|Gene name - singed
Cytological map position - 7D1-2
Function - crosslinks actin filaments
Keywords - cytoskeleton
|Symbol - sn
FlyBase ID: FBgn0003447
Genetic map position - 1-21.0
Classification - fascin
Cellular location - cytoplasmic
|Recent literature||Kelpsch, D. J., Groen, C. M., Fagan, T. N., Sudhir, S. and Tootle, T. L. (2016). Fascin regulates nuclear actin during Drosophila oogenesis. Mol Biol Cell [Epub ahead of print]. PubMed ID: 27535426
Drosophila oogenesis provides a developmental system to study nuclear actin. During Stages 5-9, nuclear actin levels are high in the oocyte and exhibit variation within the nurse cells. Cofilin and Profilin, which regulate the nuclear import and export of actin, also localize to the nuclei. Expression of GFP-tagged Actin results in nuclear actin rod formation. These findings indicate that nuclear actin must be tightly regulated during oogenesis. One factor mediating this regulation is Fascin. Overexpression of Fascin enhances nuclear GFP-Actin rod formation, and Fascin colocalizes with the rods. Loss of Fascin reduces, while overexpression of Fascin increases, the frequency of nurse cells with high levels of nuclear actin; but neither alters the overall nuclear level of actin within the ovary. These data suggest that Fascin regulates the ability of specific cells to accumulate nuclear actin. Evidence indicates Fascin positively regulates nuclear actin through Cofilin. Loss of Fascin results in decreased nuclear Cofilin. Additionally, Fascin and Cofilin genetically interact, as double heterozygotes exhibit a reduction in the number of nurse cells with high nuclear actin levels. These findings are likely applicable beyond Drosophila follicle development, as the localization and functions of Fascin, and the mechanisms regulating nuclear actin, are widely conserved.
|Otani, T., Ogura, Y., Misaki, K., Maeda, T., Kimpara, A., Yonemura, S. and Hayashi, S. (2016). IKK inhibits PKC to promote Fascin-dependent actin bundling. Development [Epub ahead of print]. PubMed ID: 27578797
Signaling molecules have pleiotropic functions and are activated by various extracellular stimuli. Protein kinase C (PKC) is activated by diverse receptors, and its dysregulation is associated with diseases including cancer. However, how the undesired activation of PKC is prevented during development remains poorly understood. Previous studies have shown that a protein kinase, IKK, is active at the growing bristle tip and regulates actin bundle organization during Drosophila bristle morphogenesis. This study demonstrated that IKK regulates the actin bundle localization of a dynamic actin cross-linker, Fascin. IKK inhibits PKC, thereby protecting Fascin from its inhibitory phosphorylation. Excess PKC activation is responsible for the actin bundle defects in ikk-deficient bristles, whereas PKC is dispensable for bristle morphogenesis in wildtype bristles, indicating that PKC is repressed by IKK in wildtype bristle cells. These results suggest that IKK prevents excess activation of PKC during bristle morphogenesis.
X-linked singed mutants were first described by Mohr (1922). The gnarled, kinky bristle phenotype is due to the lack of Actin filament bundles in both large and small bristles. With the cloning of sea urchin Fascin, it became clear that the previously cloned singed in fact codes for the Drosophila homolog of Fascin. Fascin bundles actin filaments into large tightly packed hexagonal arrays that support cellular processes including stress fibers, microvillar projections and filopodial extentions. singed is also involved in two oogenic processes: cytoplasmic streaming (in which contents of nurse cells are transferred into the developing oocyte) and follicle cell migration, in particular the migration of border cells and centripetal follicle cells (Cant, 1994).
It has recently been found that ß-Catenin, the vertebrate homolog of Armadillo, associates with Fascin. Fascin binds to the Armadillo repeat domain, a region known to associate with E-cadherin, the vertebrate homolog of Shotgun. The association of ß-cadherin with Fascin and with E-cadherin is mutually exclusive. It would thus be predicted that the biological processes of cell-cell adhesion via cadherin and filament-bundling/cell motility via fascin are coordinately regulated via the competitive titration of ß -catenin (Tao, 1996).
The interaction of Singed with the vertebrate homolog of Armadillo raises the possibility that Singed is involved in segment polarity. It is unlikely, however, that the ß-catenin - Fascin - E-cadherin interaction is involved in segment polarity, as there is no effect of singed mutation on the functioning of the wingless pathway (which involves Armadillo). Likewise, in vertebrates ß-catenin is not observed in fascin-containing stress fibers emanating from cell substrate (focal) contacts. Examples of known actin bundlers include fimbrin, alpha-actinin, alpha-catenin and fascin. It is therefore likely that different protein complexes mediate interactions between the cell surface and the cytoskeleton in different developmental contexts (Tao, 1996).
Fascin is well characterized in vitro as an actin-bundling protein and its increased expression is correlated with the invasiveness of various cancers. However, the actual roles and regulation of Fascin in vivo remain elusive. This study shows that Fascin is required for the invasive-like migration of blood cells in Drosophila embryos. Fascin expression is highly regulated during embryonic development and, within the blood lineage, is specific to the motile subpopulation of cells, which comprises macrophage-like plasmatocytes. Fascin is required for plasmatocyte migration, both as these cells undergo developmental dispersal and during an inflammatory response to epithelial wounding. Live analyses further demonstrate that Fascin localizes to, and is essential for the assembly of, dynamic actin-rich microspikes within plasmatocyte lamellae that polarize towards the direction of migration. A regulatory serine of Fascin identified from in vitro studies is not required for in vivo cell motility, but is crucial for the formation of actin bundles within epithelial bristles. Together, these results offer a first glimpse into the mechanisms regulating Fascin function during normal development, which might be relevant for understanding the impact of Fascin in cancers (Zanet, 2009).
These results show that the actin-bundling protein Fascin mediates several aspects of the cytoskeletal reorganization required for blood cell migration. Fascin is expressed specifically in the motile subpopulation of embryonic hemocytes, the plasmatocytes. Furthermore, Fascin expression is eventually lost in plasmatocytes at larval stages, when these cells become immobile (Babcock, 2008; Brock, 2008), showing that Fascin is characteristic of motile populations of blood cells. Consistent with observations in cultured cells, this study shows that Fascin is enriched in filopodia-like cellular extensions, or microspikes, within the lamellipodia of migrating plasmatocytes in vivo. The absence of Fascin impaired their migration, leading to delayed and incomplete developmental dispersal of plasmatocytes. In addition to their developmental dispersal, Drosophila plasmatocytes are rapidly responsive to epithelial wounding and are drawn to the damaged tissue where they may contribute to defense against septic infection. Interestingly, the chemotaxis of inflammatory-induced migration relies on different signaling mechanisms to those that guide developmental dispersal. Nevertheless, Fascin deprivation also disrupts the migration of plasmatocytes to a wound site, showing that Fascin exerts a general function in the motility of blood cells, beyond the differential nature of guidance cues (Zanet, 2009).
A prominent characteristic of living plasmatocytes in vivo is their polarization along their direction of migration, a feature that is generally lost in fixed specimens because of the fragility of actin-rich protrusions. Confocal movies show that the trailing edge of wild-type plasmatocytes displays a condensed organization of the cytoskeleton and cytoplasm that surround the nucleus. By contrast, the leading region organizes dynamic cell processes that are remodeled continuously during migration. These highly motile extensions are likely to contribute to exploration of the microenvironment for guidance cues as well as to forward propulsion of the cell. In the absence of Fascin, this polarized organization is disrupted and mutant plasmatocytes display an extended lamella that surrounds the whole cell. In addition, the abnormal lamella of Fascin-depleted plasmatocytes undergoes only limited reorganization over time, as compared with wild-type cells. Fascin displays a reversible and highly dynamic interaction with actin, with a half-life of 6-9 seconds as estimated in vitro (Aratyn, 2007). In vivo results suggest that this dynamic Fascin-actin interaction underlies the formation of highly motile lamellipodia/filopodia at the leading edge of migrating cells. Thus, a major role of Fascin in blood cells is to mediate the polarized organization of actin filaments at the migration front, supporting the proposed role of Fascin in invasive tumor cells (Mattila, 2008; Vignjevic, 2006; Zanet, 2009 and references therein).
Nevertheless, removal of Fascin in plasmatocytes not only prevents the formation of cell extensions but also causes a general loss of trailing versus leading edge polarity. It is proposed that Fascin is required to respond to the guidance molecules that provoke the polarization of plasmatocytes and direct their migration. Since filopodia contain receptors for diffusible signals or extra cellular matrix (ECM) molecules, it is possible that the absence of Fascin impairs efficient receptor localization or downstream signaling. Lack of Fascin might also prevent the mechanical transmission of the forces that reorganize the cytoskeleton during migration, as there is evidence (Vignjevic, 2006) that Fascin provides stiffness to actin bundles (Zanet, 2009).
Taken together, these data, collected through functional analyses in live embryos, demonstrate the importance of Fascin in dynamic filopodia assembly during the migration of Drosophila embryonic macrophages. Differential regulation of Fascin activity during development Fascin is likely to be controlled at the post-transcriptional level in Drosophila. Studies in mammalian cells have shown that following ECM-mediated signaling, Fascin can interact with protein kinase Cα (Anilkumar, 2003), which phosphorylates Fascin on a serine residue in the N-terminal actin-binding site. A phosphomimetic mutation weakens the actin-bundling activity of Fascin in vitro (Vignjevic, 2003) and reduces the number and length of filopodia when it is overexpressed in cultured cells (Vignjevic, 2006). Since this protein kinase C target site has been conserved throughout evolution, this study evaluated the importance of this serine in vivo through the substitution of endogenous Fascin with mutants preventing (S52A) or mimicking (S52D/E) its phosphorylation. Consistent with in vitro assays, the phosphomimetic mutation blocks Fascin activity in bristles. By contrast, Fascin S52A is fully active, showing that the actin-bundling activity of Fascin in bristles relies on a non-phosphorylated form. Therefore, these data demonstrate the importance of this regulatory serine in vivo (Zanet, 2009).
Unexpectedly, all Fascin forms (wild-type, S52A and S52E) display indistinguishable activities with respect to plasmatocyte migration. In both developmental and inflammatory-induced migration, the two reciprocal Fascin phosphovariants rescue plasmatocyte motility to the same extent as the wild-type protein. All Fascin variants further display a similar enrichment in filopodia and sustain the formation of a polarized lamella. Therefore, modifications of S52 do not influence Fascin activity for plasmatocyte migration (Zanet, 2009).
An intriguing question is why S52E nullifies the function of Fascin in bristles and yet has no effect on blood cell migration. One possibility is that phosphomimetic mutations specifically inactivate the bundling activity of Fascin, which might be dispensable for cell migration. This is, however, not the case because both phosphovariants appear to fulfill wild-type bundling activity, at least for the formation of actin cables in nurse cells. It is proposed that the main difference between phosphorylation-sensitive and -insensitive developmental processes is linked to architectural differences in tissue-specific actin structures that might require different kinetic properties of Fascin. The formation of bristle cell extensions is a relatively slow process that would require a stable interaction of Fascin with actin filaments, which is prevented by S52E mutations. By contrast, actin cables of nurse cells display a dynamic reorganization that is required for dumping the nurse cell cytoplasm into the oocyte. The reorganization of the actin cytoskeleton that occurs even faster during plasmatocyte migration might also be insensitive to a decreased half-life of Fascin-actin interaction. It is noteworthy that phosphomimetic forms of Fascin have also been reported to associate with dynamic filopodia in other systems (Lin-Jones, 2007; Vignjevic, 2006). Thus, this study shows unexpected complexity in Fascin regulation in vivo, whereby the regulatory activity of the conserved serine appears crucial for the formation of stable cell extensions but dispensable for the dynamic actin reorganization that occurs during invasive-like cell migration (Zanet, 2009).
Further studies in vivo will be essential to decipher the full repertoire of fascin regulation, a task that can directly benefit from genetic approaches in flies. Drosophila thus represents a valuable system in which to study how Fascin is regulated and how it functions in cells as they behave in situ, and this information will contribute to an understanding of how fascin misregulation contributes to cancer progression (Zanet, 2009).
Although prostaglandins (PGs)-lipid signals produced downstream of cyclooxygenase (COX) enzymes-regulate actin cytoskeletal dynamics, their mechanisms of action are unknown. Drosophila oogenesis, in particular nurse cell dumping, is a new model to determine how PGs regulate actin remodeling. PGs, and thus the Drosophila COX-like enzyme Pxt, are required for both the parallel actin filament bundle formation and the cortical actin strengthening required for dumping. This study provides the first link between Fascin (Drosophila Singed, Sn), an actin-bundling protein, and PGs. Loss of either pxt or fascin results in similar actin defects. Fascin interacts, both pharmacologically and genetically, with PGs, as reduced Fascin levels enhance the effects of COX inhibition and synergize with reduced Pxt levels to cause both parallel bundle and cortical actin defects. Conversely, overexpression of Fascin in the germline suppresses the effects of COX inhibition and genetic loss of Pxt. These data lead to the conclusion that PGs regulate Fascin to control actin remodeling. This novel interaction has implications beyond Drosophila, as both PGs and Fascin-1, in mammalian systems, contribute to cancer cell migration and invasion (Groen, 2012).
Prostaglandins could regulate Fascin activity in a number of ways. In human cells, protein kinase C (PKC) phosphorylates Fascin-1, blocking F-actin binding. Additionally, human Fascin-1 competes with Caldesmon and Tropomyosin for F-acti Calmodulin, and thus Ca2+/cAMP signaling, negatively regulates these two proteins, promoting Fascin-1's bundling activity. Rac, a Rho-type GTPase, also positively regulates human Fascin-1). A recent study revealed that Fascin-1 is also regulated by Rho via LIM Kinase 1 (Jayo, 2012). Notably, PGs are known to signal through all of these mechanisms. As Drosophila Fascin PKC-site phosphomutants (S52A/E) restore nurse cell dumping in fascin mutants, it is unlikely that PGs regulate Fascin in this manner during this process. However, an additional phosphorylation site (S289), associated with a bundling-independent function, has recently been identified in Drosophila (Zanet, 2012); perhaps this role of Fascin contributes to cortical actin integrity. As both cAMP and Rho GTPase regulate nurse cell dumping, it will be important to determine whether PGs signal via these pathways to regulate Fascin. It remains possible that PGs regulate Fascin by a previously unidentified means (Groen, 2012).
Singed is 35% homologous to sea urchin Fascin. Fascin was one of the first molecules identified in cytoplasmic actin gels induced to form in low Ca++ extracts of eggs from the sea urchin, and was the first actin-bundling protein to be extensively characterized. Fascin crosslinks actin filaments into hexagonally packed, linear arrays. Reconstituted actin-fascin bundles show a characteristic 11-nm periodic striping pattern. There is one fascin molecule per filament crosslink, suggesting that each fascin molecule must contain two actin filament binding sites (Bryan, 1993)
Fascin binds to ß-Catenin's Armadillo repeat domain. In vitro competition and domain-mapping experiments demonstrate that Fascin and E-cadherin utilize a similar binding site within ß-catenin, such that they form mutually exclusive complexes with ß-catenin. Fascin and ß-Catenin colocalize at cell-cell borders and the dynamic cell leading edges of epithelial and endothelial cells. In addition to cell-cell borders, cadherins colocalize with Fascin and ß-Catenin at cell leading edges. It is likely that ß-Catenin participates in modulating cytoskeletal dynamics in association with Fascin, perhaps in a coordinate manner with its functions in Cadherin and APC (adenomatous polyposis coli) complexes. Whatever the biological role of the Fascin-ß-Catenin complex, Fascin itself does not appear to be required for the function of all bundled filaments. For example, Singed mutants evince a variety of apparently normal cell activities even in embryos harboring strong singed alleles, leaving open the possibility that other bundling proteins effectively assume the role of Fascin in various contexts (Tao, 1996).
Human Fascin is phosphorylated in vivo upon treatment with TPA, a tumor promoter. With the incorporation of 0.25 mol of phosphate/mol of protein, the actin binding affinity is decreased from 6.7 x 10(6) to 1.5 x 10(6) m(-1). The actin bindling activity is also decreased. This suggests that phosphorylation of Fascin plays a role in actin reorganization (Yamakita, 1996).
Fascin is an F-actin-bundling protein shown to stabilize filopodia and regulate adhesion dynamics in migrating cells, and its expression is correlated with poor prognosis and increased metastatic potential in a number of cancers. This study identified the nuclear envelope protein nesprin-2 (see Drosophila Nesprin) as a binding partner for fascin in a range of cell types in vitro and in vivo. Nesprin-2 interacts with fascin through a direct, F-actin-independent interaction, and this binding is distinct and separable from a role for fascin within filopodia at the cell periphery. Moreover, disrupting the interaction between fascin and nesprin-2 C-terminal domain leads to specific defects in F-actin coupling to the nuclear envelope, nuclear movement, and the ability of cells to deform their nucleus to invade through confined spaces. Together, these results uncover a role for fascin that operates independently of filopodia assembly to promote efficient cell migration and invasion (Jayo, 2016).
singed contains a TATA-box deficient (TATA-less) promoter. Such promoters have a conserved sequence motif, A/GGA/TCGTG, termed the downstream promoter element (DPE), located about 30 nucleotides downstream of the RNA start site of many TATA-less promoters, including singed. DNase I footprinting of the binding of epitope-tagged TFIID to TATA-less promoters reveals that the factor protects a region extending from the initiation site sequence (about +1) to about 35 nucleotides downstream of the RNA start site. There is no such downstream DNase I protection induced by TFIID in promoters with TATA motifs. This suggests that the DPE acts in conjunction with the initiation site sequence to provide a binding site for TFIID in the absence of a TATA box to mediate transcription of TATA-less promoters (Burke, 1996).
Fascin should contain two actin binding domains, since the protein cross-links actin filaments as a monomer. Traditional biochemical approaches for mapping actin binding domains have had limited success. A human 27-kD C-terminal fragment and a mouse 3kD C-terminal fragment were generated using limited proteolysis. The C-terminal half of human or mouse Fascin appears to be able to bind, but not bundle, actin filaments and therefore contains at least one actin binding domain. A mutation that changes glycine 409 to glutamic acid results in partial inactivation of Fascin in vivo, while a mutation that changes serine 289 to asparagine almost completely inactivates Fascin in vivo. A subsequent EMS mutagenesis screen for dominant suppressors of the residue 289 mutant revealed an intragenic suppressor mutation that changed serine 251 to phenylaline and restored much of Fascin's function. These two mutations, in residue 289 and 251, draw attention to a central domain in Fascin that may prove useful in further studies on the structural basis for actin binding (Cant, 1996).
Migrating cells nucleate focal adhesions (FAs) at the cell front and disassemble them at the rear to allow cell translocation. FAs are made of a multiprotein complex, the adhesome, which connects integrins to stress fibers made of mixed-polarity actin filaments. Myosin II-driven contraction of stress fibers generates tensile forces that promote adhesion growth. However, tension must be tightly controlled, because if released, FAs disassemble. Conversely, excess tension can cause abrupt cell detachment resulting in the loss of a major part of the adhesion. Thus, both adhesion growth and disassembly depend on tensile forces generated by stress fiber contraction, but how this contractility is regulated remains unclear. This study shows that the actin-bundling protein Fascin crosslinks the actin filaments into parallel bundles at the stress fibers' termini. Fascin prevents myosin II entry at this region and inhibits its activity in vitro. In fascin-depleted cells, polymerization of actin filaments at the stress fiber termini is slower, the actin cytoskeleton is reorganized into thicker stress fibers with a higher number of myosin II molecules, FAs are larger and less dynamic, and consequently, traction forces that cells exert on their substrate are larger. It was also shown that fascin dissociation from stress fibers is required to allow their severing by cofilin, leading to efficient disassembly of FAs (Elkhatib, 2014).
Bristles are formed during pupation when the trichogen cell sends out a shaft of cytoplasm with a cytoskeletal core comprised of central microtubules and 8-12 fibrous bundles dispersed peripherally at the plasma membrane. The fibrous bundles consist of actin filaments. The morphology of the bristle appears to reflect the organization and integrity of the cytoskeletal core present at the time of cuticle deposition during bristle development (Cant, 1994 and references).
During early oogenesis, Singed protein is detected at low levels in nurse cell cytoplasm. Several migratory populations of follicle cells express very high levels of Singed. At stage 9 abundant protein is present in border cells and posterior follicle cells. The follicle cells migrating around the outside of the egg chamber do not express abundant Singed protein. In early stage 10 Singed is found abundantly in the centripetal follicle cells as they migrate along the nurse cell-oocyte interface. Singed is found near the follicle cell-nurse cell interface. Actin filaments are normally present subcortically in nurse cells, including the ring canals connecting adjacent cells. Subcortical actin-containing filaments likely support nurse cell contraction to push nurse cell cytoplasm through the ring canals into the oocyte. In late stage 10, just before the rapid phase of cytoplasm transport, actin filament bundles form in the nurse cell cytoplasm. These bundles probably have a structural role in anchoring the nurse cell nuclei in a central position away from ring canals. During stage 10 in nurse cells, there is a dramatic increase in Singed protein. During the rapid transport of nurse cell cytoplasm, Singed increases throughout nurse cell cytoplasm (Cant, 1994).
There are actually two proteins involved in cross-linking actin bundles in bristles: Forked, a protein with ankyrin repeats, and Singed. Hints as to why two species of cross-links are necessary can be gleaned from studies of bristle formation. Initially, only microbubules are contained within newly sprouted bristles. A little later in development, actin filaments appear. At early stages the filaments in the bundles are randomly packed. The Forked protein is most abundant during the earliest stages in actin bundle formation; thereafter Forked decreases, relative to Actin and Fascin. The Forked protein may be necessary early in development to tie the filaments together in a bundle so that they can be subsequently zippered together through the action of Singed (Tilney, 1995).
The singed locus was first described by Mohr in 1922. The bristles and hairs found over much of the wild-type fly's body are shortened in singed mutants, or twisted and gnarled . This phenotype is most easily seen in the large bristles (machrochaetes) on the dorsal surface of the thorax, but the smaller bristles (microchaetes) and hairs are also affected. In severe mutants, machrochaetes, microchaetes, and hairs on the head, thorax, legs, and wings are all affected to varying degrees. In addition to the bristle phenotype, many singed mutants are female sterile. Mutant singed germline clones do not make eggs, indicating a requirement for singed expression in the germline. The ovaries of female sterile singed mutants have few late stage egg chambers, and few eggs are laid. The eggs are flacid with shortened filaments, and they do not develop (Paterson, 1991 and references).
During the early stages of oogenesis, nurse cell cytoplasm flows slowly into the oocyte. In late stage 10, the rapid phase of cytoplasm transport begins. During stage 11 (final nurse cell), actin dependent cytoplasm transport takes place, resulting in a doubling of the oocyte volume in about 30 minutes and in the regression of the nurse cell cluster. In sterile singed mutants, oogenesis becomes defective at the onset of rapid cytoplasm transport. In singed mutants, cytoplasmic actin filament bundles rarely form and nurse cell nuclei become dramatically rearranged. The nuclei in the four nurse cells adjacent to the oocyte appear to be pushed into the ring canals, to extend into the oocyte, and to block the flow of cytoplasm into the oocyte. Although follicle cells continue their developmental program in singed mutants, follicle cell derived structures appear to be affected: respiratory appendages are often flattened and fused and the operculum forms at almost a right angle to the long axis of the egg. These defects are likely to be secondary consequences of the failure of nurse cell regression (Cant, 1994 and references).
Actin bundle assembly in specialized structures such as microvilli on intestinal epithelia and Drosophila bristles requires two actin bundling proteins. In these systems, the distinct biochemical properties and temporal localization of actin bundling proteins suggest that these proteins are not redundant. During Drosophila oogenesis, the formation of cytoplasmic actin bundles in nurse cells also requires two actin bundling proteins: fascin encoded by the singed gene, and a villin-like protein encoded by the quail gene. singed and quail mutations are fully recessive and each mutation disrupts nurse cell cytoplasmic actin bundle formation. P-element mediated germline transformation was used to overexpress quail in singed mutants and test whether these proteins have redundant functions in vivo. Overexpression of Quail protein in a sterile singed background restores actin bundle formation in egg chambers. The degree of rescue by Quail depends on the level of Quail protein overexpression, as well as residual levels of Fascin function. In nurse cells that contain excess Quail but no Fascin, the cytoplasmic actin network initially appears to resemble wild type, but then becomes disorganized in the final stages of nurse cell cytoplasm transport. The ability of Quail overexpression to compensate for the absence of Fascin demonstrates that Fascin is partially redundant with Quail in the Drosophila germline. Quail appears to function as a bundle initiator, while Fascin provides bundle organization (Cant, 1998).
Actin and microtubule cytoskeletons have overlapping, but distinct roles in the morphogenesis of epidermal hairs during Drosophila wing development. The function of both the actin and microtubule cytoskeletons appears to be required for the growth of wing hairs, as treatment of cultured pupal wings with either cytochalasin D or vinblastine is able to slow prehair extension. At higher doses, a complete blockage of hair development is seen. The microtubule cytoskeleton is also required for localizing prehair initiation to the distalmost part of the cell. Disruption of the microtubule cytoskeleton results in the development of multiple prehairs along the apical cell periphery. The multiple hair cells are a phenocopy of mutations in the inturned group of tissue polarity genes, which are downstream targets of the frizzled signaling/signal transduction pathway. The actin cytoskeleton also plays a role in maintaining prehair integrity during prehair development, since treatment of pupal wings with cytochalasin D, which inhibits actin polymerization, led to branched prehairs. This is a phenocopy of mutations in crinkled, and suggests mutations that cause branched hairs will be in genes that encode products that interact with the actin cytoskeleton. Several other mutant genotypes produce branched or split bristles or hairs. For example, mutations in singed, chickadee and capping protein produce bristles and/or hairs that are split, bent or stunted in ways that partially resemble cytochalasin D treatment. However, the phenotypes associated with these mutations do not resemble those seen with CD treatment as closely as the phenotype associated with crinkled (e.g. there is not hair splitting in sn mutants). The recent finding that mutations in the small G-protein rho result in an inturned-like phenotype and that the expression of a dominant negative form of rac also results in multiple hair cell phenotype is interesting with regard to the interaction of the actin and microtubule cytoskeletons. Small G-proteins of the rho and rac families are thought to interact with the actin cytoskeleton, yet they produce a wing hair phenotype that is similar to what is seen with the disruption of the microtubule cytoskeleton. This could be due to both the small G-proteins and the micotubule cytoskeleton being required for localizing a common component or activity to the vicinity of the distal vertex, or to the small G-proteins affecting the structure of the microtubule cytoskeleton, or to the microtubular cytoskeleton functioning in the localization of the small G-proteins or, alternatively, these two classes of proteins could be functioning in parallel pathways that function independently to restrict prehair initiation to the distal region of the cell. The observation that the expression of a dominant negative form of rac1 causes a disruption of the microtubule array suggests the possibility that the phenotypes associate with G-protein loss could be due to their disrupting the structure/function of the microtubule cytoskeleton and not to their being part of the frizzled signaling/signal transduction pathway (Turner, 1998).
Nurse cells are cleared from the Drosophila egg chamber by apoptosis. DNA fragmentation begins in nurse cells at stage 12, following the completion of cytoplasm transfer from the nurse cells to the oocyte. During stage 13, nurse cells increasingly contain highly fragmented DNA and disappear from the egg chamber concomitantly with the formation of apoptotic vesicles containing highly fragmented nuclear material. In mutant egg chambers that fail to complete cytoplasm transport from the nurse cells (dumpless chambers), DNA fragmentation is markedly delayed and begins during stage 13, when the majority of cytoplasm is lost from the nurse cells. These data suggest the presence of cytoplasmic factors in nurse cells that inhibit the initiation of DNA fragmentation. The dumpless mutants studied include cheerio and kelch, which both have aberrant ring canal morphology that does not permit cytoplasm to pass easily from the nurse cells to the oocytes. The chickadee, singed and quail gene products are necessary for the proper formation of cytoplasmic actin filament bundles that form in nurse cells at stage 10B, just prior to the onset of cytoplasmic transport. reeper and hid are expressed in nurse cells beginning at stage 9 and continuing throughout stage 13. The grim transcript is not expressed as strongly as rpr or hid. The negative regulators DIAP1 and DIAP2 are also transcribed during oogenesis. However, germline clones homozygous for the deficiency Df(3)H99, which deletes rpr, hid and grim, undergo oogenesis in a manner morphologically indistinguishable from wild type, indicating that genes within this region are not necessary for apoptosis in nurse cells (Foley, 1998).
Drosophila neurosensory bristle development provides an excellent model system to study the role of the actin-based cytoskeleton in polarized cell growth. Confocal fluorescence microscopy of isolated thoracic tissue was used to characterize changes in F-actin that occurred during macrochaete development in wild type flies and mutants that have aberrant bristle morphology. At the earliest stages in wild type bristle development, cortical patches of F-actin are present, but no bundles were observed. Actin bundles begin to form at 31% of pupal development and become more prominent as development progresses. The F-actin patches gradually disappear and are no longer present by 38% of pupal development. The distribution of F-actin in singed3 mutant macrochaetae is indistinguishable from wild type bristles until 35% of development when the actin bundles begin to splay and appear ribbon-like. In forked36a bristles, the mutant phenotype is evident at earlier stages of development than the singed3 mutant. Wild type tissue stained with antibodies against the Forked protein demonstrate that the Forked protein colocalize with F-actin structures found in early and late stage developing macrochaetae. Antibodies against the Singed protein show that it appears to localize with F-actin structures only at later stages in development. These data suggest that the forked gene product is required for the initiation of fiber bundle formation and the singed gene product is required for the maintenance of fiber bundle morphology during bristle development. Similar analyses of singed3/forked36a double mutants provide additional genetic evidence that the forked gene product is required before the singed gene product. Further, the analyses suggests that at least one additional crosslinking protein is present in these bundles (Wulfkuhle, 1998).
Fascin is an evolutionarily conserved actin-binding protein that plays a key role in forming filopodia. It is widely thought that this function involves fascin directly bundling actin filaments, which is controlled by an N-terminal regulatory serine residue. In this paper, by studying cellular processes in Drosophila that require fascin activity, a regulatory residue was identified within the C-terminal region of the protein (S289). Unexpectedly, although mutation (S289A) of this residue disrupted the actin-bundling capacity of fascin, fascin S289A fully rescued filopodia formation in fascin mutant flies. Live imaging of migrating macrophages in vivo revealed that this mutation restricted the localization of fascin to the distal ends of filopodia. The corresponding mutation of human fascin (S274) similarly affected its interaction with actin and altered filopodia dynamics within carcinoma cells. These data reveal an evolutionarily conserved role for this regulatory region and unveil a function for fascin, uncoupled from actin bundling, at the distal end of filopodia (Zanet, 2012).
The branched morphology of dendrites represents a functional hallmark of distinct neuronal types. Nonetheless, how diverse neuronal class-specific dendrite branches are generated is not understood. Specific classes of sensory neurons of Drosophila larvae were investigated to address the fundamental mechanisms underlying the formation of distinct branch types. The function of fascin, a conserved actin-bundling protein involved in filopodium formation, was investigated in class III and class IV sensory neurons. Terminal branchlets of different classes of neurons were found to have distinctive dynamics and are formed on the basis of molecularly separable mechanisms; in particular, class III neurons require fascin for terminal branching whereas class IV neurons do not. In class III neurons, fascin controls the formation and dynamics of terminal branchlets. Previous studies have shown that transcription factor combinations define dendrite patterns; this study found that fascin represents a downstream component of such programs, as it is a major effector of the transcription factor Cut in defining class III-specific dendrite morphology. Furthermore, fascin defines the morphological distinction between class III and class IV neurons. In fact, loss of fascin function leads to a partial conversion of class III neurons to class IV characteristics, while the reverse effect is obtained by fascin overexpression in class IV neurons. It is proposed that dedicated molecular mechanisms underlie the formation and dynamics of distinct dendrite branch types to elicit the accurate establishment of neuronal circuits (Nagel, 2012).
Anilkumar, N., Parsons, M., Monk, R., Ng, T. and Adams, J. C. (2003). Interaction of fascin and protein kinase Calpha: a novel intersection in cell adhesion and motility. EMBO J. 22: 5390-5402. PubMed Citation: 14532112
Aratyn, Y. S., Schaus, T. E., Taylor, E. W. and Borisy, G. G. (2007). Intrinsic dynamic behavior of fascin in filopodia. Mol. Biol. Cell 18: 3928-3940. PubMed Citation: 17671164
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date revised: 20 October 2016
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