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Article |
Address correspondence to Andrew C. Zelhof, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0649. Tel.: (858) 534-5423. Fax: (858) 534-8510. email: azelhof{at}biomail.ucsd.edu
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Abstract |
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Key Words: chaoptin; photoreceptor; Drosophila; amphiphysin; moesin
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Introduction |
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Biochemical analyses of brush border microvilli have provided key insights into the protein contents and organizational arrangement of the molecular components of each domain. In particular, the core of each microvillus projection consists of bundles of actin filaments (Tilney and Mooseker, 1971), which not only provide the scaffold for the overlying membrane but also create the protruding force for the generation of each microvillus. The brush border microvillus core also consists of myosin I and the actin bundling proteins villin and fimbrin (Bretscher and Weber, 1979, 1980; Coluccio and Bretscher, 1989; Mooseker and Coleman, 1989). Based on the identified components, it is not clear how or what initiates the construction of the actin cytoskeleton. For instance, villin is one of the earliest proteins to be enriched on the apical surface of enterocytes and has the ability to nucleate and collect F-actin into bundles (Friederich et al., 1990; Fath and Burgess, 1995). However, genetic knockouts of villin resulted in no discernible disruptions of microvilli ultrastructure (Pinson et al., 1998; Ferrary et al., 1999).
One set of candidates for initiating actin nucleation during microvillus formation is the ezrin/radixin/moesin family of proteins (Bretscher, 1989; Berryman et al., 1993). In the retinal pigment epithelium, the temporal and spatial expression of Ezrin corresponds with the creation of the apical microvilli, and in primary retinal pigment epithelium cultures, Ezrin antisense treatment eliminates microvillus formation (Bonilha et al., 1999). Nonetheless, how Ezrin may be coordinating microvillus biogenesis is unknown. A second possibility is that the molecular mechanisms of microvillus initiation may be similar to the formation of filopodia and lamellipodia membrane protrusions. In both cases, a signaling cue activates a small Rho GTPase upon which the rapid rearrangement of actin proceeds through the activation of Wiskott-Aldrich syndrome protein (WASp) and WAVE family members (Takenawa and Miki, 2001; Thrasher, 2002). WASp and WAVE family members can directly bind actin as well as activate the actin nucleating complex Arp 2/3 (Mullins, 2000).
In Drosophila melanogaster, the photoreceptor cells have a very extensive network of microvilli contained within the rhabdomere. The rhabdomere is a specialized organelle that forms on the photoreceptor apical surface. It is the functional equivalent of vertebrate rod and cone cell outer segments and both are responsible for housing the phototransduction proteins. Like the brush border, each rhabdomeric microvillus contains an actin cytoskeleton core but only has two parallel actin filaments (Arikawa et al., 1990). As for formation of the rhabdomere actin cytoskeleton core, only Bifocal has been implicated in its assembly. Bifocal is a novel protein that binds F-actin, and the loss of Bifocal results in irregular rhabdomere morphology (Bahri et al., 1997).
Clearly, the combination of structural and biochemical analyses have identified numerous proteins involved in the development of brush border or rhabdomeric microvilli. Nevertheless, little is known about how assembly of the actin cytoskeleton core is initiated, how the actin cytoskeleton interacts with the plasma membrane, and how the growth of each microvillus is regulated. Using Drosophila rhabdomere development as an experimental model system for understanding the biogenesis and maintenance of microvilli, we demonstrate that the Drosophila WASp is required for the correct temporal apical enrichment of F-actin and initiation of the primordial microvillar projections.
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Results |
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To further address this hypothesis, we took a biochemical approach to identify proteins that bind the SH3 domain of Amph. GST pull-down experiments using Drosophila extracts indicated several proteins that specifically interact with the SH3 domain of Amph (unpublished data). Testing a library of antibodies, one protein was identified as the Drosophila homologue of WASp. WASp is a member of an evolutionarily conserved family of proteins that act as integrators of multiple signaling pathways to direct the specific subcellular localization and polymerization of actin. The binding of WASp to Amph is dependent on the SH3 domain of Amph, and mutation of SH3 domain results in the disruption of this interaction (Fig. 1). SH3 domains bind proline-rich sequences, and within WASp there are three peptides that loosely fit the characterized consensus-binding site for the vertebrate SH3 domain of Amph-2 (PXRPXR; Owen et al., 1998; Fig. 1 B). The removal of this proline-rich domain also eliminates binding to Amph (Fig. 1 C).
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Localization of rhabdomeric proteins depends on WASp function
Based on our initial findings, we now have a mutation that affects, in a cell-autonomous fashion, the F-actin enrichment associated with the formation of the primordial microvillar projections that give rise to the rhabdomere. Numerous components involved in microvillus development have been identified, but the method of assembly or the epistatic relationships of the various proteins remain largely undefined. We can now address the question of whether the formation of the F-actin core is critical for the subsequent recruitment or stabilization of other rhabdomere proteins. For our analysis, we investigated three proteins (Amph, moesin, and chaoptin) that have been implicated in the rearrangement of the actin cytoskeleton and the required morphogenetic changes in the plasma membrane to create the microvillus protrusion.
Chaoptin is an integral membrane protein and decorates the entire photoreceptor membrane and axon (Van Vactor et al., 1988). Chaoptin is expressed as early as 24 h APF, but enrichment to the apical membrane is first observed at 48 h APF and coincident with the appearance of an organized actin cytoskeleton. Chaoptin is essential for the cross-linking of the individual microvillus projections. In WASp mutant photoreceptor cells, chaoptin is expressed and shows equal distribution along the plasma membrane and axons of both wild-type and mutant photoreceptor cells (Fig. 7 C and not depicted). In addition, using chaoptin as a marker, we do not detect any defects in axon outgrowth or synaptic pruning of the photoreceptor connections in the optic lobe in WASp mutant cells (unpublished data). Nevertheless, if the apical accumulation of chaoptin is dependent on the initiation of the cytoskeleton core, we would expect to see an impairment in chaoptin accumulation at the apical surface in mutant cells. Indeed, just as we observe for F-actin, chaoptin enrichment on the apical surface is delayed (Fig. 7 C). Importantly, the examination of chaoptin expression in photoreceptor cells demonstrates that the defects observed in WASp mutant cells are not due to the possibility that the entire photoreceptor cell is delayed in developmental time but rather more likely due to only a temporal failure in microvillus formation.
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Amph, like activated moesin, is not expressed or enriched on the apical surface until 48 h APF in wild-type photoreceptor cells (Zelhof et al., 2001). At the neuromuscular junction Amph is completely dependent on WASp for postsynaptic localization but not localization to the muscle T-tubule system (Fig. 2). In WASp mutant photoreceptor cells, Amph localization is severely hindered, whereas WASp accumulation is not affected by the loss of Amph (unpublished data). At 48 h APF, Amph accumulation is absent in distal optical sections and when present, appears discontinuous and blotchy (Fig. 7 G). In addition, the loss of one copy of amph in a WASp mutant background does not enhance the temporal delay seen in WASp mutant cells, but double mutants rarely survive to the stage in which rhabdomere initiation can be scored. Surprisingly, Amph recruitment to the apical surface, like moesin, is not completely abolished. By 72 h APF, Amph and moesin do assemble onto the extending microvilli in the absence of WASp (unpublished data). Together, our results suggest that both the function and physical presence of WASp contribute to the proper recruitment of microvillar proteins.
WASp mutant photoreceptor cells are delayed in the appearance of the primordial microvillar projections
Our immunofluorescence data imply that with the loss of WASp there is a temporal delay in the formation of the primordial microvillar projections. To corroborate this possibility, we examined the progression of microvillus formation in WASp mutant photoreceptor cells via an EM analysis. If WASp is required for the correct temporal enrichment of F-actin (initiation of the microvillar primordial projections) and the coincident removal of Armadillo from the juxtaposed apical surfaces of each photoreceptor cell, then examination of WASp mosaic clones should reveal defects in these processes. As such, we would predict, based on our immunofluorescence data, that ommatidia containing mutant cells should be recognizable by the inadequate separation of photoreceptor membranes, the lack of clear definable adherens junctions, and, finally, the lack of microvillar projections. Our analysis reveals that such deficiencies are present in our WASp mosaic clones and not present in WASp3 heterozygote ommatidia (Fig. 8 and not depicted). In an ommatidium in which all photoreceptor cells are wild type, we observe seven adherens junctions, the primordial microvillar projections are present on each photoreceptor cell, and the interrhabdomeral space is forming (Fig. 8, B and C). In adjacent ommatidia that putatively contain a mixture of wild-type and mutant cells, we cannot define all seven adherens junctions because the interrhabdomeral space is not equally distributed but, more importantly, there is a lack of primordial microvillar projections on all the photoreceptor cells (Fig. 8 D). In the case in which all cells of the ommatidium are mutant, we do not observe any adherens junctions, no separation or elimination of cellcell contacts between photoreceptor cells, and no presence of the microvillar projections (Fig. 8 E). The combination of our immunofluorescence and EM structural analysis strongly indicates that WASp is mediating a signal for the correct temporal initiation of microvillus formation in photoreceptor cells.
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Discussion |
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Our results demonstrate that WASp is expressed and localizes to the apical surface before the appearance of microvillar apical folds and before the enrichment/rearrangement of F-actin in photoreceptor cells. More importantly, the loss of WASp function results in malformed rhabdomeres. Typically, WASp mutant rhabdomeres are misshapen and a small percentage are split, which are phenotypes not observed in wild-type photoreceptor cells. Knowing WASp has been identified as a key component in the specific subcellular localization and polymerization of actin, we speculated that these phenotypes were a result of defects in establishing the F-actin cytoskeleton core.
The transformation of the apical surface into the rhabdomere is a highly coordinated event. After each apical surface involutes inward, there is an expansion of the apical surface down toward the retinal floor. As such, the examination of tangential sections through the depth of the photoreceptor cell represents a temporal profile of the photoreceptor apical surface as it is transformed into a rhabdomere. Inspection of markers for the initiation of microvillus formation (F-actin) and the separation and delimitation of each photoreceptor apical surface (Armadillo) in WASp mutant cells clearly demonstrated a temporal delay in rhabdomere formation compared with their neighboring wild-type counterparts. Furthermore, molecules that are implicated in the stabilization of the actin cytoskeleton core (moesin), the cross-linking of each microvillus (chaoptin), or the deformation of the overlying plasma membrane (Amph) are dependent on the presence of the primordial microvilliconfirmed from our EM analysisfor their recruitment/stabilization to the apical surface, and thus, are epistatic to WASp function. In the case of Amph, the direct association with WASp may aid in the recruitment of Amph to the apical surface. Even though all of our observations are consistent with the idea that WASp is coordinating the signal required for the transformation of the apical surface, surprisingly, WASp is not essential for the formation or growth of microvilli.
If WASp is responsible for integrating the signal for microvillus formation, what is the signal? Numerous studies have implicated a combination of upstream factors for the recruitment and activation of WASp, such as small Rho GTPases, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) phosphoinositide, and SH3-containing proteins that bind the proline-rich domain of WASp (Miki et al., 1996; Symons et al., 1996; Prehoda et al., 2000; Fukuoka et al., 2001; Takenawa and Miki, 2001). Our data are suggestive, but not conclusive, that Cdc42 may play a role in the activation of WASp. A similar delay in F-actin enrichment and subsequent recruitment of rhabdomeric proteins is observed in cdc42 mutant cells. This idea directly conflicts with the result that the Rho GTPase binding domain of WASp is not required for the rescue of viability in WASp mutants (Tal et al., 2002). However, the binding site for PI(4,5)P2 is also dispensable, and our own data demonstrate that the proline-rich domain is not necessary to rescue viability (Tal et al., 2002; unpublished data). Given that each individual domain in vivo is expendable, may be indicative that any one or a combination of the three domains can be sufficient for the recruitment and activation of WASp. In rhabdomere biogenesis, all three potential ways of activating WASp are present. The SH3 domain of Amph binds WASp. Mutation of Cdc42 results in defects in rhabdomere morphogenesis, and there is a coincident accrual of PI(4,5)P2 on the photoreceptor apical surface during the initiation of microvilli formation (Figs. 3 and 4). Only the combination of in vitro activation studies, further in vivo WASp structurefunction studies, and deciphering the role of PI(4,5)P2 metabolism in microvillus biogenesis will we be able to clarify the contribution of each regulatory domain of WASp in microvillus initiation.
Finally, it is evident from our results that a second pathway for initiating F-actin formation is present. WASp is not essential for microvillus formation, and clearly, F-actin accumulates and functional microvilli do form in WASp mutant cells. The molecular basis of this second pathway is unknown. One possible mechanism could involve a p21-activated kinase (PAK). Besides a role in cell differentiation and proliferation, PAK proteins have been implicated in the regulation of the cytoskeleton (Bagrodia and Cerione, 1999; Daniels and Bokoch, 1999). A recent paper has implicated Mbt (mushroom bodies tiny PAK) in photoreceptor morphogenesis (Schneeberger and Raabe, 2003). mbt mutant photoreceptor cells have malformed rhabdomeres, and Cdc42 is required for its correct localization in photoreceptor cells. That Cdc42 may be responsible for the activation of more than one pathway would be consistent with the fact that photoreceptor cell differentiation, and in particular rhabdomere formation, is more severely affected in cdc422 mutant cells compared with WASp mutant cells (unpublished data). In addition to a broader role of Cdc42, a second member of the WASp/WAVE family, SCAR, exists in Drosophila. SCAR is required for patterning of the eye ommatidium and external morphology (Zallen et al., 2002). Thus, SCAR activity could account for the assembly of actin observed in WASp mutant cells. However, it will be necessary to genetically separate the early requirement of SCAR in ommatidial patterning before examining its contribution to microvillus initiation. Overall, our work has now defined a budding network of molecules required for microvillus initiation and provided a framework in which we can further elucidate the mechanisms of microvillus biogenesis and regulation.
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Materials and methods |
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Staining of pupal photoreceptor cells
Developing whole retinas were dissected at the appropriate time and fixed in PEMFA (100 mM PIPES, 2 mM EGTA, 1 mM MgSO4, and 3.7% formaldehyde) for 1560 min. The tissue was blocked in PBTB (PBS and 0.1% Triton X-100 with 1% BSA) for 30 min, incubated in primary antibody (in PBTB) overnight at 4°C, washed in PBTB, incubated in secondary antibody for 2 h at 22°C, washed in PBT (PBS and 0.1% Triton X-100), and mounted in polyvinyl alcohol/DABCO mounting media (Sigma-Aldrich). The primary antibodies used were rabbit anti-Amph (Zelhof et al., 2001), antiphosphorylated moesin (Cell Signaling), mouse anti-chaoptin (Zipursky et al., 1984), and mouse anti-Armadillo (DSHB). Rhodamine-conjugated Phalloidin (Molecular Probes) was used for the detection of F-actin. FITC-, Red-X, and Cy5-conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories. All images were captured on a confocal microscope (model Diaphot 200 [Nikon]; model MRC1024 [Bio-Rad Laboratories]) with a 60x (1.4) oil lens (with an additional 5x zoom). The confocal Z-series were processed using Confocal Assistant software (version 4.02) and Adobe Photoshop.
Transmission EM analysis
Drosophila heads were fixed (4% formaldehyde, 3.5% glutaraldehyde, 100 mM cacodylate buffer, and 2 mM CaCl) for 3 h at 22°C, and then fixed (4% formaldehyde, 3.5% glutaraldehyde, 100 mM cacodylate buffer, 2 mM CaCl, and 1% tannic acid) overnight at 4°C. Heads were rinsed in 100 mM cacodylate buffer and postfixed in 2% osmium in 100 mm cacodylate buffer for 1 h at 22°C. The heads were dehydrated through an ethanol series and rinsed three times with propylene oxide. The heads were embedded in Spurrs for sectioning. Images of sections were captured on an electron microscope (model 1200 EX II; JEM) and photo-prints were scanned into Adobe Photoshop.
GST binding assays
Drosophila tissue was placed in extraction/binding buffer (100 mM KCl, 20 mM Hepes, 5% glycerol, 10 mM EDTA, and 0.1% Triton X-100 with the proteinase inhibitors [protease inhibitor cocktail; Roche]), homogenized, and sonicated. Transfected cells were placed in cell lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 1% NP-40, and 0.5% (wt/wt) sodium deoxycholate with proteinase inhibitors). In each case, the supernatant was collected and incubated with the appropriate GST fusion protein (Zelhof et al., 2001). The bound proteins were pelleted with glutathione Sepharose beads (Amersham Biosciences) and washed three times with extraction/binding buffer. The pellets were resuspended and equal volume of 2x sample/loading buffer was added. All extracts were resolved by SDS-PAGE and transferred to Immobilon-P (Millipore). Protein detection was done as described previously (Baker et al., 1994). Rabbit anti-WASp antibody was used at a concentration of 1:1,000 (obtained from E. Schejter).
DNA constructs and transfections
The full-length WASp cDNA (RE12101) was obtained from Research Genetics/Invitrogen. The full-length cDNA and the cDNA representing the internal deletion of amino acids (316400) were cloned into pcDNA3 (Invitrogen) and pUAST and transformed into flies. Modified HEK-293 cells (PEAKRAPID cells; Edge BioSystems) were transfected with GenePorter 2 (Gene Therapy Systems) and were harvested 48 h after transfection. The pleckstrin homology domain of PLC1 (Lemmon et al., 1995; Raucher et al., 2000) was cloned upstream and in frame with GFP in pUAST. The construct was transformed into flies and several transgenic lines were established. Expression was visualized by crossing the transgenic lines to GMR-GAL4.
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Acknowledgments |
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A.C. Zelhof was supported by National Institutes of Health (NIH) National Research Service Award (grant DC00432-02). This work was supported in part by NIH grant EY06979 to Dr. Charles S. Zuker, and C.S. Zuker is an investigator of the Howard Hughes Medical Institute.
Submitted: 8 July 2003
Accepted: 2 December 2003
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