commissureless

REGULATION

Protein Interactions

Comm sorts Robo to control axon guidance at the Drosophila midline

Axon growth across the Drosophila midline requires Comm to downregulate Robo, the receptor for the midline repellent Slit. comm is required in neurons, not in midline cells as previously thought, and it is expressed specifically and transiently in commissural neurons. Comm acts as a sorting receptor for Robo, diverting it from the synthetic to the late endocytic pathway. A conserved cytoplasmic LPSY motif is required for endosomal sorting of Comm in vitro and for Comm to downregulate Robo and promote midline crossing in vivo. Axon traffic at the CNS midline is thus controlled by the intracellular trafficking of the Robo guidance receptor, which in turn depends on the precisely regulated expression of the Comm sorting receptor (Keleman, 2002).

comm is required in commissural neurons for crossing. Conversely, forced expression of comm in ipsilateral neurons is sufficient to reroute them across the midline. It is therefore anticipated that comm is expressed in commissural neurons but not in ipsilateral neurons. Furthermore, since comm is expressed in such a dynamic pattern, it is of interest to enquire whether commissural neurons might only express comm since their axons grow across the midline. To explore these ideas, comm expression was surveyed in a set of identifiable neurons for which specific axonal markers are available, thus allowing comm expression to be correlated with growth cone behavior (Keleman, 2002).

The set of neurons examined included both commissural and ipsilateral neurons, and in each class both motor neurons and interneurons. The commissural neurons examined were (1) the RP1, 3, 4, and 5 motor neurons, which express the lim3A-taumyc reporter; (2) the cluster of 10–15 lateral EG interneurons labeled by eg-GAL4; (3) the three EW interneurons, also labeled with eg-GAL4; (4) the drlU intersegmental interneuron, identified with drlU-taumyc; and (5) the Sema2b intersegmental interneuron in each of the A4-A8 hemisegments, identified with Sema2b-taumyc. The ipsilateral neurons examined were (1) the aCC motor neuron and (2) the pCC intersegmental interneuron (both of which are labeled by anti-FasciclinII MAb 1D4) and (3) the dorsal Ap intersegmental interneuron, labeled with ap-GAL4 (Keleman, 2002).

Analysis of comm expression in these neurons reveals a striking correlation between comm expression and a contralateral projection: all of the commissural neurons and none of the ipsilateral neurons express comm. At least for this set of neurons, the correlation is perfect, with just two minor caveats: (1) for the EG neurons, and to a lesser extent the RP and EW neurons, it cannot be entirely certain that every single neuron in these clusters expresses comm, though the impression was gained that this is likely to be the case; (2) the Sema2b neuron can only be identified after its axon has crossed the midline, at which time comm expression appears to be stochastic. Stochastic expression of comm is also seen in the Ap ipsilateral neuron at later stages, although it is consistently negative for comm, as its axon first contacts the midline and turns to avoid it (Keleman, 2002).

There is a striking temporal correlaton between comm expression and midline crossing. The RP, drlU, EG, and EW neurons all extinguish their comm expression shortly after their axons have crossed the midline. Interestingly, the EG and EW neurons, which are the only commmissural neurons that could be identified before their axons reach the midline, are also clearly negative for comm prior to crossing. In particular, the EW axons grow anteriorly for a short distance before turning medially to cross the midline. These neurons do not appear to express comm until they make this medial turn (Keleman, 2002).

Ipsilateral neurons and postcrossing commissural neurons express robo but not comm, and Robo levels are high in the growth cone, whereas crossing commissural neurons express both robo and comm, and Robo levels are low. How does coexpression of Comm prevent Robo from accumulating in the growth cone? To address this question, studies sought to mimic these two situations by expressing comm and robo alone or together in cultured cells (Keleman, 2002).

In COS cells that express robo alone, Robo protein is present mainly at the plasma membrane, as well as in the Golgi and endoplasmic reticulum. Only a small amount of Robo can be detected in endosomes. In contrast, in cells that express both robo and comm, most Robo protein is in late endosomes and lysosomes, where it colocalizes with Comm. This endosomal staining of Comm is also seen in the absence of Robo and is reminiscent of the punctate distribution of endogenous Comm in neurons. Comm can also usually be detected in the Golgi but not at the plasma membrane. Plasma membrane staining is normally only seen in cells expressing particularly high levels of Comm, suggesting that the machinery that sorts Comm to the endosomal compartment can be saturated (Keleman, 2002).

It is concluded that Comm is normally sorted to the late endosomal-lysosomal system and can also recruit Robo to this compartment. To test whether this effect might be specific for Robo, it was asked whether Comm could also recruit the Netrin receptor Frazzled (Fra) to endosomes. It can not. Using Fra-Robo and Robo-Fra chimeric receptors, in which the cytoplasmic domains of the two receptors had been swapped, it was further shown that the ability of Comm to recruit Robo to endosomes requires only the extracellular and/or transmembrane domains of Robo (Keleman, 2002).

To test for a physical association between Robo and Comm, lysates from cells expressing both proteins were immunoprecipitated with antibodies against either the HA tag on Robo or the myc tag on Comm, and then probed on Western blots with anti-myc. Comm protein precipitated with anti-myc appears to exist in three major forms, one that migrates at around 40 kDa, the predicted size of the unmodified protein, and two slower-migrating forms of about 52 kDa and 55 kDa that presumably carry some posttranslational modification. Comm can also be detected in the anti-HA precipitates, indicating that Robo indeed associates with Comm. Interestingly, Robo associates preferentially with the modified forms of Comm. Using Fra and the chimeric receptors, it was shown that this association is specific and that it also requires the extracellular and/or transmembrane domains of Robo. The association between Robo and Comm does not require their colocalization in endosomes, since Robo and the Robo-Fra chimera also associate with a mutant form of Comm (L229A,P230A) that is not sorted to endosomes but is instead delivered to the plasma membrane. Additional data provide evidence that, in the presence of Comm, very little if any Robo is trafficked to endosomes via the cell surface. It is concluded that Comm does not collect Robo at the plasma membrane, but rather sorts it directly from the trans-Golgi network to late endosomes (Keleman, 2002).

Attempts were made to identify the endosomal sorting signal in the Comm cytoplasmic domain. Comm contains a predicted binding site for heterotetrameric adaptor (AP) proteins, which could potentially mediate endosomal sorting. Otherwise, there is no obvious candidate sorting signal, nor any region of significant homology to other known proteins. Additional comm-like genes were sought in Drosophila and in the mosquito Anopheles gambiae. Drosophila has two other genes with some similarity to comm, which are referred to as comm2 and comm3, and for both of these full-length cDNAs were recovered. In the Anopheles genome a single predicted gene related to comm was identified. Functional characterization of these genes is still in progress, but preliminary data indicate that Drosophila comm2 is also able to downregulate Robo proteins in vivo. These four predicted insect Comm proteins are of a similar size and structure but are poorly conserved, with only 15%–20% identity between any pair. Their cytoplasmic domains do, however, contain a highly conserved region of 22 amino acids (residues 215–236 in Comm). The putative AP binding site in Comm (YPSL, residues 251–254) is conserved in Comm2 (YPSV) but not in Comm3 or Anopheles Comm (Keleman, 2002).

To map Comm's endosomal sorting signal, a series of deletion and alanine-scanning mutations were generated within the cytoplasmic domain. The localization of these mutant Comm proteins was examined in COS cells, both in the absence and presence of Robo. In all cases where Comm was correctly targeted to endosomes, Robo goes with it, consistent with the view that the interaction between Comm and Robo does not require their respective cytoplasmic domains. These studies defined a region of 25 amino acids that, together with the 15 amino acids that were left untouched in the juxtamembrane domain, is sufficient for targeting Comm (and Robo, if coexpressed) to endosomes. This region excludes the putative AP binding site, but contains most of the highly conserved residues. Within this region, an LPSY motif is critical for endosomal sorting. If it is mutated, then Comm is found mainly at the plasma membrane. Only a minor fraction of the mutant Comm protein is found in endosomes, possibly reaching this compartment by endocytosis from the plasma membrane rather than direct trafficking from the Golgi. This LPSY motif is also present in each of the other Comms (as LPTY in Comm3). Comm3 and Anopheles Comm even have a second LPSY motif within the conserved region, where Comm has PPCY (Keleman, 2002).

Each of the mutant Comm proteins was tested for its ability to downregulate Robo and promote midline crossing in vivo. It was reasoned that if Comm also sorts Robo to late endosomes in vivo and this is its only function in midline crossing, then the LPSY sorting motif should be the only part of Comm's cytoplasmic domain needed for its function in vivo. The mutant Comm proteins that were mislocalized to the plasma membrane should all be nonfunctional in vivo, while those that are correctly sorted should still be functional, even though they lack most of the cytoplasmic domain (Keleman, 2002).

To test these predictions, flies were generated carrying UAS transgenes encoding each of the mutant Comm proteins that had been tested in COS cells. These transgenes were then expressed in all CNS neurons using the elav-GAL4 driver. These embryos were examined with anti-myc antibodies to determine the expression and localization of the transgenic Comm protein, with anti-Robo MAb 13C9 to test for the ability of the mutant Comm protein to downregulate Robo, and with MAb 1D4 to detect any misrouting of longitudinal axons across or along the midline, the hallmarks of the robo and slit loss-of-function phenotypes (Keleman, 2002).

Consistent with the hypothesis that Comm sorts Robo to endosomes in vivo, a striking correlation was found between the sorting of a mutant Comm protein to endosomes in COS cells and its function in vivo. In particular, the 25 amino acid region of Comm's cytoplasmic domain that is sufficient for endosomal sorting in vitro is also sufficient for Comm to downregulate Robo and promote midline crossing in vivo. Conversely, point mutations in the LPSY motif completely abolish Comm function in vivo, just as they prevent endosomal sorting in vitro. Comm mutants that were sorted to endosomes in COS cells also showed a punctate intracellular localization in vivo, but in general were difficult to detect, even though they were fully functional. Indeed, it was only the nonfunctional Comm proteins that could readily be detected in vivo. This seemingly paradoxical result is, however, in complete agreement with the view that the essential function of Comm in vivo is to be degraded in lysosomes, taking Robo with it. This function is critically dependent on the same LPSY motif that targets Comm (and Robo) directly from the Golgi to late endosomes and lysosomes in vitro (Keleman, 2002).

What is the basis for the specificity of Comm's action? Why are only commissural axons allowed across the midline, and why only once? Previous models have proposed that Robo levels may initially be lower in commissural neurons than in ipsilateral neurons, or that only commissural neurons might express a cell surface receptor needed for the uptake of Comm from midline glia. Analysis of comm expression in the CNS offers a much simpler explanation for its specificity: in general, only commissural neurons express comm, and only as they cross (Keleman, 2002).

These data thus suggest a simple model in which comm expression is the intrinsic switch that specifies an ipsilateral versus a contralateral projection—OFF for ipsilateral, ON for contralateral. This switch appears to be regulated not only spatially but also temporally, since comm generally goes OFF in a commissural neuron after crossing. Early markers needed to determine whether comm is usually ON or OFF before crossing are lacking, but it is noted that for the few commissural neurons that can be identified early (the EW and EG neurons), comm is initially OFF. What turns comm ON and then OFF again to allow just a single passage across the midline? One possibility is that each commissural neuron is intrinsically programmed for a brief pulse of comm expression. Alternatively, comm expression might be controlled by retrograde signals sent from the growth cone to inform the nucleus of its arrival at the midline and its successful passage across. This is an appealing idea, since such a mechanism would uncouple the ability of the growth cone to cross the midline from the precise time of arrival and duration of transit (Keleman, 2002).

Understanding Comm's function in midline crossing has also been hindered by the fact that its molecular function was unknown and its amino acid sequence provided no obvious clues. The data suggest that Comm is a sorting receptor, recognizing Robo via its lumenal and/or transmembrane domain and consigning it for delivery from the trans-Golgi network to late endosomes. Robo may not be the only cargo for Comm. From gain-of-function genetic experiments, it is inferred that Comm also selects Robo2 and Robo3 for delivery to endosomes. Analogous gain-of-function studies suggest that Comm2 also sorts Robo receptors, with a preference for Robo2, while Comm3 may not sort any of the three Robos. The Comm proteins thus define a new family of sorting receptors, the cargo of which include, but may not be limited to, the Robo family of guidance receptors (Keleman, 2002).

The view that Comm is an endosomal sorting receptor and that comm expression is a cell-autonomous switch for midline crossing leads to a model in which axon traffic at the midline is controlled by regulating the intracellular trafficking of Robo, the receptor for the midline repellent Slit. If comm is OFF, Robo is packaged into vesicles for delivery to the growth cone. The insertion of these vesicles at the growth cone confers sensitivity to Slit, thereby preventing growth across the midline. Conversely, if comm is ON, Comm sorts Robo into vesicles destined for late endosomes and lysosomes. Membrane vesicles delivered to the growth cone contain only very low levels of Robo, and so the axon can grow unimpeded across the midline by inserting these vesicles at its tip (Keleman, 2002).

One requirement for this model is that, in order to prevent a commissural axon from recrossing, Comm protein, like comm mRNA, should rapidly disappear after crossing, or at least lose its ability to sort Robo. It is believed that Comm is indeed rapidly degraded in vivo. In contrast to COS cells, very little Comm protein can be detected in vivo, even when the GAL4-UAS system is used to express high levels of comm mRNA throughout the CNS. Only if the LPSY endosomal sorting motif is mutated can Comm accumulate to appreciable levels in vivo, in this case at the plasma membrane. This suggests that, unlike other sorting receptors, Comm may not be recycled back to the Golgi for repeated rounds of sorting but instead be degraded along with its cargo in lysosomes. Other mechanisms may also exist to inactivate Comm after crossing, for example by altering the posttranslational modifications that appear to be necessary for it to recognize Robo (Keleman, 2002).

Drosophila Nedd4, a ubiquitin ligase, is recruited by Commissureless to control cell surface levels of the roundabout receptor

Crossing the midline produces changes in axons such that they are no longer attracted to the midline. In Drosophila, Roundabout reaches high levels on axons once they have crossed the midline, and this prohibits recrossing. Roundabout protein levels are regulated by Commissureless. Commissureless binds to and is regulated by the ubiquitin ligase DNedd4. The ability of Commissureless to regulate Roundabout protein levels requires an intact DNedd4 binding site and ubiquitin acceptor sites within the Commissureless protein. The ability of Commissureless to regulate Robo in the embryo also requires a Commissureless/DNedd4 interaction. These results show that changes in axonal sensitivity to external cues during pathfinding across the midline makes use of ubiquitin-dependent mechanisms to regulate transmembrane protein levels (Myat, 2002).

To identify potential components that mediate Comm activity, the intracellular region of Comm (CommIC) was used as bait in a yeast two-hybrid screen. One identified interactor has a high degree of sequence identity to vertebrate Nedd4 and has consequently been named DNedd4. DNedd4 comprises 1007 amino acids and, like its vertebrate counterpart, contains a C2 Ca2+-dependent phospholipid binding domain, three WW domains, and a C-terminal E3 hect ubiquitin ligase domain. The E3 enzymes catalyze the transfer of ubiquitin onto specific proteins, targeting cytosolic proteins for degradation by the proteasome or membrane proteins for endocytosis (Myat, 2002).

DNedd4 binds to Comm through two of its three WW domains, which recognize either of two PY motifs (PPCY or LPSY) in the intracellular domain of Comm. This identifies the PPCYTIATGLPSYDEA region of Comm as a key functional motif within the molecule. Interestingly, this region is also highly conserved in two other Comm homologs in the fly genome and several vertebrate ESTs (Myat, 2002).

Comm is ubiquitinated when coexpressed with DNedd4. In Drosophila cells, DNedd4 acts to internalize Comm within the cell; this activity requires Comm's intracellular lysines and a functional ubiquitin ligase domain within DNedd4, suggesting it is a ubiquitination-dependent event. In addition to being able to bind DNedd4, Comm also binds Robo when they are coexpressed. This raises the possibility that DNedd4 may target the Comm/Robo complex for internalization or degradation in the embryo. Comm's ability to interact with Robo and DNedd4 suggests a possible function as an adaptor protein that links Robo into the ubiquitination pathway. Currently, it is not known whether Robo itself is targeted for ubiquitination by DNedd4. Although ubiquitination of Robo could not be detected in S2 cells, this may reflect detection limits rather than real lack of ubiquitination. Nevertheless, it has been recently observed that Rsp5p, a yeast homolog of Nedd4, can internalize the plasma membrane alpha-factor receptor protein without addition of ubiquitin to the target protein. Instead, other components of the endocytic machinery are proposed to be ubiquitinated. Similarly, the growth hormone receptor is internalized by a ubiquitin-dependent event that does not involve ubiquitination of the receptor but may involve another regulatory protein that is itself ubiquitinated (Myat, 2002 and references therein).

Within the embryo, Comm protein is located both at the cell surface and within intracellular vesicles in midline cells and commissural axons. This distribution is suggestive of a protein that can move between different locations in the cell. Robo, however, is expressed on the surface of longitudinal axons. Comm can regulate Robo protein levels, and the proteins are occasionally coexpressed in the same cell in the embryo when the Robo protein is found within intracellular vesicles with Comm. Thus, Comm may internalize Robo as part of its regulation of Robo. When Comm is expressed in Drosophila S2 cells, the protein displays a similar distribution to that seen in the embryos with the majority of the protein within intracellular vesicles. Robo, as expected, is expressed on the cell surface when expressed alone in S2 cells. However, when Comm and Robo are expressed together in S2 cells, the Robo protein is no longer found at the cell surface but is now colocalized with Comm within intracellular vesicles within the cell. Thus, Comm is able to change the site of Robo localization within the cell. This ability correlates with the observation in the embryo that overexpression of Comm results in the reduction of Robo protein at the cell surface. This study shows that the normal intracellular distribution of Comm requires an interaction with DNedd4. Removal or disruption of either the DNedd4 binding sites or the intracellular lysines in Comm or the reduction of DNedd4 levels in S2 cells results in Comm accumulating at the cell surface. Comm is no longer brought into the cell and is unable to remove coexpressed Robo from the cell surface. Thus, DNedd4 is a key cofactor that allows Comm to harness the ubiquitination pathway to target its removal from the cell surface together with other membrane receptors it may bind (Myat, 2002).

To test whether DNedd4 has an important role in Comm function, neural overexpression of Comm was used as a sensitive assay. Overexpression of a single copy of comm within all CNS neurons results in the downregulation of Robo in these cells and the production of a robo phenocopy where axons recross the midline. When the level of overexpressed Comm is increased, the phenotype becomes more severe and many axons remain at the CNS midline. Overexpression of DNedd4 alongside one copy of comm produces a phenotype similar to that seen when greater levels of comm are overexpressed. The presence of additional DNedd4 makes the overexpressed Comm more effective at downregulating Robo activity, suggesting it does indeed act with Comm to regulate Robo levels in the embryo. This is supported by the observation that the overexpression of a catalytically inactive form of DNedd4 partially suppresses the ability of overexpressed comm to cause a robo phenocopy. Thus, one activity of DNedd4 in Drosophila is to function with Comm to regulate Robo protein levels. Extrapolating from S2 cell observations, it is assumed that a similar process is taking place in the embryo whereby Comm acts with DNedd4 to internalize Robo into the cell. This suggests that normally Comm and DNedd4 function together in commissural neurons to reduce Robo activity and allow axons to cross the midline. Comm accumulates within commissural axons, and recent experiments have revealed that comm is expressed within these axons with Comm protein only reaching high levels at the midline (Myat, 2002).

Nedd4 family proteins regulate the internalization of a number of cell surface proteins. Nedd4 regulates levels of the epithelial Na+ channel, while the yeast homolog Rsp5 catalyzes the internalization of a number of membrane transporters. Although Nedd4 was identified in a screen for transcripts expressed in the mouse nervous system during embryonic development, no targets for this molecule during neural development have yet been identified. DNedd4 can regulate Comm and consequently, Robo. Yet, removal of DNedd4 function in the embryo does not give rise to the same phenotype as a loss of comm. If DNedd4 was acting purely within the Comm pathway to regulate Robo protein levels, then one might expect that a loss of DNedd4 function would give rise to a comm-like phenotype, since Robo protein levels may stay high. However, inhibition of DNedd4 could result in the stabilization of Comm on neuronal membranes where it can bind Robo and possibly interfere with Robo function to produce a partial robo-like phenotype. RNA interference with DNedd4 gives rise to a phenotype where axons stall at the junction of the longitudinal and commissural axon tracts, resulting in thinner longitudinal and commissural axon tracts (i.e., neither a comm nor robo phenocopy). This phenotype suggests that DNedd4 may also regulate the cell surface levels of other axon guidance molecules. Additionally, DNedd4 may also affect neuronal fate decisions since a close homolog, Su(dx), acts as a regulator of Notch signaling. The isolation of DNedd4 loss-of-function mutations will aid full evaluation of the exact roles of DNedd4 in the embryo (Myat, 2002).

The N-terminal and transmembrane domains of Commissureless are necessary for its function and trafficking within neurons

Commissureless is a novel transmembrane molecule necessary both for commissural axons to cross the midline of the Drosophila central nervous system and normal synaptogenesis. Comm is able to reduce cell surface levels of Roundabout (Robo), a receptor for the midline repellent Slit, on commissural axons and unknown inhibitors of synaptogenesis expressed on muscle cells. Comm is expressed dynamically and is found at the cell surface and within intracellular vesicles. Comm can bind Robo and when the proteins are co-expressed Robo is found co-localized with Comm intracellularly. The ability of Comm to localize intracellularly and hence regulate Robo surface levels requires sequences in both the N-terminal and transmembrane domains. Comm can dimerize via its N-terminal domain. Furthermore, absence of the Comm N-terminal and transmembrane regions results in the protein being restricted to the neuron soma (Georgiou, 2003).

Comm is necessary for axons to cross the CNS midline in Drosophila, where it plays an important role in regulation of cell surface levels of the transmembrane receptor protein Robo. By controlling the surface levels of Robo, Comm regulates the sensitivity of axons to the midline repellent Slit. Comm is present at highest levels at the midline where it is expressed on midline cells and commissural axons, precisely the area where Robo protein is excluded. The distribution of Comm allows the downregulation of Robo on commissural axons and permits them to cross the midline. Comm also functions at the neuromuscular junction where it is expressed in the post-synaptic cell and acts to remove unknown inhibitors of synapse formation from the cell surface. Comm activity at the neuromuscular junction requires that Comm can endocytose into the cell, when it presumably co-endocytoses the synaptogenesis inhibitors. The intracellular region of Comm has been shown to be essential for function both at the midline and at the neuromuscular junction. The intracellular region of Comm includes binding sites for the ubiquitin ligase, DNedd4, and a possible adaptin binding site. The interaction between DNedd4 and Comm is necessary for Comm to localize within intracellular vesicles. In the absence of this interaction Comm is expressed at the cell surface and is unable to prevent Robo accumulation at the plasma membrane. In this paper, it has been shown that the N-terminal and transmembrane domains of Comm are also essential for its function. Both these regions of the protein appear to be necessary for Comm to localize efficiently to endosomes within the cell. In the absence of the N-terminal Comm is less efficiently targeted to the endosomes: this reduces the efficiency of the molecule to sort Robo away from the cell surface. When the N-terminal and transmembrane domains of Comm are replaced, the chimeric protein localizes to the cell surface and is unable to reduce surface levels of Robo. It is further found that the Comm transmembrane domain is required both for targeting Comm to intracellular vesicles and also its transport along CNS axons. When deletion constructs lacking the Comm transmembrane domain are expressed within neurons at the midline or in the CNS they remain in the soma and are not trafficked to the axon. When the deletion constructs CommDelta2 and CommDeltaEC are expressed at the midline, little Comm protein is seen in the axons of the midline neurons in contrast to their expression throughout the CNS when they can be seen in axons. It is suspected that these forms can reach the axons inefficiently and the protein cannot be detected in the axons when it is expressed in the small number of midline neurons as compared to when it is expressed in all CNS neurons (Georgiou, 2003).

The N-terminal domain of Comm is required for Comm's correct function in both in vivo and in vitro experiments. Comm^DeltaEC , lacking the N-terminal domain, shows some residual function but its ability to prevent Robo reaching the cell surface is severely disrupted. If increased proportions of the N-terminal are provided, an improvement in Comm function results. Comm^Delta1 is more often targeted to the same intracellular localization as Comm whereas Comm^Delta2 and Comm^DeltaEC are less often found within vesicles, suggesting an essential trafficking signal exists within the sequence between amino acids 62 and 99 (Georgiou, 2003).

When Robo is co-transfected with the Comm variants, its distribution follows that of Comm. Removal of the N-terminal of Comm results in a greater proportion of Robo localized to the plasma membrane. When driven in neurons, some Comm^DeltaEC can localize correctly to the commissure, however, the molecule's reduced function is evident in its failure to generate a strong robo phenocopy. This suggests that the appropriate trafficking of Comm requires both the DNedd4 binding site in the cytosolic domain of Comm and sequences in the N-terminal. Two models have been proposed for Comm mediated regulation of Robo whereby Comm retrieves Robo from the cell surface or prevents Robo transport to the cell surface. One model suggest that Comm acts as a protein chaperone to sort Robo away from a plasma membrane pathway to an intracellular endocytic destination. Perhaps the appropriate sorting of Comm itself to an endocytic location involves elements resident in the trans-Golgi network or endoplasmic reticulum that recognise the N-terminal region of Comm to traffic it appropriately in addition to the ubiquitin tag signal added by D-Nedd4. Alternatively, the second model suggests that the N-terminal sequence may be necessary for the receipt of an extracellular signal required for the efficient internalization of Comm via the intracellular addition of ubiquitin (Georgiou, 2003).

The normal intracellular targeting of Comm is completely disrupted if the Comm transmembrane domain is not present or is replaced. Comm variants that replace the Comm transmembrane region localize to the plasma membrane when expressed in S2 cells, at the midline or in neurons. This disruption is most striking when the proteins are expressed throughout the nervous system. Here, the mutant protein localizes to the cell surface of neuronal cell bodies and does not exit the cell bodies. This manipulation of the Comm protein disrupts two aspects of its normal intracellular localization, a vesicular distribution within the cell body and distribution to the axon (Georgiou, 2003).

In the absence of both the N-terminal and transmembrane regions of Comm, the protein cannot localize intracellularly and therefore cannot target Robo away from the cell surface. The replacement of the Comm transmembrane region results in a complete abrogation of Comm's ability to localize within intracellular vesicles, suggesting that the Comm transmembrane region provides some information necessary for its targeting in addition to that provided by the N-terminal regions (Georgiou, 2003).

Intriguingly the replacement of the normal Comm transmembrane region results in targeting specifically to the cell body plasma membrane and the manipulated Comm molecule is not transported along the axon. This suggests that Comm is translated in the cell body and requires a transport mechanism to reach distal regions of the axon. Targeting of Comm to the intracellular vesicles it normally occupies may be a requisite for Comm to extend along the axon (Georgiou, 2003).

The Comm protein has a specific spatial location within the commissural axons where it is expressed at high levels on the commissural stretches of these neurons. This is precisely the region where Robo protein levels are at their lowest throughout axon outgrowth. Even after the neurons have crossed the midline they maintain low levels of Robo protein on the commissures until stage 16 when the Comm protein levels eventually decline. This localization/stabilization of Comm at the commissures may act to ensure continued downregulation of Robo levels there and ensure Robo is only present on the distal regions of the axons. The failure to observe Robo within the commissural regions suggests that Robo may be added to the axon surface distally, perhaps at the growth cone and that a mechanism exists at the midline to prevent this protein diffusing back to the soma (Georgiou, 2003).

Neurons are highly specialized cells that can both send and receive intercellular signals at different regions within the cell. This property requires that membrane proteins be targeted to specific locations within the cell. Different proteins may be found either throughout the cell or targeted to the axon, growth cone or the somatodendritic region. Comm displays a specific distribution within neurons where it accumulates along the commissural regions at the midline. This distribution is not unique to Comm as a similar distribution is also taken up by the Drosophila Derailed protein. To date no general targeting signals or mechanisms have been identified that specify a neuronal protein's intracellular address. Studies on neuronal protein targeting have suggested that the trans-Golgi network sorts membrane proteins into different vesicles that traffic the protein to their correct location or that proteins are selectively retained in particular neuronal domains. Attempts have been made to identify the neuronal sorting signals that target proteins to the somatodendritic region or the axon. Coarse locations for these sorting signals have been defined for several neuronal proteins. The amyloid precursor protein (APP) contains a signal in its N-terminal or transmembrane domain necessary for targeting to the axon whereas Synaptobevin contains a signal in its cytoplasmic region. The transferrin receptor, polymeric immunoglobulin receptor and the low density lipoprotein receptor all have their somatodendritic sorting signals in their cytoplasmic domains. In addition to targeting signals that direct neuronal proteins to particular locations there are also selective retention signals that allow proteins to be maintained or lost from particular locations. Transcytosis can occur whereby a protein is initially located throughout the neuron but subsequently relocates via the endocytic machinery to its appropriate location, e.g., APP moves from axon to the soma. Also, diffusion barriers exist that serve to maintain localized neuronal proteins to their correct locale. In the case of Comm it appears to be specifically localized to the commissural region of the axon. Is the protein selectively trafficked to this region of the axon or do components of the endocytic machinery prevent Comm from remaining distal to the midline? Interestingly it has emerged that differential lipid domains within the membrane may also play a role in targeting proteins within a cell. Detergent-insoluble glycolipid domains or rafts may serve as sorting platforms to direct proteins such as Thy-1 to the axon. Perhaps the commissural region of axons contains a differential lipid composition that mediates Comm localization. Interestingly Nedd4 associates with lipid rafts and thus may play a role in targeting Comm (Georgiou, 2003).

Since Comm is expressed at high levels on the surface of the midline cells that contact the commissural axons at precisely the point where Comm accumulates within the axons it was wondered whether there may be an interaction between Comm on the midline cells and Comm on the commissural axons. A Comm:Comm interaction has been shown to take place within S2 cells using a co-immunoprecipitation assay. A strong interaction is dependent on the region N-terminal to the transmembrane region being intact while no interaction is observed without this region and the Comm transmembrane domain. This assay suggests that a cis-interaction between Comm molecules can take place and since these regions are necessary for Comm trafficking this interaction may also be necessary for Comm to be located intracellularly (Georgiou, 2003).

It is suggested that the Comm N-terminal region and transmembrane domain, possibly through a cis-homophilic interaction or by some other interaction, is promoting the internalization of Comm. This internal vesicular location of Comm enables it to be transported away from the cell body to the commissure. Once Comm reaches the commissural region it is sequestered there and does not travel further distally. Perhaps a signal from the midline cells triggers the sequestration of the Comm protein at the commissure. Whether this signal affects a membrane specialization of the commissural axon or endocytic trafficking processes or allows a trans-homophilic interaction between Comm on the surface of the midline cells and any Comm that reaches the surface of the axon is not yet known (Georgiou, 2003).

The intracellular domain of the Frazzled/DCC receptor is a transcription factor required for commissural axon guidance

In commissural neurons of Drosophila, the conserved Frazzled (Fra)/Deleted in Colorectal Cancer (DCC) receptor promotes midline axon crossing by signaling locally in response to Netrin and by inducing transcription of commissureless (comm), an antagonist of Slit-Roundabout midline repulsion, through an unknown mechanism. This study shows that Fra is cleaved to release its intracellular domain (ICD), which shuttles between the cytoplasm and the nucleus, where it functions as a transcriptional activator. Rescue and gain-of-function experiments demonstrate that the Fra ICD is sufficient to regulate comm expression and that both γ-secretase proteolysis of Fra and Fra's function as a transcriptional activator are required for its ability to regulate comm in vivo. These data uncover an unexpected role for the Fra ICD as a transcription factor whose activity regulates the responsiveness of commissural axons at the midline and raise the possibility that nuclear signaling may be a common output of axon guidance receptors (Neuhaus-Follini, 2015).

This study has identify the Fra ICD as a transcription factor that regulates the expression of comm, a key modulator of axonal responsiveness at the midline. γ-secretase proteolysis of Fra releases its ICD, which is capable of nuclear translocation and is sufficient to promote midline crossing and regulate comm expression in rescue and gain-of-function assays in vivo. The conserved P3 motif within the Fra ICD functions as a transcriptional activation domain and this activity is required for Fra's regulation of comm expression. Thus, in addition to its canonical role signaling locally to regulate growth cone dynamics, Fra functions as a transcription factor to regulate axonal responsiveness at the midline (Neuhaus-Follini, 2015).

comm is expressed in commissural neurons with exquisite temporal specificity. How might the transcriptional activity of the Fra ICD be regulated to contribute to comm's expression pattern? γ-secretase proteolysis is typically the second cleavage event in a proteolytic cascade, preceded by ectodomain shedding. Indeed, previous pharmacological experiments suggest that DCC's ectodomain is shed as a result of metalloprotease cleavage and that this proteolytic event is required for subsequent γ-secretase-dependent processing. Metalloprotease-dependent ectodomain shedding is often ligand-dependent, while subsequent γ-secretase processing depends on the shape of the membrane-tethered metalloprotease cleavage product. For example, metalloprotease-dependent shedding of the Notch ectodomain is stimulated by the binding of Notch ligands, and the subsequent γ-secretase cleavage of the membrane-tethered ICD is constitutive. As Fra regulates comm independent of Netrins, Fra ectodomain shedding may occur in response to the binding of a different ligand. Alternative ligands for DCC have been identified, including the vertebrate- specific proteins Draxin and Cerebellin 4. In addition, the secreted protein MADD-4 physically associates with the C. elegans ortholog of Fra/DCC, UNC-40, and guides sensory neurons and muscle arms in an UNC-40-dependent manner. The function of the Drosophila ortholog of MADD-4, Nolo, has not been investigated, nor has its ability to bind to Fra (Neuhaus-Follini, 2015).

It seems unlikely that the transcriptional activity of the Fra ICD is controlled at the level of nuclear localization. When Fra ICDDP3 (lacking a NES) was expressed in the commissural EW neurons in vivo, it accumulates in the nucleus at the earliest developmental stages that can be observed, suggesting that the Fra ICD is constitutively imported into the nucleus. Nuclear accumulation of full-length Fra ICD (with a NES) was only observed occasionally, implying that after the Fra ICD translocates to the nucleus, it is rapidly exported. The fact that Fra's NES and activation domain are both encoded by P3 raises the possibility that when Fra is engaged in transcriptional activation, the association of co-activators with P3 might prevent it from associating with nuclear export machinery, coupling Fra's nuclear activity to its nuclear retention (Neuhaus-Follini, 2015).

The finding that Fra's ability to regulate comm expression depends on its function as a transcriptional activator seems to imply that the Fra ICD can associate with chromatin, but the Fra ICD does not contain an obvious DNA-binding domain. A Neo DNA-binding domain has not been identified either, but chromatin immunoprecipitation experiments have demonstrated that the Neo ICD associates with chromatin in vitro. The Fra ICD's DNA-binding activity and specificity probably arise from associations between the Fra ICD and DNA-binding partners, as is the case with Notch. The Notch ICD has no DNA-binding activity of its own and associates with DNA as part of a complex including an obligate CSL (CBF1/ RBPjk, Su(H), Lag-1) DNA-binding partner. If the Fra ICD can associate with multiple DNA-binding proteins, it might allow the Fra ICD to regulate the expression of many different target genes, depending on which of its DNA-binding partners are expressed in particular cell types or developmental contexts (Neuhaus-Follini, 2015).

The observation that a structurally intact P3 is required for Fra-dependent transcription suggests that P3 plays another role in Fra's transcriptional output besides its function as an activation domain. One possibility is that P3 is required for Fra's association with chromatin, perhaps by functioning as a binding interface for Fra's DNA-binding co-factors. This idea is supported by the observation that FraE1354A antagonizes midline crossing in both fra mutants and heterozygotes, while FraDP3 has only a mild effect. Perhaps the ICD of FraE1354A inhibits midline crossing by occupying chromatin sites that are normally targets of both Fra and other transcriptional activators that act in a parallel pathway; the ICD of FraDP3 would not have this effect if P3 is required for Fra's association with chromatin. FraE1354A is not likely to be inhibiting endogenous Fra in rescue experiments, as fra³ is either a strong hypomorphic or null allele. This model predicts that Fra has other transcriptional targets in EW neurons that are relevant for commissural axon guidance. It will be informative to identify additional transcriptional targets of Fra both in embryonic commissural neurons and in other cell types. In the retina, R8 photoreceptor axons have targeting defects that are much milder in Netrin mutants than in fra mutants, raising the possibility that the Netrin-independent output of Fra signaling in this system might be through the transcriptional pathway that this study has identified (Neuhaus-Follini, 2015).

Cleavage of axon guidance receptors has been shown to regulate the activities of these receptors in a number of different ways. Degradation of axon guidance receptors can provide temporal control of axonal sensitivity to guidance cues. In vertebrates, this mode of regulation controls axonal responsiveness to members of the class 3 family of secreted Semaphorins (Sema3s), which signal repulsion through Neuropilin (Nrp)/ Plexin (Plex) co-receptors. Calpain proteolysis of PlexA1 in pre-crossing spinal commissural neurons reduces their sensitivity to Sema3B, which is expressed in the ventral spinal cord as these axons are growing toward the ventral midline. ADAM metalloprotease cleavage of Nrp1 reduces the sensitivity of proprioceptive sensory axons to Sema3A allowing them to terminate in the ventral spinal cord, where Sema3A expression is high. In addition, γ-secretase proteolysis of DCC in vertebrate motor neurons inhibits their responsiveness to midline-derived Netrin, preventing them from ectopically projecting toward the midline (Neuhaus-Follini, 2015).

Proteolytic processing has also been implicated as a requisite step in local repulsive Robo signaling in Drosophila. The Robo ectodomain is cleaved by the ADAM metalloprotease Kuzbanian and this proteolytic event is required for Robo's ability to transduce repulsive signals in vivo and for Slit-dependent recruitment of effectors of local Robo signaling in vitro. As γ-secretase-dependent intramembrane proteolysis is typically constitutive following ectodomain shedding, and occurs subsequent to metalloprotease processing of the human Robo1 receptor, it is likely that Drosophila Robo is cleaved to produce a soluble ICD. The observation that Robo proteolysis is required for local Slit-Robo signaling does not exclude the possibility that the Robo ICD may also have a nuclear function that contributes to axon guidance, but this possibility has not yet been explored (Neuhaus-Follini, 2015).

Proteolysis has also been identified as a regulator of contact- mediated axonal repulsion. Eph receptors signal repulsion in response to their transmembrane ephrin ligands; ephrins can also function as receptors, signaling repulsion in response to Eph binding. Metalloprotease and subsequent γ-secretase cleavage of both Ephs and ephrins have been demonstrated, providing a mechanism through which adhesive interactions can be broken to allow for repulsive signaling. The importance of this mode of regulation for axon targeting has not yet been established in vivo and a recent study using an EphA4 variant that is insensitive to metalloprotease cleavage suggests that EphA4 proteolysis is not required for EphA4-dependent motor axon targeting (Neuhaus-Follini, 2015).

This study has identified a new way in which axon guidance receptor proteolysis can influence axon responsiveness to guidance cues. γ-secretase-dependent processing of Fra releases its ICD, which translocates to the nucleus, where it functions as a transcription factor to regulate the guidance of commissural axons. It is proposed that the ability to signal from the nucleus may be a common property of axon guidance receptors and may serve as a general mechanism through which axon guidance receptors regulate their own activities or the activities of other proteins. Human Robo1 is processed by sequential metalloprotease and γ-secretase cleavage and its ICD localizes to the nucleus in vitro. It remains to be seen whether the ICDs of Ephs and ephrins, which are cleaved by γ-secretase, and of Plexins, which are proteolytically processed, but have not yet been identified as γ-secretase substrates, translocate to the nucleus as well. It will also be interesting to determine whether the ICDs of Fra and other axon guidance receptors signal from the nucleus to regulate aspects of neuronal morphogenesis and function besides axon pathfinding. Finally, recent work indicating that the cleaved C terminus of the Drosophila Wnt receptor Frizzled translocates to the nucleus and contributes to the establishment of postsynaptic structures by regulating RNA export serves as a reminder that the trafficking of cell surface receptor fragments to the nucleus may allow these fragments to signal not only by regulating transcription, but in other ways as well (Neuhaus-Follini, 2015).

DEVELOPMENTAL BIOLOGY

Embryonic

Commissureless protein colocalizes with the Golgi complex. In Drosophila, this appears as discrete vesicles located throughout the cytoplasm. A lesser amount of the Comm protein is visible in other compartments; in particular, Comm colocalizes with a late endosome marker. Cell surface localization is observed with anti-Comm antibodies in the absence of detergent. Thus, the Comm protein likely cycles from the Golgi to the surface, where some of it is endocytosed (Tear, 1996).

Expression begins during the cellular blastoderm stage in a pattern of six stripes. These stripes have a pair-rule periodicity, that is, stripes are apparent in every other parasegment, but stripe three is missing; very soon thereafter, a seventh stripe (the third pair rule stripe) appears at the position where it had been missing. By stage 6, these stripes narrow, and an interstripe of comm expression is observed resulting in a pattern of 14 stripes. This striped epidermal expression is maintained throughout germ band elongation, suggesting that the comm promoter is sensitive to regulation by segmentation genes. comm has no essential role in segmentation, since mutants have no segmentation defects. A similar patten of stripes has been reported for Prospero, which also has a function in the nervous system but no apparent role in segmentation (Tear, 1996).

During stage 10, the stripes of COMM mRNA expression begin to disappear; the dorsalmost cells lose their expression first, and this decrease continues ventralward until the segmentally repeated stripes have completely faded. At this stage, COMM transcript begins to be expressed in two longitudinal epidermal stripes, 3 to 4 cells wide, located just lateral to the neurogenic region. These longitudinal stripes extend throughout the segmented region of the embryo. Initially the dorsal limit of this stripe is irregular, but subsequently this boundary is sharpened. Simultaneously a segmentally repeated pattern of expression is observed within the emerging CNS (Tear, 1996).

COMM transcript is visible in a subset of neuroblasts and some early ganglion mother cells. No signal is seen in the neuroectoderm during the time of neuroblast segregation. The identity of the comm expressing cells has not been determined, and thus it is not clear whether the comm expressing GMCs are the progeny of comm positive neuroblasts (Tear, 1996).

By the beginning of stage 12, COMM mRNA has accumulates along the length of the midline of the CNS and in the visceral mesoderm. The midline expression remains after neuroblast segregation and division has been completed. There also appears to be transient expression in a subset of neurons immediately lateral to these midline cells, two of which are RP1 and RP3. These cells lie along the path that the first commissural axons take to reach the midline. The first axons to cross the midline are intimately associated with the cells that show comm expression. The first commissural axons are in close assocation with comm-expressing midline glia. Other than the transient expression in RP1, RP2 and several other cells just lateral to the midline during mid-stage 12, there is no other expression in CNS neurons throughout embryogenesis (Tear, 1996).

During stage 12, Comm protein expression is restricted to a subset of midline cells, mimicking the expression seen for the transcript. The dorsoventral location of these cells suggests that they are the anterior and medial pair of midline glia. There also is transient expression of Comm protein in some neurons just lateral to the midline, including RP1 and RP3. This also mimics COMM mRNA accumulation. Interestingly, the first commissural axons that contact the comm-expressing midline glia also accumulate Comm protein along their axons, despite no COMM transcript being observed in these neuronal cell body. This axonal presence of Comm protein is restricted to those axons that contact comm-expressing midline cells. It thus appears as if the entire Comm protein may be exported from the midline cells to decorate these commissural axons (Tear, 1996).

Commissureless (Comm) protein is expressed in all 30 muscles of each abdominal hemisegment in late stage embryos. Slightly lower levels of expression are seen in the most distal (dorsal) set of muscles that are targeted by the ISN motoneurons whose axons grow past all the other more proximal muscles that have high levels of Comm expression. The muscle-supplied protein is visible as early as hour 11 of embryogenesis. It precedes the contacts between motoneuron growth cone and muscles by at least 1 hour. Comm expression persists until hour 18, by which time cuticular deposition begins to block antibody penetration into whole embryonic tissues. Within muscles, Comm protein becomes localized in punctate microsome structures of approximately 0.5-1.0 mm diameter. Through a combination of vital labeling of endosomes and Comm immunocytochemistry, it was found that these Comm-positive microsomes are a subset of endosomes that undergo endocytosis during hours 11-18. About 25% of the newly formed endosomes are Comm positive. They are similar in size and distribution pattern to those that are Comm negative. No Comm immunoreactivity is detected in muscle nuclei. The presence of Comm in endosomes, combined with the presence of an Adaptin recognition site within the cytoplasmic domain of Comm, is consistent with its cytoplasmic domain associating with the Adaptin/Clathrincomplex. At hour 11, Comm is mostly found on the cell surface membrane. However, by hour 18 the majority of Comm immunoreactivity shifts to the endosomes. The Comm-positive endocytic activity peaks around hour 14, coinciding with the onset of neuromuscular synaptogenesis on the majority of muscles. As a result, when Comm protein is largely on muscle surfaces, the motoneuron growth cones that contact them extend past these muscles. However, as the bulk of the protein internalizes into muscles, those growth cones that are the normal synaptic partners of these muscles approach them and initiate synaptogenesis on respective muscles (Wolf, 1998).

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