Asymmetric division of sensory organ precursors (SOPs) in Drosophila generates different cell types of the mature sensory organ. In a genetic screen designed to identify novel players in this process, a mutation was isolated in Drosophila sec15, which encodes a component of the exocyst, an evolutionarily conserved complex implicated in intracellular vesicle transport. sec15− sensory organs contain extra neurons at the expense of support cells, a phenotype consistent with loss of Notch signaling. A vesicular compartment containing Notch, Sanpodo, and endocytosed Delta accumulates in basal areas of mutant SOPs. Based on the dynamic traffic of Sec15, its colocalization with the recycling endosomal marker Rab11, and the aberrant distribution of Rab11 in sec15 clones, it is proposed that a defect in Delta recycling causes cell fate transformation in sec15− sensory lineages. The data indicate that Sec15 mediates a specific vesicle trafficking event to ensure proper neuronal fate specification in Drosophila (Jafar-Nejad, 2005).
In a genetic screen designed to identify novel players in Drosophila sensory organ development, a mutation in sec15 was isolated that caused a pIIa to pIIb transformation phenotype. Sec15 is a component of a multiprotein complex called the exocyst or Sec6/8 complex. Mutations in exocyst components were originally isolated in a yeast screen for secretion-defective mutants. Subsequent analysis of the exocyst complex in yeast and mammalian cell culture systems has indicated that it functions in intracellular vesicle transport. In yeast, the exocyst mediates the post-Golgi to membrane targeting of exocytic cargo via an interaction with the Rab GTPase Sec4p. In Madin-Darby canine kidney (MDCK) epithelial cells, the exocyst localizes to areas of cell-cell contact and is involved in basolateral delivery of vesicles. However, none of the studies on the exocyst components have implicated these proteins in cell fate determination. The data suggest that Sec15 mediates highly specific intracellular trafficking events that promote N signaling and thereby ensure proper cell fate specification in Drosophila mechanosensory organs (Jafar-Nejad, 2005).
The various cell types that form an adult sensory organ in Drosophila are generated via asymmetric divisions of a pI and its progeny. Differential activation of the N signaling pathway between the two daughter cells of each division ensures that each sensory organ acquires the proper complement of cell types necessary to function. Sec15, a component of the evolutionarily conserved exocyst complex as reported here, is required for proper cell fate specification of the pI progeny. Studies on sec15 mutations in the eye did not reveal any fate change in the photoreceptors. Loss-of-function mutations in three other exocyst components have been reported previously: sec5 and sec6 in flies and sec8 in mice. sec8 mutant mice die at day E7.5, before the development of specific neuronal populations can be studied. Also, sec5 and sec6 mutations are cell lethal in the Drosophila eye. Therefore, this report is the first to identify a role for an exocyst component in cell fate determination. At this point, it cannot be predicted if sec5 and sec6 also play a role in neuronal cell fate specification. However, given the data obtained from studies of the fly eye, the hypothesis is favored that components of the exocyst may form more than a single functional unit and/or have subunit-specific roles (Jafar-Nejad, 2005).
Live imaging of dividing pI cells indicates that Sec15 is associated with a vesicular compartment that traffics between apical and subapical areas. In sec15− SOPs, an expanded compartment is observed that contains Spdo, N, and Dl. Unlike wt pI and pIIb cells, in which Spdo/N/Dl+ vesicles tend to reside at or above the level of septate junctions, in mutant SOPs these puncta accumulate at the basal side of the cell. Together, these observations suggest that Sec15 is involved in vesicle trafficking to the apical parts of the cell. The defect in the apical trafficking of proteins does not seem to be a general one, since localization of E-Cad and Arm at the adherens junction is not disrupted in mutant tissue. Therefore, the data link a specific vesicle trafficking event to a developmental decision made by sensory precursor cells (Jafar-Nejad, 2005).
Genetic experiments and immunohistochemical stainings strongly suggest that Sec15 and Spdo function in the same pathway in sensory cell fate determination process. It has been proposed, based on studies performed on the asymmetric divisions of Drosophila embryonic neuroblasts, that Spdo promotes N signaling at the membrane of the signal-receiving cell. In contrast, Numb and α-Adaptin in the signal-sending cell might promote endocytosis of Spdo and its removal from the membrane, thereby preventing the reception of signal by this cell. The subcellular distribution of Spdo in pIIa and pIIb cells is similar to its localization in embryonic neuroblast progeny, suggesting that this model might also apply to adult bristle formation. Notably, however, Spdo is observed at or close to the membrane of both pI progeny in sec15 clones. Therefore, while the proposed role for Spdo in promoting N signaling at the membrane of the signal-receiving cell cannot be ruled out, the data suggest a role for Spdo in Dl recycling in the signal-sending cell. It should be noted, though, that these two models are not mutually exclusive. Presence of a significantly higher number of vesicles containing both Dl and N in pIIb compared to the pIIa in wt sensory precursors has been implicated in the ability of the pIIb cell to send the Dl signal. Colocalization of Spdo with Dl in a significant fraction of these vesicles suggests that a defect in Spdo/Dl trafficking in pIIb contributes to the sec15 loss-of-function phenotype (Jafar-Nejad, 2005).
Presence of endocytosed Dl in vesicles that accumulate in sec15 clones implicates these vesicles in the endocytic traffic of Dl. This notion is further supported by the observation that in both wt and sec15− SOPs, the Spdo/Dl/N puncta show a significant colocalization with the endosomal markers Rab5 and HRS. It has recently been proposed that in order to signal, Dl needs to traffic through a specific endocytic compartment, which will lead to recycling of the protein. A defect in Dl recycling is further suggested by the aberrant accumulation of the recycling endosomal marker Rab11 in sec15 clones. The Rab11+ endosomal compartment is thought to be a central trafficking intermediate in both exocytic and endocytic pathways and is shown to control the traffic of cargo from the perinuclear recycling endosomal compartment to the membrane. Interestingly, it has been shown that Sec15 meets the criteria of being an effector for Rab11 in mammalian cell lines: Sec15 physically binds Rab11 in a GTP-dependent manner; Sec15 colocalizes with Rab11 in the perinuclear region of the cells; Sec15 labels structures containing an endocytosed protein in immuno-EM experiments. Similarly, Drosophila Sec15 and Rab11 interact physically and show a high level of colocalization in SOPs. Altogether, these data are compatible with a model in which Sec15 regulates the traffic of a subset of endocytosed Dl to the membrane of the pIIb cell via a Rab11+ recycling endosomal compartment. Sec15 traffics symmetrically in pIIa and pIIb. Therefore, it is proposed that an intrinsic difference between the endocytic traffic of Dl in pIIa and pIIb allows the pIIb cell to employ the Sec15-Rab11 machinery differentially from the pIIa cell and thereby assume the role of signal-sending cell. The most likely mechanisms for the proposed intrinsic difference are unequal segregation of Neur into the pIIb, which promotes Dl endocytosis in this cell, and asymmetric distribution of the Rab11+ recycling endosomes in the pIIb versus pIIa, which is thought to specifically mediate Dl recycling in the pIIb (Jafar-Nejad, 2005).
These data suggest that at least some of the Spdo/N/Dl-containing vesicles that accumulate in the basal areas of sec15− SOPs are of a mixed exo-endocytic nature. This is not unprecedented, since traffic from the TGN to an endosomal compartment has been documented. Accordingly, it has been proposed that some exocytic cargo might pass through the recycling endosome on its way from the TGN to the plasma membrane. Recently, it has been shown that upon exit from the Golgi apparatus, newly synthesized E-Cad fuses with a Rab11+ recycling endosomal compartment before it reaches the plasma membrane. It is interesting to note that members of the exocyst complex have been shown to localize to both the TGN and recycling endosomes in polarizing epithelial cells. Although the recycling endosome has been proposed as an intermediate to transfer the exocytic cargo to the plasma membrane, it is possible that passing through these vesicles somehow enhances the signaling ability of internalized Dl. In other words, presence of Spdo might be part of the specific environment that Dl needs to traffic through. Although Dl endocytosis and recycling are also implicated in N signaling during lateral inhibition, no lateral inhibition defects are observed in sec15 clones. It is proposed that the link to Spdo results in the specificity of the sec15 phenotype to the asymmetric divisions, since loss of spdo similarly does not affect lateral inhibition (Jafar-Nejad, 2005).
In summary, the data indicate that one component of the highly conserved exocyst complex affects the asymmetric division of the sensory precursors in the Drosophila PNS through specific vesicle trafficking events. Components of the exocyst complex are conserved from yeast to human, and several reports have shown parallels between the contribution of asymmetric divisions to Drosophila and vertebrate neurogenesis. Therefore, it is conceivable that Sec15, and perhaps other members of the exocyst complex, are involved in neural cell fate determination in other species (Jafar-Nejad, 2005).
Sec15, a component of the exocyst, recognizes vesicle-associated Rab GTPases, helps target transport vesicles to the budding sites in yeast and is thought to recruit other exocyst proteins. This study reports the characterization of a 35-kDa fragment that comprises most of the C-terminal half of Drosophila Sec15. This C-terminal domain binds a subset of Rab GTPases, especially Rab11, in a GTP-dependent manner. Evidence is provided that in fly photoreceptors Sec15 colocalizes with Rab11 and that loss of Sec15 affects rhabdomere morphology. Determination of the 2.5-Å crystal structure of the C-terminal domain revealed a novel fold consisting of ten alpha-helices equally distributed between two subdomains (N and C subdomains). The C subdomain, mainly via a single helix, is sufficient for Rab binding (Wu, 2005).
Sec15 plays multiple key roles in a variety of processes in exocytosis by interacting with vesicle-associated small Rab GTPases, assisting in targeting vesicles to budding sites and recruiting, by way of Sec10, other components of the exocyst. in vitro and in vivo studies have shed light on some of these roles. The relatively large size (85 kDa) of Sec15 ensures that there are enough docking sites, each probably residing in either a single domain or a combination of domains, for other protein components necessary for its diverse functions. These studies revealed a segment named the C-terminal domain, containing nearly the entire C-terminal half of the protein, possesses several interesting properties. This domain binds to a set of Rabs in a GTP-dependent manner. Its atomic structure is composed of two distinct subdomains, only one of which (the C subdomain) harbors the Rab-binding site. The finding of a bipartite C-terminal domain was unexpected; there was no hint of this feature even from the BLAST Conserved Domain Database search for domains. The all-helical structure of the domain, with its two different subdomains, has a novel fold. A search for overall structural similarities of the whole domain and of the N and C subdomains separately against the DALI database, a network tool for protein structure comparison, did not find any substantial matches (Wu, 2005).
Binding studies further show that Sec15 lacks stringent substrate specificity in vitro. The domain binds Rab11, Rab3, Rab8 and Rab27, but the binding data suggest that Rab11 is the major target. The ability of an effector protein to interact with different Rab proteins has been observed previously: rabphilin-3, originally identified as a Rab3-binding protein, has also been shown to interact with Rab8 and Rab27 in a cotransfection assay in COS-7 cells. In addition, Sec15 and Sec5 apparently do not have significant roles in regulating neurotransmitter release, a process in which Rab3 has been implicated. This suggests that the weaker in vitro binding of Sec15 with Rab3, Rab8 and Rab27 may have in vivo consequences. It remains to be investigated whether these interactions have any role in vivo (Wu, 2005).
The Rab-binding site is apparently confined mainly to the exposed middle three-fourths of one helix (alpha9) of the C subdomain, which contains mostly hydrophobic residues. The participation of hydrophobic residues in binding is a common feature observed in several crystal structures of complexes of effectors with their cognate small GTPases. The in vivo consequence of abolishing the Sec15-Rab11 interaction by using the Sec15 mutants is under investigation (Wu, 2005).
These studies further raise questions about the role of the ~15-kDa N subdomain, which is also composed entirely of helices, but helices with various lengths and with topology and geometry different from those in the C subdomain. The same questions could also apply to the function(s) of the ~40-kDa N-terminal half of Sec15. The combination of the N subdomain and the N-terminal half, which is approximately two-thirds of the entire Sec15 protein, seems too large for binding only the Sec10 component of the exocyst. This suggests that Sec15 may have additional partners that have yet to be identified (Wu, 2005).
There are informative differences between the phenotypes in rhodopsin1 trafficking and rhabdomere morphogenesis of rab11 and sec15 mutants. A defect in the initial delivery of rhodopsin in rab11 mutants has been associated with accumulations of rhodopsin outside the rhabdomere. No noticeable amounts of rhodopsin were observed outside the rhabdomeres in sec15 mutants. This observation, combined with the normal photoreceptor depolarization measured by electroretinography, suggests that initial delivery of rhodopsin occurs in sec15 mutants. However, there is also a significant defect in rhabdomere morphology in rab11 mutant photoreceptors. Hence, the photoreceptor staining data suggest that although targeted membrane delivery to the rhabdomere might occur in Sec15-mutant cells, defects in membrane recycling (evidenced by the strong accumulation of Rab11 in the rhabdomere) probably lead to disruptions in rhabdomere morphology over time. Together, these data suggest that the rab11 mutant phenotype is more severe than the sec15 mutant phenotype but that the two show some similarities. In summary, the interaction of Rab11 and Sec15 has functional consequences in vivo, in that Rab11 trafficking is disrupted in sec15 mutants and at least one aspect of the sec15 mutant phenotype, namely abnormal rhabdomere morphology, can be explained by loss of Rab11 function. An interaction between Sec15 and Rab11 has also been shown to have an important role in the asymmetric division of sensory organ precursors in Drosophila, in that Sec15 promotes Notch signaling during the asymmetric division of sensory organs, suggesting that Sec15/Rab11 interaction is required not only in rhodopsin targeting but also during cell-fate specification (Wu, 2005).
Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.