In many tissues, the stem cell niche must coordinate behavior across multiple stem cell lineages. How this is achieved is largely unknown. This study has identified delayed completion of cytokinesis in germline stem cells (GSCs) as a mechanism that regulates the production of stem cell daughters in the Drosophila testis. Through live imaging, a secondary F-actin ring was shown to form through regulation of Cofilin activity to block cytokinesis progress after contractile ring disassembly. The duration of this block is controlled by Aurora B kinase. Additionally, a requirement was identified for somatic cell encystment of the germline in promoting GSC abscission. It is suggested that this non-autonomous role promotes coordination between stem cell lineages. These findings reveal the mechanisms by which cytokinesis is inhibited and reinitiated in GSCs and why such complex regulation exists within the stem cell niche (Lenhart, 2015).
This first real-time analysis of GSCs through abscission has revealed surprising complexities layered in cytokinesis. First, cytokinesis is blocked after central spindle and contractile ring disassembly and before entry to the abscission phase. This block is imposed by a secondary F-actin-ring. Second, AurB regulates the transition between phase one and phase two. That transition marks a vital step in the reinitiation of cytokinesis, permitting cytoplasmic isolation and recruitment of abscission machinery. Finally, somatic cell encystment is essential to abscission. Thus, three discrete nodes of regulation are layered on top of the canonical cytokinesis program to achieve tight temporal control over daughter cell production, and thus tissue maintenance by the resident stem cells (Lenhart, 2015).
Incomplete cytokinesis is a deeply conserved feature of germ cells that establishes the syncytium necessary for robust germline development. Differentiating germ cells appear to arrest cytokinesis immediately following contractile ring ingression because the known components of stable ring canals are identical to those of the contractile ring. It was thought that delayed cytokinesis in GSCs was simply a remnant of this conserved program. In contrast, this study found that the delay is mechanistically distinct from that occurring in differentiating germ cells. GSCs complete ingression, disassemble their contractile ring F-actin, and dissolve central spindle microtubules before engaging a ROK-LimK-Cofilin pathway to regulate a secondary F-actin ring that blocks cytokinesis progression until its disassembly at the entry to phase two (Lenhart, 2015).
Interestingly, the F-actin rings of gonial cells were not disrupted by manipulation of Cofilin activity, in contrast to their precocious disassembly in GSC-Gb pairs. This functional distinction is likely tied to the different biological goal of the stem cell versus the differentiating germ cell. One must release a differentiating daughter cell while the other must communicate syncitially for differentiation to progress normally. Ultimately, because the stem cell niche confers this functional distinction, future work will investigate whether it directly controls F-actin dynamics in the stem cell by possibly modulating Cofilin, or acts indirectly through other stem cell factors to do so (Lenhart, 2015).
These data strongly indicate that the secondary F-actin ring must be disassembled for abscission to be reinitiated. This suggests that F-actin at the IC bridge inhibits abscission, and work in other cells supports this. Inhibition of the Cofilin phosphatase, activation of AurB, depletion of phosphoinositide 5-phosphatase, or of Rab35 all lead to retention of F-actin at the IC bridge and inhibit abscission. Importantly, abscission could be restored after Rab35 depletion by forcing F-actin disassembly (Lenhart, 2015).
GSC-Gb pairs depleted for aurB fail to complete abscission prior to mitotic entry and form interconnected germ cells attached to the hub. This could suggest that AurB is normally required to promote abscission. However, expressing an activated form of Svn did not induce precocious abscission as would be expected in this model. Rather, SvnS125E expression advanced the transition from phase one to two, while aurB depletion delayed it. These reciprocal effects suggest instead that AurB times the phase one-phase two transition. In this model, the lack of abscission in aurB mutants is an indirect consequence of spending a shorter fraction of the total cycle in phase two. For example, this study has shown that ESCRTIII is localized during phase two and in the apparent absence of central spindle microtubules. In aurB-depleted cells, there simply may not be enough time during the shortened phase two for the already compromised recruitment of ESCRTIII machinery to promote abscission prior to mitotic entry. It is also noted that the lack of a central spindle raises the issue of how ESCRTIII components are delivered to the IC bridge. Perhaps the midbody performs this role, as has been suggested for the C. elegans first cell division (Lenhart, 2015).
Recent studies have found that shrub is negatively regulated by AurB in female GSCs (Matias, 2015). Although the current results suggest that AurB activity should promote ESCRTIII function in the testis, it is compelling to speculate that AurB might control the phase one-phase two transition through shrub. Alternatively, AurB could directly control this transition by regulating disassembly of the secondary F-actin ring, as there is precedent for AurB controlling actin dynamics. For example, in the 'No Cut' pathway, maintenance of AurB activity late in cytokinesis is associated with persistence of F-actin at the IC bridge. Intriguingly, AurB can phosphorylate formin proteins and thereby regulate actin stress fiber formation. Although in this context AurB activity positively regulates actin polymerization, the interaction between AurB and formin suggests a direct link between CPC activity and actin dynamics. This connection is particularly compelling given that formins can also promote severing of actin filaments. Thus, it is intriguing to speculate that AurB phosphorylation of formins at the IC bridge in GSC-Gb pairs may promote severing of actin filaments in the secondary ring and thereby promote transition from phase one to phase two of delay (Lenhart, 2015).
Perhaps most excitingly, this study has identified non-autonomous control over GSC-Gb abscission by somatic cell encystment. This sheds light on the functional relevance of abscission delay. Encystment of spermatogonia by two somatic cells is required for proper germ cell differentiation. However, GSCs and their flanking CySCs do not coordinate daughter cell production by synchronizing their cell cycles. Linking abscission to encystment is an elegant alternative for promoting coordinated release of stem cell daughters from the niche (Lenhart, 2015).
Several questions are raised by the current observations, such as precisely when abscission is triggered relative to cyst cell engulfment of the Gb. It would be necessary to carry out live imaging simultaneously on germline and adjacent somatic cells to address this. However, imaging CySCs and cyst cells is fraught with difficulty due to their irregular morphology and small size. Thus, it has not yet been possible to image somatic cells with anywhere near the resolution achieved for GSC-Gb pairs (Lenhart, 2015).
Encystment could promote abscission through contact-dependent signaling, where CySCs or cyst cells produce the ligand. Alternatively, the abscission trigger might be mechanical, because tension has been suggested to regulate abscission in cultured cells. Here, as daughter cells migrated apart in culture following mitosis, tension along the bridge connecting them increased and this lengthened the time to abscission. Experimentally decreasing bridge tension triggered earlier abscission. In the current system, most Gbs are displaced some distance from the hub during phase two, with a consequent elongation of the IC bridge connecting those cells to the GSC. Perhaps movement of the Gb away from its mother GSC generates increased tension along the bridge. Symmetric encystment might relieve that tension by providing equalizing forces on both sides of the IC bridge, inducing abscission while ensuring that the Gb is properly associated with two somatic cells. In culture, increased tension delayed abscission by disrupting assembly of functional ESCRTIII complexes at the IC bridge. Therefore, it will be interesting to address whether ESCRTIII complexes in GSCs are temporally regulated by encystment. Whatever the mechanism, the cyst cells are clearly poised for intimate contact at the appropriate time, because the midbody remnant is sometimes taken up byÊencysting somatic cells after abscission (Lenhart, 2015).
This work has clarified the mechanism by which cytokinesis is delayed in GSCs, identifying three distinct regulatory events layered on top of the traditional program of cytokinesis. These events impose an appropriate delay, a timed reinitiation, and a regulated abscission in the GSCs. This stem cell-specific program assists in the coordinate release of differentiating daughter cells from the resident stem cell populations in this niche. Because similar requirements for synchronized daughter cell production between multiple stem cell populations exist in other tissues, it is enticing to speculate that regulated abscission might be used to promote coordination in other niches. Membrane scission is difficult to demonstrate in vivo in many systems, so it is not yet known if stem cells other than the germline exhibit abscission delay. As higher resolution methods are developed to visualize stem cell dynamics within endogenous niches, it will be interesting to see if abscission delay emerges as a conserved mechanism of niche-dependent control over stem cell proliferation (Lenhart, 2015).
To analyze the role of candidate proteins involved in Rho GTPase signaling and neuronal morphogenesis, the mushroom body (MB) neurons in the Drosophila central brain were used as a model. The adult MB is composed of three sets of neurons (γ, alpha′/ß′, and alpha/ß) sequentially derived from common neuroblast precursors. Each MB neuron extends a primary neurite that gives rise to dendritic branches near the cell body and a single axon that projects anteriorly and ventrally through the peduncle. Each axon of the alpha′/ß′ or alpha/ß neurons bifurcates to form a dorsal and a medial branch, whereas each γ neuron has only a medial branch. All axons terminate either medially at the proximity of the midline (for medial projections) or close to the anterior dorsal cortex (for dorsal projections) (Ng, 2004)
To analyze the functions of candidate genes, the MARCM system was used to generate wild-type or homozygous mutant single-cell or neuroblast clones that are positively labeled. Neuroblast clones allow the examination of the gross behavior of a large clone of mutant neurons and to assess the role of a given gene in cell proliferation, whereas single-cell clones enable finer resolution of axon morphology of mutant neurons (Ng, 2004).
The Rho GTPases have been shown to be essential in regulating the morphogenesis of MB neurons (Ng, 2002). What are the effector pathways that lead to cytoskeletal regulation in vivo? One candidate pathway is cofilin regulation. Cofilin and the related actin depolymerization factor (ADF) in mammals have a single homolog in Drosophila, encoded by the tsr locus (Gunsalus, 1995). To examine cofilin function, neuroblast clones in newly hatched larvae were generated and MB clones were examined in adults. Wild-type MB neuroblast clones generated under this condition typically contain approximately 400 MB neurons composed of all three classes. In contrast, tsr-/- neuroblast clones for two different null alleles of tsr (tsrN96A and tsrN121) contain 15 to 30 neurons. This cell proliferation defect is consistent with previous findings that tsr is essential for cytokinesis in the Drosophila brain (Gunsalus, 1995; Ng, 2004).
In addition to cell proliferation defects, tsr-/- neuroblast clones also exhibit severe axon growth defects, since a majority of mutant neurons fail to extend their axons beyond the peduncle. To study axon growth at a higher resolution, tsr-/- single-cell clones of γ neurons were analyzed. At the adult stage, all wild-type γ neurons extend their axons to the end of the γ lobe, to the proximity of the midline. However, 30%-40% of tsr-/- axons did not enter the medial lobe (stopping before the alpha/ß branching point) (classified as 'severe'), another 35%-40% failed to extend beyond the midpoint of the γ lobe ('strong'), and an additional 15%-20% failed to reach the end of the γ lobe ('weak'). Thus, axon growth requires cofilin cell-autonomously (Ng, 2004).
When examined at a higher magnification, tsr-/- axons exhibit characteristic morphological defects. In contrast to the relatively uniform axon shafts and terminals in wild-type neurons, most tsr-/- axons displayed multiple small protrusions and swellings along the shaft and at the terminal, reminiscent of filopodia and lamellipodia normally associated with developing growth cones. These observations are consistent with the idea that actin depolymerization is required to turn over transient growth cone filopodia and lamellipodia during axon growth and that loss of such turnover results in growth defects (Ng, 2004).
The role of tsr in MB alpha/ß neurons was analyzed by generating neuroblast clones that contain only alpha/ß neurons. Wild-type alpha/ß neurons typically extended dorsal and medial projections that end at the midline and dorsal cortex. In contrast, most tsr-/- alpha/ß axons fail to extend fully to the midline or to the dorsal cortex. Therefore, alpha/ß neuron axon growth also requires cofilin (Ng, 2004).
Cofilin function is regulated by phosphorylation in higher eukaryotes. Cofilin is active in a dephosphorylated state and becomes inhibited once it is phosphorylated at serine 3. Mutational analyses show that changing serine 3 to an alanine (S3A) generates a constitutively active cofilin, whereas changing serine 3 to a glutamic acid (S3E) inhibits cofilin activity (Agnew, 1995; Abe, 1996). To test whether cofilin phosphorylation plays an essential role in vivo, transgenic flies were generated that express wild-type, S3A, or S3E forms of cofilin under the control of the GAL4-UAS expression system. Overexpression of these transgenes in wild-type MB neurons did not result in gross axon or cell proliferation defects (Ng, 2004).
Then, using the MARCM system, transgenic rescue experiments were performed by expressing wild-type, S3A, or S3E cofilin in tsr-/- clones. The ability of various cofilin transgenes to rescue tsr-/- neuroblast proliferation defects was analyzed. Overexpression of wild-type cofilin (UAS-tsr WT) in tsr-/- neuroblast clones fully restored cell proliferation, since these clones contain a full complement of all three classes of MB neurons. Overexpression of S3A cofilin (UAS-tsr S3A) in tsr-/- clones partially rescues the cell proliferation phenotype: only the two first-born classes of neurons (γ and alpha′ß′) are generated in these neuroblast clones. This is consistent with the reduced neuronal numbers from these clones when compared to the wild-type rescue. Overexpression of S3E cofilin (UAS-tsr S3E) has no rescue effect on neuroblast proliferation; the cell numbers in these neuroblast clones remained 15 to 30 (Ng, 2004).
Using the same strategy, single-cell clones of γ neurons were examined to assay for axon growth. Overexpression of wild-type cofilin effectively rescues tsr-/- axon growth defects. Similar experiments using S3A or S3E cofilin also showed some rescue effects; however, neither of the transgenes rescued as effectively as wild-type cofilin, although S3A cofilin rescued better than S3E (Ng, 2004).
These results suggest that cofilin phosphorylation regulates both cell proliferation and axon growth. In the case of cell proliferation, the nonphosphorylatable S3A cofilin has significant rescue activity, while the S3E is completely inactive, consistent with the findings of in vitro studies in which S3A provides constitutive depolymerization activity, while S3E has little or no actin depolymerization activity (Agnew, 1995; Abe, 1996). In the case of axon growth, however, both S3A and S3E have some rescuing effects on axon growth, suggesting that phosphorylated forms of cofilin might provide some function in axon growth. Indeed, a recent study shows that, while unphosphorylated cofilin promotes actin depolymerization, phosphorylated cofilin promotes actin polymerization (Ghosh, 2004). To test this model in vivo, both S3A and S3E cofilin were coexpressed in tsr-/- neurons. Coexpression of S3E and S3A cofilin did not rescue tsr-/- phenotypes to wild-type rescue levels for cell proliferation or axon growth. This suggests that unphosphorylatable and phosphomimetic cofilins acting in trans cannot fully restore cofilin activity. These experiments then suggest that cycling between phosphorylated and nonphosphorylated cofilin is essential for optimal cell proliferation and for axon growth and that S3E cofilin may have some residual actin depolymerization activity (as suggested by Agnew, 1995) to promote axon growth (Ng, 2004).
The phenotypic and molecular characterization of twinstar, an essential gene in Drosophila, is described. Two P-element induced alleles of tsr (tsr1 and tsr2) result in late larval or pupal lethality. Cytological examination of actively dividing tissues in these mutants reveals defects in cytokinesis in both mitotic (larval neuroblast) and meiotic (larval testis) cells. In addition, mutant spermatocytes show defects in aster migration and separation during prophase/prometaphase of both meiotic divisions. The gene affected by these mutations was cloned; it encodes a 17-kD protein in the cofilin/ADF family of small actin severing proteins. A cDNA for this gene has been described by Edwards (1994). Northern analysis shows that the tsr gene is expressed throughout development, and that the tsr1 and tsr2 alleles are hypomorphs that accumulate decreased levels of tsr mRNA. These findings prompted an examination of actin behavior during male meiosis to visualize the effects of decreased Twinstar protein activity on actin dynamics in vivo. Strikingly, both mutants exhibit abnormal accumulations of F-actin. Large actin aggregates are seen in association with centrosomes in mature primary spermatocytes. Later, during ana/telophase of both meiotic divisions, aberrantly large and misshaped structures appear at the site of contractile ring formation and fail to disassemble at the end of telophase, in contrast with wild-type. These results are discussed in terms of possible roles of the actin-based cytoskeleton in centrosome movement and in cytokinesis (Gunsalus, 1995).
In larval neuroblasts of tsr mutants, the major cytological consequence mutation is the appearance of a significant proportion of polyploid cells. Other aspects of mitosis appear to be largely unaffected. The mitotic index, a parameter measuring the frequency of cells engaged in mitosis, is nearly normal. The percentages of mitotic cells that are in anaphase in mutants are very similar to wild-type. Chromosomes in mutant neuroblasts seem to be normal: no evidence is seen of irregular chromosome condensation or of chromosome breakage. The phenotype observed in both tsr brains is similar to that associated with mutations in a variety of genes that cause defects in cytokinesis. Successive failures in cytokinesis would result in cells that increase their ploidy geometrically, giving rise to large polyploid cells. The normality of other mitotic parameters suggests that these lesions in the twinstar gene do not significantly alter other mitotic processes (Gunsalus, 1995).
Male meiosis offers a particularly suitable system for the analysis of the cytological consequences of mutations affecting cell division. Primary spermatocyte nuclei are ~25 times larger than neuroblast nuclei and exhibit comparatively larger spindles that can be clearly detected by tubulin immunostaining. Because meiosis occurs in the testes of third instar larvae, it was possible to analyze the meiotic divisions in mutant tsr larvae for potential aberrations (Gunsalus, 1995).
A unique phenotypic consequence of lesions in twinstar occurs in primary spermatocytes at the prophase/prometaphase transition of meiosis I, and is revealed when fixed mutant testes are stained with antibodies directed against tubulin. In wild-type males, centrosomes reside just under the plasma membrane throughout most of the primary spermatocyte stage, during which growth and gene expression prepare the cell for subsequent morphological differentiation. Near the end of this spermatocyte maturation phase, duplicated centrosomes (each containing a pair of centrioles) migrate together to the nuclear membrane, where they nucleate prominent asters. During late prophase/early prometaphase of the first meiotic division (stage Mla), the two asters separate from each other and migrate around the periphery of the nuclear envelope, in preparation for the establishment of the bipolar spindle. By early prometaphase, asters are located directly opposite each other on either side of the nucleus, in close apposition to the nuclear membrane. In tsr spermatocytes at the Mla stage, the two asters often remain in close proximity to each other and fail to associate with the nuclear envelope. The twinstar gene was named after this characteristic aberrant arrangement of asters. This effect occurs in a very high proportion of tsr primary spermatocytes, approaching 100% in tsr mutants, but is almost never seen in wild-type. Somewhat later, in the Mlb stage, asters in tsr mutants can be seen to separate slightly, but remain unassociated with the nuclear membrane (Gunsalus, 1995).
It is remarkable that subsequent to the M1 stage, the spindles in tsr mutant spermatocytes progressively resume a relatively normal position and appearance and seem indistinguishable from wild-type throughout the remainder of the first meiotic division. However, after completion of meiosis I, tsr prophase/prometaphase secondary spermatocytes (stage M6a and M6b) exhibit the same defect in aster migration and positioning observed in primary spermatocytes. Here again, the spindles become more normal by metaphase II and retain a wild-type structure throughout the remainder of meiosis II. Although telophase II tsr spindle structures look normal, an aberrant cross-like configuration of meiotic spindles is often observed that overlap at the midbody. Most likely this arrangement reflects a failure of the first meiotic cytokinesis and the consequent occurrence of two second divisions within the same cell. The analysis of hundreds of ana-telophases of tsr mutants shows no evidence of irregular chromosome segregation during either meiotic division. Lagging chromosomes or daughter nuclei of different size are never observed. In addition, tsr 'onion stage' spermatids exhibit a uniform nuclear size. Since spermatid nuclear size is proportional to chromosome content, this observation reinforces the conclusion that tsr does not affect chromosome segregation (Gunsalus, 1995).
During both meiotic divisions in wild-type spermatocytes, mitochondria associate lengthwise along the spindle apparatus, which is pinched in half during cytokinesis, ensuring an even distribution of these organelles to each of the two daughter cells. Immediately after completion of meiosis, the mitochondria associated with each hemispindle fuse and form an interlaced conglomerate called the Nebenkern. Thus, each newly formed wild-type spermatid (referred to as the onion stage) consists of a round, phase-light nucleus associated with a single phase-dark Nebenkern of similar size. The most obvious defect in tsr mutants is seen in spermatids at the onion stage. In tsr mutant testes, a substantial fraction of onion stage spermatids contain a single Nebenkern much larger than normal, along with four normal-sized nuclei. This phenotype reflects a failure of cytokinesis at both meiosis I and II, since this would prevent proper subdivision of the mitochondrial complement into four Nebenkerns. Cells with two normal-sized nuclei are also seen in association with an intermediate-size Nebenkern, indicating a failure of cytokinesis at only one of the meiotic divisions. Because failure of cytokinesis in the first meiotic division would lead to cells containing two second division meiotic spindles that would probably encounter further difficulties in cytokinesis, it is believed that the spermatids with one Nebenkern and two nuclei are the result of second division cytokinesis failure. Finally, spermatids were observed containing one large Nebenkern plus three nuclei that are usually located in close proximity to a cell containing a normal-size Nebenkern and a single regular nucleus. These are most likely to arise if the four hemispindles in secondary spermatocytes derived from cytokinesis I failures orient in such a way that mitochondrial fusion occurs asymmetrically, yielding a small and a large Nebenkern associated with one and three nuclei, respectively (Gunsalus, 1995).
Twinfilin is a ubiquitous actin monomer-binding protein whose biological function has remained unclear. Drosophila twinfilin has been cloned and shown to be 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. 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 (Wahlstron, 2001).
In yeast, lack of twinfilin does not result in a detectable phenotype, except for slightly enlarged cortical actin patches. In combination with a temperature-sensitive cofilin allele, twinfilin causes lethality at the permissive temperature (Goode, 1998). In Drosophila, cofilin is encoded by the twinstar (tsr) gene. To investigate the possible genetic interaction between twinfilin and twinstar, the P element line tsrk05633, which has a lethal insertion in twinstar, was crossed with the twf 3701 homozygotes, and the resulting double heterozygotes were examined for a bristle phenotype. Nearly all flies had at least one macrochaete with defects at bristle tip. The bilateral posterior scutellar and anterior dorsocentral bristles on the thorax were by far the most frequently affected. These four bristles were scored for abnormalities under higher magnification. In tsrk05633/+; twf 3701/+ flies, 66% of the bristles were split, branched, or had a rough surface, whereas only 2% of the bristles on tsrk05633/+; +/+ flies and none of the bristles on +/+; twf 3701/+ flies had this phenotype. The presence of a single tsrk05633 allele in the twf 3701 homozygous background results in a more dramatic eye defect, whereas the severity of the bristle phenotype appears to be equal to the one in the twf 3701 mutant alone. These results show that twinfilin interacts genetically with the ADF/cofilin encoding gene twinstar during bristle and eye morphogenesis (Wahlstron, 2001).
A mutation in a novel gene, capulet (cap), was identified in a mosaic screen to isolate mutations that perturb actin organization in germline clones. CAPs have been shown to inhibit actin polymerization in vitro, by sequestering monomeric actin. This actin-binding activity has been mapped to the carboxy-terminal region of CAP; however, a 'verprolin homology'-related domain has been identified in all CAPs, just carboxy-terminal of the polyproline-rich domain. In members of the verprolin/WASP family, this motif binds actin monomers in vitro, but catalyses actin polymerization in vivo. Therefore, in CAP homologues, this region of the protein may be used to facilitate actin binding. Since CAP proteins have also been found associated with Abl tyrosine kinase and with adenylate cyclase, it is possible that CAP represents an intermediary in these signal transduction cascades, perhaps altering actin dynamics in response to extracellular cues (Baum, 2000).
As ectopic actin structures are formed in cap mutant egg chambers, other F-actin-rich structures are lost. In particular, cortical F-actin underlying the nurse cell membranes disappears prematurely at stages 8-9 of oogenesis. Therefore CAP may simultaneously inhibit actin polymerization at some sites and facilitate the formation of F-actin at others. If the pool of actin within the egg chamber is limited, an alternative hypothesis can be imagined, in which actin filaments are lost from nurse cell cortices to compensate for the formation of actin aggregates within the oocyte. In order to test whether the actin cytoskeleton is similarly polarized in other mutants that have excess accumulation of F-actin, twinstar germline clones were examined. twinstar inhibits actin filament formation in vivo and encodes the Drosophila homolog of an actin-severing protein, cofilin. Although ectopic actin filaments form in twinstar germline clones, as in the cap mutant, ectopic actin aggregates form at sites throughout the early twinstar mutant egg chamber. Therefore, CAP has the specific function of inhibiting actin polymerization within the oocyte (Baum, 2000).
The actin cytoskeleton orders cellular space and transduces many of the forces required for morphogenesis. Genetics and cell biological techniques have been combined to identify genes that control the polarized distribution of actin filaments within the Drosophila follicular epithelium. Profilin and cofilin regulate actin-filament formation throughout the cell cortex. In contrast, Capulet (Capt) Drosophila homologue of Adenylyl Cyclase Associated Proteins, functions specifically to limit actin-filament formation catalysed by Ena at apical cell junctions. The Abl tyrosine kinase also collaborates in this process. It is therefore proposed that Capt, Ena and Abl act in concert to modulate the subcellular distribution of actin filaments in Drosophila (Baum, 2001).
To test the specificity of the capt mutant phenotype actin organization was analyzed in twinstar (tsr) mutant cells. Because tsr encodes the actin-filament-severing protein cofilin, F-actin accumulates in tsr mutant cells, as it does in the capt mutant. In tsr mutant follicle cells, ectopic actin filaments form at apical, basal and lateral cortices, although F-actin accumulation often seems more pronounced at the basal cell surface. Frequently, tsr mutant follicle cells also exhibit an altered columnar morphology. These data suggest that cofilin functions throughout the cell cortex to catalyse the disassembly of actin filaments, whereas in contrast Capt functions in a polarized manner to regulate apical actin accumulation (Baum, 2001).
This study has used Drosophila genetics and the follicular epithelium to characterize how various actin-binding proteins act to regulate the spatial organization of F-actin. The results show that actin dynamics are regulated by distinct mechanisms within different domains of a polarized epithelial cell. Capt, Ena and Abl seem to modulate apical actin-filament formation, whereas cofilin and profilin seem to have a more global function, regulating cortical actin-filament dynamics throughout the cell. Moreover, the accumulation of F-actin at apical, basal and lateral sites in tsr mutant follicle cells and the loss of cortical actin filaments in chic mutant cells indicates that cortical actin filaments are turned over continuously throughout the cell. This being so, it is striking that F-actin becomes so highly polarized in the capt mutant (Baum, 2001).
The driving force behind cell motility is the actin cytoskeleton. Filopodia and lamellipodia are formed by the polymerization and extension of actin filaments towards the cell membrane. This polymerization at the barbed end of the filament is balanced by depolymerization at the pointed end, recycling the actin in a 'treadmilling' process. One protein involved in this process is cofilin/actin-depolymerizing factor (ADF), which can depolymerize actin filaments, allowing treadmilling to occur at an accelerated rate. Cofilin/ADF is an actin-binding protein that is required for actin-filament disassembly, cytokinesis and the organization of muscle actin filaments. There is also evidence that cofilin/ADF enhances cell motility, although a direct requirement in vivo has not yet been shown. Drosophila cofilin/ADF, which is encoded by the twinstar (tsr) gene, promotes cell movements during ovary development and oogenesis. During larval development, cofilin/ADF is required for the cell rearrangement needed for formation of terminal filaments -- stacks of somatic cells that are important for the initiation of ovarioles. It is also required for the migration of border cells during oogenesis. These results show that cofilin/ADF is an important regulator of actin-based cell motility during Drosophila development (Chen, 2001).
The phenotypes and gene-expression patterns (using a lacZ reporter) of over 1,800 P-element enhancer trap lines were studied in larval ovaries. One line analysed contains a pupal lethal mutation on its second chromosome. This mutation affects a gene that is required for terminal-filament formation and border-cell migration. This mutation maps to chromosomal region 60A7-60B6. The gene tsr, which encodes the Drosophila homologue of cofilin/ADF, also maps to this region. Complementation analysis with known tsr alleles showed that the new mutation is a loss-of-function allele of tsr. This was confirmed by the rescue of all phenotypic traits caused by the new mutation with a P-element construct carrying a 6.4-kilobase (kb) genomic fragment containing the wild-type tsr gene. Cloning and sequence analysis has shown that the new tsr allele, tsrntf (ntf for 'no terminal filaments') is not caused by a P-element mutation, but rather has an insertion of a 7.4-kb gypsy retrotransposon19 259 base pairs (bp) downstream of the 5' end of the first intron of tsr. Northern analysis using a tsr probe on total larval poly(A) + messenger RNA shows that the tsrntf allele encodes a full-length transcript that is present at 30%-40% of the amount found in wild-type larvae. The normal size of this transcript indicates that the gypsy sequences are removed from the pre-mRNA during splicing of the first intron of tsr (Chen, 2001).
This study demonstrates a requirement for cofilin/ADF in cell movements during ovary development and oogenesis. During ovary development, cells expressing terminal-filament cell markers are present in tsrntf-mutant ovaries, so the defect in filament formation does not seem to be one of cell specification or differentiation, but rather in the ability of terminal-filament cells to intercalate and form stacks. The assumption that this mutant phenotype is due to defects in cell motility is supported by the presence of unusually high levels of filamentous actin in the mutant ovary, which is consistent with a loss of cofilin/ADF, and which could alter the actin cytoskeleton sufficiently to disrupt cell movement. Cofilin/ADF has been shown to be required for migration of border cells during oogenesis. By extension, it is proposed that tsr may be required for many, perhaps all, actin-based cell movements in Drosophila. Understanding how cofilin/ADF is coordinately regulated to allow formation of terminal filaments and migration of border cells should provide insight to its function in many developmental systems. A mammalian cultured cell system has been used to show that LIM-kinase 1 (LIMK-1) phosphorylates and inactivates cofilin/ADF, and that the GTPase Rac is capable of regulating the activity of LIMK-1. Further study of systems that require cofilin/ADF for normal development may allow the determination of how these signal-transduction pathways control the dynamic alterations of the actin cytoskeleton in developing animals (Chen, 2001).
Cell migration occurs through the protrusion of the actin-enriched lamella. The effects of RNAi depletion of approximately 90 proteins implicated in actin function on lamella formation have been investigated in Drosophila S2 cells. Similar to in vitro reconstitution studies of actin-based Listeria movement, it has been found that lamellae formation requires a relatively small set of proteins that participate in actin nucleation (Arp2/3 and SCAR), barbed end capping (capping protein), filament depolymerization (cofilin and Aip1), and actin monomer binding (profilin and cyclase-associated protein). Lamellae are initiated by parallel and partially redundant signaling pathways involving Rac GTPases and the adaptor protein Nck, which stimulate SCAR, an Arp2/3 activator. RNAi of three proteins (kette, Abi, and Sra-1) known to copurify with and inhibit SCAR in vitro leads to SCAR degradation, revealing a novel function of this protein complex in SCAR stability. These results have identified an essential set of proteins involved in actin dynamics during lamella formation in Drosophila S2 cells (Rogers, 2003).
The actin-binding protein cofilin/Twinstar is essential for actin-based functions in many cell types, and in vitro and in vivo studies indicate a role for cofilin in actin filament severing and turnover. Inhibition of cofilin by RNAi prevented S2 cell spreading on con A in >95% of treated cells. These cells retained their spherical morphology, and phalloidin staining revealed a dramatic cortical accumulation of filamentous actin as well as a wrinkled "raisin-like" texture to the surface of the cell. The abnormal accumulation of filamentous actin within the cells suggests that actin turnover is inhibited in S2 cells depleted of either of these two proteins. Cofilin-inhibited S2 cells exhibited a high incidence of multinucleate cells, implicating a role in cytokinesis. This morphology and actin distribution was mimicked by RNAi inhibition of Aip1, a protein that acts cooperatively with cofilin in disassembling actin in Xenopus and budding yeast. Aip1 also produced a cytokinesis defect. These results indicate that both cofilin and Aip1 are essential for actin remodeling during lamella formation and that, despite the similarities in cell morphology produced by RNAi against either of them, these two proteins have distinct roles in actin regulation (Rogers, 2003).
Epithelial invagination is necessary for formation of many tubular organs, one of which is the Drosophila embryonic salivary gland. Actin reorganization and control of endocycle entry are crucial for normal invagination of the salivary placodes. Embryos mutant for Tec29 (Flybase designation - Btk29A), the Drosophila Tec family tyrosine kinase, show delayed invagination of the salivary placodes. This invagination delay is partly the result of an accumulation of G-actin in the salivary placodes, indicating that Tec29 is necessary for maintaining the equilibrium between monomeric actin (G-actin) and filamentous actin (F-actin) during invagination of the salivary placodes. Furthermore, normal invagination of the salivary placodes appears to require the proper timing of the endocycle in these cells; Tec29 must delay DNA endoreplication in the salivary placode cells until they have invaginated into the embryo. Taken together, these results show that Tec29 regulates both the actin cytoskeleton and the cell cycle to facilitate the morphogenesis of the embryonic salivary glands. It is suggested that apical constriction of the actin cytoskeleton may provide a temporal cue ensuring that endoreplication does not begin until the cells have finished invagination (Chandrasekaran, 2005).
Salivary placode cells must constrict apically and move their nuclei basally in order to invaginate into the embryo. Therefore, the process of salivary gland invagination is expected to require actin-myosin contractility. Tec29 is responsible for regulating the actin reorganization in the salivary placodes prior to invagination. Lack of Tec29 in the salivary placodes results in a shift of actin in the salivary placodes from F-actin to more G-actin, leading to incomplete invagination of the salivary placodes cells. This change in the balance between F-actin and G-actin is observed only on the apical surfaces of these cells, suggesting that the delayed invagination is due to aberrant apical constriction of the salivary placodes cells in Tec29 embryos. Increasing actin depolymerization further in Tec29 mutants by a mutation in chic increases the salivary invagination delay, whereas promoting actin polymerization in Tec29 mutants by mutating twinstar partially rescues the invagination delay. Thus, these results provide the first evidence that actin reorganization is necessary for salivary gland invagination (Chandrasekaran, 2005).
The p21 activated kinase (Pak) family of protein kinases are involved in many cellular functions like re-organisation of the cytoskeleton, transcriptional control, cell division, and survival. These pleiotropic actions are reflected in a plethora of known interacting proteins and phosphorylation substrates. Yet, the integration of a single Pak protein into signalling pathways controlling a particular developmental process are less well studied. For two of the three known Pak proteins in Drosophila melanogaster, D-Pak and Mushroom bodies tiny (Mbt), distinct functions during eye development have been established. This study undertook a genetic approach to identify proteins acting in the Mbt signalling pathway during photoreceptor cell morphogenesis. The genetic screen identified the actin depolymerisation factor Twinstar/Cofilin as one target of Mbt signalling. Twinstar/Cofilin becomes phosphorylated upon activation of Mbt. However, biochemical and genetic experiments question the role of the LIM domain protein kinase (Limk) as a major link between Mbt and Twinstar/Cofilin as it has been suggested for other PAK proteins. Constitutive activation of Mbt not only disturbs the actin cytoskeleton but also affects adherins junctions organisation indicating a requirement of the protein in cell adhesion dependent processes during photoreceptor cell differentiation (Menzel, 2007).
Morphogenesis is a fundamental process during the development of a multi-cellular organism, which not only requires the specification of the various cell types, but also their assembly into complex tissues by means of cell shape changes, cell movement, sorting processes, and elimination of surplus cells. Many of these events depend on precisely controlled modulation of cell-cell or cell-extracellular matrix contacts (Menzel, 2007).
Adherens junctions are specialized membrane structures that mediate adhesion between epithelial cells and provide a link to the actin cytoskeleton. The central component of adherens junctions is the cadherin-catenin complex. Cadherins constitute a family of transmembrane proteins that mediate Ca2+-dependent cell-cell adhesion. The intracellular domain of cadherin binds to β-Catenin (Drosophila: Armadillo), which in turn can associate with the actin binding protein α-Catenin. However, the prevailing dogma that cadherins at adherens junctions are linked to the actin cytoskeleton through β-Catenin and α-Catenin in a stable quaternary complex has recently been questioned by the finding that α-Catenin is associated in a mutually exclusive manner with either cadherin-β-Catenin or with actin (Menzel, 2007).
A role of adherens junctions as a focal point for intracellular signalling has emerged. Notably, the formation of cadherin-mediated cell contacts influences the activity of the RhoGTPases Cdc42, Rac and Rho. These proteins function as molecular switches, cycling between an active GTP-bound conformation and an inactive, GDP-bound state. In its active form, RhoGTPases bind to a vast number of downstream effector proteins and one of the major effects is the reorganisation of the actin cytoskeleton (Menzel, 2007).
A family of proteins, which are influenced by RhoGTPases in its activity and localisation, are the p21-activated kinases (Pak). Pak proteins are characterised by a C-terminal serine/threonine kinase domain and a N-terminal p21-binding domain (PBD) required for binding of Rac- or Cdc42-like RhoGTPases. Based on additional structural features, which determine the regulation of the kinase activity and the binding to other proteins, Pak proteins can be classified into two subgroups. From the six Pak proteins known in Homo sapiens (Hs), three (HsPak1-3) have been assigned to group 1, whereas HsPak4-6 belong to the group 2. Similar, the three Pak proteins found in Drosophila melanogaster can be classified as group 1 (D-Pak, D-Pak3) and group 2 (Mbt/D-Pak2) members, respectively. The role of Pak proteins as regulators of the cytoskeleton, cell morphology and cell motility is well established. Different Pak proteins can induce the formation of lamellipodia, filopodia, and membrane ruffles as well as the dissolution of stress fibers and the disassembly of focal adhesions. A function of Pak proteins at the centrosomes of dividing cells has been described. Deregulation of Pak protein activity can result in oncogenic transformation. The pleiotropic functions of Pak proteins are reflected in the plethora of known interacting proteins and phosphorylation substrates. This obviously raises the question of differential activation of effector pathways by different Pak proteins in a cell-type specific manner (Menzel, 2007).
The developing Drosophila eye is used as a model system to study the role of the group 2 Pak protein Mbt in tissue morphogenesis. The adult eye with its 800 single eye units (ommatidia), each containing eight photoreceptor cells as well as non-neuronal cone, pigment, and bristle cells, arises from a monolayer epithelium, the eye imaginal disc. Differentiation of the different cell types of the eye is initiated in third instar larvae, when an indentation known as the morphogenetic furrow starts to move from posterior to anterior across the eye disc. Anterior to the furrow, cells continue to proliferate, posterior to it, the photoreceptor cells and lens-secreting cone cells become specified in a sequential manner through a series of inductive cell-cell interactions. At pupal stage, pigment and bristle cells are added to complete the ommatidium structure and surplus cells are eliminated by programmed cell death. After recruitment and determination, photoreceptor cells undergo massive morphological changes to form the rhabdomeres, the light-sensitive structures. Rhabdomeres are apical membrane specializations. Alignment of rhabdomeres to the ommatidial optical axis is achieved by involution of the apical domains and adherens junctions into the epithelial layer followed by their massive expansion in distal-proximal direction (Menzel, 2007).
Loss of function studies of the Pak proteins Mbt and D-Pak provided evidence for distinct localisation and function of these proteins during photoreceptor cell development. D-Pak is required in growth cones to control guidance of photoreceptor cell axons, whereas recruitment of Mbt to adherens junctions is dependent on a functional RhoGTPase binding site (Schneeberger, 2003). In mbt mutant animals, recruitment of the photoreceptor cells appear largely normal, but their final differentiation is disturbed. The adherens junctions become highly disorganized and rhabdomeres fail to elongate properly leading to the suggestion that Mbt acts as a downstream effector of activated RhoGTPases at adherens junctions to regulate photoreceptor cell morphogenesis (Schneeberger, 2003). This study shows that activation of Mbt disturbs the co-ordinated assembly of the ommatidium structure by interfering with the actin cytoskeleton and adherens junctions. Given the multitude of potential candidate targets of Mbt, a genetic screen was establised to identify components that act downstream of Mbt to control photoreceptor cell morphogenesis. This screen identified the actin depolymerisation factor Twinstar as one target of Mbt signalling. Yet, biochemical and genetic experiments question the role of LIM domain protein kinase (Limk) as a major link between Mbt and Twinstar (Menzel, 2007).
The view of adherens junctions as static structures that maintain epithelial integrity appears to be at odds with the requirement of dynamic cell shape changes during development. The assembly of the ommaditia of the Drosophila eye from an initially unpatterned epithelium provides an example. Photoreceptor cells become specified during third larval instar, but their final morphology is established at pupal stage. Simultaneously, the non-neuronal cone, pigment, and bristle cells must form a precise cell lattice that requires cell shape changes, movements and elimination of surplus cells. In spite of these dynamic processes, the integrity of adherens junctions as a basic feature of epithelia cells has to be maintained throughout eye development. The prevailing view that E-Cadherin is stably linked via β-Catenin and α-Catenin to the actin cytoskeleton has recently been questioned. It was shown that monomeric α-Catenin binds to E-Cadherin/β-Catenin, whereas dimeric α-Catenin does not and instead binds with high affinity to actin to influence Arp2/3-mediated actin polymerisation. If it is not α-Catenin that provides a physical link between adherens junctions and the actin cytoskeleton, what other proteins could mediate such a link (Menzel, 2007)?
Genetic and biochemical evidence is presented that the Pak protein Mbt, which localizes in a RhoGTPase dependent manner to adherens junctions, could provide a link to the actin cytoskeleton during photoreceptor cell morphogenesis. Constitutive activation of Mbt in photoreceptor cells causes clustering of F-actin, which finally leads to complete disruption of the adult eye morphology. In addition, the positioning of nuclei at apical levels is disturbed. The finding that mutations in twinstar and the phosphatase-encoding slingshot act as modifiers of the eye phenotype of a hypomorphic mbt allele suggest that these proteins work together to control photoreceptor cell morphogenesis. Additional evidence comes from the phenotypic analysis of the corresponding mutants. In twinstar mutants, elevated levels of F-actin, disruption of the ommatidial architecture and misshapen rhabdomeres are observed. Loss of Slingshot function results in increased F-actin levels and defects in the apical movement of photoreceptor cell nuclei. So far, no evidence is available for a direct interaction between Slingshot and Mbt. In vertebrates, Pak4 inactivates Slingshot and activates Limk by phosphorylation. Slingshot plays a dual role. It dephosphorylates and thereby down-regulates the activity of Limk to phosphorylate Cofilin at serine 3 and it directly dephosphorylates Cofilin at serine 3. Thus Pak4 activation results in a decrease of Cofilin activity and actin filament turnover. A major role of D-Limk in Mbt signalling is questioned by biochemical and genetic experiments. Activated Mbt is able to induce Twinstar phosphorylation. However, despite binding and phosphorylation of D-Limk by activated Mbt, no strong increase of D-Limk activity was seen. Furthermore, genetic experiments with a recently identified null mutation in D-Limk show that Mbt signalling is not completely blocked in the absence of D-Limk function. Yet, the strong synergistic effect upon complete removal of Mbt and D-Limk function indicates the requirement of both proteins in eye development. If it is not Mbt, what other proteins could be involved in regulation of D-Limk function during eye morphogenesis? Recent experiments implicate a role of the Par3 protein as a regulator of vertebrate Limk-2 activity towards Cofilin. Interestingly, the Drosophila Par3 homologue Bazooka localizes to and is required for maintenance of adherens junctions during photoreceptor cell morphogenesis (Menzel, 2007).
It is also evident that regulation of Twinstar activity by Pak proteins is not restricted to eye development. Mutations in twinstar lead to proliferation defects of central brain neuroblasts and the neurons derived from these neuroblasts show axon growth defects. In this study, genetic evidence was presented that Twinstar function in axon growth is activated by Slingshot and inhibited by D-Limk, which in turn is regulated by D-Pak and Rock-kinases. In vertebrates, Limk and Slingshot control growth cone motility and morphology via Cofilin phosphorylation. Knock-out of Limk in mice leads to changes in Cofilin phosphorylation and the actin cytoskeleton resulting in abnormalities in spine morphology and in synaptic function. A dominant-negative version of mouse Pak1 causes changes in spine number and synaptic morphology. Thus, different Pak proteins might not only act as general regulators of Cofilin activity, but they could also provide spatial and temporal control even in a single cell (Menzel, 2007).
In addition to the role in re-organisation of the actin cytoskeleton, overexpression experiments indicate that Mbt is also able to influence the adhesive properties of cells. Differential adhesion is crucial to establish the final retinal pattern. It has been demonstrated that an apical protein complex consisting of Crumbs, Stardust, and DPATJ controls assembly of adherens junctions and is essential for rhabdomere maintenance. Differential adhesion, by expression of DE-Cadherin alone or together with DN-Cadherin has been suggested as a mechanism that controls cell shape changes. Expression of DN-Cadherin in cone cells (in addition to DE-Cadherin, which is expressed in all cells in an ommatidium) confers on them specific adhesion properties in order to adopt a cell shape that minimizes surface contacts with surrounding cells. Integration of other proteins into the adhesive complex could be another mechanism to modify adhesive contacts. Hibris and IrreC/rst, transmembrane proteins of the immunoglobulin superfamily, become integrated into the adhesion complex and form an intercellular link only at the interface between the presumptive primary pigment cells and secondary/tertiary pigment cells to control proper cell sorting during pupal eye development. Interference with DE-Cadherin function blocks proper localisation of IrreC/rst and results in cell sorting and survival defects. It is therefore conceivable that neuronal expression of activated Mbt influences DE-Cadherin-mediated cell adhesion of photoreceptor and bristle cells with the surrounding pigment and cone cells. In this way, motility, sorting or survival of all retinal cells can be affected. Evidence for a function of other group 2 PAK proteins in regulation of cell adhesion comes from studies with human PAK4 and Xenopus X-Pak5. Expression of an activated version of human Pak4 leads to anchorage-independent growth of fibroblasts. X-Pak5 is expressed in regions of the embryo that undergo extensive cell movements during gastrulation. It was shown that X-Pak5 localises to cell-cell junctions and regulates in a calcium-dependent manner cell-cell adhesion. An activated version of X-Pak5 decreased cell adhesiveness, while kinase-dead X-Pak5 increased adhesion. However, in all cases the molecular targets of the PAK proteins to exert these effects remain to be identified (Menzel, 2007).
Drosophila photoreceptors undergo marked changes in their morphology during pupal development. These changes include a five-fold elongation of the retinal cell body and the morphogenesis of the rhabdomere, the light sensing structure of the cell. twinstar (tsr), which encodes Drosophila cofilin/ADF (actin-depolymerizing factor), is required for both of these processes. In tsr mutants, the retina is shorter than normal, the result of a lack of retinal elongation. In addition, in a strong tsr mutant, the rhabdomere structure is disorganized and the microvilli are short and occasionally unraveled. In an intermediate tsr mutant, the rhabdomeres are not disorganized but have a wider than normal structure. The adherens junctions connecting photoreceptor cells to each other are also found to be wider than normal. It is proposed that these wide rhabdomeres and adherens junctions are secondary events caused by the inhibition of retinal elongation. These results provide insight into the functions of the actin cytoskeleton during morphogenesis of the Drosophila eye (Pham, 2008).
Analysis of tsr RS mutations suggests that tsr has an important role in retinal cell elongation and in the morphogenesis of the rhabdomere. During wild type development, retinal cells undergo five-fold elongation, whereas this elongation is defective in tsr RS mutants. Strong tsr mutants also showed additional defects in rhabdomere morphogenesis, as noted by a severe reduction in F-actin in the pupal rhabdomere, and by rhabdomere defects displayed by TEM analysis. These defects included shortened microvilli, suggesting a tsr role in microvilli morphogenesis. In addition, in many instances, a microvilli or set of microvilli appeared to 'unravel' from the rhabdomere, indicating a role for tsr in maintenance of the rhabdomere structure. It had been hypothesized that the actin rich rhabdomere terminal web is required to maintain the organization of the microvilli. The observed phenotype is consistent with tsr functioning as part of the rhabdomere terminal web to stabilize the rhabdomere structure (Pham, 2008).
Rhabdomeres in intermediate tsr RS mutants were wider than normal. While it is possible that tsr is directly involved in shaping the rhabdomere, a second possibility is presented, that the widened rhabdomeres are a secondary event caused by the lack of retinal elongation. This hypothesis suggests that there is incomplete feedback in the tsr RS mutant, preventing the cell from recognizing that it is not elongating. The tsr RS mutant photoreceptor cells therefore produce all the rhabdomere proteins necessary for a fully elongated cell, but when the mutant cell does not elongate, a short but wide rhabdomere forms. However, the relationship between length and width is not fluid, there is not a one to one correlation between a lack of rhabdomere elongation and an increased in width. For instance, in the weakest tsrRS mutant, retinal elongation is inhibited by about 50%, yet rhabdomeres from this weak mutant are wild type in width. It is therefore believed that there are cellular mechanisms that maintain that correct size and shape of the rhabdomere, and only when this mechanism is stressed beyond a certain threshold (such as in the intermediate mutant) do the widened rhabdomeres appear. As predicted by the hypothesis, the widened rhabdomeres only formed in the tsr RS mutant after retinal elongation should have begun. Widened adherens junctions were also observed in the tsr mutant, this is likely also due to a lack of retinal elongation. Interestingly, it was found that the retinal adherens junctions in a wild type 55% pupae are wider than in a wild type 73% pupae. It is therefore likely that the cell builds up adherens junction proteins in anticipation of retinal elongation, then during elongation these excess proteins converge and extend to form the longer but slimmer adult adherens junction. Many adherens junctions in the 73% pupal tsr mutant were wider than wild type, suggesting that inhibition of retinal elongation prevents this conversion and extension from occurring (Pham, 2008).
Black patches of necrotic cells are frequently observed in the strong tsr RS mutants. It is possible that this necrotic phenotype is a secondary effect due to a lack of retinal cell elongation. As the retinal cells elongate, they increase in volume. However, if the cytoskeleton cannot change its structure as the cell increases in volume, and there is no feedback telling the cell this, the result may be that the cell overgrows its support structure and bursts, committing necrosis (Pham, 2008).
Many Drosophila mutants have widened rhabdomeres as a mutant phenotype. The data suggest the possibility that these widened rhabdomeres are secondary effects due to a blockage of retinal elongation. For example, photoreceptor cells mutant for crumbs, a transmembrane apical determinant, have a complicated phenotype, but the mutant cells are shorter than wild type and also have widened rhabdomeres and adherens junctions. The results suggest that these crumbs mutant phenotypes may be related, that crumbs may be directly required for retinal elongation, and, as with tsr, that the rhabdomere and adherens junction phenotypes may be secondary events (Pham, 2008).
The above hypothesis provides an explanation for the widened rhabdomeres and adherens junctions, but does not explain why tsr is required for retinal elongation. As a first step to addressing this, it was found that mutations in three other cytoskeletal genes (ssh, capping protein β and Arc-p34) had wide rhabdomeres, suggesting an inhibition of retinal elongation, which was verified by serial section analysis of the Arc-p34 mutant. This indicates that the defect in retinal elongation is not a tsr specific mutant phenotype, but is instead a general mutant phenotype for the inability to reorganize the actin cytoskeleton during retinal elongation (Pham, 2008).
This study introduces a new kind of conditional lethal mutation that is called a repressor sensitive mutation. The main advantage of this technique is its tissue specificity, it can reduce gene activity specifically in one tissue by driving Gal4-Pc from a tissue specific promoter. This technique should be adaptable to any gene in which a genomic rescue construct is available. Theoretically, this technique should also be able to repress the expression of endogenous genes that are adjacent to a P element insertion containing UAS sites. An unexpected advantage of the repressor sensitive technique was the easily established phenotypic series. A combination of position effect and dosage compensation provided a series of mutants whose phenotypes varied from weak, to intermediate to strong. This series allowed characterization of microvilli formation in the strong mutant, and retinal elongation and the widening of the rhabdomeres and adherens junctions in the intermediate mutant. In addition, the weak tsr RS mutations are being used to study the role of tsr during phototransduction. The further development of the repressor sensitive technique will add to the already large group of genetic tools available to Drosophila researchers (Pham, 2008).
Regulation of actin assembly and depolymerization is important for the organization of epithelia. Recent studies have shown that the actin-capping proteins are required to prevent cell extrusion and inappropriate activation of Yorkie (Yki) activity in Drosophila, implicating the importance of actin regulation for epithelial integrity and Yki-dependent tissue growth. However, the role of Twinstar (Tsr), the Drosophila homolog for cofilin/actin depolymerization factor, in epithelial integrity and Hippo signaling is unknown. This study demonstrates that reduction of Tsr by RNA interference (RNAi) or mutant clones in wing disc induces not only F-actin accumulation but also ectopic expression of Wingless (Wg) and Yki target gene expanded. Knockdown of Yki in Tsr-depleted cells reduces the level of ectopic Wg expression. Reduced Tsr also leads to downregulation of cell junction proteins and extrusion of affected cells from the basal part of the epithelium. Rho is upregulated in Tsr-depleted tissue, supporting the Tsr function in the inhibition of cell extrusion from the epithelium. Tsr is also required for blocking cell death and JNK signaling. Ectopic JNK activation induces caspase activation but does not cause cell extrusion. Taken together, these data suggest that Tsr is required for cell survival and tissue growth by regulating JNK and Yki signaling while maintaining the epithelial integrity by controlling cell junctions. The study provides an insight into potential roles of ADF/cofilin in invasive cell migration and tumor suppression in higher animals (Ko, 2016). The actin bundle is an array of linear actin filaments cross-linked by actin bundling proteins, but its assembly and dynamics are not as well understood as those of branched actin network. This study used the Drosophila bristle as a model system to study actin bundle formation. Cofilin, a major actin disassembly factor of branched actin network, was found to promote the formation and positioning of actin bundles in the developing bristles. Loss-of-function of cofilin or AIP1, a cofactor of cofilin, each resulted in increased F-actin levels and severe defects in actin bundle organization, with the defects from cofilin deficiency being more severe. Further analyses revealed that cofilin likely regulates actin bundle formation and positioning by the following means. First, cofilin promotes a large G-actin pool both locally and globally, likely ensuring rapid actin polymerization for bundle initiation and growth. Second, cofilin limits the size of a nonbundled actin-myosin network to regulate positioning of actin bundles. Third, cofilin prevents incorrect assembly of branched and myosin-associated actin filament into bundles. Together, these results demonstrate that the interaction between the dynamic dendritic actin network and the assembling actin bundles is critical for actin bundle formation and needs to be closely regulated (Wu, 2016).
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