BIOLOGICAL OVERVIEW

Specialized microenvironments, or niches, provide signaling cues that regulate stem cell behavior. In the Drosophila testis, the JAK-STAT signaling pathway regulates germline stem cell (GSC) attachment to the apical hub and somatic cyst stem cell (CySC) identity. This study demonstrates that chickadee, the Drosophila gene that encodes profilin, is required cell autonomously to maintain GSCs, possibly facilitating localization or maintenance of E-cadherin to the GSC-hub cell interface. Germline specific overexpression of Adenomatous Polyposis Coli 2 (APC2) rescued GSC loss in chic hypomorphs, suggesting an additive role of APC2 and F-actin in maintaining the adherens junctions that anchor GSCs to the niche. In addition, loss of chic function in the soma resulted in failure of somatic cyst cells to maintain germ cell enclosure and overproliferation of transit-amplifying spermatogonia (Shields, 2014).

Chickadee, the only Drosophila profilin homolog, is required cell intrinsically for GSC maintenance in the testis. As profilin is a regulator of actin filament polymerization and filamentous actin (F-actin) plays a crucial role in the development and stabilization of cadherin-catenin-mediated cell-cell adhesion, profilin likely maintains attachment of Drosophila male GSCs to the hub through its effect on F-actin, which concentrates at the hub-GSC interface where localized adherens junctions anchor GSCs to hub cells. It is proposed that profilin-dependent stabilization of F-actin at the GSC cortex next to the hub may help localize E-cadherin and APC2 to the junctional region. E-cadherin and APC2 in turn may recruit β-catenin/Armadillo, stabilizing the adherens junctions that attach GSCs to the hub. Chickadee may thus facilitate maintenance of GSCs through a cascade of interactions leading to localization and/or retention of both E-cadherin and β-catenin at the hub-GSC interface (Shields, 2014).

E-cadherin plays a crucial role in maintaining hub-GSC attachment. GSC clones mutant for E-cadherin are not maintained. In addition, germline overexpression of E-cadherin delayed GSC loss in stat-depleted GSCs. The results indicate that profilin function is required in GSCs for proper localization of E-cadherin to the hub-GSC interface. Several studies have shown that the actin cytoskeleton plays a crucial role in assembly and stability of adherens junctions. A favored model in the field is that actin filaments indirectly anchor and reinforce E-cadherin-mediated cell junctions by forming an intracellular scaffold for E-cadherin molecules. Indeed, binding to F-actin stabilized E-cadherin and promoted its clustering. Furthermore, the actin cytoskeleton participates in proper localization of E-cadherin molecules to cell-cell contacts. In chic/profilin mutant GSCs, disruption of actin polymerization at the cell cortex leading to local F-actin disorganization may destabilize E-cadherin and reduce its ability to localize to the GSC-hub junction, form clusters and build adequate adherens junctions (Shields, 2014).

Destabilization of E-cadherin may contribute to the mislocalization of APC2 seen in chic mutant GSCs, as E-cadherin recruits APC2 to cortical sites in GSCs. Raising possibilities of a more direct link, actin filaments have been shown to be required for association of APC2 with adherens junctions in the Drosophila embryo and ovary. Treatment of embryos with actin-depolymerizing drugs resulted in complete delocalization of APC2 from adhesive zones and diffuse APC2 staining throughout the cell. Moreover, in ovaries of chic¹³²⁰/chic²²¹ females, APC2 was substantially delocalized from the plasma membranes of nurse cells and their ring canals, and increased levels of cytoplasmic APC2 staining were observed. Similarly, this study found that APC2 was delocalized from the hub-GSC interface in larval testes of chic¹¹/chic¹³²⁰ hypomorphs (Shields, 2014).

In several studies, delocalization of APC2 from junctional membranes correlated with detachment of β-catenin/Armadillo from adherens junctions. APC2 co-localizes with Armadillo and E-cadherin at adherens junctions of Drosophila epithelial cells, nurse cells in Drosophila ovaries and at the hub-GSC interface in Drosophila testes. Disruption of APC2 function resulting in significant reduction of junctional APC2 was accompanied by delocalization of junctional Armadillo and increased levels of free cytoplasmic Armadillo in embryonic epithelial cells and ovaries. In a previous study, which used chic¹³²⁰/chic²²¹ strong loss-of-function mutants, the delocalizing effect on junctional Armadillo was variable, presumably due to incomplete penetrance of chic mutant effects. Although this study did not observe significant disruption in Armadillo staining along the hub-GSC interface of testes from chic hypomorphs, this may be due to incomplete penetrance. In addition, the Armadillo protein detected could be localized to the cortex of hub cells rather than GSCs (Shields, 2014).

The finding that germline specific overexpression of APC2 in chic¹¹/chic¹³²⁰ hypomorphs partially rescued GSC loss is consistent with a previously proposed model that actin filaments shuttle APC2 to adherens junctions and APC2 in turn recruits cytoplasmic Armadillo to junctional membranes, reinforcing the adherens junctions. It is possible that in chic¹¹/chic¹³²⁰ hypomorphs, residual actin filaments associated with adherens junctions between the hub and GSCs are sufficient to shuttle the increased amounts of cytoplasmic APC2 to adherens junctions. This APC2 may in turn recruit free cytoplasmic Armadillo to the hub-GSC interface, locally stabilizing the adherens junctions and anchoring GSCs to their niche. Notably, however, germline specific overexpression of APC2 in testes of strong loss-of-function chic¹³²⁰/chic²²¹ mutants failed to rescue GSC loss. Thus either, adequate levels of actin filament polymerization may be required for the proposed translocation of junctional proteins to the plasma membrane, or APC2 function/localization may not be the only or even the major cell-autonomous target of profilin function important for maintaining GSCs. Indeed, loss of APC2 function did not lead to GSC loss. It is suggested that the localized cortical F-actin underlying adherens junctions at the GSC-hub interface, best candidate for the most direct target of chic function, strongly stabilizes adherens junctions between GSCs and the hub, with high levels of cortical APC2 able to in part make up for weak chic function by also stabilizing adherens junctions (Shields, 2014).

Maintenance of hub-GSC attachment appears to be a key role of STAT in GSCs. The finding that STAT binds to a site near the upstream promoter of the chic gene raises the possibility that STAT might foster GSC attachment to the hub in part by ensuring high levels of transcription of profilin in GSCs. However, activation of STAT is clearly not the only regulatory influence on profilin expression as profilin is an essential gene expressed in many cell types, including those in which STAT is not active or detected. It is likely that transcription factors other than STAT turn on profilin expression in many cell types and that STAT acts along with other regulators to reinforce profilin expression in GSCs and CySCs. Conversely, overexpression of profilin was not sufficient to re-establish attachment of stat-depleted GSCs, suggesting that STAT probably regulates a number of genes to ensure that GSCs remain within the stem cell niche (Shields, 2014).

Loss of chic function in somatic cyst cells impaired the ability of cyst cells to build and/or maintain the cytoplasmic extensions through which they embrace and enclose spermatogonial cysts. Two somatic cyst cells normally surround each gonialblast and enclose its mitotic and meiotic progeny throughout Drosophila spermatogenesis. The cyst cells co-differentiate with the germ cells they enclose. Several lines of evidence support the model that either the ability of somatic cyst cells to enclose germ cells or their ability to send signals to adjacent germ cells is important to restrict proliferation and promote differentiation of germ cells. In either case, activation of EGFR in cyst cells is required for cyst cells to enclose germ cells and/or send the signals for germ cells to differentiate. The similarities in phenotype between loss of chic function and loss of EGFR activation in somatic cyst cells raise the possibility that chic/profilin may act downstream of activated EGFR to modulate the actin cytoskeleton for the remodeling of cyst cells to form or maintain the cytoplasmic extensions that enclose germ cells. Indeed, activated EGFR is known in other systems to tyrosine phosphorylate phospholipase C-γ1 (PLC-γ1), a soluble enzyme in quiescent cells like daughter cyst cells, activating it to catalyze hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂), which binds profilin protein with high affinity, which inhibits the interaction between profilin and actin. The hydrolysis of PIP₂ by activated PLC-γ1 results in localized release of profilin and other actin-binding proteins, enabling them to interact with actin and participate in cytoskeletal rearrangement and membrane protrusion. Thus, based on biochemical analysis in other systems, a link between EGFR activation and profilin leading to local remodeling of the actin cytoskeleton is plausible in somatic cyst cells, although it remains to be directly tested (Shields, 2014).

The Drosophila insulin pathway controls Profilin expression and dynamic actin-rich protrusions during collective cell migration

Understanding how different cell types acquire their motile behaviour is central to many normal and pathological processes. Drosophila border cells represent a powerful model to address this question and to specifically decipher the mechanisms controlling collective cell migration. This study has identified the Drosophila Insulin/Insulin-like growth factor Signalling (IIS) pathway as a key regulator controlling actin dynamics in border cells, independently of its function in growth control. Loss of IIS activity blocks the formation of actin-rich long cellular extensions that are important for the delamination and the migration of the invasive cluster. IIS specifically activates the expression of the actin regulator chickadee, the Drosophila homolog of Profilin, essential for promoting the formation of actin extensions and migration through the egg chamber. In this process, the transcription factor dFoxO acts as a repressor of chickadee expression. Altogether, these results show that local activation of IIS controls collective cell migration through regulation of actin homeostasis and protrusion dynamics (Ghiglione, 2018).

This study identified the Insulin/IGF-Signalling (IIS) pathway as a key regulator of border cell migration during Drosophila oogenesis. Activation of dInR at the onset of migration promotes actin dynamics in the outer border cells, the subpopulation of cells known to drive migration. In this process, the canonical IIS pathway is shown to act through the inhibition of the transcription factor FoxO, which leads to the de- repression of chic/profilin. High levels of Profilin in turn facilitate actin polymerization and the formation of dynamic protrusions and of specific, actin long cellular extensions which are required for delamination and proper migration of the invasive cell cluster (Ghiglione, 2018).

The conserved IIS pathway couples nutritional cues with cellular metabolism, which in turn is essential for coordinating development with growth conditions. The systemic action of the IIS pathway thus makes it difficult to discriminate between chronic versus more acute or specific roles in particular cellular processes and during morphogenesis. In this context, border cells provide a powerful model to specifically address the role of the IIS pathway on cellular motility. During Drosophila oogenesis, the IIS pathway acts both in the germline and somatic cells to adjust egg chamber maturation rates to protein availability. This study used the FLP/FRT system that to show that chronic downregulation of IIS in border cells impairs their migration, a process that can be associated with metabolic defects. Interestingly, acute manipulation of IIS in border cells using the Gal4/Gal80ts system, shows that IIS downregulation can also block cluster migration specifically, a phenotype that can be rescued partly by restoring Profilin expression. These data argue for an active control of cell migration by IIS, independently of cellular fitness. This view is consistent with previous work showing that in ex vivo experiments, Insulin-containing culture medium is necessary to support egg chamber development and border cell migration (Ghiglione, 2018).

Border cells migrate towards the oocyte to make the micropyle, an opening that allows oocyte fertilization through the chorion. In this process, border cell migration needs to be synchronized with oocyte growth. It is proposed that the dual role of IIS for both egg chamber growth and border cell migration could help coordinating migratory events with organ maturation, thereby ensuring robust morphogenesis important for fertility (Ghiglione, 2018).

Actin dynamics is essential to a multitude of cellular and morphogenetic processes, therefore understanding the diverse modes of actin regulation is of prime interest. Members of the IIS pathway have been linked to actin regulation in a number of normal and pathological processes. For example, IIS plays an important role in neuronal guidance or wound healing. Additionally, PI3K has been shown to couple glycolytic flux with actin dynamics, while AKT participates to epithelial-to-mesenchymal transition required to drive mesoderm formation during gastrulation. Accumulating evidence also indicates that PI3K/AKT controls the migratory phenotype of metastatic cells. In breast cancer cells, AKT enhances cell migration and invasion through increased filopodia formation, which can be blocked with a specific AKT inhibitor. These observations suggest a model in which AKT activation potentially influences cell motility through direct modulation of actin, which is supported by studies showing that actin preferentially binds to phosphorylated AKT at pseudopodia sites. Despite these evidences, the view is fragmented and data are lacking to demonstrate a clear role of the full canonical pathway in cytoskeleton plasticity. In particular, the requirement of IIS transcriptional regulation in this process remained elusive. This report reveals that canonical IIS acts through inhibition of the transcription factor FoxO to control a major actin regulator, Profilin. These data provide a molecular mechanism as to how FoxO can control actin remodeling, which may be generalized to other processes where actin dynamics is particularly important. For example, during wound healing in Drosophila larvae, formation of an acto-myosin cable has been shown to depend on PI3K activation and redistribution of the transcription factor FoxO (Ghiglione, 2018).

In conclusion, these findings establish the canonical IIS pathway as a gene regulatory network important for collective cell migration. The data also provide a novel mechanism by which actin homeostasis and organization is regulated transcriptionally in a dynamic migratory process. In this mechanism, the formation of actin-rich protrusions is constitutively and negatively controlled by the transcription factor FoxO, whose inhibition by IIS signalling can generate peak levels of actin polymerization required for delamination and migration. It will be interesting to establish whether the control of Profilin expression through IIS signalling represents a general mechanism controlling actin remodeling in cell and tissue morphogenesis (Ghiglione, 2018).

Earlier Summaries

Chickadee, the Drosophila homolog of Profilin, a protein involved in actin polymerization, was initially characterized in a search for genes that function during cytoplasmic flow from nurse cells to the oocyte. Cytoplasmic flow along actin-based cytoskeletal microfilaments is responsible for transferring cytoplasmic components from nurse cells to oocytes. This process is critical: it ensures that the oocyte is supplied with sufficient stores for initial zygotic development. In chickadee mutants, females are sterile and there is a disruption of the nurse cell cytoplasmic flow and lack of nurse cell cytoplasmic actin networks. Thus chickadee is a perfect candidate to study as a gene involved in actin based dynamics in oocytes (Cooley, 1992)

Chickadee is implicated in several stages of bulk transport (known as 'dumping') of nurse cell contents into the oocyte. In chickadee mutants some nurse cell cytoplasm flows into the oocyte, although the transfer is incomplete. As the microtubule based cytoskeleton is implicated in nurse cell dumping (See beta1 tubulin), it is presumed that both actin based microfilaments and tubulin based microtubules (Theurkauf, 1994) are involved in dumping. What aspect of the actin based cytoskeleton is involved in cytoplasmic dumping? It is likely that sub-cortical actin-based nurse-cell microfilaments play a role in cytoplasmic flow into the oocyte. (There are there are more than three types of cytoskeleton: for example, actin based microfilaments, microtubules, and subcortical microfilaments. See cytoskeleton for a more detailed description of these three). A nonmuscle myosin is found to associate with subcortical actin but not with cytoplasmic networks. These subcortical actin filaments are very sensitive to cytochalasin treatment. Thus, contraction of the subcortical actin could play a role in the bulk movement of nurse cells into the oocyte (Cooley, 1992 and references).

The actin based microfilament cytoskeleton plays an additional role in cytoplasmic dumping. At stage 11, the nurse cells dump their contents into the oocyte through cytoplasmic bridges termed ring canals. Microfilament bundles form in the nurse cells during this process and are apparently required to hold the nurse cell nuclei in place so that they do not obstruct the ring canals and allow rapid flow of nurse cell cytoplasm into the oocyte. It is thought that these cytoplasmic microfilament bundles are non-contractile and serve a structural function (Mahajan-Miklos, 1994). Mutants in chickadee, quail and singed affect actin bundle formation. Profilin, encoded by chickadee, is presumably required for the polymerization of the actin filaments that compose the bundles (Cooley, 1992), while a villin-related protein encoded by quail and a fascin-related protein encoded by singed are thought to be required to cross-link the actin filaments to form the actin bundles. Two components of the actin-lined ring canals have also been identified - an adducin-like protein encoded by hu-li tai shao and a protein containing scruin repeats encoded by kelch (Mahajan-Milos, 1994 and Manseau, 1996 and references).

The actin and tubulin based microfilament components of the cytoskeleton are intimately associated in oocytes; any discussion of one without the other is clearly incomplete. The rapid cytoplamic streaming that occurs during the microfilament-dependent rapid transfer of cytoplasm from nurse cells into the oocytes is dependent on microtubules. This is known since streaming is inhibitable by colcemid, which functions to disrupt microtubules. Mutations in cappuccino and spire repress this microtubule-based ooplasmic streaming. In capu and spir mutants, the bundling of the microtubules at the cortex of the oocyte and streaming of the oocyte cytoplasm occurs prematurely. The effects on capu and spir mutations suggest that these genes are involved in microtubule processes. However, chickadee mutants share the premature streaming phenotype with capu and spir. The mutant phenotype of these three genes is due to a premature bundling of microtubules. Normally microtubules are found at the cortex of the oocyte from stages 8 through 10. In chic and capu mutants, long tubulin-staining fibers are found throughout the oocyte. It is concluded that a protein that interacts with the actin based cytoskeleton, Chickadee, is also involved in maintainence of the tubulin based cytoskeleton. In fact, mutations in chic result in the mislocalization of Staufen, which normally localizes to the posterior pole. Although the phenotype is quite variable, there is a close relationship between the effects of chic on the distribution of microtubules and on the distribution of Staufen (Manseau, 1996).

Therefore, Chickadee, a protein involved in actin cytoskeletal dynamics, is involved in maintainence of the tubulin based cytoskeleton. How can this been? It has been found that Cappuccino interacts directly with profilin. The fact that Capu and Chickadee interact directly, suggests that Capu and Chic may affect the same processes through this interaction. If this is true, then these two proteins may serve as the interface between the actin and tubulin based cytoskeletons (Manseau, 1996).

Another candidate for an protein that interacts with Chicadee may be Diaphanous, which functions in Drosophila to promote cytokinesis, the final separation process that occurs between daughter cells in mitosis. A mammalian protein, p140mDia, has been identified as a downstream effector of Rho, a small GTPase that regulates cell morphology, adhesion and cytokinesis through the actin cytoskeleton. (For more information see Drosophila Rac1). p140mDia is a mammalian homolog of Diaphanous. p140mDia binds selectively to the GTP-bound form of Rho and also binds to profilin, the homolog of Chickadee in mammals. The interactions among Rho, Diaphanous and profilin suggest that Rho regulates actin polymerization by targeting profilin via p140mDia beneath the specific plasma membranes, and that Diaphanous and profilin play a joint role in cytokinesis (Watanabe, 1997).

In Drosophila, Chickadee is involved in proliferation of germ-line cells in both males and females. In the adult female, the germarium is devoid of germline material, resulting in empty follicle cells stacks. Testes of mutant flies contain a few spermatid bundles; testes from older males are markedly smaller than wild type and appear agametic (Verheyen, 1994).

Both macrochaete and microchaete bristles on the head, thorax, legs and wings are affected by chickadee mutation. The bristle shaft is formed as a cytoplasmic extension of the trichogen cell. Its structure is provided by a core of microtubules surrounded by fiber bundles that are in fact actin filament bundles. The ridges seen on the bristle cuticle are formed by cytoplasm of the trichogen cell protruding between the actin filament bundles: therefore these ridges are indicative of the number of bundles formed. chic mutant bristles are thicker and shorter than normal with sharp bends, kinks and forked ends. The ridges on mutant bristles are often thinner, more numerous and disorganized. Mutant bristles contain abundant actin filament bundles, but they are more numerous and somewhat thinner than wild type. Thus the aberrant external ridge morphology correlates with the condition of the underlying actin filament bundles (Verheyen, 1994).

How does profilin/Chickadee function to regulate the actin cytoskeleton? To approach this question one must know something about actin, the substrate of profilin in mammals. Actin is an ATPase that goes through a cycle of nucleotide binding and hydrolysis. ATP hydrolysis is associated with actin polymerization, and ATP-actin polymerizes faster and at a lower critical concentration than ADP-actin. Profilin binding to an actin monomer decreases 1000-fold the affinity of actin for its bound nucleotide. Since ATP is generally present in large excess over ADP, this will have the effect inside cells of replacing bound ADP with ATP. In the absence of profilin, nucleotide exchange on an actin monomer is relative slow. These observations led to a model in which profilin can locally promote actin filament growth (Theriot, 1993).

Profilin functions in another pathway to promote filament growth. It has been shown that the profilin-actin complex adds directly onto the barbed end of growing actin filaments. Since profilin has a relatively low affinity for the barbed end of filaments, the profilin dissociates, leaving the actin filament one subunit longer. It is though that the pathway for barbed-end elongation involving profilin is more thermodynamically favored than the pathway without it; consequently, the final amount of free unpolymerized actin monomer is lower in the presence of profilin than in its absence (Theriot, 1993).

Profilin was originally identified as a component of cell extracts that inhibit actin filament growth in vitro. It was initially assumed that profilin was a major sequestering factor in most cells, and sequestering was considered to be profilin's primary function. However, there is not nearly enough profilin in a typical cell for this, and another protein that occurs in higher abundance (thymosin beta4) may be responsible for monomer sequestration (Theriot, 1993).

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

Profilin is a well-characterized protein known to be important for regulating actin filament assembly. Relatively few studies have addressed how profilin interacts with other actin-binding proteins in vivo to regulate assembly of complex actin structures. To investigate the function of profilin in the context of a differentiating cell, an instructive genetic interaction between mutations in profilin (chickadee) and capping protein beta (cpb) was studied. Capping protein is the principal protein in cells that caps actin filament barbed ends. When its function is reduced in the Drosophila bristle, F-actin levels increase and the actin cytoskeleton becomes disorganized, causing abnormal bristle morphology. chickadee mutations suppress the abnormal bristle phenotype and associated abnormalities of the actin cytoskeleton seen in cpb mutants. Furthermore, overexpression of profilin in the bristle mimics many features of the cpb loss-of-function phenotype. The interaction between cpb and chickadee suggests that profilin promotes actin assembly in the bristle and that a balance between capping protein and profilin activities is important for the proper regulation of F-actin levels. Furthermore, this balance of activities affects the association of actin structures with the membrane, suggesting a link between actin filament dynamics and localization of actin structures within the cell (Hopmann, 2003).

Capping protein loss of function leads to dramatic increases in F-actin in the fly bristle, resulting in aberrant organization of the actin cytoskeleton. Reduction of profilin suppresses the disorganized actin phenotype caused by reduction of capping protein function, suggesting that profilin promotes actin assembly in the elongating bristle. These results emphasize the idea that the balance of activities of actin-binding proteins is critical for assembling actin structures that are organized and positioned properly (Hopmann, 2003).

Numerous studies have demonstrated the importance of the actin cytoskeleton for the normal elongation and morphogenesis of the fly bristle. Inhibitors of actin polymerization significantly decreases the elongation rates of bristles whereas inhibitors of microtubule polymerization have little effect. The morphology of bristle actin bundles is affected by changes in the amount of cross-linking proteins as well as mutations in genes that encode regulators of actin dynamics, including ADF/cofilin (twinstar, twinfilin), and ADF/cofilin phosphatase (slingshot). Yet many of these alterations do not cause severely displaced and disoriented actin bundles. In contrast, mutations in capping protein strongly affect not only the amount of F-actin but also the position and orientation of actin structures. In this regard, the phenotype of twinfilin (twf) mutants is particularly noteworthy. Twinfilin is a monomer-sequestering protein that is structurally related to ADF/cofilin. In twf mutant bristles, F-actin levels are increased and the actin bundles are very disorganized, as they are in cpb mutants. Furthermore, the actin bundles show the same dramatic displacement from the membrane in twf as they do in cpb. This contrasts with the phenotype of chic bristles, which do not show displacement of bundles, and underscores the fact that although twinfilin and profilin both have sequestering activity in vitro, they clearly have different roles in vivo (Hopmann, 2003).

What the analysis of individual mutant phenotypes does not reveal is how the different actin regulatory proteins work together to generate normal actin bundles. Analysis of cpb chic double mutants demonstrates this clearly. Because the original phenotypic characterization of cpb and chic single mutants suggested that they both led to increased levels of F-actin, the original expectation was that chic loss of function would enhance cpb loss of function. Instead, the opposite effect was observed. This approach has yielded valuable insights regarding the importance of the balance of capping protein and profilin activities in normal cells. In other cases, mutant combinations do exhibit predictable phenotypes. For example, double heterozygous combinations of twf and tsr, which encode ADF/cofilin, exhibit a moderate bristle phenotype even though the single mutant heterozygotes show little or no bristle phenotype. This is consistent with the proposed function of both proteins: reduction of twinfilin leads to increases in F-actin assembly due to reduced sequestering activity, and reduction of ADF/cofilin leads to a decreased rate of actin depolymerization. Thus, it is expected that the two mutations behave synergistically and cause an increase in F-actin. It is anticipated that additional mutant combinations will be equally informative about the complex interplay of activities required to construct normal actin bundles. At present, formulating a model that incorporates the many different actin regulators is difficult because there is limited data of this type available (Hopmann, 2003).

The results support the idea that profilin has polymerization-promoting activity. Expression of vertebrate or plant profilins in mammalian tissue culture cells leads to increases in F-actin and profilin null clones in the developing Drosophila eye exhibit greatly reduced levels of F-actin (Hopmann, 2003 and references therein).

However, the observation that profilin acts in an opposite manner as that of capping protein, seeming to stimulate actin polymerization in the fly bristle, seems at first difficult to reconcile with the original characterization of the chic bristle phenotype. In chic mutants, the elongating bristle seemed to have an increased number of actin bundles that are thinner than wild-type bundles. This phenotype was thought to reflect an overall increase in the amount of F-actin, which is consistent with a monomer-sequestering role for profilin. Two possible explanations are suggested for this seeming paradox. First, biochemical data on profilin activity have shown that its activity is dependent on the state of the barbed ends. Profilin-actin can add to free barbed ends but not to capped ones. Thus, in wild-type bristles, barbed ends may be maximally capped (except at the growing tip) and profilin's primary function would be to sequester monomer. In a chic mutant bristle, reduction in profilin-mediated sequestering activity might lead to the observed increase in F-actin. It would then be predicted that when capping protein is reduced, barbed ends are not maximally capped and thus, profilin's polymerization-promoting activity would predominate, which is consistent with current observations (Hopmann, 2003).

Another interpretation of the chic bristle phenotype is suggested by the results of inhibitor studies performed on cultured Drosophila pupae. Exposure of cultured pupae to cytochalasin D, an inhibitor of actin polymerization, causes the actin bundles in elongating bristles to fall apart by splitting into thinner subbundles, reminiscent of chic mutant bristles that exhibit an increased number of thinner bundles. The similarity of these two phenotypes suggests that continued actin polymerization is required to maintain the integrity of actin bundles, and reductions in actin polymerization cause the actin bundles to 'unravel'. Although it is clear that profilin can promote actin polymerization, the mechanism by which it does this is less well understood. Studies in yeast have demonstrated that profilin's nucleotide exchange activity is required for its function. Because ATP-actin is more readily incorporated onto barbed ends of filaments, this activity can explain profilin's effects on actin assembly. However, there is reason to believe Drosophila profilin may not work this way. Plant profilins do not catalyze nucleotide exchange, and some even seem to repress it. A comprehensive mutational analysis of profilin in fission yeast has identified tyrosine79 as critical to its ability to stimulate nucleotide exchange. When tyrosine79 is mutated to arginine, S. pombe profilin loses its exchange activity. Notably, the majority of plant profilins naturally contain arginine at the comparable position, whereas all characterized vertebrate profilins, which tend to have very high exchange activity, contain aspartate. Thus, there is a correlation between arginine at position 79 and low activity, tyrosine and moderate activity, and aspartate and high activity. Interestingly, Drosophila has arginine: it is the only nonplant profilin, besides that of shrimp, known to have arginine at this position. The exchange activity of Drosophila profilin is unknown, but it seems reasonable to predict that Drosophila profilin has low activity (Hopmann, 2003).

Although plant profilins do not enhance nucleotide exchange by actin monomers, some stimulate actin polymerization in vitro in thymosin-ß4/actin solutions. Thymosin-ß4 is a true monomer sequestering protein in that T-ß4-actin cannot add to a growing filament, whereas profilin-actin adds readily to the barbed ends of actin filaments. Profilin is thought to shuttle monomer out of the T-ß4 pool, and this may be the relevant mechanism in other cell types. Studies in Drosophila may prove useful in elucidating the details as well as the physiological relevance of alternate mechanisms of profilin activity (Hopmann, 2003).

This article, as well as previous work, has demonstrated that a reduction of capping protein function leads to increased F-actin and abnormal actin organization. It is likely that the aberrant actin cytoskeleton underlies all of the defects observed in the adult bristle such as decreased length, bending, branching, and abnormal groove patterns. Although some of the correlations between the actin abnormalities and adult phenotypes are fairly obvious, it may seem counterintuitive that increases in F-actin levels would lead to shorter bristles. One might expect increased F-actin polymerization to give rise to longer bristles. Indeed, treatment of cultured pupae with jasplakinolide, a drug that stabilizes F-actin, increases the growth rate of the bristle shaft. However, these experiments were done for 6-7 h, whereas bristle elongation takes ~16 h at 25°C. Perhaps the increased growth rate would not be maintained were it possible to expose the growing bristle to drug for the entire elongation period. It is hypothesized that in cpb mutants, actin is overpolymerized at the beginning of bristle elongation. Some component required for actin bundle assembly may be limiting in the bristle; therefore, in a cpb mutant bristle, the limiting component would be prematurely depleted due to the increase in F-actin. Comparing the growth rates of wild-type and mutant bristles can test this idea (Hopmann, 2003 and references therein).

Although the data demonstrate that reduction of capping protein function leads to increases in F-actin, these changes were not quantified. It would have been desirable to measure the concentrations of F-actin in the various mutant genotypes directly, but technical limitations prevented doing so in a controlled manner. Phalloidin staining often varies greatly between experiments, so the subtle differences that were expected between different genotypes could be obscured. Quantitative methods are currently being developed for measuring actin in situ (Hopmann, 2003).

One of the most puzzling features of the cpb mutant phenotype is the displacement of actin bundles from the membrane. An increase in the amount of F-actin in the bristle does not, by itself, seem to explain this phenotype. In bristles where the cross-linking protein fascin is overexpressed, F-actin amounts are increased and bundles are considerably larger, but they do not show significant displacement from the membrane. A structural function of capping protein in physically linking the bundles to the plasma membrane would explain this phenotype. Previous studies in chicken myoblasts have uncovered a structural requirement for capping protein in organizing actin filaments within the sarcomere. However, a structural role seems unlikely given that the displacement of bundles is suppressed when profilin dosage is reduced. Instead, the proper regulation of actin assembly may be important for the positioning of actin bundles. twf mutant bristles also exhibit this displacement phenotype. Because capping protein and twinfilin are known to associate in yeast, this raises the interesting possibility that these two proteins work together in regulating actin assembly such that the association of bundles with the membrane is established and/or maintained. Intriguingly, treatment of cultured pupae with okadaic acid, an inhibitor of protein phosphatases, causes a similar displacement of actin bundles, suggesting the phosphorylation status of one or more proteins may be relevant (Hopmann, 2003).

This article shows that the balanced activities of capping protein and profilin are essential in the regulation of actin dynamics and organization in the elongating Drosophila bristle. The data are consistent with the emerging idea that the activity of profilin is context dependent, and that in many cells, profilin promotes actin assembly. The data also suggest that perturbations of actin dynamics in the bristle lead to a striking displacement of actin bundles from the membrane. In the future, it is hoped the role of capping protein in the bristle will be clarified, resulting in a better understand of how capping protein is integrated with the many other actin regulators functioning in the bristle, such that actin bundles are correctly assembled and positioned (Hopmann, 2003).

GENE STRUCTURE

Two transcripts, of 1.0 and 1.2 kb are present. The complementary DNAs reveal two different 5' exons, suggesting that the two transcripts are products of transcription from alternative promoters. The longer transcript is present in wild-type males and females and also present in RNA isolated from chickadee mutant flies. It is abundant in ovaries but also present in ovarectomized females. The shorter 1.0 kb transcript, however, is virtually ovary specific. It is concluded that the phenotype displayed by female-sterile alleles of chickadee is caused by the absence of the ovary-specific transcript of a gene that is also transcribed from a different promoter (Cooley, 1992). chickadee is immediately adjacent to the eukaryotic initiation factor, eIF4A (Verheyen, 1994).

Exons - Four, with alternative first exons

Bases in 3' UTR - 347

PROTEIN STRUCTURE

Amino Acids - 126

Structural Domains

Drosophila Chickadee is 40% identical to profilins from Saccharomyces cerevisiae, Physarum and Acanthamoeba. The homology increases to greater tha 60% similarity when conserved amino acid substitutions are considered. Profilin from mouse and human is 15 amino acids longer than the protein from lower eukaryotes. Alignment allowing gaps in the proteins shows 25% identity and 50% similarity of the fly protein to profilin of mouse and humans. The homology extends throughout the protein including very high conservation at the carboxy terminus. The region of Acanthamoeba profilin (from amino acids 95-125) has been shown to contain an actin binding site (Cooley, 1992 and references).

date revised: 22 November 2022  

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