Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension

Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin.     

Regulation of actin filament cross-linking and bundle shape in Drosophila bristles

Previous studies demonstrate that in developing Drosophila bristles, two cross-linking proteins are required sequentially to bundle the actin filaments that support elongating bristle cells. The forked protein initiates the process and facilitates subsequent cross-linking by fascin. Using cross-linker-specific antibodies, mutants, and drugs we show that fascin and actin are present in excessive amounts throughout bundle elongation. In contrast, the forked cross-linker is limited throughout bundle formation, and accordingly, regulates bundle size and shape. We also show that regulation of cross-linking by phosphorylation can affect bundle size. Specifically, inhibition of phosphorylation by staurosporine results in a failure to form large bundles if added during bundle formation, and leads to a loss of cross-linking by fascin if added after the bundles form. Interestingly, inhibition of dephosphorylation by okadaic acid results in the separation of the actin bundles from the plasma membrane. We further show by thin section electron microscopy analysis of mutant and wild-type bristles that the amount of material that connects the actin bundles to the plasma membrane is also limited throughout bristle elongation. Therefore, overall bundle shape is determined by the number of actin filaments assembled onto the limited area provided by the connector material. We conclude that assembly of actin bundles in Drosophila bristles is controlled in part by the controlled availability of a single cross-linking protein, forked, and in part by controlled phosphorylation of cross-links and membrane actin connector proteins.     

Expression of the 53 kD forked protein rescues F-actin bundle formation and mutant bristle phenotypes in Drosophila.

forked mutations affect bristle development in Drosophila pupae, resulting in short, thick, gnarled bristles in the adult. The forked proteins are components of 200-300-microm-long actin fiber bundles that are present transiently during pupal development [Petersen et al., 1994: Genetics 136:173-182]. These bundles are composed of segments of 3-10 microm long, and forked protein is localized along the actin fiber bundle segments and accumulates at the junctions connecting them longitudinally. In the forked mutants, f(36a) and f(hd), F-actin bundles are greatly reduced in number and size, and bundle segmentation is absent. The p-element, P[w(+), falter] contains a 5.3-kb fragment of the forked gene that encodes the 53-kD forked protein [Lankenau et al., 1996: Mol Cell Biol 16:3535-3544]. Expression of only the 53-kD forked protein is sufficient to rescue the actin bundle and bristle phenotypes of f(36a) and f(hd) mutant flies. The 5.3-kb forked sequence, although smaller than the 13-kb region previously shown to rescue forked mutants [Petersen et al., 1994: Genetics 136:173-182], does contain the core forked sequence that encodes actin binding and bundling domains in cultured mammalian cells [Grieshaber and Petersen, 1999: J Cell Sci 112:2203-2211]. These data show that the 53-kD forked protein is sufficient for normal bristle development and that the domains shown previously to be important for actin bundling in cell culture may be all that are required for normal actin bundle formation in developing Drosophila bristles.     

Capping protein and the Arp2/3 complex regulate nonbundle actin filament assembly to indirectly control actin bundle positioning during Drosophila melanogaster bristle development

Drosophila melanogaster bristle development is dependent on actin assembly, and prominent actin bundles form against the elongating cell membrane, giving the adult bristle its characteristic grooved pattern. Previous work has demonstrated that several actin-regulating proteins are required to generate normal actin bundles. Here we have addressed how two actin regulators, capping protein, a barbed end binding protein, and the Arp2/3 complex, a potent actin assembly nucleator, function to generate properly organized bundles. As predicted from studies in motile cells, we find that capping protein and the Arp2/3 complex act antagonistically to one another during bristle development. However, these proteins do not primarily act directly on bundles, but rather on a dynamic population of actin filaments that are not part of the bundles. These nonbundle filaments, termed snarls, play an important role in determining the number and spacing of the actin bundles. Reduction of capping protein leads to an increase in snarls, which prevents actin bundles from properly attaching to the membrane. Conversely, loss of an Arp2/3 complex component leads to a loss of snarls and accumulation of excess membrane-attached bundles. These results indicate that in nonmotile cells dynamic actin filaments can function to regulate the positioning of stable actin structures. In addition, our results suggest that the Arpc1 subunit may have an additional function, independent of the rest of the Arp2/3 complex.     

Actin filament turnover regulated by cross-linking accounts for the size, shape, location, and number of actin bundles in Drosophila bristles

Drosophila bristle cells are shaped during growth by longitudinal bundles of cross-linked actin filaments attached to the plasma membrane. We used confocal and electron microscopy to examine actin bundle structure and found that during bristle elongation, snarls of uncross-linked actin filaments and small internal bundles also form in the shaft cytoplasm only to disappear within 4 min. Thus, formation and later removal of actin filaments are prominent features of growing bristles. These transient snarls and internal bundles can be stabilized by culturing elongating bristles with jasplakinolide, a membrane-permeant inhibitor of actin filament depolymerization, resulting in enormous numbers of internal bundles and uncross-linked filaments. Examination of bundle disassembly in mutant bristles shows that plasma membrane association and cross-bridging adjacent actin filaments together inhibits depolymerization. Thus, highly cross-bridged and membrane-bound actin filaments turn over slowly and persist, whereas poorly cross-linked filaments turnover more rapidly. We argue that the selection of stable bundles relative to poorly cross-bridged filaments can account for the size, shape, number, and location of the longitudinal actin bundles in bristles. As a result, filament turnover plays an important role in regulating cytoskeleton assembly and consequently cell shape.     

The role actin filaments play in providing the characteristic curved form of Drosophila bristles

Drosophila bristles display a precise orientation and curvature. An asymmetric extension of the socket cell overlies the newly emerging bristle rudiment to provide direction for bristle elongation, a process thought to be orchestrated by the nerve dendrite lying between these cells. Scanning electron microscopic analysis of individual bristles showed that curvature is planar and far greater near the bristle base. Correlated with this, as development proceeds the pupa gradually recedes from the inner pupal case (an extracellular layer that encloses the pupa) leading to less bristle curvature along the shaft. We propose that the inner pupal case induces elongating bristles to bend when they contact this barrier. During elongation the actin cytoskeleton locks in this curvature by grafting together the overlapping modules that comprise the long filament bundles. Because the bristle is curved, the actin bundles on the superior side must be longer than those on the inferior side. This is accomplished during grafting by greater elongation of superior side modules. Poor actin cross-bridging in mutant bristles results in altered curvature. Thus, the pattern of bristle curvature is a product of both extrinsic factors-the socket cell and the inner pupal case--and intrinsic factors--actin cytoskeleton assembly.     

Actin organization, bristle morphology, and viability are affected by actin capping protein mutations in Drosophila

Regulation of actin filament length and orientation is important in many actin-based cellular processes. This regulation is postulated to occur through the action of actin-binding proteins. Many actin-binding proteins that modify actin in vitro have been identified, but in many cases, it is not known if this activity is physiologically relevant. Capping protein (CP) is an actin-binding protein that has been demonstrated to control filament length in vitro by binding to the barbed ends and preventing the addition or loss of actin monomers. To examine the in vivo role of CP, we have performed a molecular and genetic characterization of the beta subunit of capping protein from Drosophila melanogaster. We have identified mutations in the Drosophila beta subunit-these are the first CP mutations in a multicellular organism, and unlike CP mutations in yeast, they are lethal, causing death during the early larval stage. Adult files that are heterozygous for a pair of weak alleles have a defect in bristle morphology that is correlated to disorganized actin bundles in developing bristles. Our data demonstrate that CP has an essential function during development, and further suggest that CP is required to regulate actin assembly during the development of specialized structures that depend on actin for their morphology.     

Mutational analysis of Stubble-stubbloid gene structure and function in Drosophila leg and bristle morphogenesis

The Stubble-stubbloid (Sb-sbd) gene is required for ecdysone-regulated epithelial morphogenesis of imaginal tissues during Drosophila metamorphosis. Mutations in Sb-sbd are associated with defects in apical cell shape changes critical for the evagination of the leg imaginal disc and with defects in assembly and extension of parallel actin bundles in growing mechanosensory bristles. The Sb-sbd gene encodes a type II transmembrane serine protease (TTSP). Here we use a Sb-sbd transgenic construct to rescue both bristle and leg morphogenesis defects in Sb-sbd mutations. Molecular characterization of Sb-sbd mutations and rescue experiments with wild-type and modified Sb-sbd transgenic constructs show that the protease domain is required for both leg and bristle functions. Truncated proteins that express the noncatalytic domains without the protease have dominant effects in bristles but not in legs. Leg morphogenesis, but not bristle growth, is sensitive to Sb-sbd overexpression. Antibody localization of the Sb-sbd protein shows apical expression in elongating legs. Sb-sbd protein is found in the base and shaft in budding bristles and then concentrates at the growing tip when bristles are elongating rapidly. We propose a model whereby Sb-sbd helps coordinate proteolytic modification of extracellular matrix attachments with cytoskeletal changes in both legs and bristles.     

F actin bundles in Drosophila bristles. I. Two filament cross-links are involved in bundling

Transverse sections though Drosophila bristles reveal 7-11 nearly round, plasma membrane-associated bundles of actin filaments. These filaments are hexagonally packed and in a longitudinal section they show a 12-nm periodicity in both the 1.1 and 1.0 views. From earlier studies this periodicity is attributable to cross-links and indicates that the filaments are maximally cross-linked, singed mutants also have 7-11 bundles, but the bundles are smaller, flattened, and the filaments within the bundles are randomly packed (not hexagonal); no periodicity can be detected in longitudinal sections. Another mutant, forked (f36a), also has 7-11 bundles but even though the bundles are very small, the filaments within them are hexagonally packed and display a 12-nm periodicity in longitudinal section. The singed-forked double mutant lacks filament bundles. Thus there are at least two species of cross-links between adjacent actin filaments. Hints of why two species of cross-links are necessary can be gleaned by studying bristle formation. Bristles sprout with only microtubules within them. A little later in development actin filaments appear. At early stages the filaments in the bundles are randomly packed. Later the filaments in the bundles become hexagonally packed and maximally cross-linked. We consider that the forked proteins may be necessary early in development to tie the filaments together in a bundle so that they can be subsequently zippered together by fascin (the singed gene product).     

Drosophila Kelch regulates actin organization via Src64-dependent tyrosine phosphorylation

The Drosophila kelch gene encodes a member of a protein superfamily defined by the presence of kelch repeats. In Drosophila, Kelch is required to maintain actin organization in ovarian ring canals. We set out to study the actin cross-linking activity of Kelch and how Kelch function is regulated. Biochemical studies using purified, recombinant Kelch protein showed that full-length Kelch bundles actin filaments, and kelch repeat 5 contains the actin binding site. Two-dimensional electrophoresis demonstrated that Kelch is tyrosine phosphorylated in a src64-dependent pathway. Site-directed mutagenesis determined that tyrosine residue 627 is phosphorylated. A Kelch mutant with tyrosine 627 changed to alanine (KelY627A) rescued the actin disorganization phenotype of kelch mutant ring canals, but failed to produce wild-type ring canals. Electron microscopy demonstrated that phosphorylation of Kelch is critical for the proper morphogenesis of actin during ring canal growth, and presence of the nonphosphorylatable KelY627A protein phenocopied src64 ring canals. KelY627A protein in ring canals also dramatically reduced the rate of actin monomer exchange. The phenotypes caused by src64 mutants and KelY627A expression suggest that a major function of Src64 signaling in the ring canal is the negative regulation of actin cross-linking by Kelch.     

F-actin bundles in Drosophila bristles are assembled from modules composed of short filaments

The actin bundles in Drosophila bristles run the length of the bristle cell and are accordingly 65 microns (microchaetes) or 400 microns (macrochaetes) in length, depending on the bristle type. Shortly after completion of bristle elongation in pupae, the actin bundles break down as the bristle surface becomes chitinized. The bundles break down in a bizarre way; it is as if each bundle is sawed transversely into pieces that average 3 microns in length. Disassembly of the actin filaments proceeds at the "sawed" surfaces. In all cases, the cuts in adjacent bundles appear in transverse register. From these images, we suspected that each actin bundle is made up of a series of shorter bundles or modules that are attached end-to-end. With fluorescent phalloidin staining and serial thin sections, we show that the modular design is present in nondegenerating bundles. Decoration of the actin filaments in adjacent bundles in the same bristle with subfragment 1 of myosin reveals that the actin filaments in every module have the same polarity. To study how modules form developmentally, we sectioned newly formed and elongating bristles. At the bristle tip are numerous tiny clusters of 6-10 filaments. These clusters become connected together more basally to form filament bundles that are poorly organized, initially, but with time become maximally cross-linked. Additional filaments are then added to the periphery of these organized bundle modules. All these observations make us aware of a new mechanism for the formation and elongation of actin filament bundles, one in which short bundles are assembled and attached end-to-end to other short bundles, as are the vertical girders between the floors of a skyscraper.     

Twinfilin is required for actin-dependent developmental processes in Drosophila.

The actin cytoskeleton is essential for cellular remodeling and many developmental and morphological processes. Twinfilin is a ubiquitous actin monomer-binding protein whose biological function has remained unclear. We discovered and cloned the Drosophila twinfilin homologue, and show that this protein is ubiquitously expressed in different tissues and developmental stages. A mutation in the twf gene leads to a number of developmental defects, including aberrant bristle morphology. This results from uncontrolled polymerization of actin filaments and misorientation of actin bundles in developing bristles. In wild-type bristles, twinfilin localizes diffusively to cytoplasm and to the ends of actin bundles, and may therefore be involved in localization of actin monomers in cells. We also show that twinfilin and the ADF/cofilin encoding gene twinstar interact genetically in bristle morphogenesis. These results demonstrate that the accurate regulation of size and dynamics of the actin monomer pool by twinfilin is essential for a number of actin-dependent developmental processes in multicellular eukaryotes.     

The serine/threonine kinase dPak is required for polarized assembly of F-actin bundles and apical-basal polarity in the Drosophila follicular epithelium

During epithelial development cells become polarized along their apical-basal axis and some epithelia also exhibit polarity in the plane of the tissue. Mutations in the gene encoding a Drosophila Pak family serine/threonine kinase, dPak, disrupt the follicular epithelium that covers developing egg chambers during oogenesis. The follicular epithelium normally exhibits planar polarized organization of basal F-actin bundles such that they lie perpendicular to the anterior-posterior axis of the egg chamber, and requires contact with the basement membrane for apical-basal polarization. During oogenesis, dPak becomes localized to the basal end of follicle cells and is required for polarized organization of the basal actin cytoskeleton and for epithelial integrity and apical-basal polarity. The receptor protein tyrosine phosphatase Dlar and integrins, all receptors for extracellular matrix proteins, are required for polarization of the basal F-actin bundles, and for correct dPak localization in follicle cells. dpak mutant follicle cells show increased beta(Heavy)-spectrin levels, and we speculate that dPak regulation of beta(Heavy)-spectrin, a known participant in the maintenance of membrane domains, is required for correct apical-basal polarization of the membrane. We propose that dPak mediates communication between the basement membrane and intracellular proteins required for polarization of the basal F-actin and for apical-basal polarity.     

A subset of dynamic actin rearrangements in Drosophila requires the Arp2/3 complex

The Arp2/3 complex has been shown to dramatically increase the slow spontaneous rate of actin filament nucleation in vitro, and it is known to be important for remodeling the actin cytoskeleton in vivo. We isolated and characterized loss of function mutations in genes encoding two subunits of the Drosophila Arp2/3 complex: Arpc1, which encodes the homologue of the p40 subunit, and Arp3, encoding one of the two actin-related proteins. We used these mutations to study how the Arp2/3 complex contributes to well-characterized actin structures in the ovary and the pupal epithelium. We found that the Arp2/3 complex is required for ring canal expansion during oogenesis but not for the formation of parallel actin bundles in nurse cell cytoplasm and bristle shaft cells. The requirement for Arp2/3 in ring canals indicates that the polymerization of actin filaments at the ring canal plasma membrane is important for driving ring canal growth.     

Analysis of PINCH function in Drosophila demonstrates its requirement in integrin-dependent cellular processes

Integrins play a crucial role in cell motility, cell proliferation and cell survival. The evolutionarily conserved LIM protein PINCH is postulated to act as part of an integrin-dependent signaling complex. In order to evaluate the role of PINCH in integrin-mediated cellular events, we have tested directly the in vivo function of PINCH in Drosophila melanogaster. We demonstrate that the steamer duck (stck) alleles that were first identified in a screen for potential integrin effectors represent mutations in Drosophila pinch. stck mutants die during embryogenesis, revealing a key role for PINCH in development. Muscle cells within embryos that have compromised PINCH function display disturbed actin organization and cell-substratum adhesion. Mutation of stck also causes failure of integrin-dependent epithelial cell adhesion in the wing. Consistent with the idea that PINCH could contribute to integrin function, PINCH protein colocalizes with betaPS integrin at sites of actin filament anchorage in both muscle and wing epithelial cells. Furthermore, we show that integrins are required for proper localization of PINCH at the myotendinous junction. The integrin-linked kinase, ILK, is also essential for integrin function. We demonstrate that Drosophila PINCH and ILK are complexed in vivo and are coincident at the integrin-rich muscle-attachment sites in embryonic muscle. Interestingly, ILK localizes appropriately in stck mutant embryos, therefore the phenotypes exhibited by the stck mutants are not attributable to mislocalization of ILK. Our results provide direct genetic evidence that PINCH is essential for Drosophila development and is required for integrin-dependent cell adhesion.     

Asymmetric Microtubule Function Is an Essential Requirement for Polarized Organization of the Drosophila Bristle

While previous studies have shown that microtubules (MTs) are essential for maintaining the highly biased axial growth of the Drosophila bristle, the mechanism for this process has remained vague. We report that the MT minus-end marker, Nod-KHC, accumulates at the bristle tip, suggesting that the MT network in the bristle is organized minus end out. Potential markers for studying the importance of properly polarized MTs to bristle axial growth are Ik2 and Spindle-F (Spn-F), since mutations in spn-F and ik2 affect bristle development. We demonstrate that Spn-F and Ik2 are localized to the bristle tip and that mutations in ik2 and spn-F affect bristle MT and actin organization. Specifically, mutation in ik2 affects polarized bristle MT function. It was previously found that the hook mutant exhibited defects in bristle polarity and that hook is involved in endocytic trafficking. We found that Hook is localized at the bristle tip and that this localization is affected in ik2 mutants, suggesting that the contribution of MTs within the bristle shaft is important for correct endocytic trafficking. Thus, our results show that MTs are organized in a polarized manner within the highly elongated bristle and that this organization is essential for biased bristle axial growth.Polarized cell growth, manifested as cellular growth biased toward one pole of a cell, is the result of dynamic developmental processes that require an extensive reorganization of the cytoplasm in response to both intracellular and extracellular signals. Essentially, all cells can polarize in response to internal and/or external cues, such as matrix components, cell-cell contacts, or chemical gradients. Eukaryotic cells generally interpret these cues by assembling a polarized actin cytoskeleton at the cortex, which in turn coordinates with microtubules to guide internal membranes. This network ultimately polarizes events that occur internally and at the cell surface (10). A critical issue in this respect concerns how the cytoskeleton responds to those cues that lead to polarized growth.During development, Drosophila epidermal cells form a variety of polarized structures. These include the epidermal hairs that decorate much of the adult cuticular surface, the shafts of the bristle sense organs, the lateral extensions of the arista, and the larval denticles. These cuticular structures are produced by cytoskeleton-mediated outgrowths of the epiderma (13, 16). Since alterations in bristle morphology are easy to detect in living flies and since small changes in the actin cytoskeleton, as induced by drugs or mutations, often result in an easily detectable phenotype, the growth of the bristle cell is used to define the role of the cytoskeleton in polarized cell growth.Bristle cells sprout during metamorphosis and elongate over the course of ∼18 h. Growth is driven by actin filament polymerization (41). The actin bundles in bristle sprouts begin as microvilli (45) and are cross-bridged into modular bundles 1 to 5 μm in length by at least two cross-linking proteins, forked and fascin (43, 45, 46). These modules are then grafted together by end-to-end joining into stiff bundles (15) which run longitudinally along the bristle shaft, attached to the plasma membrane (40), to support the cell extension as well-spaced ribs. Bundles are tapered, with the largest cross-sectional area of individual bundles found at the base, containing >500 filaments (40). In Drosophila pupae, developing bristles contain 7 to 11 (microchaeta) or 12 to 18 (macrochaeta) bundles of cross-linked actin filaments and a large population of microtubules (MTs) that run longitudinally along the bristle shaft. It was suggested that bristle MTs are highly stable, forming at the start of elongation and then moving out along the shaft as the cell elongates (44). Inhibitor studies suggest that MTs are essential for maintaining bristle axial growth, since injection of microtubule antagonists, such as vinblastine, into pupae resulted in short and fat bristles (13).It was previously demonstrated that mutations in the Drosophila ikkɛ homologue, ik2, and in the novel gene spindle-F (spn-F), which is not conserved outside insect species, affect both egg chamber polarity and bristle development (1, 37). During oogenesis, both ik2 and spn-F affect mRNA localization due to their effects on actin and MT minus-end organization. Moreover, we were able to show that Ik2 and Spn-F form a complex that regulates cytoskeleton organization during Drosophila oogenesis, with Spn-F serving as the direct regulatory target for Ik2 kinase activity (11). Further evidence for the role of ik2 in cytoskeleton-related processes comes from its interaction with the Drosophila inhibitor of apoptosis 1 (DIAP1). It was suggested that ik2 acts as a negative regulator of F-actin assembly and maintains the fidelity of polarized elongation during cell morphogenesis by modulating DIAP1 levels (22, 29). Recently it was shown that ik2 regulates the dendrite pruning involved in MT disassembly (23).Since ik2 and spn-F affect bristle polarity organization, we investigated the role of these genes in shaping bristle morphology. We report that MTs within the bristle are organized in a polarized manner, minus-end out. We also demonstrate that both the Spn-F and Ik2 proteins are localized to the bristle tip. Close examination during the bristle elongation period revealed that mutations in either gene affect cytoskeleton organization. Specifically, upon mutation of ik2, the MT minus-end marker is no longer accumulated at the bristle tip. Moreover, we found that the Hook protein is localized at the bristle tip and that such localization is affected in spn-F and ik2 mutants, suggesting that MT functionality within the bristle is essential for recruitment of components of the endocytic trafficking to the tip of the bristle. Thus, we suggest that ik2 and spn-F affect MT functions which are required for the biased axial shape of the bristle. This, in turn, affects the localization of the endocytic trafficking machinery to the bristle tip.     

Drosophila quail, a villin-related protein, bundles actin filaments in apoptotic nurse cells

Drosophila Quail protein is required for the completion of fast cytoplasm transport from nurse cells to the oocyte, an event critical for the production of viable oocytes. The abundant network of cytoplasmic filamentous actin, established at the onset of fast transport, is absent in quail mutant egg chambers. Previously, we showed that Quail is a germline-specific protein with sequence homology to villin, a vertebrate actin-regulating protein. In this study, we combined biochemical experiments with observations in egg chambers to define more precisely the function of this protein in the regulation of actin-bundle assembly in nurse cells. We report that recombinant Quail can bind and bundle filamentous actin in vitro in a manner similar to villin at a physiological calcium concentration. In contrast to villin, Quail is unable to sever or cap filamentous actin, or to promote nucleation of new actin filaments at a high calcium concentration. Instead, Quail bundles the filaments regardless of the calcium concentration. In vivo, the assembly of nurse-cell actin bundles is accompanied by extensive perforation of the nurse-cell nuclear envelopes, and both of these phenomena are manifestations of nurse-cell apoptosis. To investigate whether free calcium levels are affected during apoptosis, we loaded egg chambers with the calcium indicator Indo-1. Our observations indicate a rise in free calcium in the nurse-cell cytoplasm coincident with the permeabilization of the nuclear envelopes. We also show that human villin expressed in the Drosophila germline could sense elevated cytoplasmic calcium; in nurse cells with reduced levels of Quail protein, villin interfered with actin-bundle stability. We conclude that Quail efficiently assembles actin filaments into bundles in nurse cells and maintains their stability under fluctuating free calcium levels. We also propose a developmental model for the fast phase of cytoplasm transport incorporating findings presented in this study.     

Dusky-like functions as a Rab11 effector for the deposition of cuticle during Drosophila bristle development

The morphogenesis of Drosophila sensory bristles is dependent on the function of their actin and microtubule cytoskeleton. Actin filaments are important for bristle shape and elongation, while microtubules are thought to mediate protein and membrane trafficking to promote growth. We have identified an essential role for the bristle cuticle in the maintenance of bristle structure and shape at late stages of bristle development. We show that the small GTPase Rab11 mediates the organized deposition of chitin, a major cuticle component in bristles, and disrupting Rab11 function leads to phenotypes that result from bristle collapse rather than a failure to elongate. We further establish that Rab11 is required for the plasma membrane localization of the ZP domain-containing Dusky-like (Dyl) protein and that Dyl is also required for cuticle formation in bristles. Our data argue that Dyl functions as a Rab11 effector for mediating the attachment of the bristle cell membrane to chitin to establish a stable cuticle. Our studies also implicate the exocyst as a Rab11 effector in this process and that Rab11 trafficking along the bristle shaft is mediated by microtubules.     

A balance of capping protein and profilin functions is required to regulate actin polymerization in Drosophila bristle

Profilin is a well-characterized protein known to be important for regulating actin filament assembly. Relatively few studies have addressed how profilin interacts with other actin-binding proteins in vivo to regulate assembly of complex actin structures. To investigate the function of profilin in the context of a differentiating cell, we have studied an instructive genetic interaction between mutations in profilin (chickadee) and capping protein (cpb). Capping protein is the principal protein in cells that caps actin filament barbed ends. When its function is reduced in the Drosophila bristle, F-actin levels increase and the actin cytoskeleton becomes disorganized, causing abnormal bristle morphology. chickadee mutations suppress the abnormal bristle phenotype and associated abnormalities of the actin cytoskeleton seen in cpb mutants. Furthermore, overexpression of profilin in the bristle mimics many features of the cpb loss-of-function phenotype. The interaction between cpb and chickadee suggests that profilin promotes actin assembly in the bristle and that a balance between capping protein and profilin activities is important for the proper regulation of F-actin levels. Furthermore, this balance of activities affects the association of actin structures with the membrane, suggesting a link between actin filament dynamics and localization of actin structures within the cell.