To determine the expression pattern and subcellular localization of Sec15, a polyclonal antibody was generated against a fragment of Sec15. The antibody is specific to Sec15; staining of sec15 homozygous mutant eye disc clones display a reduction of staining to background levels. During initial target recognition with glia (5% of pupal development) and during cartridge formation (30%), Sec15 immunoreactivity is highly enriched in the developing optic lobe neuropils, including the lamina and medulla. Costaining with mAb24B10 shows that photoreceptor terminals contain Sec15, as do synaptic terminals of other cells that contribute to the neuropil. In the second half of pupation, synapse formation commences in the lamina, and Sec15 immunoreactivity decreases in all neuropils. However, low levels of Sec15 immunoreactivity persist into adulthood in a very distinct punctate staining pattern. Sec15 punctae are localized within the glial border of each cartridge, indicating that they are present in photoreceptor terminals as well as pre- and post-synaptic endings of other cell types. These punctae mostly do not colocalize with n-synaptobrevin and synaptotagmin I and are located near active zones. In summary, these data indicate that Sec15 is present at the correct time and place during development to account for the neuronal targeting defects in the mutants and may serve other functions into adulthood by specifying distinct subdomains of synaptic terminals (Mehta, 2005).
To isolate new genes that play a role in synapse development or function, an F1 screen was carried out in the Drosophila visual system. Using the eyFLP system, 210,000 flies were created with eyes homozygous for a randomly induced chemical mutation while keeping the rest of the body heterozygous. Two assays were employed to identify mutations that cause a failure to evoke a postsynaptic response. To identify mutations that affect the accuracy of synaptic contacts (synaptic specificity), neuronal targeting defects were assessed with light microscopy for 450 mutants and synapse formation with electron microscopy for 40 complementation groups (Mehta, 2005).
Flies were selected with grossly normal eye morphology that phototax poorly or not at all. Control flies consistently walk toward light, while flies that do not synthesize the neurotransmitter histamine fail to phototax. Two independently isolated mutations that failed to complement each other, 3R411 and 3R412, display an aberrant response to light (red and blue). Electroretinograms (ERGs) were performed to identify mutations that cause a lack of 'on' and 'off' responses but display a normal depolarization profile. The lack of an on and off response is thought to indicate a lack of, or aberrant communication between, pre- and post-synaptic cells. This can be caused by (1) a defect in neurotransmission or (2) a developmental defect in synapse formation. To test for developmental defects at the level of light microscopy, adult brains were stained with the photoreceptor-specific antibody against chaoptin, mAb 24B10. The Drosophila compound eye consists of 800 unit eyes, called ommatidia, each with a complement of eight photoreceptor cells. mAb 24B10 staining reveals the morphology of photoreceptor terminals R1-R6 in the first optic neuropil, the lamina, as well as R7-R8 in the second optic neuropil, the medulla. 3D visualization of the R7/8 terminal field in the medulla of a control animal reveals a highly regular array of terminals. The terminals of R7 and R8 synapse in separate layers in the medulla. In contrast, 3R411 mutant photoreceptors display loss of the regular array of terminals in the medulla and highly aberrant R7 and R8 target layering. It was next analyzed whether these morphological disruptions are the result of long-range growth cone guidance defects or short-range wiring disruptions within the correct brain areas. Visualization of the adult optic neuropils with the synaptic marker N-cadherin revealed strong morphological disruptions of neuropil shape, but no alteration of their arrangement or size, indicating morphological disruptions only within the neuropils in mutant optic lobes. Visualization of only the R7 photoreceptor using R7-specific GFP expression in an eyFLP 3R41 mutant background revealed that all observable R7 terminals project into the distal medulla. While gross defects in R7 axon outgrowth were not detected, it cannot be rule out that tere are more subtle defects that are beyond the resolution of the described analyses. However, the data indicate that axons are not affected in long-range axonal pathfinding, axon extension, and the recognition of the correct brain areas or neuropils. Based on ERG results, mutant photoreceptors are able to sense light and depolarize normally after stimulation. Taken together, these data suggest that 3R41 mutant neurons do not exhibit disruptions in general cellular processes, but exhibit a severe and specific defect of neuronal terminals in establishing a normal, local wiring pattern within their correct neuropils (Mehta, 2005).
Sec15 is conserved from yeast to humans over the length of the protein. The second instar lethality associated with the loss of sec15 was rescueable using a 5 kb genomic fragment. Using expression of the sec15 cDNA in eyes in an eyFLP; sec15 background, both the R7–R8 terminal layering defect and the on and off transients of the ERG were rescued. These data show that phenotypes observed in the mutants are due to loss of Sec15. The identification of mutations in sec15 in a screen for synaptic specificity defects was unanticipated, given the proposed role of the exocyst in cellular polarization (Nelson, 2003) and neurite outgrowth, as well as the cell lethality associated with the loss of sec5 (Murthy, 2003) in photoreceptors (Mehta, 2005).
To determine whether R1-R6 photoreceptors display defects in synapse morphology, synapse formation, or vesicle morphology/number, wild-type and mutant laminae were analyzed by transmission electron microscopy (TEM). R1-R6 terminals from different ommatidia are organized into synaptic units called cartridges. During late larval stages, axons of R1-R6 invade the developing lamina plexus, where their growth cones are halted by interactions with glial cells. Initially, photoreceptors from the same ommatidium travel together in bundles. Later, during the first half of pupation, the R1-R6 terminals defasciculate and organize themselves into cartridges. In this sorting process, photoreceptors that receive light from the same point in space but reside in different ommatidia sort into a single cartridge according to the principle of neural superposition. Synaptogenesis does not start before cartridge formation is complete at the beginning of the second half of pupation. Quantitative ultrastructural studies of 1-day-old adults was performed to assess cartridge formation and synapse formation. Cartridges consist of six photoreceptor terminals that surround the processes of the L1 and L2 postsynaptic lamina monopolar cells. Photoreceptor terminals were identified based on the presence of capitate projections. The cartridges are delimited by epithelial glia. In eyFLP; 3R411 and eyFLP; 3R412 mutant laminae, cartridges are easily identifiable, but they contain highly variable numbers of photoreceptor terminals. Quantitative analysis of terminal number per cartridge revealed that eyFLP; 3R41 mutant laminae have a much broader distribution of photoreceptor terminals per cartridge than wild-type, indicating a defect in cartridge formation (Mehta, 2005).
Whether synapses are formed in these mis-sorted cartridges was analyzed. Drosophila photoreceptors form tetrad synapses in which the presynaptic active zone makes contacts with four postsynaptic dendrites from lamina monopolar and amacrine cells. The presynaptic active zone is identified by an electron-dense structure known as the 'T bar', and at least two of the four postsynaptic dendrites are identifiable in an ultrathin section at most angles. The typical configuration of tetrad synapses in which two T bars face each other and share postsynaptic processes is observed. In eyFLP; 3R41 mutant laminae, synapses appear morphologically normal and occur with a similar frequency as in controls, indicating that 3R41 mutants have no defect in synapse assembly. The synaptic vesicle content of mutant terminals appears to be normal, and immunohistochemical analyses of synaptic vesicle proteins including synaptotagmin and neuronal synaptobrevin revealed no altered distribution. It is concluded that 3R41 mutants exhibit a defect in synaptic specificity, since qualitatively and quantitatively normal synapses are formed in cartridges containing an incorrect complement of photoreceptor terminals (Mehta, 2005).
Homozygous mutant eyFLP; 3R41 eyes are generally smooth with only occasional irregularities in the ommatidial array. To ensure that the observed defects are not due to secondary defects in photoreceptor specification or differentiation, third instar larval imaginal discs containing marked 3R41 mutant clones at the time of axonal outgrowth were labeled with a variety of markers. No obvious defects were observed in patterning. Since early developing mutant photoreceptors are indistinguishable from wild-type and photoreceptors are able to respond to light stimuli (as evidenced by normal depolarization of the ERG), it is concluded that the targeting defects are not due to neuronal differentiation defects (Mehta, 2005).
The eyFLP system generates homozygous mutant photoreceptors, as well as homozygous mutant lamina and medulla cells. This implies that some aspects of the sec15 mutant phenotype that were observe may not be caused by loss of Sec15 in photoreceptors. The finding that driving the sec15 cDNA only in eye tissue largely rescues the R7/R8 targeting defects as well as the ERG defect in eyFLP; sec15 mutant optic lobes indicates that Sec15 plays a critical role in photoreceptors. To further investigate the cell type-specific aspects of the observed phenotypes, two sets of experiments were devised (Mehta, 2005).
(1) The eyFLP system was used in combination with the MARCM technique to generate laminae in which 50% of the photoreceptors were homozygous mutant for sec15 in a random distribution. In these laminae, mutant photoreceptors express GFP, while other mutant optic lobe cells are not marked. Strong morphological disruptions are invariably and selectively seen in clones with marked mutant photoreceptors. Interestingly, areas with no mutant photoreceptors have very subtle or no morphological defects. These areas may contain mutant optic lobe cells despite not having any mutant photoreceptor terminals. Since an eye-specific driver was use to express GFP in mutant cells, the lamina cells that are mutant will not be marked. This finding indicates that the contribution of these cells to the overall morphological phenotype is minor. Also elevated levels of chaoptin were observed in isolated mutant terminals at the clone borders. To quantify this effect, Twelve cartridges at clone boundaries containing single mutant photoreceptor terminals were analyzed. 3D reconstructions were made of mutant terminals with one adjacent control terminal in each cartridge and pairwise mean fluorescence level ratios between mutant and control terminals were calculated. Isolated mutant terminals displayed a 62.3% (±11.7%) increase in chaoptin levels, indicating a cell-autonomous upregulation of chaoptin in sec15 mutant photoreceptor terminals (Mehta, 2005).
(2) In a second set of experiments, use was made of a new eyeless FLPase system developed by Iris Salecker and colleagues, called ey3.5FLP. This system uses a specific eyeless enhancer that only drives FLPase expression in eye imaginal discs (I. Salecker, personal communication to Mehta, 2005). This ensures that the only mutant terminals in the lamina are from photoreceptors. It was found that ey3.5FLP; sec15 mutant optic lobes still exhibit neuronal targeting defects. However, the ERGs exhibit on and off transients, albeit at reduced size. This clearly indicates that neurotransmitter release persists in photoreceptors lacking Sec15, even though TEM of the laminae of these flies revealed cartridges with abnormal numbers of terminals. However, the distribution of terminals per cartridge for sec15 mutants was less broad using the ey3.5FLP system compared to the eyFLP system. These data indicate that Sec15 is required for neuronal targeting in photoreceptors and also serves a function in other neurons. Since on and off transients in ERGs are field potential recordings of the synchronized firing of postsynaptic cells in the lamina, it is suspected that the loss of on and off transients in eyFLP; sec151 flies are secondary to the morphological defects. If only photoreceptors are made mutant using the ey3.5FLP system, the miswiring is less severe, causing small on and off responses to return. Likewise, when only nonphotoreceptor optic lobe cells are made mutant in the photoreceptor-specific sec15 cDNA rescue of the eyFLP; sec151 phenotype, on and off responses also persist and the R7/R8 targeting defect is greatly reduced. Sec15 must be removed from both populations of neurons (as in the eyFLP system) in order to eliminate the on and off responses, indicating that the loss is a cumulative effect secondary to morphological disruptions. These data argue that Sec15 is required in photoreceptors for correct neuronal targeting, but does not play an important role in regulating neurotransmission (Mehta, 2005).
Elevated levels of chaoptin in photoreceptor terminals have been described for another vesicle-trafficking mutant, the vesicle-SNARE neuronal-synaptobrevin (n-syb). This mutant also exhibits neuronal targeting defects. This observation raises the possibility that vesicle-dependent trafficking of transmembrane or other signaling molecules might be responsible for the neuronal targeting defects of sec15 mutant photoreceptors. Recently, Zhang (2004) identified Rab11 as an interacting partner of Sec15 in mammalian cell culture and proposed that Sec15 is an effector for some but not all Rabs. Indeed, an accumulation or upregulation of Rab11 immunoreactivity was seen in sec15 mutant photoreceptors, consistent with Rab11-positive vesicles failing to fuse with their target sites. To further test this hypothesis, the localization of cell adhesion and signaling molecules was examined in mutant photoreceptor cell bodies as well as terminals during photoreceptor development, precisely when target selection and cartridge formation occur (between P + 5% to P + 40% referring to time after pupation). Proteins were examined that have either been shown to be required for photoreceptor target selection, such as Dlar, N-cadherin, flamingo, and IrreC-rst, or that are likely to be required, based on work in other systems, such as Armadillo, Chaoptin, and Fasciclin II (Mehta, 2005).
Fasciclin II (Fas2) localization was examined in sec15 mutant photoreceptors, since chaoptin upregulation coincides with elevated levels of Fas2 in n-syb mutant photoreceptors. Fas2 appears to be present in aggregates in sec15 mutant photoreceptor cell bodies at P + 20%, in contrast to wild-type photoreceptors. In addition, the neuronal connections of the cell bodies exhibit Fas2 aggregated along the length of the mutant axons. Similarly, overexpression of Fas2 in photoreceptors causes neuronal targeting defects between P + 20% and P + 40%. In contrast to n-syb, however, no elevated levels of Fas2 are observed later in development. Hence, the data suggest that an aberrant localization of Fas2 in a specific developmental time window may at least partially underlie the observed phenotypes (Mehta, 2005).
Similar mislocalization phenotypes in photoreceptor cell bodies were also observed for other cell adhesion molecules such as Dlar and IrreC-rst during the developmental time window of photoreceptor target selection. Dlar is normally restricted apically in developing wild-type photoreceptors, at the center of the ommatidial array. In sec15 mutant photoreceptors it appears much more randomly distributed, such that a basal optical section through the eye shows Dlar at higher levels in mutant ommatidia. Although these results show mislocalization of cell adhesion molecules in the correct cell at the time when they are known to be required for proper target selection, no obvious defects were detected in the localization of Dlar or IrreC-rst in the developing lamina. This leaves open the question of whether mislocalization of Dlar and IrreC-rst beyond the resolution limit of confocal microscopy additionally contributes to the observed targeting defects (Mehta, 2005).
In vertebrates, Lar is known to localize to adherens junctions. Hence, a possible explanation for the mislocalization of Fas2, IrreC-rst, and Dlar in mutant photoreceptor cell bodies is a defect of adherens junctions. The subcellular localization of the adherens junction markers N-cadherin and armadillo was examined in the cell bodies as well as the terminals of mutant photoreceptors, but no mislocalization of N-cadherin was detected in either compartment. However, armadillo displayed localization defects selectively in the developing lamina, but not the photoreceptor cell bodies. Several other cell adhesion and signaling molecules, including flamingo, Crumbs, and Bazooka, were examined, all of which did not display aberrant localization at the level of light microscopy. It is concluded conclude that a specific subset of proteins is mislocalized in sec15 mutants (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).
Loss of function of the Drosophila exocyst components in epithelial cells results in E-Cadherin (Shotgun) accumulation in an enlarged Rab11 recycling endosomal compartment and inhibits Shotgun delivery to the membrane. Rab11 and Armadillo interact with Sec15 and Sec10, respectively. These results support a model whereby the exocyst regulates E-Cadherin trafficking, from recycling endosomes to sites on the epithelial cell membrane where Armadillo is located (Langevin, 2005).
In budding yeast, the exocyst has been proposed to tether post-Golgi vesicles to the membrane of the growing bud prior to fusion. This model is supported by several observations. (1) Exocyst components localize both on post-Golgi vesicles and on the bud membrane (Boyd, 2004). Analogously in Drosophila, Sec5 and Sec15 localize along the lateral membrane and on the REs. (2) Mutations in genes encoding components of the exocyst complex lead to the accumulation of post-Golgi vesicles (Novick, 1980). Analogously, Sec5, Sec6, and Sec15 loss of function leads to an enlargement of the recycling endosome (RE) compartment; this enlargement interpreted as an accumulation of RE vesicles. (3) The localization of Sec8p and Exo70p at the growing bud, i.e., the site of polarized exocytosis, depends on the function of the other exocyst components. Analogously, Sec5 is localized along the lateral membrane, where E-Cadherin delivery is affected, and its localization along the cortex depends on Sec6. It is therefore proposed that in Drosophila epithelial cells, Sec5, Sec6, and Sec15 act by tethering vesicles originating from the recycling endosomal compartment to the lateral membrane of epithelial cells, as a prerequisite for their exocytosis (Langevin, 2005).
In epithelial cells, Arm and E-Cadherin colocalize to the AJs of the ZA as well as along the lateral membrane. In the absence of Sec5, Sec6, and Sec15 function, E-Cadherin trafficking is affected and E-Cadherin accumulates in the RE. Similarly, in the absence of arm, E-Cadherin fails to localize at the membrane and localizes in the RE. The identification of an interaction between Arm and Sec10 is therefore consistent with a model whereby this interaction provides a landmark at the site where Arm is enriched in order to deliver E-Cadherin from the recycling endosomes. Nevertheless, Arm may play an additional role in stabilizing E-Cadherin at the AJs. A direct demonstration of the function of Arm in regulating the delivery of E-Cadherin will therefore require the identification of arm mutant alleles that do not perturb its function as a regulator of E-Cadherin stabilization and only affects its interaction with Sec10 (Langevin, 2005).
In the absence of Sec5, Sec6, or Sec15 function, E-Cadherin delivery to the lateral membrane is inhibited and E-Cadherin accumulates in the REs. Furthermore, E-Cadherin was found to transcytose in a Sec5-dependent manner from the lateral membrane of epithelial cells to the apical AJs. Therefore, this study reveals at least a role of the exocyst in the recycling of E-Cadherin from the lateral membrane to the apical AJs. Furthermore, the strong reduction of E-Cadherin present on the lateral membrane is interpreted as a failure to recycle E-Cadherin from the lateral membrane back to the lateral membrane, which cannot be compensated for by the delivery of newly synthesized E-Cadherin to the lateral membrane. The loss of E-Cadherin on the lateral membrane may also lead to a reduction of E-Cadherin delivery at the AJs. This may have also contributed to the loss of epithelial cell polarity observed in some of the sec5 mutant epithelial cells (Langevin, 2005).
In polarized MDCK cells, the apical REs are well known as a site of sorting during endocytic and transcytotic transport. The REs have also been shown to serve as an intermediate during the transport of newly synthesized proteins from the Golgi to the plasma membrane in nonpolarized MDCK cells. Similarly, upon overexpression of GFP-E-Cad in HeLa cells, E-Cad transits from the Golgi to the Rab11 endosomes (Lock, 2005). Nevertheless, the existence of such a pathway remains to be established in polarized MDCK cells. In fact, the overexpression of a dominant-negative form of Rab11 leads to sequestration of E-Cadherin in the REs, but whether sequestered E-Cadherin represented newly synthesized or recycled E-Cadherin was not determined (Lock, 2005; Miranda, 2001). The existence of such a Golgi-to-RE pathway also remains to be established in Drosophila epithelial cells. If so, a role of the exocyst in regulating the delivery of newly synthesized E-Cadherin from the Golgi to the lateral membrane via the REs remains plausible (Langevin, 2005).
Whether the exocyst regulates E-Cadherin localization in mammalian cells has not been directly analyzed. However, E-Cadherin is proposed to act as a regulator of the localization of the exocyst complex in polarizing mammalian cells since E-Cad- and Nectin-2α-dependent cell-cell contacts were proposed to recruit the exocyst complex in order to promote the growth of the lateral epithelial cell domain (Yeaman, 2004). The current study suggests that upon the recruitment of the exocyst complex by E-Cadherin, the exocyst promotes the delivery of more E-Cadherin to the lateral membrane during the establishment of apico-basal polarity. In fact, several reports can be reconciled with a function of the exocyst in regulating the transport of E-Cadherin in mammalian cells. Thus, polarized exocytosis of E-Cad to the lateral membrane is dependent upon its interaction with Arm. And, as stated above, REs have shown to serve as an intermediate during the transport of E-Cad from the Golgi to the lateral membrane where E-Cadherin, β-Catenin, and α-Catenin form the AJs (Lock, 2005). Furthermore, the overexpression of a dominant-negative form of Rab11 impairs the delivery of E-Cadherin to the lateral membrane (Lock, 2005). Consistent with the exocyst regulating trafficking from the REs, exocyst components also localize on the REs, and Sec15 is an effector of Rab11 (Folsch, 2003: Prigent, 2003 ; Zhang, 2004). Finally, E-Cadherin and catenins are associated with exocyst components (Yeaman, 2004; Langevin, 2005).
In conclusion, this work provides evidence for a conserved role of the exocyst in regulating the delivery of E-Cadherin from REs to sites on the plasma membrane and in thereby contributing to the maintenance of epithelial cell polarity (Langevin, 2005).
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date revised: 12 March 2006
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