InteractiveFly: GeneBrief

Sec5 : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The exocyst (Sec6/8) complex is necessary for secretion in yeast and has been postulated to establish polarity by directing vesicle fusion to specific sites along the plasma membrane. The complex may also function in the nervous system, but its precise role is unknown. Exocyst function was investigated in Drosophila with mutations in one member of the complex, sec5. Null alleles die as growth-arrested larvae, whose neuromuscular junctions fail to expand. In culture, neurite outgrowth fails in sec5 mutants once maternal Sec5 is exhausted. Using a trafficking assay, impairments were found in the membrane addition of newly synthesized proteins. In contrast, synaptic vesicle fusion was not impaired. Thus, Sec5, although not involved in vesicle transport, nevertheless differentiates between two forms of vesicle trafficking at the membrane: trafficking for cell growth and membrane protein insertion depend on sec5, whereas transmitter secretion does not. In this regard, Sec5 differs from the homologs of other yeast exocytosis genes that are required for both neuronal trafficking pathways (Murthy, 2003). Sec5 is also required for membrane traffic and polarity in the Drosophila ovary. During oogenesis, Sec5 localization undergoes dynamic changes, correlating with the sites at which it is required for the traffic of membrane proteins. Germline clones of sec5 possess defects in membrane addition and the posterior positioning of the oocyte. Additionally, the impaired membrane trafficking of Gurken, the secreted ligand for the EGF receptor, and Yolkless, the vitellogenin receptor, results in defects in dorsal patterning and egg size. However, the cytoskeleton is correctly oriented. It is concluded that Sec5 is required for directed membrane traffic, and consequently for the establishment of polarity within the developing oocyte (Murthy, 2004).

Directed membrane traffic is essential for many developmental processes, including cell growth, cytokinesis and signaling between cells. Such processes require membrane traffic to particular domains of the cell surface, in order to insert proteins at restricted regions of the membrane, to enlarge particular regions of the cell membrane, or to signal asymmetrically to neighboring cells. The identification, therefore, of the molecules required for directed membrane traffic will be important for understanding organismal development and cell-cell signaling (Murthy, 2004).

The exocyst complex, a set of eight proteins first identified from secretory mutants in yeast, is an attractive candidate for mediating directed traffic. Yeast cells use an anisotropic secretory apparatus for polarized growth at a selected bud site. While the bud is growing, there is almost no increase in the surface area of the mother cell, indicating that all membrane addition occurs at the bud tip. Later, secretion is redirected to the neck between mother and bud. The exocyst complex marks these areas of membrane addition, localizing to the bud tip of a growing daughter cell and the bud neck at the time of cytokinesis. Mutations in each member of the exocyst complex block the polarized trafficking that allows the bud to grow, but do not disrupt bud site selection. Thus, the exocyst complex in yeast may provide a model for the directed membrane traffic of developing cells in higher organisms (Murthy, 2004 and references therein).

The components of the exocyst complex, Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84, are conserved from yeast to mammals. In multicellular organisms, though less extensively studied, these proteins are implicated in establishing cell polarity. In epithelial cells, E-cadherin mediated adhesion is sufficient to initiate the segregation of apical from basolateral membrane proteins. The exocyst localizes to these sites of adhesion and is required for the polarized transport of proteins to the basolateral domain (Grindstaff, 1998). Similarly, overexpression of Sec10 in MDCK cells increased delivery of a membrane protein to the basal-lateral, but not apical, domain (Lipschutz, 2000). Thus, as in yeast, the mammalian complex may mediate fusion at a spatially restricted domain (Murthy, 2004 and references therein).

Neurons generate their polarity by directing membrane traffic to growing neurites and growth cones, and by sorting proteins differentially between the axon and dendrites. The exocyst has a punctate distribution in processes of cultured primary hippocampal neurons and localizes near, but not precisely with, synaptic vesicles (Hsu, 1996; Kee, 1997). A role for the complex in mammalian synapse formation and neurite outgrowth has been suggested by the early presence of Sec6/Sec8 immunoreactivity at sites of synaptogenesis in culture (Hazuka, 1999) and the impairment of neurite outgrowth with dominant-negative forms of sec10 and sec8 in PC12 cells (Vega, 2001). Though abundant in adult brain, the function of the complex in neurons after development remains uncertain. The exocyst, like SNARE complexes, may be needed for all fusions at the plasma membrane both in developing and mature neurons, or it may be required only for particular forms of traffic (Murthy, 2003 and references therein).

Thus far, resolving the neuronal function of the exocyst complex has been hampered by the absence of a functional genetic analysis. In the mouse, a sec8 mutant has been identified, but these mice die shortly after gastrulation of the embryo, precluding a more detailed analysis of the role of the complex (Friedrich, 1997). sec5 mutations have been identified and characterized in Drosophila in order to delineate the role of the protein in neurons and particularly at synapses. This protein is required for many aspects of membrane traffic within neurons, including the elaboration of neurites, but the release of neurotransmitter at the synapse is independent of this exocyst component (Murthy, 2003).

Within neurons, multiple pathways are known to transport proteins and transmitters to the cell surface. In some cases, the exocytotic vesicles derive from the Golgi apparatus and consist of newly synthesized materials, while in others the vesicles derive from the plasma membrane and cycle locally. Another distinction that has been drawn contrasts the constitutive and the regulated pathways to distinguish the ongoing transport of protein and lipid to the cell surface from the ability to secrete hormones and transmitters in response to specific stimuli. Subsequently, further distinctions in trafficking pathways have been invoked to explain different forms of regulation and fusion at different domains of the cell. Two important pathways were examined in the current study: (1) a basic pathway of exocytosis that brings new proteins to the cell surface and permits the cell to grow, and (2) synaptic transmission, a specialized form of exocytosis, regulated by Ca²⁺ entry, in which vesicles already present at synapses fuse with the membrane and recycle locally (Murthy, 2003).

The mechanistic basis for these distinct pathways remains obscure. Presently, all membrane trafficking steps are thought to possess a similar underlying mechanism for membrane fusion, and yet clear distinctions in these trafficking steps must also occur. In many cases, synaptic transmission is thought to consist of the basic exocytotic pathway with additional regulatory processes superimposed. This study has found a distinction between these two trafficking pathways in which the basic pathway depends on a protein, Sec5, that does not appear to be required for synaptic transmission (Murthy, 2003).

Mutations were isolated in sec5, a component of the Drosophila exocyst complex; the basic exocytotic pathway is impaired in these mutants, and yet vesicle fusion at the synapse persists. Despite a small amount of maternal protein remaining in the mutant at the end of its life span, dramatic phenotypes were observed with respect to membrane addition. Muscles lacking Sec5 do not increase their surface area and motor neurons do not increase their arborizations on the muscle. When cultured, neurons lacking Sec5 survived but proved incapable of extending neurites. This defect is not due to a general malaise of the mutant neurons: neurons at this time point were capable of synthesizing new proteins, conducting action potentials, and releasing neurotransmitter, and they are equally viable in culture. Instead, the likely mechanism for this deficit in cell growth is due to a specific defect in membrane traffic, as revealed in the trafficking assay with which the cell surface insertion of a newly made transmembrane protein was followed in first instar larvae. Endogenous proteins insert into the cell surface during the time the maternal contribution persists, however, when CD8-GFP is induced only after the maternal contribution is no longer functionally adequate, CD8-GFP fails to accumulate in the plasma membrane. These dramatic phenotypes in membrane addition are consistent with data in yeast (Novick, 1980, 1981), where exocyst mutants fail to add new membrane during cell division and accumulate vesicles in the presumptive daughter bud (Murthy, 2003).

What step in membrane trafficking requires Sec5? Members of the exocyst complex have been shown to be recruited to budding vesicles in the trans-Golgi network (Liljedahl, 2001), and antibodies to Sec6 and Sec8 are reported to interfere with the ability of these proteins to exit the Golgi (Yeaman, 2001). In the sec5 null mutants, however, normal levels of synthesis of the CD8 and Synaptotagmin-GFP constructs were observed. They were both distributed throughout the cytoplasm and transported down the axon with equal efficiency to wild-type. The requirement for Sec5 would therefore appear to lie at a later step in membrane traffic, most likely at docking to and fusion with the plasma membrane. This finding is consistent with the phenotype of exocyst mutants in yeast (Novick, 1980, 1981), where vesicles are appropriately transported into the bud but fail to fuse with the membrane (Murthy, 2003).

In marked contrast to the arrest of growth and the impairment of the pathway for membrane protein addition, synaptic transmission continued to be robust. Each action potential that arrived at the neuromuscular junction of a 96 hr AEL sec5^E10 homozygous larva caused an average of 33 ± 8 vesicles to fuse. Though Sec5 protein declined between 24 and 96 hr in these null mutants, the quantal content increased and, if anything, attained a level slightly higher than would be expected for a junction with the observed number of boutons. This persistence of vesicle fusion cannot be attributed to a residuum of maternally derived Sec5, since there were severe defects in the other assays conducted at these times. At the same time point that the larvae are capable of repeatedly secreting numerous synaptic vesicles, the lack of Sec5 so effectively prevented net membrane addition that almost no neurites could be extended and that little CD8 was successfully expressed on the cell surface. Thus, the synaptic response in larva lacking Sec5 would appear to derive from the combination of a defect in neuronal growth and an unaltered capacity for synaptic vesicle fusion. The growth defect prevented the creation of the normal number of boutons and release sites for a 96 hr animal; however, those boutons that had formed remained fully functional (Murthy, 2003).

What is reported for mutations in sec5 is likely to represent the function of the entire exocyst complex. From biochemical studies in yeast and mammalian cells, the component proteins of the exocyst are found primarily in a complex (Guo, 1999b; Hsu, 1996; TerBush, 1995). This model is consistent with the similarities of the mutant phenotypes for each component of the complex in yeast. Moreover, Sec5p binds in the center of the complex (Guo, 1999a), and its absence causes Sec10p and Exo84p to be dissociated from Sec8p (Guo, 1999b). It is hypothesized that loss of this individual component will be equivalent to loss of most or all exocyst function (Murthy, 2003).

Heretofore, synaptic transmission could be viewed as an elaboration of the basic exocytosis pathway, in which regulatory mechanisms are superimposed on a scaffold equivalent to the exocytotic machinery of yeast. Thus far, all of the genes studied at the nerve terminal that are orthologs of genes isolated in yeast for defects in the final steps of exocytosis have essential roles in synaptic vesicle fusion (Lin, 2000; Rothman, 1994). These genes include the proteins of the SNARE complex, syntaxin (yeast Sso1p), SNAP-25 (yeast Sec9p), and VAMP (yeast Snc1p), as well as nsec1 (yeast Sec1p), rab (yeast Sec4p), NSF (yeast Sec17p), and α-SNAP(yeast Sec18p). The same screen in yeast identified six of the eight members of the exocyst complex, but, as described above, the exocyst appears to be an exception to this established pattern: it is essential for exocytosis in yeast but dispensable for synaptic vesicle fusion (Murthy, 2003).

Why is vesicle fusion at the synapse distinct from the vesicle fusion required for neurite outgrowth or the addition of CD8 in the membrane? The data indicate that the synapse has evolved away from a need for the exocyst in fusion. In yeast, the exocyst complex is known to be required before the assembly of the SNARE complex and the fusion of secretory vesicles with the plasma membrane (Grote, 2000), which would place the exocyst complex upstream of the final stage of vesicle fusion. At the synapse, the complicated cytoskeletal network at the active zone may take the place of the exocyst complex in tethering vesicles at the release site and priming them for fusion. Such active zone proteins as Munc-13 and RIM, likely regulators of SNARE complex formation, which do not have homologs in yeast, may fulfill at the active zone the role assigned to the exocyst elsewhere in the cell. A second possibility is that the fundamental distinction between exocyst-dependent and-independent pathways in neurons depends on the source of the vesicle; the local cycling of vesicles not derived primarily from the TGN, but directly from the plasma membrane or through an endosome, may be independent of the exocyst complex. In this case, exocyst-independent fusions of endosome-derived vesicles may occur at many places in the cell and not just at synapses. However, TGN-derived vesicles, exemplified in this study by the CD8-GFP containing vesicles, would require the exocyst. It remains to be determined whether peptide-containing dense core vesicles, which fuse in a regulated manner like synaptic vesicles, but which, unlike synaptic vesicles, bud directly off of the TGN, can fuse with the membrane in the absence of Sec5. A recent study (Moskalenko, 2002) may point in this direction; inhibition of Ral function, which binds to the N terminus of Sec5, blocks the stimulated release of human growth hormone, contained in dense core vesicles, from PC12 cells (Murthy, 2003).

After the run down of maternal contribution in sec5 mutants, newly synthesized vesicles labeled with Synaptotagmin-GFP do not concentrate at the synapse. Though the boutons contain a large population of preexisting synaptic vesicles, as evidenced by immunostaining with the vesicle marker CSP and by the electrophysiology of the terminals, the boutons do not accumulate Synaptotagmin-GFP. This is attributed to the nature of the biosynthetic pathway for synaptic vesicles. The current model is that transport vesicles containing synaptic vesicle proteins fuse with the plasma membrane via the constitutive pathway of exocytosis from the TGN. Thereafter, they are internalized and sorted in endosomes to become mature synaptic vesicles. This model is supported by studies following the movement of radioactively labeled synaptophysin in neuroendocrine cells. It seems likely that in sec5 mutants, transport vesicles have not fused with the membrane and are not matured into synaptic vesicles. Therefore, the Synaptotagmin-GFP, though transported down and visible in the axon, does not enter the pool of mature synaptic vesicles that are clustered in the bouton. The transport vesicles that fail to fuse with the plasma membrane likely remain in the axon or are sent retrogradely back to the soma (Murthy, 2003).

Neurite outgrowth fails in the absence of Sec5. This role for the exocyst complex was predicted from its localization to the tips of neurites in cultured hippocampal neurons (Hazuka, 1999) and from studies with a dominant-negative sec10 construct in cultured PC12 cells (Vega, 2001). Neurite outgrowth requires a substantial increase in the surface area of the neuron and, not surprisingly, requires membrane addition. This membrane addition has been shown primarily to occur at the growth cone rather than in the soma or along the length of the neurite. Thus, as in yeast, the localization of the exocyst complex, both in Drosophila and hippocampal cultures (Hazuka, 1999), is consistent with a role in targeting vesicles to the site of membrane addition (Murthy, 2003).

The surface expression of CD8 in neuronal cell bodies and along the axon is impaired by loss of Sec5. These defects in traffic to the cell surface have parallels in studies of the MDCK epithelial cell line. In MDCK cells, the exocyst complex localizes to tight junctions between cells, which are thought to be the sites of membrane addition. Antibodies to Sec8 block the delivery of proteins to the basolateral membrane, with little effect on apical membrane addition. With regard to the trafficking and sorting of proteins, parallels have been drawn between the neuronal axon and the apical domain of epithelial cells. In the case of Sec5, however, that equation would not appear to apply, in that removing Sec5 affects surface expression of CD8 along the axon and in nerve terminals as well as in the soma. Alternatively, there may have been an exocyst dependence of apical transport in MDCK cells in the Grindstaff (1998) study that was resistant to the blockade by the antibody protocol. The examination of polarized transport in the epithelia of the sec5 mutants may clarify the question of the exocyst complex and apical transport (Murthy, 2003).

Intriguingly, the motor neurons of sec5 mutants failed to form new synaptic boutons after the maternal contribution of Sec5 was gone. This observation, and the apparent requirement for the exocyst complex in membrane addition but not synaptic transmission, suggests that sec5 and the exocyst complex may play a very particular role in synaptic plasticity. Anatomical plasticity, the formation of a distinct synaptic contact between a neuron and its targets, may require the exocyst protein to be targeted to the site from which the new branch must sprout. By recruiting new membrane and synaptic components to this area, the exocyst may permit the growth of a new synapse that will subsequently function independently of the exocyst. The identification of mutations in sec5 and other components of the complex should permit tests of this hypothesis (Murthy, 2003).

REGULATION

The NSL complex regulates housekeeping genes in Drosophila

MOF is the major histone H4 lysine 16-specific (H4K16) acetyltransferase in mammals and Drosophila. In flies, it is involved in the regulation of X-chromosomal and autosomal genes as part of the MSL and the NSL complexes, respectively. While the function of the MSL complex as a dosage compensation regulator is fairly well understood, the role of the Non-Specific Lethal (NSL) complex (Raja, 2010) in gene regulation is still poorly characterized. This study reports a comprehensive ChIP-seq analysis of four NSL complex members (NSL1, NSL3, MBD-R2, and MCRS2) throughout the Drosophila melanogaster genome. Strikingly, the majority (85.5%) of NSL-bound genes (a sample of which include Bap170, CG6506, sec5, CG15011, Ent2, Incenp, tho2 and Patj) are constitutively expressed across different cell types. An increased abundance of the histone modifications H4K16ac, H3K4me2, H3K4me3, and H3K9ac in gene promoter regions was found to be characteristic of NSL-targeted genes. Furthermore, these genes have a well-defined nucleosome free region and broad transcription initiation patterns. Finally, by performing ChIP-seq analyses of RNA polymerase II (Pol II) in NSL1- and NSL3-depleted cells, it was demonstrated that both NSL proteins are required for efficient recruitment of Pol II to NSL target gene promoters. The observed Pol II reduction coincides with compromised binding of TBP and TFIIB to target promoters, indicating that the NSL complex is required for optimal recruitment of the pre-initiation complex on target genes. Moreover, genes that undergo the most dramatic loss of Pol II upon NSL knockdowns tend to be enriched in DNA Replication-related Element (DRE). Taken together, these findings show that the MOF-containing NSL complex acts as a major regulator of housekeeping genes in flies by modulating initiation of Pol II transcription (Lam, 2012).

This study has revealed that the majority of the NSL-complex-bound targets are housekeeping genes in Drosophila. While chromatin-modifying complexes that regulate tissue-specific genes, such as SAGA, polycomb and trithorax complexes, have been studied extensively, global regulators of housekeeping genes are poorly understood. The NSL complex is the first identified major regulator of housekeeping genes (Feller, 2012; Lam, 2012).

The promoters of NSL target genes exhibit prominent enrichment of certain histone modifications (H4K16ac, H3K9ac, H3K4me2, H3K4me3) as well as specific core promoter elements (such as DRE, E-box and motif 1). Furthermore, these genes display distinct nucleosome occupancy and dispersed promoter configuration characterized by multiple transcription start sites. The correlation between these promoter characteristics (well-defined chromatin marks, TATA-less DNA sequences and broad initiation patterns) was previously identified for housekeeping genes in mammals and flies, but how these promoter features are translated into gene transcription had remained elusive. This study now conclusively demonstrates that the NSL complex modulates transcription at the level of transcription initiation by facilitating pre-initiation complex loading onto promoters. Therefore, it is proposed that the NSL complex is a key trans-acting factor that bridges the promoter architecture, defined by the DNA sequence, histone marks and higher chromatin structures with transcription regulation of constitutive genes in Drosophila (see Summary model: NSL-dependent Pol II recruitment to promoters of housekeeping genes) (Lam, 2012).

Excitingly, the enrichment of DNA motifs on NSL target gene promoters in combination with the genome-wide Pol II binding data has established functional links between the motifs enriched on housekeeping genes and the NSL-dependent Pol II binding to promoters. The abundance of DRE motifs, for example, was found to be positively associated with the magnitude of Pol II loss upon NSL knockdowns. The DRE binding factor (DREF) interacts tightly with TRF2 to modulate the transcription of DRE-containing promoters in a TATA-box-independent fashion (Hochheimer, 2002). It is tempting to speculate that the NSL complex might also cooperate with the TRF2 complex to facilitate transcription in a specific manner, rendering DRE-containing promoters more sensitive to NSL depletions. As the NSL-bound promoters are associated with a large variety of transcription factors, it will be of great interest to study whether the NSL complex communicates with different transcription regulators, perhaps making use of distinct mechanisms (Lam, 2012).

In contrast to DRE, motif 1 showed an opposing effect on Pol II recruitment to NSL-complex-bound genes as the presence of strong motif 1 sequences was associated with decreased Pol II loss upon NSL depletion. The mechanistic reasons for this remain unclear. However, one can envisage several possible scenarios. It is possible that motif 1 may recruit another transcription factor, which can also function to recruit the transcription machinery. Alternatively, the turnover of the transcription machinery might be slower on promoters containing strong motif 1 sequences. There is precedent for the transcription machinery having various turnover rates on different promoters. For example, in yeast, it has been shown that TBP turnover is faster on TATA-containing than on TATA-less promoters. It is therefore possible that certain levels of the initiation complexes may still be maintained on motif-1-containing promoters, even though the recruitment of the transcription machinery will be compromised in the absence of NSL complex. Further work is required to understand the importance of sequence determinants for NSL complex recruitment and the analysis sets the grounds for targeted experiments in the future (Lam, 2012).

Taking MOF-mediated H4K16 acetylation into consideration, a putative role of the NSL complex might be to coordinate the opening of promoter architecture by histone acetylation and the assembly of PIC. Coupling of histone acetylation and PIC formation has been described before. For example, TAF1, a component of TFIID, is a histone aceyltransferase. The SAGA complex, which contains Gcn5 and can acetylate H3K9, is reported to interact with TBP and other PIC components to regulate tissue-specific genes and the recruitment of P300 to the promoter and H3 acetylation have been shown to proceed binding of TFIID in a coordinated manner. H4K16ac is also well-known for its role in transcription regulation of the male X chromosome, yet how H4K16 acetylation and PIC assembly are coordinated remains elusive. Interestingly, absence of the NSL complex does not severely abolish H4K16ac from target genes. Since the turnover of H4K16ac on target promoter is unknown, it remains possible that H4K16ac could remain for some time at the promoter after the NSL complex is depleted. Further studies will be crucial in unraveling the functional relevance of H4K16 acetylation and NSL complex function on housekeeping genes (Lam, 2012).

Protein Interactions

Polarized exocytosis plays a major role in development and cell differentiation but the mechanisms that target exocytosis to specific membrane domains in animal cells are still poorly understood. This characterized Drosophila Sec6, a component of the exocyst complex that is believed to tether secretory vesicles to specific plasma membrane sites. sec6 mutations cause cell lethality and disrupt plasma membrane growth. In developing photoreceptor cells (PRCs), Sec6 but not Sec5 or Sec8 shows accumulation at adherens junctions. In late PRCs, Sec6, Sec5, and Sec8 colocalize at the rhabdomere, the light sensing subdomain of the apical membrane. PRCs with reduced Sec6 function accumulate secretory vesicles and fail to transport proteins to the rhabdomere, but show normal localization of proteins to the apical stalk membrane and the basolateral membrane. Furthermore, Rab11 forms a complex with Sec5 and Sec5 interacts with Sec6 suggesting that the exocyst is a Rab11 effector that facilitates protein transport to the apical rhabdomere in Drosophila PRCs (Beronja, 2005).

Two recent findings suggest that the small GTPase Rab11 interacts with exocyst proteins in Drosophila PRCs. (1) Depletion of Rab11 function in PRCs causes a mutant phenotype similar to that seen in sec6(pr) (for partial rescue) flies that is characterized by a massive accumulation of Rh1 containing secretory vesicles and small rhabdomeres. (2) Physical interactions between the exocyst protein Sec15 and Rab11 were found in mammalian culture cells. To test for interactions between Rab11 and exocyst proteins in Drosophila PRCs GFP-tagged Rab11 was expressed in adult eyes and immunoprecipitated with anti-GFP antibodies. These precipitates contained Sec5 but Sec6 was not detected. Similar results were obtained with embryonic extracts. Together, the accumulation of rhabdomere-specific secretory vesicles seen in Rab11 and sec6(pr) mutant PRCs and the physical interactions between Rab11 and Sec5 suggest that the exocyst is a Rab11 effector complex in PRCs and possibly other Drosophila tissues (Beronja, 2005).

Rab11 localizes to the RE in many different cell types. This raises the possibility that rhabdomere proteins are taking not a direct route from the Golgi to the apical membrane, but are delivered first to the basolateral membrane and then transcytosed to the apical membrane. This scenario seems unlikely as the expression of dominant-negative Rab5, which interferes with an early step in endocytotic trafficking, does not prevent Rh1 delivery to the rhabdomere. To further corroborate a direct Golgi to rhabdomere route for Rh1 endocytosis was compromised by disrupting the function of Dynamin. Flies that carried a temperature sensitive allele of shibire (shi^ts2), which encodes Drosophila Dynamin, and were maintained at the restrictive temperature (29°C) for 2 or 3 d during late PD showed normal localization of Rh1 to the rhabdomere. In contrast, shi^ts2 sec6(pr) double mutants show cytoplasmic accumulation of Rh1 similar to sec6(pr) mutants, suggesting that the cytoplasmic accumulation of Rh1 is not the result of increased endocytosis. In summary, these findings strongly argue for a direct Rab11/exocyst-dependent biosynthetic transport of Rh1 from the Golgi to the apical rhabdomere (Beronja, 2005).

Rab11-depleted PRCs accumulate secretory vesicles similar to sec6(pr) mutant PRCs, and Sec5 coimmunoprecipitates with Rab11::GFP from PRC and embryo lysates. These findings suggest that Rab11 takes the place of yeast Sec4p as the transport vesicle-associated small GTPase that recruits the exocyst. Although Sec6 was detected in Sec5 immunoprecipitates, no Sec6 was detected in Rab11::GFP precipitates. Two explanations for this discrepancy are envisioned. First, Rab11 may predominantly associate with a subcomplex of the exocyst that includes Sec5 but not Sec6. Second, in the yeast exocyst, Sec6p links to Sec4p through Sec15p, Sec10p, and Sec5p, suggesting that the Sec6 Rab11 interaction may involve several intermediates including Sec5 and therefore is more difficult to detect. Both explanations are consistent with the model that Sec5 connects Sec6 to Rab11, a relationship that is similar to the interactions of yeast exocyst components and Sec4p. These results suggest that the exocyst is a Rab11 effector complex in PRCs (Beronja, 2005).

Sec15 is required for the localization of some but not all exocyst members to neuronal terminals

The exocyst is a complex of proteins originally identified in yeast that has been implicated in polarized exocytosis/secretion. Components of the exocyst have been implicated in neurite outgrowth, cell polarity, and cell viability. An exocyst component, sec15, has been isolated in a screen for genes required for synaptic specificity. Loss of sec15 causes a targeting defect of photoreceptors that coincides with mislocalization of specific cell adhesion and signaling molecules. Additionally, sec15 mutant neurons fail to localize other exocyst members like Sec5 and Sec8, but not Sec6, to neuronal terminals. However, loss of sec15 does not cause cell lethality in contrast to loss of sec5 or sec6. The data suggest a role for Sec15 in an exocyst-like subcomplex for the targeting and subcellular distribution of specific proteins. The data also show that functions of other exocyst components persist in the absence of sec15, suggesting that different exocyst components have separable functions (Mehta, 2005).

Does Sec15 exert an exocyst-dependent function at the neuronal terminal? To date, only one other mutant has been reported that affects a component of the exocyst in Drosophila, namely sec5 (Murthy, 2003). Murthy showed that Sec5 is required for cell polarization in the developing oocyte and neurite outgrowth in cell culture. In vivo, homozygous mutations in sec5 are lethal in photoreceptor neurons (Murthy, 2003), as are mutations in sec6 (S.B. and U.T., unpublished data). In contrast, it was observed that sec15 homozygous mutant photoreceptor neurons are viable, even in aged flies. Either Sec15 exerts a function independent of the exocyst at the neuronal terminal, or its developmental role only represents a specialized task of the complex or subcomplex. To distinguish between these two possibilities, the localization of Sec5, Sec6, and Sec8 in developing neuropil was examined as well as in sec15 mutant clones (Mehta, 2005).

In the developing lamina of the late third instar larva, Sec5 and Sec15 colocalize. Both are highly enriched in the developing neuropil, whereas immunoreactivity in the functional larval central brain is much lower. In the adult lamina, Sec5 and Sec15 are coexpressed in cartridges. Sec5 colocalizes with Sec15 to a larger extent than any of the other markers tested, including plasmalemmal, synaptic vesicle, or active zone markers. However, the colocalization of Sec15 and Sec5 is not perfect, leaving subdomains marked only by anti-Sec15 or anti-Sec5. These data suggest that Sec5 and Sec15 may have common as well as separate functions (Mehta, 2005).

The expression patterns of two other presumed core members of the exocyst, Sec6 and Sec8, were examined using two newly generated polyclonal antibodies. In adult lamina cartridges, Sec6 immunoreactivity exhibits a very specific pattern that exactly matches the localization of the postsynaptic lamina monopolar cells. In contrast, the antibody against Sec8 exhibits a punctate staining pattern throughout the cartridges that is similar to Sec15. Likewise, Sec6 and Sec8 antibodies have both specific but different staining patterns in the developing brain: Sec6 strongly colocalizes with Sec5 in developing neuropil, whereas Sec8 is enriched in cell bodies but is almost completely excluded from the developing neuropil. Finally, stainings were performed to examine the localization of Sec15, Sec6, and Sec8 at the third instar larval neuromuscular junction. Sec15 is present in both boutons and muscle cells, but seems enriched at boutons. In contrast, Sec 6 is highly enriched at the Z bands of muscle cells and very weakly present in boutons, while Sec8 is not present in muscle cells or neurons, but is in a highly punctate distribution in unidentified processes that may be glial projections. These data are not consistent with a single functional Sec6/8 complex (Mehta, 2005).

To test whether Sec15 at the photoreceptor terminals affects Sec5, Sec6, and Sec8, the presence of these proteins was investigated in sec15 mutant clones. Sec5 immunoreactivity in sec15 mutant clones of photoreceptor terminals in the lamina is markedly reduced and possibly absent in the terminals. Likewise, Sec8 immunoreactivity is reduced in sec15 mutant clones of photoreceptor terminals. However, the levels of Sec6 appear to be unaffected by mutations in sec15. This is likely because Sec6 is enriched in postsynaptic cells in the lamina and because it seems to be absent presynaptically. In addition, the specific developmental and adult staining patterns of Sec8, as well as its downregulation in sec15 mutant clones, suggest common and separable functions compared to sec5, sec6, and sec15 at different points in development. Since loss of sec5 in photoreceptors causes cell lethality, the loss or downregulation of Sec5 in sec15 mutant terminals is unlikely to reflect a global loss of the protein. These data rather suggest that Sec15 is required for localization of Sec5 and Sec8, but not Sec6 to the presynaptic photoreceptor terminal. These data suggest that Sec15 may recruit or stabilize a complex that includes some but not all exocyst members in photoreceptor terminals in a spatiotemporally regulated manner (Mehta, 2005).

dEHBP1 controls exocytosis and recycling of Delta during asymmetric divisions

Notch signaling governs binary cell fate determination in asymmetrically dividing cells. A forward genetic screen identified the fly homologue of Eps15 homology domain containing protein-binding protein 1 (dEHBP1) as a novel regulator of Notch signaling in asymmetrically dividing cells. dEHBP1 is enriched basally and at the actin-rich interface of pII cells of the external mechanosensory organs, where Notch signaling occurs. Loss of function of dEHBP1 leads to up-regulation of Sanpodo, a regulator of Notch signaling, and aberrant trafficking of the Notch ligand, Delta. Furthermore, Sec15 and Rab11, which have been previously shown to regulate the localization of Delta, physically interact with dEHBP1. It is proposed that dEHBP1 functions as an adaptor molecule for the exocytosis and recycling of Delta, thereby affecting cell fate decisions in asymmetrically dividing cells (Giagtzoglou, 2012).

This study describes the identification of dEHBP1 as a novel, positive regulator of Notch signaling in asymmetrically dividing cells in the ESO lineage in Drosophila. In the absence of dEHBP1, external cell types, such as socket and shaft cells, are transformed into internal cell types, i.e., neuron and sheath cells, one of the hallmarks of loss of Notch signaling. EHBP1 has been previously studied in mammalian cell culture systems and in vivo in C. elegans. In mammalian adipocytes, EHBP1 affects endocytosis and recycling of the glucose transporter GLUT4 in the context of insulin signaling, depending on its interaction via the NPF motifs present in its N-terminal region with EHD2 or EHD1, respectively. However, the fly and worm EHBP1 lack the NPF motifs, suggesting that the EHD-EHBP1 interaction may have emerged later in evolution. In C. elegans, EHBP1 was shown to impair rab10-mediated endocytic recycling of clathrin-independent endocytosed cargoes, such GLR-1 glutamate receptor. This study shows that dEHBP1 is required in the exocytosis and recycling of Delta, a ligand of the Notch receptor. Notch signaling defects were not reported in C. elegans ehbp1 mutants. Therefore, it would be interesting to investigate whether EHBP1 and its homologues play an evolutionarily conserved role of EHBP1 in Notch signaling (Giagtzoglou, 2012).

dEHBP1 is a ubiquitous protein that is associated with the plasma membrane, enriched at the lateral and basal surface of pII cells, where it colocalizes with F-actin. Live imaging with mCherry-dEHBP1 and immunofluorescent stainings with anti-dEHBP1 antisera also reveal dEHBP1-positive, punctate, intracellular structures within ESO lineages. An extensive analysis with a diverse array of intracellular markers revealed that these punctae colocalize with Rab8, indicating their exocytic nature. Importantly, in C. elegans, EHBP1 physically interacts and colocalizes with Rab8 and Rab10, and controls the recruitment of Rab10 in recycling endosomal structures. However, in the current studies, overexpression of dominant-negative forms of Rab10 or Rab8 in the ESO lineages as well as thoracic clones of a newly identified Rab8 loss-of-function allele do not confer any cell fate phenotypes. Furthermore, no interaction was detected between dEHBP1 and Rab8 or Rab10 in a yeast two-hybrid analysis. Therefore, it is believed that loss of either Rab8 or Rab10 function does not underlie the dEHBP1 mutant phenotypes that are describe (Giagtzoglou, 2012).

Notably, many key players that affect cell polarity or mark subcellular compartments, including Arm, Rab11, Sec15, and F-actin, are not affected by the loss of dEHBP1. In addition, cell fate determinants Numb and Neuralized are correctly segregated upon asymmetric cell division in dEHBP1 mutant cells. However, loss of dEHBP1 specifically affects the abundance and localization of Spdo, a regulator of Notch signaling in asymmetrically dividing ESO cells, and the exocytosis and trafficking of Delta (Giagtzoglou, 2012).

Spdo facilitates reception of Notch signal at the plasma membrane of the signal-receiving cell. Therefore, accumulation of Spdo in dEHBP1^−/− ESO clusters and its presence in the plasma membrane should result in a Notch gain of function, instead of the loss-of-function phenotype that was observed. No effects have been observed of Spdo overexpression upon cell fate acquisition in the ESO lineage. Alternatively, the accumulation of Spdo in the absence of dEHBP1 in these cells may reflect defects in its trafficking and membrane localization, which render the activation of Notch signaling more difficult (Giagtzoglou, 2012).

dEHBP1 mutations cannot suppress the gain of function phenotype of overexpressed ligand-independent, activated Notch intracellular domain. In addition, dEHBP1 does not affect the steady-state levels of Notch protein, as well as its endocytosis. Therefore, it is concluded that dEHBP1 functions at a level upstream of presenilin-mediated S3 cleavage of Notch during reception of the signal. Although it cannot be excluded that dEHBP1 functions in the signal-receiving cell, where it may control the trafficking and localization of Spdo, it is concluded that dEHBP1 also functions in the sending of the signal. This conclusion is based on the fact that dEHBP1 mutations are able to suppress the gain of function of Notch phenotype conferred by the overexpression of DaPKC^ΔN. Overexpressed constitutively active DaPKC^ΔN places Spdo at the plasma membrane, enabling the activation of Notch signaling. This study found that upon loss of dEHBP1, Spdo is still found at the plasma membrane under conditions of overexpression of DaPKC^ΔN. Therefore, the suppression of the overexpression phenotype of DaPKC^ΔN by loss of dEHBP1 may be because of other defects, such as loss of the ability of Delta to signal. Furthermore, loss of dEHBP1 leads to development of additional neurons despite the concomitant ectopic expression of Delta^R+, a variant of Delta, in clones within pupal nota at 36 h APF. Because the steady-state levels of Delta are not affected in dEHBP1^−/− ESO lineages, whether dEHBP1 affects Delta trafficking in the signal-sending cell was examined. Upon loss of dEHBP1, the abundance of Delta at the cell surface is significantly reduced, suggesting that exocytosis is defective. Importantly, most of the remaining extracellular Delta protein localizes at the basal side of the signal-sending cell. This suggests that in addition to affecting exocytosis of Delta, dEHBP1 may also play a role in basal-to-apical trafficking of Delta. This leads to a reduced level of Delta at the signaling interface, which interferes with proper Notch signaling in the cell receiving the signal. Although the results do not exclude a possible role of dEHBP1 in other aspects of Delta trafficking, such as endocytosis, reduced exocytosis of Delta should mask an endocytic defect in the assays. The enrichment of dEHBP1 in the basal and lateral area of the plasma membrane, its colocalization with F-actin at the actin-rich structure at the interface of the pIIa and pIIb cells, the reduction of Delta exocytosis in mutant cells, and the absence of Delta at the interface and the apical surface of the ESO cluster in mutant cells indicate a role of dEHBP1 in the Sec15/Rab11 recycling pathway. Indeed, the colocalization of dEHBP1 and Delta in sec15^−/− ESO lineages implies that the exocyst component, Sec15, controls exocytosis of Delta, Spdo, and dEHBP1 to the apical plasma membrane through a common compartment. Because loss of dEHBP1 does not affect the localization of either Rab11 or Sec15, it is concluded that sec15 lies more upstream in the trafficking pathway regulating the localization of multiple components, while dEHBP1 functions during the later stages of intracellular trafficking. Furthermore, the physical interaction between dEHBP1 and Sec15 as well as Rab11 suggest a mechanism how dEHBP1 may regulate the membrane localization of Delta via its interaction with Sec15 and Rab11 at the pII cells interface, even though such interaction was detected under transient overexpression conditions. It is proposed (see Model of dEHBP1 function) that dEHBP1 is an adaptor of the Rab11/Sec15-positive, Delta-bearing vesicles required for exocytosis (Giagtzoglou, 2012).

The identification of dEHBP1 provides further compelling evidence that the exocytosis and recycling pathway of Delta during asymmetric divisions is tightly regulated. The recycling pathway of Delta appears to be context dependent, i.e., it is not required in all cells that use Notch signaling. Still, the discovery of dEHBP1 as a novel player in Notch signaling provides the opportunity to test its role in Notch-related neurobiological behaviors, such as sleep and addiction, as well as in Notch-related diseases, as for example in Wiskott-Aldrich syndrome, an immunodeficiency characterized by abnormal differentiation and function of T cell lineages. Furthermore, because the anthrax toxins lethal factor (LF) and edema factor (EF) inhibit the Sec15/Rab11-dependent Delta-recycling pathway in flies and endothelial cells, it would be interesting to hypothesize whether they target dEHBP1 to mediate their toxicity (Giagtzoglou, 2012).

DEVELOPMENTAL BIOLOGY

Embryonic

sec5 transcripts are present at rather constant levels throughout development, including the first 2 hr of embryogenesis, before zygotic transcription begins. This was confirmed by analysis of Sec5 protein (Murthy, 2003).

Mouse polyclonal 5AL (raised against a GST fusion protein containing aa 1-321), mouse polyclonal 1RN (raised against a GST fusion protein containing aa 634-894), and a monoclonal antibody 22A2 each recognized a band of 100 kDa on immunoblots, consistent with the predicted mass of Drosophila Sec5. The intensity of the band sharply decreases in homozygous null mutant larvae at 48 or 72 hr AEL, relative to heterozygous mutants from the same stock. Crossreacting bands observed with either unpurified antiserum 5AL or 1RN were unaffected by the mutation, consistent with the identification of the 100 kDa band as the sole product of the sec5 locus (Murthy, 2003).

The amount of maternally derived protein (maternal contribution) remaining in sec5^E10 null mutants between hatching (24 hr AEL) and 96 hr AEL was quantified with antibody 5AL on immunoblots by means of a fluorophore-coupled secondary antibody, employed in the linear range of detection. In wild-type, Sec5 protein levels remained constant relative to total protein between 24 and 96 hr AEL. At 24 hr, sec5^E10 homozygotes had ~29% of Sec5 protein (normalized) that was present in equivalently aged wild-type larvae (p = 0.02). By 48 hr AEL, when the growth of the larva ceased, the protein level in homozygous mutants had dropped to 11% of the 24 hr control. Sec5 protein continues to decline thereafter, and by 72 or 96 hr the remaining Sec5 appears almost completely gone (3% and 2.5% of the 24 hr control, respectively). The nearly complete absence of Sec5 in homozygous E10 alleles was confirmed by immunocytochemistry with monoclonal 22A2. While Sec5 immunoreactivity appears to be present ubiquitously at low levels, within the nervous system, Sec5 is enriched in the neuropil, the synapse-rich region of the ventral nerve cord. In sec5^E10 mutants at 72 hr AEL, immunoreactivity is barely detectable. Thus, sec5^E10 larvae, between 72 and 96 hr, provide a suitable system in which to study the consequences of a loss of Sec5 (Murthy, 2003).

Oogenesis

Oocyte polarity and egg chamber organization depend on the addition of particular proteins to subregions of the plasma membrane. The localization of these proteins is likely to involve multiple levels of regulation, including the localization of transcripts, the transport of proteins to particular cytoplasmic regions and the retention of proteins in defined membrane domains. If selective, localized insertion into the plasma membrane is to be a factor in the asymmetric distribution of membrane proteins, then a component of the exocytotic machinery must be appropriately localized. Such a role is proposed for Sec5 and the exocyst complex. No other protein involved in membrane trafficking is known to have a suitably regulated distribution within the oocyte. The cognate pairing of SNAREs is reported to be significant in distinguishing intracellular compartments for vesicle targeting. Syntaxin, however, is expressed all along the oocyte membrane and at constant levels, and therefore cannot direct polarizing events. In contrast, by concentrating the exocyst complex at different regions of the membrane during oocyte development, the oocyte may target the trafficking of proteins (Murthy, 2004).

The distribution of Sec5 during oogenesis correlates with where it is required. Sec5 is initially present on all membranes within the egg chamber. At this stage, all the cells of the egg chamber are growing and removing sec5 from the germline disrupts membranes. Glycoproteins and Syntaxin, which are normally found in the plasma membrane, accumulate instead within the cytosol of the egg chamber. At early stages, the exocyst thus appears to mediate general membrane traffic for cell growth. Sec5 is most abundant at this time at the boundary between the oocyte and posterior follicle cells, the site of reciprocal signaling that governs oocyte position. Making either the germline or the posterior follicle cells mutant for sec5 results in an abnormally anterior position for the oocyte. In addition to its requirement in general membrane growth, Sec5 is therefore likely to be required at this cell boundary for signals and adhesion molecules on the oocyte and follicle cell surfaces and thereby for establishing the anteroposterior axis. At stage 8, its widespread distribution closely parallels the sites of Yolkless insertion, a Sec5-dependent process. Beginning at stage 7 and culminating at stage 10, Sec5 is increasingly concentrated at anterior corners, when Gurken is inserted at that site. Owing to an inability to traffic Gurken efficiently to the membrane, females with sec5^E13 germline clones lay eggs with dorsal patterning defects (Murthy, 2004).

During the early stages of oogeneis Sec5 concentrates posteriorly, at the boundary between the follicle cells and the oocyte. Particularly at stage 5, sec5 resides at the apical end of the two posterior polar cells. At stage 6, Sec5 localization changes; although still enriched in the polar cells and expressed at low levels ubiquitously, Sec5 concentrates at the oocyte membrane. At this time, the oocyte grows at a faster rate than the nurse cells and the enrichment in the oocyte membrane may reflect the greater need for membrane addition there. During stage 7, when the microtubule cytoskeleton reorients and the nucleus moves to the dorsoanterior corner of the oocyte, Sec5 appears enriched along this anterior rim, at the corners where the lateral and anterior membranes of the oocyte meet, although still expressed all along the membrane. This pattern continues through stage 8. Finally, at stage 10, Sec5 is highly concentrated at the anterolateral margins of the oocyte, with less detectable towards the posterior end of the cell. To determine if this distribution was shared by other plasma membrane-associated components of the membrane-trafficking apparatus, Sec5 labeling was compared with that of the t-SNARE Syntaxin. Syntaxin is present along the length of the oocyte membrane, including the posterior region (Murthy, 2004).

Because Sec5 becomes enriched at the anterior membrane of the oocyte at the time when the microtubule cytoskeleton rearranges within the oocyte and because the exocyst has been shown to associate with the cytoskeleton, whether the localization of Sec5 was dependent on the cytoskeleton was tested. In oocytes from females treated with colcemid, a microtubule-depolymerizing drug, Sec5 continues to be concentrated at the stage-appropriate domains of the oocyte membrane, including the anterior corners at stage 10 (Murthy, 2004).

Hexagonal packing of Drosophila wing epithelial cells by the planar cell polarity pathway

The mechanisms that order cellular packing geometry are critical for the functioning of many tissues, but they are poorly understood. This problem was investigated in the developing wing of Drosophila. The surface of the wing is decorated by hexagonally packed hairs that are uniformly oriented by the planar cell polarity pathway. They are constructed by a hexagonal array of wing epithelial cells. Wing epithelial cells are irregularly arranged throughout most of development, but they become hexagonally packed shortly before hair formation. During the process, individual cell boundaries grow and shrink, resulting in local neighbor exchanges, and Cadherin is actively endocytosed and recycled through Rab11 endosomes. Hexagonal packing depends on the activity of the planar cell polarity proteins. It is proposed that these proteins polarize trafficking of Cadherin-containing exocyst vesicles during junction remodeling. This may be a common mechanism for the action of planar cell polarity proteins in diverse systems (Classen, 2005).

How might Cadherin or other junctional material be added to a growing boundary? In other epithelia, Cadherin is dynamically endocytosed and recycled to modulate cell adhesion. To test whether this might happen in the pupal wing, the temperature-sensitive shibire (shi) mutation of dynamin was used. Dynamin is required for scission of endocytic vesicles and vesicles formed from Rab11 recycling endosomes. A total of 30-45 min after shifting to 34°C, gaps form in junctional E-Cadherin in shi mutant wings that are not found in wild-type control wings, even after 3 hr of temperature shift. Similar results are obtained in clones of shi mutant cells. The gaps form exclusively in intervein regions, and they occur primarily at or adjacent to vertices. Similar results were obtained for Armadillo, another adherens junction protein. In contrast, the septate junction protein Coracle and basolaterally localized CD2GFP were undisturbed by loss of Dynamin. After 3 hr at 34°C, shi mutant cells show even larger gaps in Cadherin. By 6 hr, cell-free areas are seen in the intervein region by Cadherin staining. After these animals are restored to 18°C, emerging adults have holes in wing intervein regions. None of these changes are observed when temperature shifts are performed on third instar larvae, even for longer times. Loss of Cadherin is not a consequence of cell death; Cadherin is lost before Caspase is found in the nucleus. These data suggest that Dynamin is required to maintain uniform localization of adherens junctions, but not septate junctions or basolateral proteins, during repacking. Development of holes in intervein regions where Cadherin gaps form suggests that the loss of junctional proteins disturbs epithelial integrity (Classen, 2005).

To precisely define the stage at which Dynamin is required to maintain Cadherin, shi mutants were systematically shifted to 34°C during a sliding 6 hr window starting just after pupariation and ending after hair formation. The frequency and placement of holes in the adult wing were quantified as a read-out because antibody penetration is prevented by the cuticle throughout much of pupal development. Although a variety of phenotypes were observed, only temperature shifts initiated between pupal stage P2A and mid-P2C (before hair formation) cause holes in the wing. These data show that epithelial repacking is temporally coincident with the requirement for Dynamin (Classen, 2005).

To confirm that Cadherin enters the endocytic pathway at the time of hexagonal repacking, GFP-Cadherin-expressing pupal wings (stage P2B) were stained with FM4-64. FM4-64 labels the plasma membrane and endosomes that form after its addition. The majority of pupal wing cells contain multiple internal spots of GFP-Cadherin that colocalize with FM4-64 after 15-30 min. Thus, Cadherin is actively endocytosed during repacking (Classen, 2005).

To ask which type of endosomes contained Cadherin, flies that ubiquitously expressed YFPRab11 or CFPRab5 at low levels were used. Rab11 labels recycling endosomes, and Rab5 marks early endosomes. Cadherin was observed in both types of endosomes, supporting the idea that it is endocytosed and recycled (Classen, 2005).

In MDCK cells, Cadherin is delivered through Rab11 endosomes (Lock, 2005). To ask whether this occurs in the wing, Rab11 function was disturbed by short-term expression of the dominant-negative Rab11SN. A total of 3 hr after initiating Rab11SN expression, Cadherin begins to be lost from the junctional region -- a phenotype similar to that of the shi mutant. These cells are not apoptotic. No gaps form when Rab11SN is expressed for similar times in larval wing discs. Thus, Rab11 is required to deliver Cadherin to junctions, and this requirement is acute during epithelial repacking. Loss of junctional E-Cadherin in dynamin mutant cells may reflect Dynamin's function at Rab11 endosomes (Classen, 2005).

The exocyst is a multiprotein complex that mediates polarized membrane delivery from recycling endosomes and from the golgi in many different cell types. In the thorax, E-Cadherin delivery from recycling endosomes to the zonula adherens depends on exocyst components (Langevin, 2005). To test whether E-Cadherin was recycled via the exocyst during repacking in the wing, a mutation was utilized in Sec5 (sec5^E13) that has been suggested to preferentially perturb recycling. Cadherin accumulates in internal vesicles and along the plasma membrane in sec5^E13 mutant cells. Accumulation of internal vesicles suggests that delivery of Cadherin is slowed. It is not known whether higher levels of peripheral Cadherin staining reflect accumulated unfused vesicles, or whether Sec5 may also function at some other step in Cadherin trafficking (Classen, 2005).

To ask whether perturbing endocytosis and recycling causes defective cell packing, shi mutant wings were examined shortly after the shift to the restrictive temperature. Compared with wild-type shifted to the same temperature, shi tissue was less hexagonal and had a higher variability in the length of individual cell contacts. This is consistent with the possibility that Dynamin-dependent recycling of junctional components is needed to remodel junctions; however, packing may have been perturbed by some other Dynamin-dependent process (Classen, 2005).

To test whether turnover of Cadherin itself was required for hexagonal packing, expression of an E-Cadherin:α-Catenin fusion protein was induced at the time of repacking. A similar vertebrate construct is not regulated by β-catenin, causes abnormally stable adhesiveness, and inhibits motility in L cells. Expression of this construct disrupts hexagonal packing and increases the variability of cell contact lengths. This is consistent with the idea that junction remodeling depends on the disassembly of E-Cadherin-mediated contacts, although additional effects mediated by irreversible linkage to the actin cytoskeleton cannot be ruled out (Classen, 2005).

A link between the PCP pathway and epithelial repacking is suspected, because repacking occurs at the time that these proteins are thought to polarize. Therefore neighbor number and junction length variability was quantified at the time of hair outgrowth in different PCP mutants. For prickle (pk-sple^13/26), neighbor number was quantitated over time (Classen, 2005).

pk-sple^13/26 wings begin repacking at the same time as wild-type; however, the process is less successful. Whereas wild-type wings reduce the percentage of pentagonal cells from 34% to 13% by the time that hairs begin to emerge, pk-sple^13/26 wings retain 21%. Thus, about 40% of the pentagonal cells that normally assemble boundaries with new neighbors (and become hexagonal) fail to do so in pk-sple mutants. Consistent with this, pk-sple wing epithelia contain abnormally high numbers of four-way vertices between cells. pk¹ mutant wings are even more irregularly packed than pk-sple^13/26 wings. A total of 62% of the pentagonal cells that would normally become hexagonal fail to assemble boundaries with new neighbors in pk¹ wings. Even four-sided cells accumulate significantly in pk¹ mutant wings. Individual cell contact lengths are also much more variable; while pk-sple^13/26 boundary lengths were 9% more variable than wild-type, those of pk¹ were 42% more variable. These data are consistent with the earlier observation that adult pk wings frequently contain pentagonal cells. These data suggest that the assembly of new cell boundaries and regularization of junction length do not occur efficiently in the absence of products of the Pk-Sple locus (Classen, 2005).

Packing defects of the hypomorphic Flamingo (fmi) allele, fmi(stan)³, are mild but significant. The null allele fmi^E59 produces much stronger defects. The variability of individual junctional lengths in these cells is more than twice that of wild-type, and only 69% of fmi^E59 mutant cells become hexagonal, compared with 78% in wild-type. Pentagonal cells persisted in fmi^E59 mutants (27% compared with 13% in wild-type). This suggests that the majority of pentagonal cells fail to assemble boundaries with new neighbors when Fmi is missing (Classen, 2005).

The packing geometry was examined of two different frizzled (fz) alleles, fz^R52 and fz^P21. fz^P21 mutant wings fall into two classes. While the majority of wild-type and PCP mutant wings initiate hair formation by 42 hr after puparium formation (APF) (at 22°C), a subset of fz^P21 mutant wings does not. Since these wings were not apoptotic (as indicated by Caspase staining), they were included in the analysis and quantified separately. Even at 50 hr APF, their packing is much more irregular than that of wild-type . Defects in fz^P21 mutant wings that do initiate hair formation by 42 hr APF are milder but still significant. fz^R52 homozygotes do not produce viable pupae in these experiments, and homozygous mutant clones are small. These clones have even stronger packing defects than those of fz^P21, suggesting that little repacking occurs in fz^R52 homozygous tissue. Thus, Fz is needed to develop regular hexagonal packing (Classen, 2005).

stbm⁶ and dgo³⁸⁰ mutant wings have milder, but significant, alterations in the ratio of pentagons, hexagons, and heptagons and of four-way vertices. Both mutants, however, affect junction length variability more strongly than pk-sple^13/26. Taken together, these data indicate that PCP mutant cells fail to efficiently assemble boundaries with new neighbors and cannot regularize their packing geometry (Classen, 2005).

To ask whether interfering with PCP polarity could alter the geometry of packing in wild-type cells, cells were examined surrounding PCP mutant clones with either autonomous (fmi^E59) or nonautonomous (fz^R52) effects on polarity. The frequency of pentagons, hexagons, and heptagons was examined in fz^R52 and fmi^E59 mutant clones, and in the areas of disturbed and normal Fmi polarity surrounding both. The mutant cells within both fz^R52 and fmi^E59 clones are abnormally packed. However, whereas the packing defects caused by Fmi clones are predominantly restricted to the clone and directly adjacent cells, Fz clones alter packing over long distances in wild-type tissue in the same regions where Fmi polarity is disturbed. The abnormal packing of wild-type cells surrounding fz^R52 clones is unlikely to be a consequence of altered cell packing within the mutant clone, because fmi^E59 mutant clones pack just as abnormally, but do not perturb packing in the surrounding tissue. This suggests that dominant reorientation of Fmi polarity by frizzled mutant clones disturbs the repacking of wild-type cells (Classen, 2005).

To investigate how the PCP proteins were localized during repacking, pupal wings were imaged for Fmi before, during, and after hexagonal packing. Since it is thought that PCP proteins do not polarize until shortly before hair formation, it was surprised to find that the subcellular distribution of Fmi is polarized in many areas of the wing before junction remodeling is initiated, even in late third instar wing discs and prepupal wings. Fz-GFP is distributed similarly. This polarity may have been missed because it exhibits less long-range coherence in imaginal discs and prepupal wings than it does later (Classen, 2005).

In prepupal wings, Fmi polarity is roughly proximal-distal in the region surrounding L3. Coherent Fmi polarity is lost at the beginning of the pupal period: this is exactly the time at which junction remodeling initiates. Although polarity is not coherent, Fmi is not uniformly distributed along cell boundaries. This can be clearly seen when Fmi localization is compared to that of E-Cadherin (Classen, 2005).

At pupal time TP1, Fmi polarization begins in vein cells as they contract their apical cross-section. Intervein regions contain only small groups of cells with coherent polarity, and the axes of these groups are not always proximal-distal. By TP2, Fmi polarity is coherent between larger groups of cells, although the axis of polarity is still mixed. Fmi polarity is aligned in large coherent domains along the proximal-distal axis by TP4, when hexagonal packing is completed, and it remains unchanged at TP5 when hairs emerge. In summary, PCP proteins polarize during larval and prepupal stages, alignment of polarity between cells is disturbed when junction remodeling begins, and long-range polarity is reestablished as hexagonal packing is completed. Early polarization of PCP proteins is consistent with the genetic requirement for fz and ds activity at this time to determine the axis of polarity, and it suggests that the feedback loop that organizes coupled proximal and distal domains probably acts during these early stages (Classen, 2005).

It was asked whether PCP proteins might affect packing by influencing recycling of junctional components. Therefore, it was asked whether PCP mutants enhance the hole formation caused by shi loss of function. Double mutant pupae were shifted to a subrestrictive temperature that never causes holes to form in shi mutants or in PCP mutants. When shi is combined with dgo³⁸⁰, stbm⁶, stbm¹⁵³, stbm^D, stan³, pk-spl¹, or pk¹, hole formation occurs even under these mild conditions. This raises the possibility that PCP proteins may worsen Cadherin recycling defects in dynamin mutant cells. Consistent with this, gaps in Cadherin arise more frequently in double shi;pk¹ or shi;dgo³⁸⁰ mutant wings than in wings mutant for shi alone. This suggests that Cadherin is recycled less efficiently in the absence of PCP proteins (Classen, 2005).

Despite this enhancement, no striking abnormalities in Cadherin distribution were seen in most PCP mutants. fz^P21 mutant cells sometimes show gaps in E-Cadherin that are similar to, but much less frequent than, those of shi mutants. In fmi^E59 mutant cells, E-Cadherin levels are elevated, but no gaps in localization are observed. These observations suggest that PCP proteins are not required for delivery of Cadherin to cell contacts during remodeling. Nevertheless, the PCP mutants enhance Cadherin recycling defects caused by loss of Dynamin. One model consistent with this shows that PCP proteins bias Cadherin recycling to specific places on the cortex. Reducing both the rate of recycling and its elevation at a particular site could exacerbate the failure of Cadherin delivery to growing cell boundaries (Classen, 2005).

To test whether exocyst components were polarized by PCP proteins, Sec5 localization was examined during repacking of the wing epithelium. At this time, cell shapes are irregular, and Fmi polarity is not coherent between cells. Nevertheless, Fmi accumulates preferentially on specific regions of the cortex. Although Sec5 vesicles are seen throughout the cell, they are particularly enriched near Fmi-positive cell boundaries. Enrichment persists as Fmi polarity becomes aligned (Classen, 2005).

To test whether Fmi plays an active role in recruiting Sec5, Fmi was overexpressed and Sec5 localization was examined. Overexpressed Fmi is present uniformly around the cortex and in large punctate structures within the cell. Sec5 dramatically accumulates in cells overexpressing Fmi and is recruited to sites of Fmi localization. Large internal structures positive for Fmi and Sec5 also contain Cadherin. These observations indicate that Fmi can recruit Sec5-positive vesicles containing E-Cadherin, and they suggest that PCP proteins may promote hexagonal packing by polarizing membrane trafficking (Classen, 2005).

The conserved cassette of PCP proteins controls a variety of seemingly different developmental processes, and no common cell biological mechanism has ever been proposed for their action. Polarizing membrane trafficking by recruiting Sec5 is a basic function that could be utilized in many different contexts, and it may help explain the requirement of PCP proteins in a divergent set of processes. Both rotation of photoreceptor clusters and convergent extension movements depend on the ability of cells to make and break intercellular contacts, as they do during hexagonal packing in the wing. Consistent with this, Silberblick (Wnt-11) acts through the PCP pathway and appears to affect endocytic trafficking of Cadherin during zebrafish gastrulation. Recruitment of exocyst components might also be a plausible mechanism to explain the ability of PCP proteins to bias Notch Delta signaling between R3 and R4 photoreceptors, since Delta delivery is dependent on the exocyst. In the future, identifying the chain of events that leads from PCP protein localization to exocyst recruitment may increase the understanding of these important processes (Classen, 2005).

EFFECTS OF MUTATION

The exocyst component Sec5 is required for membrane traffic and polarity in the Drosophila ovary

The directed traffic of membrane proteins to the cell surface is crucial for many developmental events. Sec5, a member of the exocyst complex, directs membrane traffic in the Drosophila oocyte. During oogenesis, Sec5 localization undergoes dynamic changes, correlating with the sites at which it is required for the traffic of membrane proteins. Germline clones of sec5 possess defects in membrane addition and the posterior positioning of the oocyte. Additionally, the impaired membrane trafficking of Gurken, the secreted ligand for the EGF receptor, and Yolkless, the vitellogenin receptor, results in defects in dorsal patterning and egg size. However, the cytoskeleton is correctly oriented. It is concluded that Sec5 is required for directed membrane traffic, and consequently for the establishment of polarity within the developing oocyte (Murthy, 2004).

Oogenesis in Drosophila requires the establishment and maintenance of cellular asymmetry within the developing oocyte, and provides a system in which to study directed membrane traffic. Within the egg chamber, which consists of 16 germline cells interconnected by ring canals and surrounded by somatic follicle cells, membrane ligands, adhesion proteins and transmembrane receptors are called upon to signal within particular domains of the cell surface. These signals allow the oocyte to migrate to the posterior end of the egg chamber, induce reorganization of the microtubule cytoskeleton, establish thereby asymmetries within the oocyte, and induce differentiations of the adjacent follicle cells. These events all rely on the directed trafficking of proteins, including E-cadherin, Gurken and the EGFR, to the plasma membrane, so as to establish polarity within the oocyte and its surrounding cells. In addition, the localization of some non-membrane determinants of polarity, such as Oskar, may be indirectly dependent on membrane trafficking. Sec5 is dynamically localized during oogenesis in a manner that corresponds with the changing needs of the egg chamber for directed membrane traffic. Furthermore, Sec5 is required both for growth of the germline cells and for membrane trafficking necessary for establishment of the anteroposterior axis and dorsoventral pattern (Murthy, 2004).

The FLP-dominant female sterile technique was used to generate maternal germlines homozygous for sec5^E10, a null allele that contains an early stop codon and is cell lethal when homozygous in the eye (Murthy, 2003). When homozygous in oocytes, sec5^E10 impairs development and no eggs are laid. These defects are entirely due to the loss of Sec5, because the viability and fertility of homozygous sec5^E10 females can be restored by a sec5 transgene. The presence in the ovaries of sequentially staged homozygous egg chambers made it possible to investigate the timing and manner of the developmental arrest (Murthy, 2004).

Wild-type development of the egg chamber has been subdivided into a series of 14 stages. During stages 2-6, after the egg chamber exits the germarium, the 15 nurse cells and one oocyte grow at similar rates. The oocyte occupies the posterior-most position among the group of sixteen cells. By stage 7, the microtubule cytoskeleton within the oocyte reorients and the nucleus moves to an anterior corner position, i.e. along the circular rim where the lateral and anterior surfaces of the oocyte meet, and thereby specifies this region as the dorsal side. Thereafter, the oocyte grows disproportionately to the nurse cells (Murthy, 2004).

sec5^E10 mutant germlines arrest at approximately stage 6 with striking structural changes. Normally, F-actin resides just beneath the membrane of each of the 16 cells and in the actin-rich ring canals that provide cytoplasmic bridges between the germ cells. In sec5^E10 germlines, actin, visualized with rhodamine-phalloidin, no longer separates the nuclei and the ring canals cluster together in the center of the egg chambers. Defects are apparent much earlier, however, with many egg chambers failing to exit the germarium and ring canals clumping together by stage 2. By stage 5 or 6, the follicle cell layer, which is heterozygous for the mutation, begins to disintegrate as well, a likely secondary consequence of the germline phenotype. The absence of phalloidin-staining suggested that the membranes that normally separate the cells of the cyst were absent. To test this hypothesis, egg chambers were labeled with a fluorescein-conjugated tomato lectin which binds to glycoproteins in membranes. In the control, the fluorescein-conjugated lectin labels the plasma membrane and therefore co-localizes with phalloidin. In sec5^E10 mutant egg chambers, either no plasma membrane was detected between nuclei, or a membrane was only observed between some nuclei and not others. Large dispersed pools of lectin-stained puncta resided within the cytoplasm of mutant, but not control, egg chambers. These puncta could either represent proteins that were not trafficked to the plasma membrane, or remnants of a disrupted plasma membrane. Egg chambers were also stained for Syntaxin, an additional marker of the plasma membrane, where it is required for vesicle fusion. In sec5^E10 germlines, Syntaxin-labeled membranes were frequently absent between nuclei and Syntaxin immunoreactivity was concentrated within the cytoplasm, surrounding the clumps of ring canals. These findings suggest that the primary defects in oogenesis are due to a block in membrane trafficking. Since the heterozygous follicle cells expand, the surface area of the mutant germline cells probably fails to increase at an adequate pace, causing the membranes dividing these cells to fall apart. Consequently, ring canals clump together, while membrane fragments and possibly unincorporated transport vesicles remain within the cytosol of the large multinucleate cells (Murthy, 2004).

Additionally, sec5^E10 mutant germlines display polarity defects. Normally, the oocyte occupies the posterior-most position, owing to E-cadherin-based differential adhesion between the oocyte and the posterior follicle cells. In sec5^E10 germlines, the oocyte, labeled with antibodies to Dynein Heavy Chain (Dhc), is often mispositioned. In addition, the polar cells, which are important for establishing initial polarity cues within the egg chamber, are often mis-positioned within the heterozygous follicle epithelium. In sec5^E10 germline clones, the development was observed of compound follicles with multiple germline cysts enclosed within a single follicle epithelium, a phenotype common to mutants that cause a loss of polar cell identity. These phenotypes indicate that sec5 is important for the initial establishment of anteroposterior polarity within the egg chamber (Murthy, 2004).

Because germline clones of sec5^E10 are lethal early in oogenesis, it was not possible to assess the subsequent roles of Sec5 during cytoskeletal rearrangement and establishment of the anteroposterior and dorsoventral axes. However, germline clones of the hypomorphic allele E13 (truncated at position 361) are not lethal (Murthy, 2003), and these females lay eggs (Murthy, 2004).

sec5^E13 phenotypes in the germline are diverse, with some egg chambers resembling those of the control. Others possess defects similar to those of sec5^E10: phalloidin-marked membranes are missing between cells, nurse cell nuclei appear to fall into the oocyte where the membrane between them has broken down and ring canals clump together. However, all eggs laid by sec5^E13 mothers show dorsoventral patterning defects, similar to those caused by hypomorphic mutations in the Gurken and EGF receptor signaling pathway. Dorsal appendages are either too closely spaced or are fused (Murthy, 2004).

To determine the role of Sec5 in the posterior follicle cells, mutant follicle cell clones were generated, marked by an absence of green fluorescent protein (GFP). It was not possible to generate clones of the cell-lethal E10 allele, but the hypomorphic E13 allele could be used. The follicle epithelium appeared disorganized in large clones of sec5^E13. However, even small homozygous clones induced a phenotype when they included posterior follicle cells; the oocyte no longer migrated to the posterior-most position among the germline cells, much as was seen when the oocyte was mutant for sec5. Thus, mutations in sec5 in either posterior follicle cells or the germline prevented development of the proper anteroposterior axis (Murthy, 2004).

The shift in Sec5 localization from the posterior of the oocyte to the anterior during stage 7 parallels a shift in the directed secretion of Gurken. Secreted at the posterior margin before stage 7, Gurken thereafter signals from an anterior corner of the oocyte to adjacent follicle cells. Those cells that receive the highest levels of Gurken repress the differentiation of the dorsal lateral follicle cells, thus creating a space between two lateral patches of cells that will form the appendages. Because females with sec5^E13 germlines lay eggs with fused dorsal appendages, a role for Sec5 in Gurken signaling was hypothesized (Murthy, 2004).

In early stages, both wild type and sec5^E13 germlines appropriately accumulated Gurken in the oocyte. After stage 7, however, Gurken is mislocalized in granules throughout the mutant oocytes. In stage 10 egg chambers, when Gurken is present at the dorsoanterior membrane of the oocyte in wild type, a substantial amount of Gurken is observed in granules scattered throughout the cytoplasm of sec5^E13 oocytes. Much Gurken remains in the vicinity of the nucleus, but very little is present in the membrane. The cytoplasmic Gurken in sec5^E13 oocytes is not coincident with a marker for the ER, Boca , indicating that the block in the directed trafficking of Gurken is at a later step of the pathway (Murthy, 2004).

Eggs derived from sec5^E13 homozygous germlines, are typically flaccid, small and, by Nomarski optics, devoid of yolk granules. Yolk proteins, however, are synthesized in fat bodies and follicle cells (which were not homozygous for the mutation) and are subsequently imported into the oocyte by endocytosis after binding to the vitellogenin receptor, Yolkless. A defect in the trafficking of Yolkless to the oocyte surface might therefore explain the decreased yolk content of the sec5 oocytes (Murthy, 2004).

In wild-type germlines, Yolkless is diffusely distributed until stage 8, whereupon, induced by an unknown signal, Yolkless translocates from the ooplasm to the cortex. At stage 7, Yolkless was detectable within both control and sec5^E13 oocytes. At stage 8 in the mutant, however, the majority of the receptor does not go to the surface, and remains cytoplasmic through stage 10. The mistrafficking of Yolkless, like the general disruption of membranes in the sec5 null allele, indicates that Sec5 is not only required for Gurken localization, but rather is of general significance for the membrane trafficking of many germline proteins (Murthy, 2004).

Although the Gurken and Yolkless mislocalizations are probably due to a defect in membrane trafficking, these phenotypes might be secondary to a defect in the concurrent reorganization of the oocyte, which includes the reorientation of the microtubule cytoskeleton, the movement of the oocyte nucleus to the anterior cortex of the oocyte, and the localization of Gurken mRNA and protein near the nucleus (Murthy, 2004).

To investigate this possibility, the localization was examined of several proteins restricted to the posterior pole of the oocyte: Oskar, Par-1 and a kinesin-ß-gal fusion. In both control and sec5^E13 germlines, all three proteins accumulate properly at the posterior pole in stage 8-10 oocytes. Dynein Heavy Chain (Dhc), also localizes to the posterior end of late stage oocytes. This marker also was normal in the mutants, accumulating first in the oocytes of early stage egg chambers and after stage 8 at the posterior end of the oocyte (Murthy, 2004).

To examine directly the polarity of the microtubules, sec5^E13 oocytes were imaged that expressed in the germline a marker for the minus ends of microtubules, a fusion of the head domain of Nod (no-distributive disjunction) to GFP. At stages 7 and 10, Nod-GFP is concentrated at the anterior end of the oocyte in both wild type and mutant. The correct positioning of the minus ends in sec5^E13 was also demonstrated with FITC-conjugated alpha-tubulin. Thus, the defective trafficking of Gurken and Yolkless cannot be secondary to microtubule defects (Murthy, 2004).

It was observed, however, that the overexpression of Nod-GFP in sec5^E13 oocytes enhances the phenotype of the sec5^E13 allele alone: the oocyte nucleus is often displaced from the cortex, membranes between cells are absent, the development of the follicle epithelium is disturbed and no eggs of this genotype are laid. These defects are never observed in Nod-GFP expressing lines that are not mutant for sec5. It is possible that the overexpression of the Nod motor domain impairs microtubule-based transport, thereby enhancing the sec5^E13 phenotype by further slowing the delivery of membrane to the cell surface (Murthy, 2004).

Examining sec5^E13 egg chambers, it was noted that the oocyte nucleus was sometimes mislocalized. The nucleus invariably moved to the anterior, as in wild type, but was not closely associated with the dorsoanterior plasma membrane. A three dimensional composite image was assembled from individual z sections of stage 10 egg chambers and rotated to reveal the relationship of the nucleus to the plasma membrane. This analysis confirmed that the nucleus was not always adjacent to the dorsal membrane: eight out of 39 (21%) sec5^E13 oocytes had a mispositioned nucleus, but none of 40 wild-type oocytes (Murthy, 2004).

Bicaudal-D (Bic-D) is a cytosolic protein that interacts with the dynein-dynactin complex, and participates in the cortical anchoring of the nucleus. Bic-D localized normally in sec5^E13 oocytes throughout oogenesis. In early stages, Bic-D is at the microtubule minus ends at the oocyte posterior, and by stage 6 relocalizes to the anterior rim, preceding the arrival of the nucleus. Subsequently, Bic-D concentrates above the nucleus. Even when the oocyte nucleus is displaced from the dorsal cortex, Bic-D remaines near the nucleus, indicating that its nuclear association is not sufficient to attach the nucleus to the dorsal cortex. Because no alteration of the microtubule cytoskeleton was in sec5^E13 germline clones, nor gross mislocalization of Bic-D, the lack of a tight association of the nucleus with the membrane must have other causes (Murthy, 2004).

The trafficking of the Gurken protein provides at present the best example of an identified membrane protein whose selective, Sec5-dependent localization is crucial to proper development. The final deposition of Gurken is likely to arise from a combination of mechanisms, including the transport of the oocyte nucleus to an anterior corner, the nearby localization of Gurken mRNA, the microtubule-dependent transport of Gurken protein to the cortex, and the insertion of both pre-existing and newly synthesized Gurken into the plasma membrane by vesicle fusion. The presence of displaced Gurken protein in the posterior regions of the mutant ooplasm may be an indirect result of blocked membrane fusion after which Gurken-containing vesicles may drift away from their normal target. Gurken trafficking, however, also indicates that Sec5 and the exocyst cannot be the only cues that direct vesicle fusion: Sec5 localizes along the entire anterior lateral rim of the oocyte, but Gurken is inserted only at that section adjacent to the nucleus. Furthermore, when the nucleus and Gurken transcripts are mislocalized by cytoskeletal changes, some Gurken signaling occurs ectopically, near the misplaced nucleus, and away from the major concentration of Sec5. Thus, the localization of Sec5 should be viewed as one of several layers of likely mechanisms for directing membrane proteins (Murthy, 2004).

In mediating the traffic of multiple membrane proteins, including both Gurken and Yolkless, Sec5 is clearly in a distinct category from Cornichon and Boca, proteins that act in the ER. These proteins are needed for the correct transport of individual proteins and appear to act at earlier trafficking steps. Gurken is retained inside the cell in cornichon mutants, although vitellogenesis proceeds normally. Boca, however, is required for the trafficking of Yolkless and other LDL receptor family proteins to the membrane, but does not influence Gurken traffic. These highly specific deficits, which are likely to occur upon exiting from the ER, are distinct from the more general disruption of traffic in sec5 mutants (Murthy, 2004 and references therein).

Many forms of membrane traffic to the cell surface now appear to depend on the exocyst. In multicellular organisms, these include vesicles derived from the trans-Golgi network (TGN) carrying newly synthesized proteins or mediating neurite outgrowth. However, not all forms of exocytosis depend on the exocyst. The fusion of synaptic vesicles at nerve terminals persists in sec5 mutants in which other trafficking events are blocked (Murthy, 2003) and apical protein delivery in MDCK cells is resistant to a block by antibodies to exocyst components. The essential differences between exocyst dependent and independent exocytotic events remain unclear (Murthy, 2004 and references therein).

In addition to an established role for the exocyst in targeting or fusion at the plasma membrane (Grote, 2000; Guo, 1999b), there is also evidence to suggest a role at earlier stages of protein traffic. The exocyst may associate with microtubules and a septin protein, Nedd5, and thereby promote transport of post-Golgi vesicles to target membranes (Vega, 2001 and 2003). Members of the complex have been observed on perinuclear compartments in the cell (Shin, 2000; Vega, 2001), and Sec6 and Sec8 are recruited to budding vesicles in the TGN, where antibodies against these components interfere with the ability of cargo to exit the Golgi (Yeaman, 2001). Recently, the exocyst component Sec10 has been found associated with Sec61ß, a component of the ER translocon complex (Lipschutz, 2003). Genetic interactions of sec61beta with members of the exocyst complex have also been found (Lipschutz, 2003; Toikkanen, 2003). Indeed, mutations in Drosophila sec61ß (Valcarcel, 1999) can cause abnormal dorsal appendages very similar to those observed here for sec5, probably owing to defects in Gurken translocation (Murthy, 2004).

No exocyst functions have been detected in Drosophila at stages before exocytosis. Sec5 is concentrated only at the plasma membrane. Microtubule polarity and the polarized localization of cytosolic components are unaffected by the mutations. Perinuclear Gurken, a pool that is likely to represent protein in the ER and Golgi, is present in both wild-type and mutant stage 10 oocytes, but the mislocalized cytoplasmic granules of Gurken that characterize sec5^E13 oocytes do not colocalize with the ER marker Boca. The early lethality of E10 clones, however, requires that the analysis of Gurken and Yolkless trafficking be performed on the hypomorphic allele, E13. It is therefore possible that residual Sec5 function is sufficient for transport through the ER but insufficient at the plasma membrane (Murthy, 2004).

Although some aspects of the sec5 phenotype can be ascribed to defects in the transport of particular membrane proteins, such as Gurken and Yolkless, others cannot, and these phenotypes may imply the existence of as yet unidentified oocyte proteins. An example is the altered location of the nucleus in late stage oocytes: whereas control nuclei were inevitably tightly associated with the anterior membrane, in sec5^E13 germline clones, the nucleus is frequently displaced. Members of the dynein-dynactin complex are probably important for the association. However, Bic-D, a component of the dynein-dynactin complex, is anteriorly transported and properly localized near the nucleus in sec5^E13 clones. Therefore, the existence is hypothesized of an as yet unidentified membrane protein that tethers the oocyte nucleus to the cortex via the dynein-dynactin complex. If the directed membrane traffic of this unidentified protein is compromised in the sec5 mutants, the displacement of the oocyte nucleus could be explained in a manner consistent with the other actions of Sec5 in the oocyte (Murthy, 2004).

Owing to defects in directed traffic to the plasma membrane, aspects of the anteroposterior axis and dorsoventral axis develop incorrectly in sec5 mutant germlines. The requirement for Sec5 in directed membrane traffic is consistent with previous studies in cells that use a polarized secretory apparatus for cell growth and the transport of certain cargoes, such as growing neurites, MDCK cells and yeast (Murthy, 2004 and references therein).

The spatial correlation of membrane traffic with the position of the exocyst raises the crucial question of how the exocyst acquires its localization. This microtubule- and actin-independent mechanism remains elusive. The membrane receptor for the exocyst is not currently known, but in yeast Sec3p may be the exocyst component closest to the membrane and its localization may be controlled by Rho1p and Cdc42p. In the Drosophila oocyte, the localization mechanism must undergo developmental regulation to account for the shift in localization observed between stages 5 and 10. The mechanism that targets the exocyst to the membrane and regulates the changes in its localization is likely to be crucial to patterning and polarization in the germline (Murthy, 2004).

Mutations in Sec5 disrupt neuronal membrane traffic, but neurotransmitter release persists

To understand the role of the exocyst complex in synapse formation, the Drosophila genome database was searched for homologs to members of the rat exocyst complex. The genome contained only one isoform of each member of the exocyst complex. By in situ hybridization to polytene chromosomes, preexisting chromosomal deficiencies that removed sec5 were identified. Df(2L)tim⁰² and Df(2L)P22 remove the sec5 locus, which is located at 23F. Because Sec5 is a central component of the yeast exocyst complex (Grote, 2000; Guo, 1999a; Jantti, 1999), efforts were concentrated on sec5 (Murthy, 2003).

An EMS mutagenesis screen for lethals uncovered by Df(2L)P22 generated candidate sec5 mutations. The sec5 gene resides in an 11 kb Kpn fragment of genomic DNA from 23F that had been previously introduced into flies as a transgene (P[Kpn]) and that also contained the genes msl-2 and CG3246 (Bashaw, 1995). This transgene was used to identify candidate sec5 alleles among the lethal mutations isolated from the screen. In addition to msl-2, two complementation groups were rescued with the P[Kpn] transgene. The open reading frame of sec5 was sequenced in all alleles from both of these complementation groups and nonsense mutations in sec5 were found in alleles E10 (R31 to STOP) and E13 (Q361 to STOP) from one complementation group, and no mutations were found in the coding sequence of sec5 in alleles of the other complementation group (Murthy, 2003).

Allele E10, which contains an early stop codon in sec5, meets the genetic criterion for a null allele-either in combination with Df(2L)tim⁰² or homozygous, it has the same lethal phase. sec5^E10 mutants die within 96 hr of egg laying, as morphologically first instar larvae. These larvae hatch at the same time as their heterozygous siblings, at approximately 24 hr after egg laying (AEL), and their growth between 24 and 48 hr is comparable to that of wild-type larvae. However, sec5^E10 mutants do not grow after 48 hr AEL and remain late first instar larvae, whereas by 96 hr AEL, wild-type larvae progress to the early third instar stage (Murthy, 2003).

Eyes composed exclusively of cells homozygous for a mutation can be generated in an otherwise heterozygous animal by means of mitotic recombination that is induced during development of the eye disc. When this method was applied to either sec5^E10 or sec5^E13, the eye was completely ablated. The presence of the P[Kpn] transgene restores eye development in these flies, demonstrating that the cell lethality is due to the mutations in sec5 and not to second site mutations on the chromosome. This apparent requirement for sec5 in cell viability raises the question of why the homozygous null embryos develop and survive for up to 96 hr AEL. This survival can be attributed to maternally deposited sec5 mRNA and protein. sec5 transcripts are present at rather constant levels throughout development, including the first 2 hr of embryogenesis, before zygotic transcription begins. This was confirmed by analysis of Sec5 protein (Murthy, 2003).

Maternal contribution of Sec5 is essential for appropriate embryonic development-when attempts were made to remove it by generating maternal germlines homozygous for either sec5^E10 or sec5^E13, no fertilized eggs were produced. However, in sec5^E10 homozygotes, the maternal contribution is sufficient to allow the nervous system to form. The role of the exocyst complex in neuronal vesicle trafficking could thus be studied in a nervous system that had subsequently run out of Sec5 (Murthy, 2003).

The neuromuscular junction (NMJ) of Drosophila consists of a string of boutons from a small number of axon branches that form a pattern characteristic for each muscle in each abdominal segment. In wild-type, the motor neuron first contacts the muscle at about 14 hr AEL, and these contacts mature into synapses by 16-17 hr AEL. After hatching at 24 hr AEL, both the muscle and the motor neuron increase in size. In wild-type, the size of muscles increases 10-fold between first and third instar development, with a concurrent increase in synaptic bouton number. In sec5^E10 homozygous embryos, neuromuscular junctions develop with an apparently normal morphology, and the NMJs of sec5^E10 mutants are size-matched to wild-type until 48 hr AEL. However, sec5^E10 mutant larvae do not grow substantially between 48 and 96 hr AEL. Because maternal protein decreases dramatically during larval development in the mutant, aberrations in the postembryonic maturation of the NMJ were sought (Murthy, 2003).

The size of muscle 6 was first measured for both mutant and control. While the size of muscle 6 increased 6-fold between 48 and 96 hr AEL in wild-type, there was no growth of muscle 6 in the sec5^E10 mutant during this time. The number of synaptic boutons at the NMJ of muscles 6 and 7 was then determined with an antibody to the synaptic vesicle marker Cysteine String Protein (CSP). The bouton number at the wild-type NMJ increased 2.5-fold between 48 and 96 hr AEL, whereas there was no change in the mutant. By immunocytochemistry, diffuse Sec5 staining was observed in segmental muscles 6 and 7 and along the nerves that innervate these fibers. The presence of Sec5 in the muscles, however, obscured the detection of Sec5 in the nerve terminals of the NMJ (Murthy, 2003).

The observed defect in muscle growth and bouton addition may reflect the inability of the muscle and nerve cells to insert new membrane. However, assayed in vivo, it was also possible that the failure of these cells to grow might be secondary to malnutrition of the animals, the absence of a secreted growth signal, or a similar confounding phenotype. Moreover, because the increase in bouton number is thought to be tightly coupled to muscle size, it was not possible to determine in these experiments whether the inability of the synapse to grow was secondary to the failure of the muscle cells to expand. To address more directly the potential requirement of the exocyst in the neuron, neurite extension was studied in vitro (Murthy, 2003).

The ventral nerve cord and some adhering tissues from either control larvae at 48 hr AEL or sec5^E10 mutant larvae at 48 or 72 hr AEL were dissociated and placed in culture for 1 day. To distinguish neurons in these cultures from other contaminating cell types, both control and mutant cultures were made from Drosophila that expressed Tau-GFP under the control of the neuron-specific elav promoter. GFP-positive cells were scored for the presence of neurites and the lengths of any processes were measured. The viability of neurons in culture was judged by the integrity of their nuclei, stained with Hoechst 33342. Neurons that were not healthy were excluded from further analysis. The survival of cells in culture was also judged by staining with ethidium homodimer-1, which only enters cells with damaged membranes and undergoes a 40-fold enhancement of fluorescence upon binding to DNA. The presence or absence of Sec5 did not alter the overall viability of the 1 day cultures, with comparable numbers of cells from either genotype surviving. In control cultures from +/+ larvae at 48 hr AEL, 69% of the neurons had extended neurites. In contrast, neurites were scarce in sec5^E10 cultures: only 4% of neurons cultured at 48 hr AEL extended neurites, and in cultures made at 72 hr AEL, less than 1% of healthy neurons possessed neurites. The few neurites present in sec5 mutant cultures are significantly smaller than the neurites observed in control cultures. It is concluded that Sec5 is required for neurite outgrowth (Murthy, 2003).

In yeast, exocyst proteins have been shown to mark the site of membrane addition (Finger, 1998), and so the immunolocalization of Sec5 was undertaken in these cultures. Consistent with the findings of Hazuka (2000) in mammalian cultured neurons, Drosophila Sec5 immunoreactivity is present in the cell bodies of neurons, in puncta along developing neurites, and at the tips of neurites. Membrane addition in growing neurites occurs primarily at growth cones, and therefore the presence of Sec5 at neurite tips may reflect sites of membrane addition. The widespread distribution of Sec5 in the cell, however, may reflect a broader role in vesicle traffic (Murthy, 2003).

To look directly for defects in membrane trafficking, an assay was developed that would allow for a determination of the efficacy with which newly synthesized protein could be added to the neuronal surface in larvae in which little or no Sec5 remained. To this end, the Geneswitch system was used to activate a reporter gene at 48 or 72 hr AEL. The neuron-specific elav promoter was used to express the Geneswitch product, an inactivated form of Gal4. Upon feeding larvae RU486, the Geneswitch is activated and can bind to an upstream activating sequence, or UAS, which results in transcriptional activation of the transgene. For this assay, a transmembrane protein, murine CD8 fused to GFP at its cytoplasmic end, served as the reporter transgene. An anti-mCD8 antibody was used that recognizes an extracellular epitope. In the absence of Triton X-100, this antibody will recognize exclusively the subset of the CD8 reporter gene that has been expressed on the cell surface, whereas the GFP fluorescence will represent both surface and internal pools of the protein. Larvae were fed RU486 for 12 hr, which successfully turned on the transgene. When dissected and immunolabeled in the presence of Triton X-100, the GFP signal colocalized with staining for mCD8 throughout the cell. As expected, without detergent, mCD8 immunolabeling was restricted to the cell surface (Murthy, 2003).

sec5^E10 mutant larvae at 72 hr AEL were fed RU486 and compared with either similarly sized control larvae fed at 48 hr AEL or similarly aged controls fed at 72 hr AEL. Larvae were dissected 12 hr after introduction to the drug and stained in the absence of detergent. Both control and mutant larvae showed strong expression of the transgene in the central nervous system, indicating that despite a lack of Sec5, protein synthesis was not impaired. Attention was focused on lateral bipolar dendrite (bd) sensory neurons, because their cell bodies and axons are easily visualized due to their isolation in the periphery and because they lie close to the surface of the dissected larva. In the absence of Triton, the anti-mCD8 antibody is able to access the neuron and label its surface reliably. No difference was observed between mutant and control in the expression of the reporter gene as determined by GFP fluorescence in the somata of these cells (Murthy, 2003).

To examine the transport of the mCD8 reporter gene to the surface of the bd neurons, antibody staining was performed in the absence of Triton and examined in confocal sections. Total anti-mCD8 labeling of the surface of the cell body was reduced in the mutant to 13% of control. Animals were costained with an anti-HRP antibody that labels a surface antigen in all neurons so that the mCD8 signal could be normalized to the surface area of the cell. The immunostaining for the HRP-like antigen appeared to decrease in the mutant, and this could result from thinner axons or from a defect in the addition of the epitope to the surface. Normalization of CD8 surface staining to this parameter was therefore conservative and may have underestimated the extent to which insertion of the CD8 reporter was impaired in the mutant. The loss of Sec5 reduced the surface mCD8 immunoreactivity to 17% of control when normalized to the HRP signal. Therefore, between 72 and 84 hr AEL in sec5^E10 mutants, less of the newly synthesized mCD8 is inserted at the membrane, demonstrating a defect in this membrane trafficking pathway. The difference between mutant and control cannot be attributed to the different ages of the animals (72 hr AEL for sec5^E10 versus 48 hr AEL for +/+) because control larvae fed RU486 at 72 hr AEL also efficiently transported the mCD8 reporter to the plasma membrane (Murthy, 2003).

Membrane traffic was also examined in the axons of the bd neurons. In the axons, the GFP signal was equivalent between mutant and control, demonstrating that Sec5 was not required for axonal transport of the CD8-containing vesicles. Similar to the cell soma, however, the surface mCD8 immunoreactivity was reduced to 13% of control (Murthy, 2003).

In addition, the synaptic boutons of the NMJ were examined to analyze integral membrane protein insertion at the same nerve endings that had failed to increase with age and at which electrophysiological studies were conducted. In these terminals, there was also a dramatic reduction in surface expression of mCD8. Thus, in these terminals, transport vesicles carrying the newly synthesized protein do not appear to fuse with the plasma membrane. Interestingly, a decrease in GFP signal relative to control boutons was also observed, although the axons entering the muscle had abundant GFP. It is likely, therefore, that transport vesicles that fail to fuse with the membrane are not retained in the terminal but may return to the axon for retrograde transport (Murthy, 2003).

The axonal transport of post-Golgi vesicles was examined further with a second reporter gene, a Synaptotagmin (Syt)-GFP fusion. In the absence of Sec5, vesicles containing newly synthesized Syt-GFP are present along the length of the axon but are rarely seen in the synaptic boutons of the NMJ. However, synaptic vesicles, visualized with antibodies either to Synaptotagmin or to CSP, continue to be concentrated at the synapse. It is concluded that the Syt-GFP-labeled vesicles fail to fuse with the membrane and are not retained in the mutant terminals (Murthy, 2003).

Does the fusion of synaptic vesicles at the terminal depend on Sec5? The strength of synaptic transmission at the NMJ was examined when the maternal contribution was no longer adequate to support other forms of membrane traffic. Synaptic transmission persisted during this period despite the decline in Sec5 protein. At 96 hr AEL, although the size of the NMJ had not changed and Sec5 protein represented 3% of control, the evoked response was actually increased 2.5-fold over its amplitude at 48 hr (Murthy, 2003).

The amplitude and frequency of spontaneous synaptic events (mEJCs or minis) was examined. The amplitude of minis was unchanged in the mutant, indicating that the number of postsynaptic receptors and the amount of transmitter per vesicle was not significantly altered in the mutant between 48 and 96 hr AEL (Murthy, 2003).

From the size of the nerve-evoked responses and the size of the individual minis, the quantal content, the number of vesicles released per stimulus (see Experimental Procedures), could be calculated. Consistent with the robust evoked responses in the mutants, the quantal content of the homozygous null mutants was also observed to increase even as the maternally derived protein declined. To take into account the difference in size between the neuromuscular junctions of different stages and genotypes, quantal content was normalized to bouton number. Despite the decline in Sec5 protein, sec5 mutants between 48 and 96 hr AEL secrete equivalent amounts of neurotransmitter per bouton to wild-type animals throughout larval development. Finally, it was found that the frequency of minis is somewhat increased in the mutants between 48 and 96 hr AEL, although not significantly. The persistence of minis indicates that synaptic vesicle fusions that are not driven by action potentials can also persist at rates appropriate to the anatomical size of the synapse. In conclusion, these data demonstrate that sec5 is not required for the exocytosis of synaptic vesicles (Murthy, 2003).

Sec5, Sec6 and Sec8 act as a complex, each member dependent on the others for proper localization and function

To allow a detailed analysis of exocyst function in multicellular organisms, sec6 mutants were generated in Drosophila. These mutations were used to compare the phenotypes of sec6 and sec5 in the ovary and nervous system, and they were found to be similar. Sec5 is mislocalized in sec6 mutants. Additionally, an epitope-tagged Sec8 was generated that is localized with Sec5 on oocyte membranes and is mislocalized in sec5 and sec6 germ-line clones. This construct further revealed a genetic interaction of sec8 and sec5. These data, taken together, provide new information about the organization of the exocyst complex and suggest that Sec5, Sec6 and Sec8 act as a complex, each member dependent on the others for proper localization and function (Murthy, 2005).

The distribution of Sec5 has been examined most closely in the ovary. In this tissue, it was present on all membranes early in the development of the egg chamber. At late stages, however, Sec5 acquires a characteristic distribution not reported for any other cellular component -- a progressive enrichment at the anterior end of the lateral oocyte membranes. HA-Sec8 has now been found to be similarly concentrated in this area, suggesting that several (and perhaps all) exocyst components will be similarly localized (Murthy, 2005).

Mutations in one complex member appear to disrupt the localization of others. Thus, in sec5^E13 homozygous oocytes, HA-Sec8 is no longer membrane bound or concentrated at the anterior sites. Instead, it appears to fill the cytoplasm diffusely. Similarly, Sec5 is mislocalized within the nervous system of sec6 mutant larvae and Sec5 and HA-Sec8 are both mislocalized within germ lines homozygous for sec6. The mislocalization of Sec5 and HA-Sec8 in sec6 germ lines, however, is not identical to the mislocalization of HA-sec8 in sec5 germ lines. Whereas the latter involves a diffuse filling of the cytoplasm with immunoreactivity, the mislocalized Sec5 and HA-Sec8 remain punctate within the sec6 egg chambers. Because these puncta resemble syntaxin and lectin-staining in sec5 germ lines, it seems likely that they represent fragments of membrane or transport vesicles that have not fused with the plasma membrane. The difference in these two phenotypes might arise from any of several causes, including the perdurance of some Sec6 in the sec6^Ex15 mutant germ lines. It is tempting to speculate, however, that the difference reflects the organization of proteins within the complex. Sec3p has been shown in yeast to bind to the plasma membrane at the bud tip even when other complex members are absent. This has been interpreted as indicating that Sec3p binds directly to a membrane protein and that the localization of other complex members is dependent on Sec3p. Sec5p is thought to bind directly to Sec3p and so it is plausible that, in the present study, Sec5 remains membrane bound via its direct interaction with Sec3 even in the absence of Sec6. Sec8, however, is not thought to interact directly with Sec3. Because Sec8 appears to remain membrane-associated in sec6 but not sec5 mutants, it is hypothesized that a partial complex consisting of Sec3, Sec5 and Sec8 remains on the membrane even in the absence of Sec6. The disposition of the remaining complex members in the sec5 and sec6 mutants must remain speculative until suitable reagents have been obtained for their localization (Murthy, 2005).

The interdependence of the complex members is also evident in the genetic interaction of Sec8 and Sec5: although germ-line expression of HA-tagged Sec8 has no phenotype of its own, it enhances the germ-line phenotype of sec5^E13, making this partial loss-of-function allele more similar to the null allele. This observation requires that the epitope-tagged transgene be used with caution, because its expression might interfere with exocyst function owing either to an influence of the epitope tag or to unphysiological expression levels. Indeed, phenotypes have been associated with the overexpression of Sec10, another complex member (Murthy, 2005).

The phenotypes of sec6 and sec5 mutants can be compared in several regards. Like sec5, sec6 caused lethality at approximately 96 hours AEL and these larvae were stunted in their growth and do not progress beyond the first instar. In an assay of membrane-protein transport to the cell surface of identified neurons, trafficking defects were found for sec6 that were akin to those of sec5. In the germ line, it was found that membranes between cells disintegrate in sec6 clones, a phenotype observed for the null allele of sec5. For sec5, it is hypothesized that, as the cells of the germ line grow and expand, membrane addition cannot keep pace, and that membranes between nurse cells and the oocyte consequently fall apart. A similar explanation is likely for sec6. The mispositioning of the oocyte within the sec6 germ line was also observed. This phenotype occurs when either the germ line or the posterior follicles are mutant for sec5. Because the positioning of the oocyte is dependent on E-cadherin and cell-cell signaling between the oocyte and follicle cells, it is likely that this phenotype arises from a defect in the expression of E-cadherin or other signaling molecules on the oocyte surface. In fact, E-cadherin and Nectin 2a have been recently shown to be binding partners for the exocyst complex in MDCK cells (Murthy, 2005).

Although the similarities of their phenotypes suggest that Sec5 and Sec6 share functions, some differences are observed in the mutant phenotypes. sec6^Ex15 larvae are smaller than sec5^E10 larvae but germ-line clones of sec5^E10 have a more severe phenotype in the ovary, arresting earlier and with fewer remaining membranes. The most intriguing difference arose in the mCD8-GFP expression assay: whereas sec5^E10 larvae are capable of synthesizing the protein but not of expressing it at the cell surface, sec6^Ex15 larvae express only low levels of the protein, which also appear to be blocked in their transport to the surface. Finally, whereas HA-Sec8 protein is mislocalized in both sec5 and sec6 germ-line clones, the patterns of mislocalized protein are distinct. The differences in the mutant phenotypes might arise from minor factors such as the degree of perdurance of protein in the homozygous germ-line clones or the amount or stability of maternal protein deposited in the egg. However, they might also represent legitimate functional distinctions. The most pronounced difference, the different levels of expression of the mCD8-GFP reporter protein, might reflect the fact that Sec6 is required at an earlier step in the synthesis of membrane proteins, in addition to its requirement (along with Sec5) for insertion at the plasma membrane. Such a role would be consistent with findings that Sec6 and Sec8 have been observed in the TGN, that Sec8 and Sec10 associate with proteins at the TGN and ER, and that overexpression of Sec10 alters membrane-protein synthesis. The general similarities between and severity of the sec6 and sec5 phenotypes also do not exclude the possibility that other components will have more restricted roles, particularly given that several GTPases have emerged as binding partners of particular members of the complex and might be either effectors or regulators of those components (Murthy, 2005).

In contrast to the cell lethality of the sec5 and sec6 phenotypes, a Sec10 RNA-interference construct in Drosophila has very little effect in most tissues, possibly affecting only the secretions of the ring gland cells. However, because no antibody is available for Drosophila Sec10 and because maternally contributed protein would be unaffected by this construct, the RNA interference might have been ineffective at reducing endogenous Sec10 levels. In light of the broad phenotypes of dominant negative and overexpressed Sec10 in other cell types, this is a likely explanation of the discrepancy (Murthy, 2005).

In summary, the similarity of localization of Sec5 and HA-Sec8, the interdependency of the complex members for proper localization in this study, the genetic interaction between HA-Sec8 and sec5, and the general similarity of the sec5 and sec6 phenotypes all suggest that Sec5, Sec6 and Sec8 associate as a complex in Drosophila, acting in concert, and that each is crucial for the function of the complex at the membrane. It will be important to examine the localization and phenotypes of the other complex members to determine whether all the complex members do indeed function primarily as part of the intact exocyst. Furthermore, the mutations in sec5 and sec6 should provide a useful genetic background for structure function studies with which to test the significance of their individual binding partners and regulators (Murthy, 2005).

Rab6 mediates membrane organization and determinant localization during Drosophila oogenesis

The Drosophila body axes are defined by the precise localization and the restriction of molecular determinants in the oocyte. Polarization of the oocyte during oogenesis is vital for this process. The directed traffic of membranes and proteins is a crucial component of polarity establishment in various cell types and organisms. This study investigated the role of the small GTPase Rab6 in the organization of the egg chamber and in asymmetric determinant localization during oogenesis. Exocytosis is affected in rab6-null egg chambers, which display a loss of nurse cell plasma membranes. Rab6 is also required for the polarization of the oocyte microtubule cytoskeleton and for the posterior localization of oskar mRNA. In vivo, Rab6 is found in a complex with Bicaudal-D, and Rab6 and Bicaudal-D cooperate in oskar mRNA localization. Thus, during Drosophila oogenesis, Rab6-dependent membrane trafficking is doubly required; first, for the general organization and growth of the egg chamber, and second, more specifically, for the polarization of the microtubule cytoskeleton and localization of oskar mRNA. These findings highlight the central role of vesicular trafficking in the establishment of polarity and in determinant localization in Drosophila (Coutelis, 2007).

During polarized exocytosis, secretory vesicles emerging from the TGN are targeted via molecular motors and cytoskeletal tracks to the plasma membrane, where they are tethered. Subsequently, their fusion with the plasma membrane permits the secretion of the vesicle contents, as well as the incorporation of vesicular lipids and proteins into the plasma membrane, allowing membrane growth and the establishment of specific domains. The exocyst complex plays a crucial role in the incorporation of particular membranes and membrane proteins at specific sites or in active domains of the plasma membrane. Consistent with this, Drosophila sec5 mutant egg chambers display mislocalization of other exocyst components, cytoplasmic clusters of actin and a loss of plasma membranes. Thus, Sec5 protein is at the core of the exocyst complex in Drosophila, as is the case in yeast and in mammals (Coutelis, 2007).

Both sec5 null (sec5^E10) and strongly affected rab6^D23D egg chambers display actin and general organization defects, and arrest development during early oogenesis. Similarly, sec5 hypomorphic (sec5^E13) and rab6^D23D egg chambers that develop past stage 7 display phenotypes ranging from wild type to a loss of nurse cell cortical actin and the concomitant presence of ring canal clusters in the nurse cell cytoplasm. The striking parallel between the rab6 and sec5 phenotypes, together with the finding that a loss of Rab6 affects Sec5 localization, suggests that the varying degrees of membrane loss observed in rab6^D23D egg chambers reflects the relative reduction of exocyst-complex function in the egg chamber. Thus, during Drosophila oogenesis, Rab6 promotes Sec5 localization and therefore appears to be important for exocyst-complex organization and function. However, consequent to loss of rab6 function, a striking difference was observed between nurse cells and oocyte in the severity of plasma membrane collapse and Sec5 mislocalization. It is hypothesized that the oocyte acts as a major source of membrane in rab6^D23D egg chambers and/or that multiple exocytic pathways cooperate within the germline cyst to promote cyst development (Coutelis, 2007).

Differences in membrane content between the oocyte and the nurse cells, as well as between the individual nurse cells, are observed as early as the germarium stage in wild-type egg chambers. The fusome, a membranous Spectrin-rich structure derived from the spectrosome, which itself is a precursor organelle present in the germline stem cells, grows asymmetrically through the ring canals during the divisions of the germline cyst, linking each cystocyte. It is thought that the oocyte is the four-ring-canal cell that retains the greater part of fusome during the first division. Furthermore, a Drosophila Balbiani body has recently been discovered, which, together with the fusome, organizes the specific enrichment of organelles in the oocyte throughout oogenesis. It is therefore possible that, in rab6 clones, in which the fusome appears normal, such a mechanism of enrichment of organelles in the oocyte concomitantly ensures that the concentration in the oocyte of any perduring Rab6 protein, thus privileging the growth of the plasma membrane of the oocyte over that of the nurse cells. Supporting this notion is the observation that GFP-tagged Rab6 expressed in the germline is enriched in the oocyte from the early stages of oogenesis (germarium region 2) onwards. Together, the combined actions of a residual Rab6-dependent and of additional Rab6-independent pathways might also permit most rab6^D23D oocytes to maintain sufficient vesicular trafficking to develop past stage 7 (Coutelis, 2007).

The stereotypic organization of affected rab6^D23D egg chambers at mid-oogenesis is striking. The oocyte is connected to open syncytia via its four ring canals, suggesting that the membranes linking nurse cells and oocyte are the most resistant. Furthermore, the growth of the remaining membranes indicates that additional vesicular material is delivered and incorporated into these plasma membranes. This suggests that, in these rab6^D23D egg chambers, sustained vesicle trafficking in the oocyte causes new membrane addition to the oocyte plasma membrane. It is hypothesized that, due to the continuity of the plasma membrane defining the cyst, the oocyte acts as a source of membrane that spreads by lateral diffusion throughout the plasma membrane of the cyst, allowing its growth (Coutelis, 2007).

It appears that Rab6-independent exocytic pathways also contribute to the delivery of vesicular material to the plasma membrane in the Drosophila egg chamber. Indeed, Syx1A is detected on the remaining plasma membrane of both rab6-null and sec5 egg chambers, supporting the existence of a Rab6- and Sec5-independent exocytic pathway mediating protein export. This selective loss of Sec5 from nurse cell membranes in rab6 open syncytia, together with the known functions of the exocyst, suggest a simple explanation for the defects caused by a lack of Rab6 function in oogenesis. It is hypothesized that Rab6-dependent and -independent pathways might differ qualitatively in the proteins whose traffic they mediate, or quantitatively in their relative contributions to the delivery of the same cargo between nurse cells and oocyte. These differences may account for the observed differential requirement for Rab6 in the localization of Sec5 in nurse cell, versus oocyte, plasma membranes (Coutelis, 2007).

Our analysis has revealed two separate functions of Rab6: one is a general role in the organization and growth of the egg chamber, and the other is its specialized role in MT cytoskeleton polarization and oskar mRNA localization. This second function appears specific to Rab6 because, in sec5 mutant egg chambers, Staufen localization is normal and the MT cytoskeleton is correctly organized. Only oskar mRNA, and not Oskar protein, is ectopically detected in rab6^D23D egg chambers. This suggests an impairment of oskar mRNA localization, rather than a defect in its anchoring, in which case Oskar protein would be detected with the detached RNA. Defects in oskar mRNA localization, which relies on MT polarity, could be due to a failure in the focusing of the MT cytoskeleton that is observed in rab6 egg chambers (Coutelis, 2007).

In Drosophila and mammalian cells, BicD is known to regulate MT organization. At mid-oogenesis, Rab6 and BicD cooperation could direct MT organization and/or promote the vesicular transport necessary for oocyte polarization and oskar mRNA localization. Given the implication of membrane trafficking in the asymmetric localization of mRNAs, it also possible that polarized membrane transport along the oocyte MT network directs oskar mRNA to the posterior of the oocyte, by hitch-hiking along trafficking vesicles (Coutelis, 2007).

In MDCK cells, definition of apical and basolateral plasma membrane domains is required during polarization for the arrangement of MT along an apical-basal axis. Vesicular trafficking is crucial to establish, specify and maintain these membrane domains. By analogy, at stage 7, the polarizing signal from the posterior follicular cells to the Drosophila oocyte that causes repolarization of the MT cytoskeleton might do so by inducing the definition of anterior-lateral and posterior membrane domains. It is therefore possible that, in rab6^D23D oocytes, as in epithelia, defects in vesicular trafficking and TGN sorting underlie the observed defects in MT-network organization. Consistent with this idea, a mispolarized MT cytoskeleton is also observed in oocytes lacking Rab11. Thus, vesicular trafficking and the specification of membrane domains may be required for repolarization of the MT network and for the localization of molecular determinants in the Drosophila oocyte at mid-oogenesis (Coutelis, 2007).

Sec5, a member of the exocyst complex, mediates Drosophila embryo cellularization

Cellularization of the Drosophila embryo is the process by which a syncytium of ~6000 nuclei is subdivided into discrete cells. In order to individualize the cells, massive membrane addition needs to occur by a process that is not fully understood. The exocyst complex is required for some, but not all, forms of exocytosis and plays a role in directing vesicles to appropriate domains of the plasma membrane. Sec5 is a central component of this complex, and this study reports the isolation of a new allele of sec5 that has a temperature-sensitive phenotype. Using this allele, whether the exocyst complex is required for cellularization was investigated. Embryos from germline clones of the sec5^ts1 allele progress normally through cycle 13. At cellularization, however, cleavage furrows do not invaginate between nuclei and consequently cells do not form. A zygotically translated membrane protein, Neurotactin, is not inserted into the plasma membrane and instead accumulates in cytoplasmic puncta. During cellularization, Sec5 becomes concentrated at the apical end of the lateral membranes, which is likely to be the major site of membrane addition. Subsequently, Sec5 concentrates at the sub-apical complex, indicating a role for Sec5 in the polarized epithelium. Thus, the exocyst is necessary for, and is likely to direct, the polarized addition of new membrane during this form of cytokinesis (Murthy, 2010).

The exocyst complex was identified in yeast as essential for post-Golgi secretion and for the separation of the daughter and mother cells. In higher organisms, unlike yeast, some forms of exocytosis appear to be independent of exocyst components: the transport of vesicles to the apical surface of MDCK cells is not blocked by antibodies to Sec6 and Sec8 and the release of neurotransmitter at the synapse persists in Drosophila sec5 mutants. In yeast, the exocyst is required for septum growth and separation during cytokinesis, whereas in higher organisms RNAi studies indicate that the exocyst is only needed for the resolution of the intracellular bridge during late stages of cytokinesis. These studies, however, are constrained because complete loss of exocyst function is lethal to cells and so earlier phases of cytokinesis may persist due to residual exocyst function. The present study circumvented the requirement for the exocyst for cell viability by isolating a sec5 allele that gives rise to embryos with minimal Sec5 function (Murthy, 2010).

The cellularization of the Drosophila blastoderm is clearly dependent on the exocyst component Sec5 and, as in yeast, the exocyst marks the locations of membrane addition. In embryos containing only the truncated sec5^ts1-encoded protein, embryo development arrested when membrane addition should have begun. The arrest was abrupt and specific; prior stages of embryogenesis, including fertilization, nuclear divisions, nuclear migration to the cortex, nuclear divisions in the periphery, metaphase furrow formation and actin cap formation, occurred properly. Although the density of peripheral nuclei was normal relative to the control and the microtubule network appeared normal, the mutant embryos failed to cellularize. Nuclei did not elongate, furrow canals did not invaginate correctly, F-actin became disorganized, and nuclei fell away from the periphery. Because the complete lack of Sec5 is cell-lethal in the germline and arrests the development of the oocyte, it would appear that this new allele of sec5 permits a degree of exocytosis that at 15°C is only enough for oocyte formation but, at either 15°C or 25°C, is inadequate to support the massive membrane addition during cellularization. Even in the absence of most cell membranes, many mutant embryos attempted gastrulation. The defect thus appears to be focused on membrane invagination itself (Murthy, 2010). During the slow phase of membrane growth, the cellularization front progresses ~7 microm basal to the surface of the embryo. The front never progressed this far in sec5^ts1 embryos at 15°C, although shorter furrows were seen in patches. Defects were also apparent in the persistence of apical actin in 4- to 6-hour-old embryos, with a failure of nuclei to elongate and the inclusion of several nuclei within a partially formed cell. The hallmarks of the fast phase of cellularization, i.e. the transition of F-actin from hexagons to rings and the rapid invagination that results in cells of 35 microm, never occurred in sec5^ts1 embryos. It is concluded that during both the slow and fast phases of cellularization, the residual function of sec5^ts1 cannot support proper membrane growth. The small degree of membrane invagination sometimes observed at 15°C could be completely prevented by shifting the embryos to 25°C, whereas that shift did not change cellularization in control embryos. These defects appear to be more severe than those observed in conventional cytokinesis in higher organisms, in which the exocyst is only required for late stages. This discrepancy could be explained by the greater amount of membrane addition needed in cellularization compared with other types of cytokinesis or because the allele that was used more completely disrupts exocyst function than the RNAi constructs used previously (Murthy, 2010).

Membrane addition during cellularization is supported by Golgi-derived vesicles and the exocyst is needed for post-Golgi trafficking to the plasma membrane. The failure of newly synthesized Nrt to be trafficked and inserted into the plasma membrane in sec5^ts1 embryos is consistent with this role. Because significantly more Nrt in the mutant embryos was found in Rab11-positive endosomes than in GM130-positive Golgi membranes, a direct movement of Golgi proteins to Rab11 endosomes, without previous plasma membrane insertion and endocytosis, is likely. This direct route from the Golgi to recycling endosomes has been suggested previously and the current data provide further support for the existence of this pathway (Murthy, 2010).

The phenotype of sec5^ts1 embryos is distinct from those of several other early embryonic mutations. Cellularization proceeds normally in shotgun, armadillo (β-catenin), bazooka (par-3), stardust and crb mutants, but the formation of the AAJ is disrupted and the epithelium is transformed into a multilayered sheet of cells. In slow as molasses (slam) embryos, membrane invagination does not occur in the slow phase, Nrt is not inserted into the plasma membrane, and the furrow canal remains at the apical end, much like sec5^ts1 embryos. However, slam embryos are able to initiate the fast phase of cellularization and slam nuclei elongate. Also, in slam mutants, Nrt is still transported through the cytoplasm and accumulates just under the apical membrane, whereas in sec5^ts1 embryos Nrt remains mostly in the basal cytoplasm (Murthy, 2010).

Although Syntaxin and Sec5 localize differently during cellularization, the Syntaxin 1 phenotype is most similar to that of sec5. Ovaries homozygous for the hypomorphic allele Syntaxin^L266 yield embryos that complete nuclear divisions but fail to cellularize correctly, producing large acellular patches. Because Syntaxin is essential for vesicle fusion, the similar phenotypes suggest that Sec5 also mediates vesicle fusion at the embryo plasma membrane (Murthy, 2010).

Although it acts in post-Golgi transport, the exocyst is also required for some steps of endocytosis and post-endocytic traffic. During cellularization, apical membranes, which begin cellularization with microvilli, are internalized, pass through an endosome and are then reinserted to form the growing lateral membranes. Because of the extreme failure of membrane growth in sec5 mutant embryos, it seems likely that the addition of endosomally trafficked, apically derived membrane is blocked. Thus, Sec5 might also be involved in the fusion of endosomally derived vesicles (Murthy, 2010).

Three sec5 truncations give rise to temperature-sensitive phenotypes: (1) sec5^ts1 as reported in this study; (2) sec5^E13, which has a modest temperature sensitivity but less activity at permissive temperatures than sec5^ts1; and (3) the yeast allele sec5-24. It is speculated that this temperature sensitivity might arise from thermal instability of exocyst complexes that incorporate the truncated proteins, rather than from the temperature-dependent unfolding of the Sec5 protein itself. The novel F1 screen that yielded sec5^ts1 should be of wide utility for obtaining new alleles of a gene for which there is a transgenic rescue construct (Murthy, 2010).

The sec5^ts1 phenotype does not preclude functions for Sec5 in addition to membrane addition. In particular, it was observed that Nrt-containing membranes were not transported to the apical region and nuclei failed to elongate in sec5^ts1. These phenotypes could reflect cytoskeletal defects involving microtubule organization and prior studies have found biochemical interactions between exocyst components and microtubules. However, the failure in cellularization is unlikely to be due to a defect in microtubules because the microtubule network remains intact in sec5^ts1 embryos and other microtubule-dependent processes, such as nuclear migration to the cortex, are likewise intact. Actin localization was normal in sec5^ts1 embryos prior to the arrest of cellularization; there was no apparent defect in actin cycling between caps and metaphase furrows during mitotic divisions preceding cellularization. The late actin phenotypes are therefore likely to arise as an indirect consequence of a membrane traffic defect. However, exocyst interactions with actin-regulating Rho family GTPases might contribute to the phenotypes (Murthy, 2010).

The exocyst appears to correlate with, and is likely to define, particular domains of the plasma membrane at which vesicle fusion occurs. Once established as requisite for membrane addition in cellularization, Sec5 localization could serve as an index of the site of membrane insertion. Of the two prevalent models for cellularization, i.e. addition at the tip of the furrow canal or addition at the apicolateral membrane, the data clearly favor the latter. Indeed, the correspondence is striking between the dynamic localization of Sec5 and the sites of membrane addition. The redistribution of exocyst proteins to correlate with changing sites of membrane fusion has precedent and this study offers a further example of the exocyst complex providing a spatial cue for directing post-Golgi vesicles to the plasma membrane. The resulting membrane addition is crucial to the cellularization of the embryo. The identification of the molecules that are responsible for the localization and relocalization of the exocyst will be key to understanding the mechanism of localized membrane addition (Murthy, 2010).

REFERENCES

Search PubMed for articles about Drosophila Sec5

Bashaw, G. J. and Baker, B. S. (1995). The msl-2 dosage compensation gene of Drosophila encodes a putative DNA-binding protein whose expression is sex specifically regulated by Sex-lethal. Development 121: 3245-3258. 7588059

Beronja, S., Laprise, P., Papoulas, O., Pellikka, M., Sisson, J. and Tepass, U. (2005). Essential function of Drosophila Sec6 in apical exocytosis of epithelial photoreceptor cells. J. Cell Biol. 169(4): 635-46. 15897260

Classen, A.-K., Anderson, K. I., Marois, E. and Eaton, S. (2005). Hexagonal packing of Drosophila wing epithelial cells by the planar cell polarity pathway. Dev. Cell 9(6): 805-17. 16326392

Coutelis, J. B. and Ephrussi, A. (2007). Rab6 mediates membrane organization and determinant localization during Drosophila oogenesis. Development 134(7): 1419-30. Medline abstract: 17329360

Feller, C., Prestel, M., Hartmann, H., Straub, T., Soding, J. and Becker, P. B. (2012). The MOF-containing NSL complex associates globally with housekeeping genes, but activates only a defined subset. Nucleic Acids Res 40: 1509-1522. PubMed ID: 22039099

Finger, F. P. and Novick, P. (1997). Sec3p is involved in secretion and morphogenesis in Saccharomyces cerevisiae. Mol. Biol. Cell 8: 647-662. 9247645

Friedrich, G. A., Hildebrand, J. D., and Soriano, P. (1997). The secretory protein Sec8 is required for paraxial mesoderm formation in the mouse. Dev. Biol. 192: 364-374. 9441674

Fukai, S., Matern, H. T., Jagath, J. R., Scheller, R. H. and Brunger, A. T. (2003). Structural basis of the interaction between RalA and Sec5, a subunit of the sec6/8 complex. EMBO J. 22(13): 3267-78. 12839989

Giagtzoglou, N., Yamamoto, S., Zitserman, D., Graves, H. K., Schulze, K. L., Wang, H., Klein, H., Roegiers, F. and Bellen, H. J. (2012). dEHBP1 controls exocytosis and recycling of Delta during asymmetric divisions. J Cell Biol 196: 65-83. PubMed ID: 22213802

Grindstaff, K. K., Yeaman, C., Anandasabapathy, N., Hsu, S. C., Rodriguez-Boulan, E., Scheller, R. H. and Nelson, W. J. (1998). Sec6/8 complex is recruited to cell-cell contacts and specifies transport vesicle delivery to the basal-lateral membrane in epithelial cells. Cell 93: 731-740. 9630218

Grote, E., Carr, C. M. and Novick, P. J. (2000). Ordering the final events in yeast exocytosis. J. Cell Biol. 151: 439-452. 11038189

Guo, W., Grant, A. and Novick, P. (1999a). Exo84p is an exocyst protein essential for secretion. J. Biol. Chem. 274: 23558-23564. 10438536

Guo, W., Roth, D., Walch-Solimena, C., and Novick, P. (1999b). The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis. EMBO J. 18: 1071-1080. 10022848

Hazuka, C. D., Foletti, D. L., Hsu, S. C., Kee, Y., Hopf, F. W. and Scheller, R. H. (1999). The sec6/8 complex is located at neurite outgrowth and axonal synapse-assembly domains. J. Neurosci. 19: 1324-1334. 9952410

Hochheimer, A., Zhou, S., Zheng, S., Holmes, M. C. and Tjian, R. (2002). TRF2 associates with DREF and directs promoter-selective gene expression in Drosophila. Nature 420: 439-445. PubMed ID: 12459787

Hsu, S. C., Ting, A. E., Hazuka, C. D., Davanger, S., Kenny, J. W., Kee, Y., and Scheller, R. H. (1996). The mammalian brain rsec6/8 complex. Neuron 17: 1209-1219. 8982167

Jantti, J., Lahdenranta, J., Olkkonen, V. M., Soderlund, H., and Keranen, S. (1999). SEM1, a homologue of the split hand/split foot malformation candidate gene Dss1, regulates exocytosis and pseudohyphal differentiation in yeast. Proc. Natl. Acad. Sci. 96: 909-914. 9927667

Kee, Y., Yoo, J. S., Hazuka, C. D., Peterson, K. E., Hsu, S. C., and Scheller, R. H. (1997). Subunit structure of the mammalian exocyst complex. Proc. Natl. Acad. Sci. 94: 14438-14443. 9405631

Lam, K. C., Muhlpfordt, F., Vaquerizas, J. M., Raja, S. J., Holz, H., Luscombe, N. M., Manke, T. and Akhtar, A. (2012). The NSL complex regulates housekeeping genes in Drosophila. PLoS Genet 8: e1002736. PubMed ID: 22723752

Langevin, J., et al. (2005). Drosophila exocyst components Sec5, Sec6, and Sec15 regulate DE-Cadherin trafficking from recycling endosomes to the plasma membrane. Dev. Cell. 9(3): 355-76. 16224820

Liljedahl, M., Maeda, Y., Colanzi, A., Ayala, I., Van Lint, J., and Malhotra, V. (2001). Protein kinase D regulates the fission of cell surface destined transport carriers from the trans-Golgi network. Cell 104: 409-420. 11239398

Lin, R. C. and Scheller, R. H. (2000). Mechanisms of synaptic vesicle exocytosis. Annu. Rev. Cell Dev. Biol. 16: 19-49. 11031229

Lipschutz, J. H., Guo, W., O'Brien, L. E., Nguyen, Y. H., Novick, P. and Mostov, K. E. (2000). Exocyst is involved in cystogenesis and tubulogenesis and acts by modulating synthesis and delivery of basolateral plasma membrane and secretory proteins. Mol. Biol. Cell 11: 4259-4275. 11102522

Lipschutz, J. H., Lingappa, V. R. and Mostov, K. E. (2003). The exocyst affects protein synthesis by acting on the translocation machinery of the endoplasmic reticulum. J. Biol. Chem. 278: 20954-20960. 11102522

Mehta, S. Q., et al. (2005). Mutations in Drosophila sec15 reveal a function in neuronal targeting for a subset of exocyst components. Neuron 46(2): 219-32. 15848801

Moskalenko, S., Henry, D. O., Rosse, C., Mirey, G., Camonis, J. H. and White M. A. (2002). The exocyst is a Ral effector complex. Nat. Cell Biol. 4(1): 66-72. 1174049

Moskalenko, S., Tong, C., Rosse, C., Mirey, G., Formstecher, E., Daviet, L., Camonis, J. and White, M. A. (2003). Ral GTPases regulate exocyst assembly through dual subunit interactions. J. Biol. Chem. 278(51): 51743-8. 14525976

Mott, H. R., Nietlispach, D., Hopkins, L. J., Mirey, G., Camonis, J. H. and Owen D. (2003). Structure of the GTPase-binding domain of Sec5 and elucidation of its Ral binding site. J. Biol. Chem. 278(19): 17053-9. 12624092

Murthy, M., Garza, D., Scheller, R. H. and Schwarz, T. L. (2003). Mutations in the exocyst component Sec5 disrupt neuronal membrane traffic, but neurotransmitter release persists. Neuron 37(3): 433-47. 12575951

Murthy, M. and Schwarz, T. L. (2004). The exocyst component Sec5 is required for membrane traffic and polarity in the Drosophila ovary. Development. 131(2): 377-88. 14681190

Murthy, M., et al. (2005). Sec6 mutations and the Drosophila exocyst complex. J. Cell Sci. 118: 1139-1150. 15728258

Murthy, M., Teodoro, R. O., Miller, T. P. and Schwarz, T. L. (2010). Sec5, a member of the exocyst complex, mediates Drosophila embryo cellularization. Development 137: 2773-2783. PubMed Citation: 20630948

Novick, P., Field, C., and Schekman, R. (1980). Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell 21: 205-215. 6996832

Novick, P., Ferro, S., and Schekman, R. (1981). Order of events in the yeast secretory pathway. Cell 25: 461-469. 7026045

Pelissier, A., Chauvin, J. P. and Lecuit, T. (2003). Trafficking through Rab11 endosomes is required for cellularization during Drosophila embryogenesis. Curr. Biol. 13: 1848–1857. PubMed Citation: 14588240

Prigent, M., et al. (2003). ARF6 controls post-endocytic recycling through its downstream exocyst complex effector. J. Cell Biol. 163: 1111-1121. 14662749

Raja, S. J., et al. (2010). The nonspecific lethal complex is a transcriptional regulator in Drosophila. Mol. Cell. 38: 827-841. PubMed Citation: 20620954

Riggs, B., et al. (2003). Actin cytoskeleton remodeling during early Drosophila furrow formation requires recycling endosomal components nuclear-fallout and Rab11. J. Cell Biol. 163: 143–154. PubMed Citation: 14530382

Rothman, J.E. (1994). Mechanisms of intracellular protein transport. Nature 372: 55-63. 7969419

Shin, D. M., Zhao, X. S., Zeng, W., Mozhayeva, M. and Muallem, S. (2000). The mammalian Sec6/8 complex interacts with Ca(2+) signaling complexes and regulates their activity. J. Cell Biol. 150: 1101-1112. 10973998

Sjolinder, M., Uhlmann, J. and Ponsting, H. (2002). DelGEF, a homologue of the Ran guanine nucleotide exchange factor RanGEF, binds to the exocyst component Sec5 and modulates secretion. FEBS Lett. 532(1-2): 211-5. 12459492

Sugihara, K., Asano, S., Tanaka, K., Iwamatsu, A., Okawa, K. and Ohta, Y. (2002). The exocyst complex binds the small GTPase RalA to mediate filopodia formation. Nat. Cell Biol. 4(1): 73-8. 11744922

Takaya, A., Ohba, Y., Kurokawa, K. and Matsuda, M. (2004). RalA activation at nascent lamellipodia of EGF-stimulated Cos7 cells and migrating MDCK cells. Mol Biol Cell. 15(6): 2549-57. 15034142

TerBush, D. R. and Novick, P. (1995). Sec6, Sec8, and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae. J. Cell Biol. 130: 299-312. 7615633

Toikkanen, J. H., Miller, K. J., Soderlund, H., Jantti, J. and Keranen, S. (2003). The beta subunit of the Sec61p endoplasmic reticulum translocon interacts with the exocyst complex in Saccharomyces cerevisiae. J. Biol. Chem. 278: 20946-20953. 12665530

Valcarcel, R., Weber, U., Jackson, D. B., Benes, V., Ansorge, W., Bohmann, D. and Mlodzik, M. (1999). Sec61beta, a subunit of the protein translocation channel, is required during Drosophila development. J. Cell Sci. 112: 4389-4396. 10564656

Vega, I. E. and Hsu, S. C. (2001). The exocyst complex associates with microtubules to mediate vesicle targeting and neurite outgrowth. J. Neurosci. 21: 3839-3848. 11356872

Vega, I. E. and Hsu, S. C. (2003). The septin protein Nedd5 associates with both the exocyst complex and microtubules and disruption of its GTPase activity promotes aberrant neurite sprouting in PC12 cells. NeuroReport 14: 31-37. 12544826

Wang, L., Li, G. and Sugita, S. (2004). RalA-exocyst interaction mediates GTP-dependent exocytosis. J. Biol, Chem. [Epub ahead of print] 14978027

Yeaman, C., Grindstaff, K. K., Wright, J. R. and Nelson, W. J. (2001). Sec6/8 complexes on trans-Golgi network and plasma membrane regulate late stages of exocytosis in mammalian cells. J. Cell Biol. 155: 593-604. 11696560

date revised: 15 March 2013

Home page: The Interactive Fly © 2017 Thomas Brody, Ph.D.