courtship. It is concluded that a cluster of cells, some excitatory and some inhibitory, in the lateral protocerebrum regulates courtship initiation in Drosophila. These cells are likely to be an integration center for the multiple sensory inputs that trigger male courtship (Broughton, 2004).

Male courtship is elicited by visual and chemosensory cues, either of which is sufficient to initiate courtship behavior in the presence of a virgin female. Projections from the antennal lobes to the lateral protocerebrum, independent of the mushroom bodies, are essential for courtship initiation. These data, which are consistent with the normal initiation of courtship in mushroom body-ablated and mushroom-body impaired males, suggest that courtship is initiated via a mushroom body-independent mechanism (Broughton, 2004).

In the current study, only those GAL4 lines expressed in a common region of the lateral, posterior protocerebrum had a specific effect on courtship initiation. Of particular significance is the reciprocal effect on initation seen in MJ286 and MJ146 when expressing a transgene suppressing neuronal activity as compared with an enhancing transgene. The implication is that these cells exert an activating effect on courtship initiation. This region has previously been implicated sex specifically in male courtship, physiologically in male courtship, and in mediating the plasticity associated with courtship conditioning. As the recipient of many different kinds of sensory projections, it is likely to carry out a variety of integrative functions, as already suggested by a study of its olfactory inputs (Broughton, 2004).

These lateral protocerebral cells in enhancer trap lines MJ286 and MJ146 lie just ventral to the anatomical neighborhood previously identified as the sex-specific focus for courtship initiation: the dorsal posterior brain. In fact, the marking technique used in the earlier studies detected cell bodies whose neuronal processes may well project into the region identified in the current study. The presence in MJ286 of sexually dimorphic function suggests that its dorsal, lateral, and protocerebral cells may even be part of the sex-specific focus for initiation, as well as being required for its physiological realization. The lack of FRU^M expression in these cells may reflect the incomplete overlap in expression patterns between FRU^M and the male-specific product of another gene in the sex determination cascade, doublesex (DSX^M), each of which distinctively influences male courtship behavior. MJ146 does not show sex-specific function, despite its overlap, albeit limited, with FRU^M expression (Broughton, 2004).

In contrast, the one case in which the transgene that enhances neuronal activity exerted a suppressing effect on courtship, MJ63, is suggestive of an inhibitory circuit, though it is not associated with the GABA-ergic marker GAD-dsRED. In this instance, the effect was unidirectional: blocking neuronal activity in the same cells had no effect. One interpretation of this result is that this region normally acts in a modulatory fashion and does not act to continuously inhibit courtship behavior. When MJ63 is used to drive expression of a CaMKII inhibitory peptide during the courtship conditioning assay, memory is disrupted and regions defined by this line have been suggested to be involved in enabling memory of the inhibition by the mated female. These data raise the possibility that the inhibitory regions identified by MJ63 may normally act to mediate experience-dependent inhibition of male courtship behavior and the inappropriate activation of them suppresses courtship, thus mimicking the conditioning paradigm (Broughton, 2004).

The opposing effects of c747/UAS-shi^ts1 and c309/UAS-shi^ts1 on wing extension and vibration, are likely to be due to the fact that the broader expression pattern in c747 disrupts inhibitory sites that are intact in c309 and that c309 affects excitatory sites. If this excitation were due to the mushroom body expression in c309, which would then be couteracted by additional expression in c747, it would be consistent with the previous finding of a sex-specific effect of this structure on the performance of wing extension and vibration. The song itself is controlled in the mesothoracic ganglion. Although the mushroom bodies have been suggested to be involved in mate discrimination, a recent finding that expression of UAS-shi^ts1 in the mushroom bodies did not induce male-male courtship behavior shows that mushroom body activity is not required for the recognition of sex-specific pheromones that inhibit male-male courtship. Mushroom bodies have been implicated, however, in modulating other kinds of motor output (Broughton, 2004).

Mapping the neural elements of male courtship is an essential step in understanding the functional circuitry and neural basis for this evolutionarily critical behavior. The fact that courtship consists of a series of stereotypical steps offers great advantages for assigning roles to particular circuits and will facilitate the merging of findings from studies of the sexual dimorphisms underlying courtship with those of genetic perturbations of physiology (Broughton, 2004).

Dpp morphogen movement is independent of dynamin-mediated endocytosis but regulated by the glypican members of heparan sulfate proteoglycans

The Drosophila transforming growth factor ß homolog Dpp acts as a morphogen that forms a long-range concentration gradient to direct the anteroposterior patterning of the wing . Both planar transcytosis initiated by Dynamin-mediated endocytosis and extracellular diffusion have been proposed for Dpp movement across cells. In this work, it was found that Dpp is mainly extracellular, and its extracellular gradient coincides with its activity gradient. A blockage of endocytosis by the dynamin mutant shibire does not block Dpp movement but rather inhibits Dpp signal transduction, suggesting that endocytosis is not essential for Dpp movement but is involved in Dpp signaling. Furthermore, Dpp fails to move across cells mutant for dally and dally-like (dly), two Drosophila glypican members of heparin sulfate proteoglycan (HSPG). These results support a model in which Dpp moves along the cell surface by restricted extracellular diffusion involving the glypicans Dally and Dly (Belenkaya, 2004).

One new observation in this work is that the extracellular Dpp is broadly distributed in the wing disc. Consistent with these findings, previous biochemical analysis demonstrated that the majority of mature Dpp signaling molecules are extracellular. Importantly, the overall shape of the extracellular Dpp gradient coincides well with its activity gradient, suggesting that the extracellular Dpp gradient contributes to Dpp activity gradient in the wing disc. The observation of broadly distributed extracellular Dpp led to a re-examination of the role of Dynamin-mediated endocytosis in Dpp movement and signaling. These analyses argue that Dynamin-mediated endocytosis is not essential for Dpp movement: (1) both Dpp signaling activity and extracellular GFP-Dpp levels are not reduced in the wild-type cells behind the shi^ts1 clones that are defective in endocytosis; (2) the extracellular GFP-Dpp is also broadly distributed in endocytosis-defective wing discs homozygous for shi^ts1 at nonpermissive temperature. These data demonstrate that Dpp molecules are able to move across Dynamin-defective cells. Finally, it was found that extracellular Dpp accumulates on the cell surface of shi^ts1 mutant clones, suggesting that Dpp is able to move into shi^ts1 mutant cells and that Dynamin-mediated endocytosis is normally involved in downregulating levels of the extracellular Dpp. No accumulation of extracellular Dpp on wild-type cells was observed in front of shi^ts1 mutant clones; this would be expected if endocytosis were required for Dpp movement (Belenkaya, 2004).

While Dynamin-mediated endocytosis does not appear to be critical for Dpp movement, Dpp signaling activity is reduced cell autonomously in shi^ts1 mutant cells. This result argues that Dynamin-mediated endocytosis is an essential process for Dpp signaling. Studies in mammalian cell culture system have demonstrated the critical role of Dynamin-mediated internalization of activated TGF-β receptors in TGF-β signaling. SARA (Smad anchor for receptor activation), a FYVE finger protein enriched in early endosomes is involved in this process. Although the exact mechanism of endocytosis-mediated TGF-β signaling is still unclear, current data suggest a role of early endosomes as a signaling center for TGF-β. Consistent with this view, it has been shown that ectopic expression of the dominant-negative form of Rab5 (DRab5S43N) using engrailed-Gal4 leads to a reduction of Dpp signaling, while overexpression of Rab5 broadens the Dpp signaling. Rab5 localizes in early endosomes and is required for endosome fusion. Taken together, it is proposed that dynamin-mediated endocytosis is not directly involved in Dpp movement but is essential for Dpp signaling. Furthermore, Dynamin-mediated endocytosis can downregulate extracellular Dpp levels, thereby shaping the Dpp morphogen gradient (Belenkaya, 2004).

To investigate the role of HSPGs in Dpp morphogen gradient formation, Dpp signaling and its extracellular distribution was examined in sulfateless (sfl) and dally-dly mutant clones. dally and dly are shown to be required and partially redundant in Dpp signaling and movement in the wing disc. Two lines of evidence support the role of Dally and Dly in Dpp movement across cells: (1) Dpp signaling activity is reduced in cells behind sfl or dally-dly mutant clones; (2) extracellular Dpp levels are diminished in cells behind sfl or dally-dly mutant clones. Importantly, it was found that sfl or dally-dly mutant clones only a few cells wide can effectively block GFP-Dpp movement, suggesting that Dpp movement does not occur through 'free diffusion', by which extracellular Dpp would be expected to move across sfl or dally-dly mutant cells. Based on these observations, it is proposed that Dpp moves from cell to cell along the epithelium sheet through restricted diffusion involving Dally and Dly (Belenkaya, 2004).

If the HSPGs Dally and Dly are indeed involved in Dpp movement, observation of extracellular GFP-Dpp accumulation in front of sfl, dally-dly mutant clones would be expected. Indeed, extracellular GFP-Dpp accumulation is visible in front of sfl or dally-dly mutant clones. Consistent with this observation, Hh has been observed to accumulate abnormally in clones mutant for tout-velu (ttv) and brother of tout-velu (botv), two Drosophila EXT members involved in HS GAG chain biosynthesis. Both Wg and Dpp accumulation in front of ttv-botv clones are also observed, albeit less pronounced, compared with the case of Hh. Similarly, extracellular GFP-Dpp accumulation is relatively weak, compared with Hh accumulation. One possibility is that extracellular Dpp molecules bound by Dally and Dly in wild-type cells can still be internalized by adjacent sfl or dally-dly mutant cells through cell-cell contact, leading to a reduction of extracellular Dpp accumulation in front of sfl or dally-dly mutant cells. Consistent with this view, it was noticed that, within sfl or dally-dly mutant clones, the first row of the mutant cells immediately adjacent to wild-type cells and facing Dpp-expressing cells is still capable of transducing Dpp signaling (Belenkaya, 2004).

In addition to being required for Dpp movement, Dally and Dly are also essential for Dpp signaling in its receiving cells. Dpp signaling is reduced in sfl or dally-dly mutant cells. Reduced levels of extracellular Dpp were observed in sfl or dally-dly mutant clones. Consistent with the results in this work, clones mutant for ttv or botv as well as sister of tout-velu (sotv), members of Drosophila EXT, led to reductions in Dpp signaling and its ligand distribution when analyzed by a conventional staining protocol that revealed both extracellular and intracellular Dpp. Collectively, these data suggest that the main function of Dally and Dly in Dpp signaling is to maintain and/or concentrate the extracellular Dpp available for Dpp receptors (Belenkaya, 2004).

This study has shown that Dynamin-mediated endocytosis is not essential for Dpp movement. Dpp movement is through a cell-to-cell mechanism involving the HSPGs Dally and Dly. On the basis of these findings, it is proposed that secreted Dpp molecules in the A-P border are immediately captured by the GAG chains of Dally and Dly on the cell surface located in either the A or P compartments. The differential concentration of Dpp on the cell surface of producing cells and receiving cells drives the displacement of Dpp from one GAG chain to another toward more distant receiving cells. Alternatively, Dpp molecules bound by Dally or Dly could also move along the cell surface through a GPI linkage that is inserted in the outlet leaflet of the plasma membrane and is enriched in raft domains. In the receiving cells, Dally and Dly may present Dpp to its receptor, Tkv, that transduces Dpp signal through the Dynamin-mediated internalization process, which further downregulates extracellular Dpp levels and cell surface Tkv. Based on this model, extracellular Dpp and its receptor, Tkv, would be accumulated on the surface of Dynamin-deficient cells, and extracellular Dpp would be able to move across Dynamin-deficient cells to reach more distal cells. In sfl or dally-dly mutant clones, extracellular Dpp molecules can not be attached on the cell surface and therefore can not be transferred further to more distal cells. In this model, endocytosis is not directly involved in Dpp movement; however, through receptor-mediated internalization, Dynamin-mediated endocytosis can downregulate extracellular Dpp levels, thereby shaping the Dpp morphogen gradient. It remains to be determined how Dpp is transferred from one cell to another by the GAG chains of Dally and Dly and whether Dally and Dly play a role in preventing extracellular Dpp from degradation. Further studies are needed to determine whether other mechanisms are also involved in Dpp movement (Belenkaya, 2004).

Dpp gradient formation by dynamin-dependent endocytosis: receptor trafficking and the diffusion model

Developing cells acquire positional information by reading the graded distribution of morphogens. In Drosophila, the Dpp morphogen forms a long-range concentration gradient by spreading from a restricted source in the developing wing. It has been assumed that Dpp spreads by extracellular diffusion. Under this assumption, the main role of endocytosis in gradient formation is to downregulate receptors at the cell surface. These surface receptors bind to the ligand and thereby interfere with its long-range movement. Recent experiments indicate that Dpp spreading is mediated by Dynamin-dependent endocytosis in the target tissue, suggesting that extracellular diffusion alone cannot account for Dpp dispersal. A theoretical study of a model for morphogen spreading was performed based on extracellular diffusion, which takes into account receptor binding and trafficking. Profiles of ligand and surface receptors obtained in this model were compared with experimental data. To this end, the pool of surface receptors and extracellular Dpp was monitored directly with specific antibodies. It is concluded that current models considering pure extracellular diffusion cannot explain the observed role of endocytosis during Dpp long-range movement (Kruse, 2004).

Three points lead to this conclusion. (1) A 'diffusion, binding and trafficking' DBT model of the 'shibire shadow assay' generates permanent shadows (depletion of Dpp behind the clone), whereas the experimental shadows are transient. (2) The DBT model with saturating cell surface receptor concentration (DBTS) model can generate transient shadows, but only if the surface receptor levels in the clone increase dramatically. This leads to a strong increase in the levels of extracellular ligand in the clone. Using receptor antibodies in the 'shibire shadow assay', these higher levels of surface receptors in the clone were not observed. Similarly, the levels of extracellular ligand were not increased in the clone. (3) In the DBTS model for the 'shibire rescue assay', the levels of both the extracellular Dpp and the surface receptors are dramatically increased in the endocytosis-defective target cells as compared with the WT source. Such an increase is not seen experimentally. Instead, extracellular Dpp enters the receiving tissue over a distance of only 4-5 cells in steady-state. This is in contrast to both DBT and DBTS models of the 'shibire rescue assay' in which ligand can enter the tissue over large distances. Therefore, in addition to downregulating surface receptors, endocytosis is likely to play additional roles in the transport of ligands during gradient formation (Kruse, 2004).

These three caveats of the DBT/DBTS models are actually not caused by the choice of a particular set of parameters. The parameter values used in the calculations were chosen in such a way, that the typical distance over which the ligand gradient extends as well as the characteristic time to reach steady state are consistent with the experimentally observed profiles. Furthermore, if possible, parameters were chosen similar to values measured for the EGF receptor in a cell culture system. The results showing that a high surface receptor concentration inside the clone is required for shadows to appear is independent of any choice of parameters. Furthermore, convincing shadows appear in the DBT and DBTS models only for values of k_off, which are small compared with those typically measured in related systems. It will be necessary to estimate the actual parameter values for Dpp during wing morphogenesis in order to ultimately understand its mechanism of spreading (Kruse, 2004).

Therefore, neither the DBT nor the DBTS model can explain the observed ligand and receptor profiles during Dpp spreading in the wing disc. Why should these models fail even though they incorporate many essential phenomena such as ligand diffusion, internalization and resurfacing via receptor recycling? The essential point of both the DBT and the DBTS model is that ligand transport is solely because of diffusion. In other words, this means that ligand bound to the surface receptors when internalized can only resurface at the same position on the cell surface where it was internalized. Only in this case is equation justified and the intracellular transport of the ligand would not contribute to the current of the ligand in the tissue. This implies that simple reaction diffusion models ignore that, in principle, ligand could also be transported by traveling through cells and resurface at other positions on the cell surface when receptors are recycled (Kruse, 2004).

The fact that the DBT and DBTS models (which ignore these effects), cannot account for observed Dpp spreading suggests that contributions of receptor trafficking to transport and ligand current may indeed play an important role. The DBT/DBTS models are currently being generalized to incorporate all relevant transport phenomena (diffusion and transcytosis) in the ligand current as well as the possibility of extracellular degradation of the ligand (Kruse, 2004).

The working hypothesis is that two phenomena contribute to the Dpp current in the developing wing epithelium: extracellular diffusion and intracellular trafficking (i.e. endocytosis plus resecretion). What is the relative importance of these two phenomena to the spreading of the morphogen? Both might be important. Limited by binding to the extracellular matrix and/or degradation, extracellular transport of the morphogen may only account for the spreading of the ligand across a few cell diameters. Intracellular trafficking in turn accounts for the movement of the morphogen across one cell diameter. Both phenomena together then lead to the long-range spreading of the morphogen (Kruse, 2004).

Although it is expected that extracellular diffusion plays a role during morphogen spreading, it has been argued that extracellular diffusion alone is insufficient to understand the reliability and precision of the formed gradient. The important role of intracellular trafficking has been uncovered in experiments in which endocytosis is blocked during morphogenetic signaling. When endocytosis is blocked in the receiving tissue, Dpp spreading does occur, but generates a short-range gradient and thereby signaling responses only within 3 to 5 cells. In particular, in a thermosensitive alpha-adaptin mutant, Dpp activates transcription of its target gene spalt only within 4-5 cells from the source, instead of within 15 cells in WT. Similar results Were obtained by expressing a dominant-negative Rab5 mutant, which impairs endocytosis and endosomal dynamics. These results do not exclude a role for endocytosis in the transduction, rather than on the spreading of Dpp. However, in the 'shibire rescue assay', the reduced range of the extracellular Dpp gradient indicates that impaired endocytosis restricts the spreading of Dpp (Kruse, 2004).

This report is a theoretical and experimental study to address whether diffusion as the sole transport mechanism can explain the spreading of Dpp. The role of different transport mechanisms for Dpp spreading is currently being studying. It has been argued that the rates of endocytosis and recycling known for the EGF receptor in cultured cells are too small to allow for a sufficiently rapid transport by transcytosis. Indeed, the first results based on generalized models (including diffusion and transport by planar transcytosis) show that the parameter values used in this work do not produce consistent gradients during reasonable times. In particular, these models require a faster rate of endocytosis and recycling than those known for the EGF receptor in cultured cells. Therefore, it is essential to measure directly the different dynamic parameters, including the extracellular diffusion coefficient as well as the rates of endocytosis, degradation and recycling of Dpp in the developing wing. To estimate these parameters in situ, photoactivatable fusion proteins are currently being monitored in different cellular locations (extracellular versus endosomal) in the very context of the developing wing epithelium (Kruse, 2004).

shibire mutations reveal distinct dynamin-independent and -dependent endocytic pathways in primary cultures of Drosophila hemocytes

A primary cell culture system derived from embryonic and larval stages of Drosophila allows for high-resolution imaging and genetic analyses of endocytic processes. This study investigated endocytic pathways of three types of molecules: an endogenous receptor that binds anionic ligands (ALs), glycosylphosphatidylinositol (GPI)-anchored protein (GPI-AP), and markers of the fluid phase in primary hemocytes. The endogenous AL-binding receptor (ALBR) is internalized into Rab5-positive endosomes, whereas the major portion of the fluid phase is taken up into Rab5-negative endosomes; GPI-APs are endocytosed into both classes of endosomes. ALBR and fluid-phase-containing early endosomes subsequently fuse to yield a population of Rab7-positive late endosomes. In primary culture, the endocytic phenotype of ALBR internalization in cells carrying mutations in Drosophila Dynamin (dDyn) at the shibire locus (shi^ts) parallels the temperature-sensitive behavior of shi^ts animals. At the restrictive temperature in shi^ts cells, receptor-bound ALs remain completely surface accessible, localized to clathrin and α-adaptin-positive structures. On lowering the temperature, ALs are rapidly sequestered, suggesting a reversible block at a late step in dDyn-dependent endocytosis. By contrast, GPI-AP and fluid-phase endocytosis are quantitatively unaffected at the restrictive temperature in shi^ts hemocytes, demonstrating a constitutive dDyn and Rab5-independent endocytic pathway in Drosophila (Guha, 2003).

Metazoan systems like Caenorhabditis and Drosophila provide a unique opportunity to extract molecular details about the mechanisms of endocytosis as well as a broader idea about the role of endocytic trafficking in the development and physiology of multicellular animals. For example, numerous genetic screens in Drosophila for mutations that result in reversible temperature-sensitive paralysis have led to the discovery of molecules affecting various aspects of neural transmission. Such behavioral screens, originally conceived as a means of isolating conditional mutations affecting muscle physiology, have largely identified molecules that affect axonal conduction (para) and synaptic vesicle recycling (shibire, stoned, comatose, syntaxin). Complementary reverse-genetic approaches have also identified other molecules affecting nervous system function (cysteine string protein, α-adaptin). Whereas specialized modes of trafficking such as synaptic vesicle recycling have been extensively investigated in Caenorhabditis and Drosophila, the diversity of endocytic pathways in these systems has yet to be characterized (Guha, 2003).

Not surprisingly, essential mediators of synaptic endocytosis like α-adaptin and the shibire (shi) gene product Drosophila Dynamin (dDyn), which is the only reported Drosophila homolog of vertebrate Dyn, are not restricted to the nervous system. dDyn is known to regulate endocytosis in a variety of fly tissues (Kosaka, 1983a; Kosaka, 1983b; Tsuruhara, 1990). Zygotic null mutations at the shi locus are lethal and the mutants show neural hyperplasia (Poodry, 1990). Electronmicroscopy (EM) studies that addressed the phenotypes of temperature-sensitive alleles at the shibire locus (shi^ts) in larval garland cells showed an accumulation of 'coated pits' at the plasma membrane and reduced uptake of fluid-phase tracers like horse radish peroxidase (HRP) (Kosaka and Ikeda, 1983b). These studies suggested that dDyn is required for all pathways of endocytosis in the cell types examined (Guha, 2003).

By contrast, over-expression of an analogous temperature-sensitive mutant of Dyn (Dyn^ts) in HeLa cells led to an initial reduction of fluid-phase uptake at 39°C that subsequently recovered (Damke, 1995); the internalization of human transferrin was completely inhibited at this temperature. These results argued for pathways of fluid-phase uptake in mammalian cells that are induced or upregulated when Dyn function is perturbed. Glycosylphosphatidylinositol-anchored proteins (GPI-APs) and the D2 dopamine receptor continue to be internalized into mammalian cells upon expression of dominant-negative Dyn isoforms. GPI-APs are endocytosed in a Dyn-independent manner into distinct endocytic compartments that contain a majority of the internalized fluid phase . These studies suggest that mammalian cells exhibit constitutive Dyn-independent pathways of endocytosis for both membrane and fluid-phase markers. However, mammals have three distinct genes encoding Dyn and as many as 25 splice variants (Cao, 1998), leaving open the possibility that alternate forms of Dyn might be involved in these endocytic events (McNiven, 2000). By contrast, in Drosophila dDyn, a multi-domain protein is encoded by a single locus that has six splice variants (Staples, 1999; van der Bliek, 1991). The availability of shi^ts alleles that map to domains conserved across all the splice variants makes Drosophila an attractive system to dissect the involvement of dDyn in different endocytic processes (Guha, 2003). Drosophila macrophages and hemocytes are a part of the innate immune system of the animal and are capable of internalizing a variety of ligands by a scavenger receptor-mediated (dSR) pathway. These cells are also phagocytic and have been shown to engulf both apoptotic cells and microbes; cells of this lineage in other metazoa are known to have multiple pathways of endocytosis (Guha, 2003).

This study establishes a methodology to reproducibly obtain primary cultures of macrophages and hemocytes from wild-type and mutant Drosophila embryos and larvae, respectively. The larval hemocytes have an endogenous anionic-ligand binding receptor (ALBR) with a similar ligand-binding specificity as dSR. In this system, the existence of multiple endocytic pathways was probed. Specifically, it was asked whether cells from embryonic and larval stages of wild-type and temperature-sensitive shibire animals are capable of dDyn-dependent and -independent endocytosis. It was found that, first, the endocytic phenotype of ALBR-mediated endocytosis in cells from temperature-sensitive shibire mutants parallels the behavior of mutant flies; ALBR-mediated internalization is reversibly blocked by raising the temperature. Second, at the restrictive temperature, cell-surface, ALBR-bound ligands remain completely accessible to relatively large molecules (70 kDa proteins); upon shifting to low temperatures, the arrested structures are internalized in a surge, restoring 'endocytic competence'. This is suggestive of a block in a late step of endocytosis in shi^ts mutants. By contrast, fluid-phase and GPI-AP endocytosis, which occurs via distinct endosomal structures, remains unaffected at the restrictive temperature in the shi^ts mutants, providing evidence for a constitutive dDyn-independent pathway (Guha, 2003).

Targeted expression of shibire^ts and semaphorin 1a reveals critical periods for synapse formation in the giant fiber of Drosophila

In order to determine the timing of events during the assembly of a neural circuit in Drosophila, expression of the temperature-sensitive shibire gene was targeted to the giant fiber system and then endocytosis was disrupted at various times during development. The giant fiber retracts its axon or incipient synapses when endocytosis is blocked at critical times, and four phases were perceived to giant fiber development: an early pathfinding phase, an intermediate phase of synaptogenesis, a late stabilization process and, finally, a mature synapse. By co-expressing shibire^ts and semaphorin 1a, evidence was provided that Semaphorin 1a is one of the proteins being regulated by endocytosis and its removal is a necessary part of the program for synaptogenesis. Temporal control of targeted expression of the semaphorin 1a gene shows that acute excess Semaphorin 1a has a permanent disruptive effect on synapse formation (Murphey, 2003).

The giant fiber (GF) system of the fly consists of a large interneuron that controls the visually evoked escape behavior through its synaptic contacts with thoracic motoneurons. Anatomical studies have provided the outlines of the development of this circuit. The dendrites of the jump motoneuron (TTMn) grow into the target area at the beginning of pupal development, before the axon of the GF reaches the thorax. The GF initiates growth in the late larval stage and the axon leaves the brain and reaches the target area in the second thoracic neuromere by ~25% of pupal development. The growth cone of the GF appears to contact the TTMn dendritic growth cone at this time, and during a synaptogenic phase (~25%-50% of pupal time) the GF elaborates a lateral 'bend' along the TTMn that becomes the presynaptic terminal and by 40% of pupal development the two neurons are dye-coupled (Murphey, 2003).

Blocking endocytosis seems on first examination to be a relatively blunt instrument for the analysis of nervous system development because of the wide variety of functions it might disrupt. However, two distinct effects have helped dissect pupal development of the GF system. First, blocking endocytosis causes retraction and subsequent regeneration of the GF, which alters the timing of axon growth and disrupts the resulting structure and function. Second, a role for membrane trafficking was highlighted by an interaction between sema1a and shibire^ts; this interaction created large vesicles in the axon terminal. Blocking endocytosis at the time of synapse formation appears to enhance the disruptive effect of Sema1a on synapse formation. This supports the idea that Sema1a, in its role as a repulsive receptor, must be removed from the growth cone during synaptogenesis. The interaction with shibire^ts suggests that this is likely to be regulated by dynamin-dependent endocytosis (Murphey, 2003).

Targeted blockade of endocytosis has direct effects on axon growth and retraction, presumably by disrupting the recycling of membrane in a rapidly growing axon terminal. It has been shown that neurons from mutant shi^ts1 animals grown in culture and then shifted to the restrictive temperature undergo collapse of growth cones, cessation of axon outgrowth and axon retraction. Shifting back to the permissive temperature leads to a resumption of growth and a rebound of growth rate. The temperatures used and the temperature shift paradigms employed in vitro are identical to those used to assay the timing of developmental events of the giant fiber in vivo (Murphey, 2003).

When endocytosis is blocked during pathfinding the axons retract during the temperature shift and when returned to the permissive temperature regenerate and overgrow the target area (phase I). By contrast, temperature shifts which correspond to the time that the GF is being transformed from growth cone to synapse (phase II) produce a different effect. GFs retract but when examined in adults the axons did not overgrow the target area but rather stopped in the target area and lacked the lateral bends. The initial effects induced by blocking endocytosis during both phase I and II are likely to be caused by the retraction of the axon and the subsequent heterochronic growth of the GF. However, the difference of the responses (overgrowth versus bendless-like) between phase I and II cannot be attributed directly to the block of endocytosis, but are more likely to be attributed to the different developmental states of the GF when heterochronic regeneration occurs. One relevant difference may be that in phase I most GFs have not contacted the target area and in phase II most GFs have contacted the targets. This means that the heterochronic growth induced in phase I results in naïve GFs that approach the target area with a delay, while heterochronic growth in phase II results in the re-generation of 'experienced' GFs. Possibly the GF loses its ability to regenerate the GF-TTMn synapse after it has contacted the target resulting in the bendless-like phenotype (Murphey, 2003).

Finally, blockade of endocytosis in phase III reveals a distinct defect. The function of the synapse is disrupted by temperature shifts although the structure remained normal. This distinguishes a stabilized synapse from a mature synapse. Possibly the block of endocytosis during phase III disrupts trafficking of receptors/ligands that are involved in maturation of the giant synapse. For example, Fasiclin 2 and Wingless/Frazzled have been shown to be required for maturation of the neuromuscular junction and correct dynamin-dependent trafficking is required for normal synaptogenesis (Murphey, 2003).

It has been suggested that Sema1a must be removed from the presynaptic terminal in order for synaptogenesis to proceed correctly, but the exact timing and mechanism for these events have not been examined. The acute presence of Sema1a during synapse formation is shown to have a lasting effect that prevents the regeneration of a functional synapse. The temporal aspects of Sema1a function, independent of endocytosis, were examined by taking advantage of the temperature sensitivity of the UAS constructs. Overexpression of Sema1a during synapse formation (phase II) causes the majority of axons to terminate in bendless-like structure and exhibit weak synaptic connections, while the acute presence of Sema1a in phase I or phase III has only minor effects. This suggests that removal of Sema1a is crucial for synaptogenesis. The sensitivity to Sema1a overexpression overlaps the time that the GF first contacts its targets and becomes dye-coupled to them, suggesting that Sema1a plays a role in the transition from growth cone to synapse. Interestingly, the bendless mutant causes phenotypes similar to those seen when Sema1a is overexpressed in the present experiments. When bendless was cloned and shown to be a ubiquitin conjugase it was speculated that the bendless mutant may be affecting the lifetime of Sema1a on the GF growth cone. The finding that Sema1a trafficking is involved in the assembly of the GF-TTMn synapse and the recent realization that ubiquitin can function to regulate trafficking of membrane proteins suggest that Sema1a trafficking may be regulated by Bendless (Murphey, 2003).

Endocytosis plays an important role in ligand-dependent receptor responses that serve as a mechanism for the regulation of signal strength in a variety of signaling pathways. It is proposed that during the transition from growth cone to synapse, Sema1a, which functions as a receptor on the GF growth cone, encounters its ligand and this slows the progress of the growth cone as a first step in the transition. However, the repulsive signaling of Sema1a must be downregulated because it is disruptive for subsequent events in the formation of the synapse and it is therefore normally removed through a dynamin-dependent receptor-mediated endocytosis. When UAS-sema1a is combined with UAS-shibire^ts in a genetic interaction experiment, simultaneous overexpression of Sema1a and the block of endocytosis exaggerates the disruptive effects of Sema1a. One effect is greater retraction of the axon presumably by enhancing the total amount of the repulsive receptor (Sema1a) present on the surface of the presynaptic cell. A second effect is the accumulation of large vesicles in the axon terminal. An interpretation is that the unusually high levels of Sema1a at the surface activates excessive receptor-mediated endocytosis. This may cause a vesicular 'traffic jam' in the growth cone, thereby disrupting the ability to carry out normal functions. These vesicular traffic jams are consistent with other experiments on the GF system that show similar phenotypes. For example, blocking retrograde transport by expression of a truncated version of the P150^Glued component of the dynein/dynactin motor also causes the formation of large vesicles in the GF terminal. Although the vesicles seen in these various genotypes cannot be directly linked to each other, the common phenotype makes it seem likely that a common membrane trafficking pathway involved in synapse formation is being interrupted. Markers for various aspects of the endosomal system in Drosophila will eventually allow the identification of the origin of these vesicles and link the various genotypes together in a model of receptor trafficking and synapse formation (Murphey, 2003).

In vertebrate neurons, semaphorin signaling has been linked to endocytosis during growth cone guidance and growth cone collapse. Sema3a serves as a ligand for the plexin/neuropilin receptor complex and has been shown to stimulate endocytosis during growth cone collapse. Moreover this is a Rac1-mediated process because Sema3a and Rac1 are associated with vesicles after Sema3a treatment and Rac1 is required for endocytosis of growth cone membrane during growth cone collapse. Although Sema3a is working as a ligand in vertebrate neurons and Sema1a is working as a receptor in the GF, there are a number of striking parallels between the vertebrate work and the Drosophilawork. In both cases, semaphorin and endocytosis are linked and in both cases Rac1 is involved in growth cone structure and behavior. Overexpression of the small GTPase Rac1 disrupts the termination of the GF and caused the accumulation of large vesicles in the terminal. Although Rac1 has not been directly linked to the semaphorin effects, the similarity between the GF phenotypes in these various experiments is consistent with the vertebrate work. The involvement of semaphorins, Rac1 and endocytosis in growth cone repulsion in vertebrate neurons and in the transition to synapse formation in the DrosophilaGF system highlights the similarities between the systems. Since synapse formation requires that growth cones slow or stop as they invade a target region, it seems likely that the growth cone guidance machinery has been commandeered to regulate the initial stages of synaptogenesis (Murphey, 2003).

Surprisingly, the appearance of large vesicles in the GF in the interaction experiment between Sema1a and endocytosis is delayed with respect to the temperature shift since no vesicles are detected immediately after the temperature shift but rather the vesicles emerge as pupal development proceeds. There are numerous suggestions that defects in membrane trafficking are linked to neurodegeneration and that these vesicles in the GF may be a prelude to synaptic degeneration; this possibility is being explored (Murphey, 2003).

Drosophila awd, the homolog of human nm23, regulates FGF receptor levels and functions synergistically with shi/dynamin during tracheal development

Human nm23 has been implicated in suppression of metastasis in various cancers, but the underlying mechanism of such activity has not been fully understood. Using Drosophila tracheal system as a genetic model, this study examined the function of the Drosophila homolog of nm23, the abnormal wing disc (awd) gene, in cell migration. Loss of Drosophila awd results in dysregulated tracheal cell motility. This phenotype can be suppressed by reducing the dosage of the chemotactic FGF receptor (FGFR) homolog, breathless (btl), indicating that btl and awd are functionally antagonists. In addition, mutants of shi/dynamin show similar tracheal phenotypes as in awd and exacerbate those in awd mutant, suggesting defects in vesicle-mediated turnover of FGFR in the awd mutant. Consistent with this, Btl-GFP chimera expressed from a cognate btl promoter-driven system accumulate at high levels on tracheal cell membrane of awd mutants as well as in awd RNA duplex-treated cultured cells. Thus, it is proposed that awd regulates tracheal cell motility by modulating the FGFR levels, through a dynamin-mediated pathway (Dammai, 2003. PubMed ID: Full text of article).

Unique biochemical and behavioral alterations in Drosophila shibire^ts1 mutants imply a conformational state affecting dynamin subcellular distribution and synaptic vesicle cycling

Dynamin is a GTPase protein that is essential for clathrin-mediated endocytosis of synaptic vesicle membranes. The Drosophila dynamin mutation shi^ts1 changes a single residue (G273D) at the boundary of the GTPase domain. In cell fractionation of homogenized fly heads without monovalent cations, all dynamin was in pellet fractions and was minimally susceptible to Triton-X extraction. Addition of Na(+) or K(+) can extract dynamin to the cytosolic (supernatant) fraction. The shi^ts1 mutation reduced the sensitivity of dynamin to salt extraction compared with other temperature-sensitive alleles or wild type. Sensitivity to salt extraction in shi^ts1 was enhanced by GTP and nonhydrolyzable GTP-gammaS. The shi^ts1 mutation may therefore induce a conformational change, involving the GTP binding site, that affects dynamin aggregation. Temperature-sensitive shibire mutations are known to arrest endocytosis at restrictive temperatures, with concomitant accumulation of presynaptic collared pits. Consistent with an effect upon dynamin aggregation, intact shi^ts1 flies recovered much more slowly from heat-induced paralysis than did other temperature-sensitive shibire mutants. Moreover, a genetic mutation that lowers GTP abundance (awd^msf15), which reduces the paralytic temperature threshold of other temperature-sensitive shibire mutations that lie closer to consensus GTPase motifs, did not reduce the paralytic threshold of shi^ts1. Taken together, the results may link the GTPase domain to conformational shifts that influence aggregation in vitro and endocytosis in vivo, and provide an unexpected point of entry to link the biophysical properties of dynamin to physiological processes at synapses (Chen, 2002).

Wingless gradient formation in the Drosophila wing

Although Wingless, Hh and Dpp have been shown to act directly on distant cells in the developing limbs of the Drosophila, little is known about how ligand gradients form in vivo. Wg protein is found in vesicles in Wg-responsive cells in the embryo and imaginal discs. It has been proposed that Wg may be transported by a vesicle-mediated mechanism. A novel method to visualize extracellular Wg protein was used to show that Wg forms an unstable gradient on the basolateral surface of the wing imaginal disc epithelium. Wg movement does not require internalization by dynamin-mediated endocytosis. Dynamin activity is, however, required for Wg secretion. By reversibly blocking Wg secretion, it was found that Wg moves rapidly to form a long-range extracellular gradient. It is concluded that the Wg morphogen gradient forms by rapid movement of ligand through the extracellular space, and depends on continuous secretion and rapid turnover. Endocytosis is not required for Wg movement, but contributes to shaping the gradient by removing extracellular Wg. It is proposed that the extracellular Wg gradient forms by diffusion (Strigini, 2000).

Using conventional antibody labeling methods, Wg protein has been detected in Wg-expressing cells at the dorsoventral (DV) boundary of the wing disc and in an irregular pattern of spots in nearby cells. The intensity and number of spots decreases with distance from the source of Wg, providing indirect evidence that Wg protein forms a gradient across the disc. The conventional antibody labeling protocol involves incubating anti-Wg antibody with fixed and permeabilized wing discs. When discs were incubated with anti-Wg antibody before fixation, a gradient of Wg protein was observed that appeared broader, shallower and less punctate than that observed with the conventional protocol. Control experiments showed that tubulin, an abundant intracellular protein, is readily visualized using the conventional protocol, but is not detected using the extracellular staining protocol. Thus, Wg visualized in this way reflects the distribution of the secreted extracellular protein (Strigini, 2000).

The distribution of extracellular and conventionally labeled Wg protein was compared by sequentially labeling discs using anti-Wg antibodies produced in different species. To determine what proportion of Wg protein is extracellular, intact wing discs were treated with proteinase K. Digestion of Wg was compared with digestion of Fasciclin II, a glycosyl phosphatidyl inositol (GPI)-linked membrane protein, and with cytoplasmic tropomyosin in immunoblots of total disc extracts. In the absence of detergent, Fasciclin II was completely digested, and cytoplasmic tropomyosin was not digested. Wg levels were reduced considerably. Wg and cytoplasmic tropomyosin were completely digested when disc cells were permeabilized with detergent during the protease treatment. These observations indicate that much of the Wg protein in the discs is extracellular and accessible to protease digestion or to antibody binding. The remaining Wg protein is presumably intracellular (Strigini, 2000).

Imaginal discs consist of a single-layered sac of polarized epithelial cells, with the apical surface of the cells oriented towards the lumen of the disc. The polarity of the epithelial cells can be visualized using antibody to Coracle, which labels the junctional complex that separates apical from basolateral surfaces. Using the conventional labeling protocol, most of the Wg appears to be concentrated above the nuclei, near the junctional complex, in Wg-expressing cells. In contrast, extracellular Wg is mainly associated with the basolateral surface of cells. The spots of Wg visualized by conventional labeling in cells away from the source appear to reflect vesicles of internalized Wg protein (Strigini, 2000).

The observation that extracellular Wg appears to be concentrated on the basolateral surface of Wg-expressing and nearby cells prompted an investigation to see whether Wg moves across the apical (that is, lumenal) or basolateral surface of the epithelium. To distinguish between these possibilities, use was made of the observation that overexpression of the Drosophila Wg receptor Frizzled 2 (Dfz2) causes accumulation of Wg on cells at a distance from the DV boundary. Wg accumulation was compared in cells expressing full-length Dfz2 and cells expressing the Wg-binding domain of Dfz2 as a GPI-anchored protein (GPI-Dfz2). Full-length Dfz2 is expressed uniformly on the apical and basolateral surfaces of the epithelium, whereas GPI-anchored proteins localize to the basolateral surface of the imaginal disc cells and so can only accumulate Wg on the basolateral surface. In both cases, Wg accumulates to high levels on the basolateral surface of the epithelial cells but is absent from the region above the nuclei, where most Wg protein was found in Wg-expressing cells. These observations suggest that the extracellular Wg gradient forms on the basolateral surface of the wing disc (Strigini, 2000).

The extracellular Wg gradient correlates with a graded distribution of intracellular Wg vesicles. Does Wg internalization play a role in gradient formation? Previous studies have suggested a role for Shibire-dependent endocytosis in Wg transport in the embryo; shibire encodes the Drosophila homolog of the GTPase dynamin. Dynamins have been implicated in the internalization of clathrin-coated endocytic vesicles and in the internalization of caveolae. To determine whether shibire-dependent vesicle traffic is required for Wg gradient formation, Wg distribution was examined in wing discs carrying clones of shibire temperature sensitive mutant cells. Clones were allowed to grow under conditions in which the temperature-sensitive Shibire protein is active (18ƒC). The larvae were then shifted to 32ƒC for 3 hours to inactivate Shibire. Under these conditions, punctate Wg wis not observed in the mutant cells using the conventional labeling protocol, indicating that dynamin activity is required for Wg internalization. Although spots of internalized Wg were not seen in shibire mutant cells, Wg is internalized by wild-type cells adjacent to the clone. The presence of Wg in these cells may reflect movement of Wg across the mutant tissue to reach wild-type cells. In support of this view, extracellular Wg was observed on the surface of shibire mutant cells. The level of extracellular Wg was higher than on nearby wild-type cells. The interpretation that Wg can move across the shibire mutant tissue depends on the assumption that extracellular Wg is not simply stabilized in the shibire mutant tissue. To determine whether removing dynamin function stabilizes extracellular Wg, Wg distribution was examined in shibire mutant discs. At 18ƒC, extracellular Wg distribution in shibire mutant discs is comparable to that in the wild type. When Shibire is inactivated at 32ƒC for 3 hours, little or no extracellular Wg is detected. This indicates that extracellular Wg turns over rapidly when the entire disc is shibire mutant. It is concluded that the Wg on the shibire mutant clones reflects Wg secreted by nearby wild-type tissue that has moved across the clone and that the local accumulation reflects impaired endocytosis. Thus, dynamin-mediated internalization does not appear to play a role in Wg transport in the wing disc. In contrast, dynamin-mediated endocytosis appears to play a role in removing secreted Wg from the extracellular space and, therefore, may help to maintain a steep gradient (Strigini, 2000).

The loss of extracellular Wg from homozygous mutant shibire discs suggested that Wg secretion might be impaired by removing dynamin activity. Conventional staining showed an intense band of intracellular Wg accumulation at the DV boundary under these conditions, which resembles the accumulation of Wg in clones of porcupine mutant cells. The porcupine gene encodes a protein that resides in the endoplasmic reticulum and is required for post-translational processing and secretion of Wg in the embryo. Comparable Wg accumulation is observed in shibire mutant clones that include Wg-expressing cells. Wg accumulates only in Wg-expressing mutant cells, even when the clone is small and most of the surrounding cells are wild-type. No intracellular Wg accumulation is seen in shibire mutant clones that abut but do not include Wg-expressing cells (Strigini, 2000).

As shibire encodes the only Drosophila Dynamin identified to date, these results suggest that Shibire protein may have a role in the formation of transport vesicles at the trans-Golgi network, comparable to that reported for dynamin-2, one of Shibire's vertebrate homologs. If so, the traffic of vesicles from the trans-Golgi to the plasma membrane might be blocked in shibire mutant cells. Alternatively, blocking Shibire-dependent endocytosis might impair membrane traffic, and indirectly reduce Wg secretion (Strigini, 2000).

The temperature-sensitive defect in dynamin function caused by the shibire temperature sensative mutation is rapidly reversed by shifting flies back to the permissive temperature. This provides a means to reversibly block Wg secretion. The extracellular Wg gradient was depleted within 3 hours in shibire^ts mutant discs at 32^oC, indicating that extracellular Wg is rapidly lost or degraded even in the absence of endocytosis. Furthermore, internalized Wg is almost entirely cleared from the disc by 3 hours at 32^oC. Shifting shibire mutant larvae back to the permissive temperature provides the opportunity to monitor the time course of gradient formation when Wg secretion is reinitiated. With 15 minutes of recovery at 18^oC, spots of internalized Wg protein can again be detected across the disc. By 30-60 minutes, the Wg distribution resembles that in control discs kept at 18^oC, indicating that Wg protein has traveled across the disc and been internalized by distant cells. These observations indicate that Wg moves rapidly through the tissue to form a gradient covering at least 50 µm in approximately 30 minutes. For comparison, in Xenopus explants, the signaling protein Activin can form a gradient over more than 250 µm by diffusion in a few hours. Thus, the rate of Wg movement is compatible with diffusion through the extracellular space (Strigini, 2000).

Wg binds avidly to glycosaminoglycans. Mutations that affect glycosaminoglycan biosynthesis phenocopy weak wg mutations and genetically interact with wg mutant alleles. Recent reports have implicated the GPI-anchored proteoglycan Dally as a cofactor in Wg signaling. Dally might facilitate Wg signaling by improving retention and movement of Wg along the basolateral surface of the epithelium. No significant alteration could be detected in the distribution of extracellular Wg in dally mutant discs or in dally mutant clones. Overexpression of Dally using the patched promoter to drive expression of the GAL4-encoded transcriptional activator (ptc-GAL4) causes little or no additional accumulation of Wg in cells near the DV boundary. This contrast with the effects of overexpressing Dfz2 using dpp-GAL4 (a weaker GAL4 driver). These observations suggest that Dally may have a relatively low capacity to bind Wg in vivo or that it is present in excess, and that Dfz2 is the limiting factor in Wg binding. These observations strengthen the proposal that Dally might serve as a coreceptor with Dfz2, and suggest that Dally does not play a significant role in shaping the Wg gradient. Clones of cells mutant for sugarless were also examined and no effect on Wg distribution was seen. These observations leave open the question of whether other proteoglycans might contribute to Wg gradient formation (Strigini, 2000).

Directionality of wingless protein transport influences epidermal patterning in the Drosophila embryo

Wg and Wnt molecules tightly associate with membrane and extracellular matrix and appear not to be readily soluble. Thus, it is unlikely that these proteins freely diffuse through extracellular spaces. Rather, Wg appears to be transported via active cellular processes. This phenomenon was first demonstrated using the shibire^ts (shi^ts) mutation to block endocytosis. shi encodes the fly dynamin homolog, a GTPase required for clathrin-coated vesicle formation. Rather than the broad, punctate Wg protein distribution normally found over several cell diameters on either side of the wg-expressing cells, shi mutant embryos show high level accumulation of Wg around the wg-expressing cells. Structure/function analysis of the Wg molecule further supports the idea that active transport of the ligand is essential. Four mutations within wg have been isolated that specifically disrupt Wg transport without abolishing signaling activity. These mutant molecules generate a restricted response within the segment, as assayed by both cuticular pattern elements and molecular events. Homozygous mutant embryos produce naked cuticle but little denticle diversity, and show narrowed domains of Wg protein distribution and Arm stabilization. Three of these four mutations are single amino acid substitutions; each affects a residue that is highly conserved throughout the Wnt family, suggesting that ligand transport may be an important general aspect of Wnt function (Moline, 1999 and references).

To assess the functional consequences of this broad Wg distribution, a means has been devised of perturbing endocytosis in spatially restricted domains within the embryo. A transgene expressing a dominant negative form of shibire (shi), the fly dynamin homolog, was constructed. When this transgene is expressed using the GAL4-UAS system, Wg protein distribution within the domain of transgene expression is limited and Wg-dependent epidermal patterning events surrounding the domain of expression are disrupted in a directional fashion. These results indicate that Wg transport in an anterior direction generates the normal expanse of naked cuticle within the segment and that movement of Wg in a posterior direction specifies diverse denticle cell fates in the anterior portion of the adjacent segment (Moline, 1999).

Interfering with posterior movement of Wg rescues the excessive naked cuticle specification observed in naked (nkd) mutant embryos. It is proposed that the nkd segment polarity phenotype results from unregulated posterior transport of Wg protein and therefore that wild-type Nkd function may contribute to the control of Wg movement within the epidermal cells of the segment (Moline, 1999).

Using en-Gal4-driven shi^D expression to reduce posterior movement of Wg suppresses the phenotype of the segment polarity mutation, naked. nkd mutant embryos secrete denticle belts that have essentially normal denticle type diversity but that are replaced to varying degrees by naked cuticle. This excess naked cuticle depends upon Wg activity levels. The wg;nkd double mutant shows no naked cuticle across the ventral region and reducing the dosage of wg in a nkd mutant restores denticle belts. Thus wild-type nkd gene function appears to be involved in limiting Wg signaling activity within the segment. Consistent with this idea, Wg target genes become ectopically expressed in nkd mutant embryos. The en expression domain expands 2-3 cell diameters during stage 9, and an ectopic stripe of wg expression arises at stage 10, in the row of cells posterior to this expanded en domain. The posterior expansion of en expression suggests that nkd might play a role in restricting the movement of Wg protein in a posterior direction. Indeed, when nkd mutant embryos were generated that express shi^D at moderate levels in the en domain, there was a dramatic reduction in the amount of naked cuticle specified. These embryos are very similar in appearance to wild-type embryos in which en-Gal4 drives shi^D expression except that the nkd mutant head defect is not fully rescued. en-Gal4-driven shi^D expression also prevents the ectopic activation of en expression in nkd mutants. The stripes of en expression in the thoracic and abdominal segments are restored to the normal width, although some expansion is still observed in the head segments (Moline, 1999).

Since wild-type Wg signaling activity is required for stabilization of en expression, En stripes of normal width indicate that sufficient functional Wg contacts both rows of en-expressing cells to produce normal target gene regulation. This result demonstrates that expression of shi^D does not interfere with Wg signal transduction and supports the idea that moderate level shi^D expression reduces, but does not eliminate, transport of Wg across the affected domain. In contrast, embryos expressing high level shi^D in the en domain show a narrowed stripe of En antibody staining, suggesting that Wg can no longer traverse the first row of en-expressing cells to stabilize en in the second row. However, because of the severe effects of a more complete endocytotic block, these embryos do not secrete cuticle properly and so the effects on cuticle pattern are not interpretable (Moline, 1999).

During early stages of wild-type embryogenesis, Wg protein can be detected at high levels in cells both anterior and posterior to the wg-expressing row of cells. Diversity of denticle types, as well as stabilization of en expression in the adjacent cells, are specified by Wg activity during these early stages of embryonic development. By mid-stage 10, when Wg is no longer required for denticle specification or en stabilization, the Wg protein distribution shifts and Wg appears to be excluded from the en-expressing cells. This exclusion is not observed in nkd mutants at the same stage. Rather, Wg protein continues to be detected in cells on either side of the wg-expressing row of cells and the levels become substantially higher due to the ectopic stripe of wg expression. These results suggest that nkd gene function may play a role in the posterior restriction of Wg protein that occurs during stage 10. Hence the mutant phenotype is rescued dramatically when this restriction is produced artificially, by expressing shi^D in the en-expressing row of cells. All stage 11 and 12 embryos derived from this cross show posterior restriction of Wg protein, indicating that the nkd homozygotes do not exhibit excess posterior movement of Wg under these conditions. It is suspected that, in wild-type embryos, this restrictive function is not limited to the en-expressing cells. If this were the case, then one would expect to observe excess naked cuticle replacing denticle belts when wg+ is expressed in the en domain. Instead, en-Gal4-driven wg+, either alone or when co-expressed with shi^D, does not produce substantial amounts of ectopic naked cuticle. Thus, it seems likely that some ability to restrict posterior Wg movement during later stages is shared by the rows of cells at the anterior of each segment (Moline, 1999).

It is believed that this analysis of Wg transport by perturbing endocytosis is physiologically relevant because a similar inhibition of transport can be produced by overexpressing Dfz2, the cognate receptor for Wg. It is presumed that these effects result from sequestering ligand, because pattern defects are observed only when Wg levels are limiting. No change from the wild-type cuticle pattern is detected when Dfz2 is driven at ubiquitous high levels of expression with E22C-Gal4. However, in embryos heterozygous for a null mutation of wg, significant pattern defects are observed at a frequency of 60%. Ectopic denticles appear in the domain of cells that normally secrete naked cuticle, similar to what is observed in segments where anterior Wg transport is perturbed by shi^D. These pattern defects caused by Dfz2 overexpression are accompanied by a restricted Wg protein distribution and by a narrowed domain of Arm stabilization. However, it is not possible to directly compare Dfz2 with shi^D in this experiment. E22C-Gal4-driven expression of shi^D , even with UAS lines that express at low levels, results in cell death and failure to secrete cuticle as was the case with the original shi^ts mutation at restrictive temperature (Moline, 1999).

Ligand endocytosis drives receptor dissociation and activation in the Notch pathway

Receptor and ligand processing have recently come under scrutiny as critical elements in the regulation of Notch signaling. Delta is proteolytically processed to yield at least four isoforms; however, the functional significance of this processing is currently unclear. Processing of Delta may be necessary for signal activation or for downregulation, or may result from protein degradation following clearance of ligand from the cell surface. Notch is processed in a complex manner that is thought to be required for genesis and activation of the receptor. First, Notch is cleaved during transport through the Golgi at a site amino-proximal to the transmembrane domain ('site 1' or 'S1') by a furin-like convertase. Following this cleavage, Notch is 'reassembled' and transported to the cell surface as a heterodimeric receptor. Another cleavage event (termed 'S3') within the intracellular domain has also been shown to occur. The S3 cleavage of Notch is ligand-dependent and produces a Notch intracellular domain fragment that may act, in conjunction with Su(H), in the nucleus as the primary Notch signal transducer. The mechanism that triggers the intracellular domain cleavage is unknown. However, the Notch/lin-12 repeats (LNRs) within the receptor extracellular domain may contribute to regulation of this cleavage. When the LNRs are removed, intracellular domain cleavage occurs in a constitutive manner in the absence of ligand. This has led to the hypothesis that binding of Delta to Notch may result in a cleavage event (termed 'S2') in the Notch extracellular domain. This cleavage would uncouple the LNRs from the remainder of the receptor, allowing the intracellular domain cleavage (S3) to occur constitutively. Recently, Notch S2 cleavage has been demonstrated in mammalian cells (Mumm, 2000 in press, cited in Parks, 2001). S2 cleavage in these cells occurs in response to ligand binding and blocking S2 cleavage results in loss of S3 cleavage, consistent with a proteolytic cascade model of Notch activation (Parks, 2000).

Endocytosis of the ligand Delta (by the signaling cell), possibly by inducing cleavage of the receptor at the S2 site, is required for activation of the receptor Notch during Drosophila development. The Notch extracellular domain (NotchECD) dissociates from the Notch intracellular domain (NotchICD) and is trans-endocytosed into Delta-expressing cells in wild-type imaginal discs. Reduction of dynamin-mediated endocytosis in developing eye and wing imaginal discs reduces Notch dissociation and Notch signaling. Furthermore, dynamin-mediated Delta endocytosis is required for Notch trans-endocytosis in Drosophila cultured cell lines. Endocytosis-defective Delta proteins fail to mediate trans-endocytosis of Notch in cultured cells, and exhibit aberrant subcellular trafficking and reduced signaling capacity in Drosophila. It is suggested that endocytosis into Delta-expressing cells of NotchECD bound to Delta plays a critical role during activation of the Notch receptor and is required to achieve processing and dissociation of the Notch protein (Parks, 2000).

The separation of NotchECD from NotchICD would relieve LNR-mediated repression of the S3 cleavage, which would then occur constitutively to release a non-membrane bound, activated form of NotchICD. Several predictions of this model are borne out. Dl alleles that encode endocytosis-defective ligands are loss-of-function mutations, and these defective ligands fail to support Notch trans-endocytosis in cultured cells and Delta-mediated signaling in vivo. Dynamin function, which is necessary for Notch signaling, is required for Delta endocytosis and for Notch trans-endocytosis in cultured cells, and for dissociation of NotchECD from NotchICD in developing imaginal tissues. In addition, the third epidermal growth factor-like repeat within the Delta extracellular domain is required for Delta endocytosis and Notch trans-endocytosis, and for Delta-dependent signaling during development (Parks, 2000).

Three alternative mechanisms are proposed by which endocytosis may induce receptor activation. (1) After binding of Delta to Notch, molecular strain imparted to Notch by endocytosis in the signaling and receiving cells results in a conformational change that permits access by processing enzyme(s) to the S2 site. (2) The S2 site is masked by proteins that interact with Notch to form a complex. Following Delta binding, endocytosis of Delta and Notch alters intramolecular interactions within the complex, unmasking the S2 site and making it available for cleavage. (3) Endocytosis is not required for Delta-induced S2 cleavage, but is instead required to separate NotchECD and associated proteins from the remainder of the Notch protein, thus relieving inhibition of S3 cleavage by NotchECD. The fact that Serrate-expressing cultured Drosophila cells mediate Notch trans-endocytosis at frequencies similar to those observed for Delta-expressing cells suggests that trans-endocytosis of NotchECD is one aspect of the Notch activation mechanism that is common to Notch ligands (Parks, 2000).

Role of Drosophila alpha-Adaptin in presynaptic vesicle recycling

Rapid flow of information in the nervous system involves presynaptic vesicle recycling by clathrin-mediated endocytosis, an event triggered by the alpha-Adaptin-containing AP2 complex. A Drosophila alpha-Adaptin is expressed in the garland cells, imaginal discs, and the CNS. In presynaptic terminals, alpha-Adaptin defines a network-like membrane structure to which the GTPase dynamin is recruited. alpha-Adaptin is necessary for the formation of clathrin-coated pits and participates in the dynamin-dependent release of coated vesicles from the membrane surface. These results suggest an alpha-adaptin-dependent control of the vesicle cycle that maintains the balance between the amount of vesicle- and surface-associated membranes (González-Gaitán, 1997).

alpha-Adaptin also acts downstream of Numb in the determination of alternative cell fates in asymmetric cell division. During asymmetric cell division in sensory organ precursor cells, Numb protein localizes asymmetrically and segregates into one daughter cell, where it influences cell fate by repressing signal transduction via the Notch receptor. Numb acts by polarizing the distribution of alpha-Adaptin, a protein involved in receptor-mediated endocytosis. alpha-Adaptin binds to Numb and localizes asymmetrically in a Numb-dependent fashion. Mutant forms of alpha-Adaptin that no longer bind to Numb fail to localize asymmetrically and cause numb-like defects in asymmetric cell division. These results suggest a model in which Numb influences cell fate by downregulating Notch through polarized receptor-mediated endocytosis, since Numb also binds to the intracellular domain of Notch (Berdnik, 2002).

Adaptins are subunits of adaptor protein (AP) complexes involved in the formation of intracellular transport vesicles and in the selection of cargo for incorporation into the vesicles. The term 'adaptin' was coined to designate a group of ~100 kDa proteins that copurify with clathrin upon isolation of clathrin-coated vesicles. The ~100 kDa-proteins are subunits of heterotetrameric adaptor protein (AP) complexes, and the term 'adaptin' has been extended to all subunits of these complexes. Four basic AP complexes have been described: AP-1, AP-2, AP-3, and AP-4. Each of these complexes is composed of two large adaptins (one each of g/alpha/d/e and ß1-4, respectively, 90-130 kDa), one medium adaptin (µ1-4, ~50 kDa), and one small adaptin (s1-4, ~20 kDa). [See the figure in the Hill review (2001) for information on the AP structure]. The analogous adaptins of the four AP complexes are homologous to one another (21-83% identity at the amino acid level). In general, the subunits of different AP complexes are not interchangeable, with the exception of some nonmammalian ß1/2 hybrid proteins, and possibly mammalian ß1 andß2, which can be components of both AP-1 and AP-2. Some of the adaptins occur as two or more closely-related isoforms encoded by different genes. Additional diversity arises from alternative splicing of adaptin mRNAs. Thus, cells that express several of these adaptin variants have the potential to assemble a diverse array of AP complexes. AP-1, AP-2, and AP-3 are expressed in all eukaryotic cells examined to date. AP-4, on the other hand, is ubiquitously expressed in man (Homo sapiens), mouse (Mus musculus), chicken (Gallus gallus), and the plant Arabidopsis thaliana, but not in the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, and the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe (Hill, 2001 and references therein).

AP complexes are components of protein coats that associate with the cytoplasmic face of organelles of the secretory and endocytic pathways. The complexes participate in the formation of coated vesicular carriers, as well as in the selection of cargo molecules for incorporation into the carriers. AP-2 mediates rapid endocytosis from the plasma membrane, while AP-1, AP-3, and AP-4 mediate sorting events at the trans-Golgi network (TGN) and/or endosomes. AP-1 and AP-2 function in conjunction with clathrin, whereas AP-4 is most likely part of a nonclathrin coat. Mammalian (but not yeast) AP-3 has been shown to interact with clathrin, but the functional significance of this interaction is still unclear. The AP complexes have the overall shape of a 'head' with two protruding 'ears' connected to the head by flexible 'hinge' domains (Hill, 2001 and references therein).

Synaptic transmission requires the repeated release of neurotransmitters at high frequency at the nerve terminal. The current understanding of this rapid communication process between the nerve cells and their targets is primarily based on the biochemistry of the synaptic vesicles, protein–protein interaction studies, structural analyses, electrophysiology, and imaging. These studies suggest that the repeated release of transmitters involves a synaptic vesicle cycle that includes the formation of neurotransmitter-filled vesicles, the release of the neurotransmitter by exocytosis, recycling of both the membrane and the release machinery by endocytosis, and, finally, the regeneration of vesicles that undergo a new round of exocytosis/endocytosis. Each step of the vesicle cycle involves a number of identified components, and their assembly is likely to be mediated by distinct protein–protein interactions (González-Gaitán, 1997 and references therein).

While recent studies have focused on the proteins involved in exocytosis, it is not clear whether (or to what extent) the vesicle membrane recycles via clathrin-coated vesicles, or whether the membrane is directly retrieved by a fast endocytotic process. Biochemical studies lead to the proposal that the AP2 complex, a heterotetramer containing alpha-adaptin, plays an essential role in orchestrating different steps of endocytosis at the synapse. AP2 is able to bind to the cytoplasmic tail of a number of membrane receptors including Synaptotagmin, a transmembrane protein that controls the Ca²⁺-dependent membrane fusion during exocytosis. Synaptotagmin interaction with AP2 is consistent with its proposed function during endocytosis, which is based on a synaptotagmin endocytotic mutant phenotype. This would argue that synaptotagmin plays a dual role in the vesicle cycle by acting in the final step of exocytosis and the initial step of endocytosis, thereby coupling the two processes at the plasma membrane. Once recruited to the inner surface of the plasma membrane, AP2 is likely to initiate the formation of clathrin-coated pits by triggering the assembly of clathrin triskelion subunits into a polygonal lattice that causes a bending of the membrane into the coated pit structure. Clathrin-coated pits detach from the plasmalemma by a GTP-dependent fission reaction that is mediated by the GTPase dynamin, and the resulting coated membrane vesicles become internalized. Dynamin was shown to bind the AP2 complex in vitro, and to be functionally required for the detachment of the clathrin-coated vesicles from the membrane. After internalization, the clathrin-coated vesicles shed their coats, a process that involves a number of proteins, including auxilin, Hsp-70, and the cystein string protein (CSP), which may function in a chaperone-like manner to unfold the clathrin lattice at the outer surface (González-Gaitán, 1997 and references therein).

While there is little doubt that synaptic vesicle membranes are endocytosed by clathrin-coated vesicles, it remains to be established whether this pathway is essential for synaptic transmission. The most direct evidence is derived from an analysis of the only endocytotic mutant isolated to date, the Drosophila mutant shibire (shi), which affects the gene coding for Dynamin. In a temperature-sensitive shi mutant, animals develop normally at the permissive temperature. At the restrictive temperature, however, release of transmitter ceases, leading to rapid paralysis. Examination of the shi mutant phenotype by electron microscopy indicated that endocytosis is normally initiated and clathrin-coated pits are formed but remain trapped in the collared pit stage. Thus, vesicle recycling and, consequently, transmitter release are blocked when the function of dynamin is impaired (González-Gaitán, 1997 and references therein).

Mutations in a Drosophila alpha-Adaptin gene (D-alphaAda) have been used to study recycling of synaptic vesicles. At the larval neuromuscular junction, synaptic vesicle recycling is impaired in weak alpha-Ada mutants and blocked in alpha-adaptin-deficient embryos. alpha-adaptin is confined to areas of the presynaptic plasma membrane that are distinct from the sites containing the active zones of exocytosis. Furthermore, alpha-adaptin is required for the recruitment of dynamin to the endocytotic sites (González-Gaitán, 1997).

Cloning of alphaAdaptin was initiated through a P element enhancer trap insertion found in a screen for genes expressed in the embryonic nervous system. The corresponding P element maps to cytological position 21C1-2. A genomic DNA fragment adjacent to the P element insertion site was identified and a genomic walk was constructed encompassing the region that turned out to be the alphaAdaptin transcription unit. Northern blot analysis with polyA⁺ RNA of embryos and adults revealed that alphaAdaptin codes for two transcripts of about 4.8 and 6.3 kb. Both transcripts appear with similar intensities in the polyA⁺ RNA of embryos, while in adults, the smaller transcript appears to be enriched over the longer one (González-Gaitán, 1997).

Rapid synaptic transmission in the nervous system takes advantage of the general property of cells to recycle the membrane by endocytosis. Consistently, vertebrate alpha-Adaptins have been found to be expressed ubiquitously, with the exception of an alpha-Adaptin-A isoform that is specifically enriched in neural cells. Likewise, Drosophila dynamin, which is required during synaptic transmission, is ubiquitously expressed and required. Evidence is provided for an essential role of a Drosophila alpha-Adaptin in recycling of synaptic vesicles. The highly restricted expression patterns of the Drosophila alpha-Adaptin and the specific defects in aAdaptin mutants exclude a general role for alpha-Adaptin in endocytosis in all cells. Also, the mutant defect indicates that the lack of alphaAdaptin activity in the nervous system cannot be compensated for by a redundant action of other members of the alpha-Adaptin family, which must exist in the Drosophila genome to function in endocytosis outside of the nervous system (González-Gaitán, 1997).

In vitro studies have shown that alpha-Adaptin triggers the formation of a clathrin lattice that turns into empty coat structures. This finding, and the position of AP2 between the clathrin lattice and the vesicle membrane, is consistent with the proposal that the formation of coated vesicles is initiated by an alpha-Adaptin-dependent recruitment of clathrin to the membrane. The lack of vesicles and the corresponding increase of the presynaptic plasma membrane in the aAdaptin mutant establish that alpha-Adaptin is indeed required for the formation of clathrin-coated pits. The resulting lack of membrane recycling causes an increase of the membrane surface, indicating that the pool of vesicle membranes is fused with the presynaptic plasma membrane of the alpha-Ada mutant (González-Gaitán, 1997).

At the presynaptic terminal, vesicles dock and fuse to release neurotransmitters by exocytosis. In Drosophila, active zones of transmitter release at the plasma membrane are a discrete electron-dense structure, the so-called dense bodies. At the neuromuscular junctions, such active zones are found in patterns of interspersed islands. The restricted network-like structure of alpha-Adaptin in both wild-type and shi mutant presynaptic terminals, where vesicles formation is blocked at the collared pit stage, leaves such islands void of alpha-Adaptin. This observation suggests that alpha-Adaptin defines regions within the presynaptic membrane that are complementary to the distribution of active zones. If this inference is correct, it would imply that exocytosis and endocytosis events occur in different locations at the presynaptic membrane (González-Gaitán, 1997).

The compositions of the plasma membrane and the synaptic vesicle membranes are different. This supports the argument that the membranes of exocytotic vesicles never fuse completely with the plasma membrane and instead open up at the fusion site to release the transmitter, closing immediately thereafter. Such a mechanism implies that active zones of exocytosis and the site of vesicle recycling would coincide. However, most recent ultrastructural analysis of shi mutant synaptic terminals provides evidence for two distinct pathways for vesicle re-formation. One pathway emanates from the active zone of exocytosis and has a fast time course. It involves small clusters of vesicles that are observed at the active zones. The formation of these vesicles does not include intermediate structures, such as coated pits, coated vesicles, or cisternae, and might be accomplished by a direct pinch-off at the plasma membrane. The second pathway emanates from sites away from the active zones and results in the re-formation of the rest of the vesicle population throughout the terminal. It has a slower time course and involves coated collared pits (González-Gaitán, 1997).

The distinct staining pattern of alpha-Adaptin, in a network-like array, suggests that alpha-Adaptin acts preferentially, if not exclusively, in the second pathway involving coated vesicles outside the active zones. Furthermore, the lack of vesicles and a corresponding expansion of the plasma membrane in alpha-Adaptin-deficient embryos are consistent with a recycling mechanism that builds upon a complete fusion between the vesicle and plasma membrane compartments during exocytosis, and the re-formation of vesicle membranes at separate centers of endocytosis (González-Gaitán, 1997).

Dynamin, originally identified as a microtubule-binding protein, has been shown to have GTPase activity in vitro, containing three GTP-binding consensus motifs. Introducing mutations that interfere with GTP binding has been found to have no effect on microtubules in transfected cells, but endocytosis is blocked. In shi mutants, the clathrin-coated pits are normally formed at the plasma membrane but fail to pinch off the membrane. In contrast, the coated pits fail to form in the membrane of Drosophila alpha-Adaptin mutants. These observations formally argue that alpha-Adaptin and dynamin act in two distinct steps during vesicle recycling, and they are not consistent with the proposed direct interaction between AP2 and dynamin as revealed by in vitro studies (Wang, 1995). However, an increased temperature sensitivity of the shi mutation caused by the lack of one functional copy of the alpha-Adaptin gene strongly suggests that the two molecules act in linked processes, and, therefore, that alpha-Adaptin is also required for the dynamin-dependent internalization of the vesicles. This finding, together with the subcellular localization of alpha-Adaptin in wild type and the colocalization of alpha-Adaptin and dynamin in shi mutant synapses, is therefore consistent with a function of the alpha-Adaptin-containing AP2 complex in recruiting the GTPase dynamin to the assembled clathrin-coated pits (Wang, 1995). The genetic interaction shown in this study also indicates that alpha-Adaptin directly or indirectly interacts with dynamin. This strongly suggests that alpha-Adaptin functions upstream of dynamin, since it acts at an earlier step of endocytosis than dynamin (as indicated by their respective mutant phenotypes), and since the subcellular localization of alpha-Adaptin is not affected in shi mutants. These findings indicate a molecular link between the formation of clathrin-coated pits and the detachment of coated vesicles, coordinating two consecutive steps of the process of endocytosis (González-Gaitán, 1997).

The alpha-Adaptin mutant phenotype indicates that the lack of vesicle formation affects not only physiological aspects of synapse function but also causes a corresponding increase of the plasma membrane complement. This suggests that membrane fractions present in the vesicle and plasma membrane compartments, and their rapid exchange by exocytosis/endocytosis events, are regulated to maintain the surface area of the synapse and the pool of available synaptic vesicles. This control obviously does not involve exocytosis, since it proceeds to cause an expansion of the membrane surface in the alpha-Adaptin mutant synapses. Neither is vesicle internalization the controlling step, because in the dynamin mutant shi, the membrane can be processed into clathrin-coated pits that remain attached to the plasma membrane. Furthermore, collared coated pits are rarely observed in wild type, suggesting that they represent a very transient intermediate, and, therefore, that vesicle internalization by dynamin is not a limiting step (González-Gaitán, 1997).

One can envision a scenario where the recruitment of the AP2 complex to the plasma membrane is a rate-limiting step, which in turn could be controlled by membrane-associated AP2 receptors that are released from exocytotic vesicles. Such a molecular link between exocytosis and endocytosis events would guarantee exocytosis-dependent membrane retrieval, as suggested by the temporal link of exocytosis and endocytosis and by the in vitro interaction between AP2 and synaptotagmin (Zhang, 1994). The results of this study show, however, that synaptotagmin does not colocalize with alpha-Adaptin in shi mutants. This suggests that the role of synaptotagmin and AP2 association (Zhang, 1994) is more likely to serve the recycling of synaptotagmin from the membrane, returning it to the cytoplasmic pool of synaptic vesicles to take part in the subsequent exocytosis event. Alternatively, synaptotagmin could be one of several functionally redundant receptors to anchor the AP2 complex to the membrane to initiate a new vesicle cycle (González-Gaitán, 1997).

A dynamin 1-, dynamin 3- and clathrin-independent pathway of synaptic vesicle recycling mediated by bulk endocytosis

The exocytosis of synaptic vesicles (SVs) elicited by potent stimulation is rapidly compensated by bulk endocytosis of SV membranes leading to large endocytic vacuoles ('bulk' endosomes). Subsequently, these vacuoles disappear in parallel with the reappearance of new SVs. This study used synapses of dynamin 1 and 3 double knock-out neurons, where clathrin-mediated endocytosis (CME) is dramatically impaired, to gain insight into the poorly understood mechanisms underlying this process. Massive formation of bulk endosomes was not defective, but rather enhanced, in the absence of dynamin 1 and 3. The subsequent conversion of bulk endosomes into SVs was not accompanied by the accumulation of clathrin coated buds on their surface and this process proceeded even after further clathrin knock-down, suggesting its independence of clathrin. These findings support the existence of a pathway for SV reformation that bypasses the requirement for clathrin and dynamin 1/3 and that operates during intense synaptic activity (Wu, 2014).

Search PubMed for articles about Drosophila Shibire

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date revised: 19 June 2024

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