A genome-wide analysis reveals that the Drosophila transcription factor Lola promotes axon growth in part by suppressing expression of the actin nucleation factor Spire

The phylogenetically conserved transcription factor Lola is essential for many aspects of axon growth and guidance, synapse formation and neural circuit development in Drosophila. To date it has been difficult, however, to obtain an overall view of Lola functions and mechanisms. Expression microarrays were used to identify the lola-dependent transcriptome in the Drosophila embryo. lola was found to regulate the expression of a large selection of genes that are known to affect each of several lola-dependent developmental processes. Among other loci, it was found that lola is a negative regulator of spire, an actin nucleation factor that has been studied for its essential role in oogenesis. spire was shown to be expressed in the nervous system and is required for a known lola-dependent axon guidance decision, growth of ISNb motor axons. It was further shown that reducing spire gene dosage suppresses this aspect of the lola phenotype, verifying that derepression of spire is an important contributor to the axon stalling phenotype of embryonic motor axons in lola mutants. These data shed new light on the molecular mechanisms of many lola-dependent processes, and also identify several developmental processes not previously linked to lola that are apt to be regulated by this transcription factor. These data further demonstrate that excessive expression of the actin nucleation factor Spire is as deleterious for axon growth in vivo as is the loss of Spire, thus highlighting the need for a balance in the elementary steps of actin dynamics to achieve effective neuronal morphogenesis (Gates, 2011).

The transcription factor Lola is required for a variety of axon growth and guidance events in the developing fly embryo. Expression microarray analysis of lola mutant embryos now reveals that, rather than producing large changes in the levels of a restricted number of major-effect downstream targets, Lola appears to exert its influence via the cumulative effects of small, quantitative changes in a broad spectrum of downstream loci. One key Lola target is spire, which encodes an actin nucleation factor that has been studied intensively for its role in regulating cytoskeletal structure in the developing fly oocyte. spire, like lola, is required for development of ISNb motor axons, its level goes up in lola mutants, and reduction of spire dosage suppresses, but does not eliminate, the ISNb mutant phenotype of lola (Gates, 2011).

Previous analysis of candidate genes implicated in various lola-dependent axon guidance processes identified several whose expression was subtly affected by lola, but none that were dramatically altered. This and other observations led to the proposition that Lola might execute its effects by fine-tuning the expression levels of genes that contribute quantitatively to various guidance decisions, and not simply by turning these genes ON versus OFF. As an unbiased test of this hypothesis, expression microarrays were used to perform a genome-wide comparison of the embryonic transcriptome of wild-type and lola zygotic mutant embryos. RNA isolated from animals 10 to 12 hours after egg laying was analyzed, at a time when a large number of lola-dependent axons are extending. By this analysis, the expression of no single-copy Drosophila gene was altered more than four-fold by lola, and few were altered more than 2.5-fold. It is possible that this is an underestimate due to the compression of expression ratios in microarray experiments, but qRT-PCR results were largely consistent with the array data. It is also possible that expression of some genes may have been altered by a greater factor in just a small subset of expressing cells, but it is noted that most lola isoforms are themselves expressed very broadly, making this possibility less likely. Finally, some genes can be affected oppositely by different lola isoforms, or in different tissues, so it may be that a small net change in expression of a lola target gene hides larger but counteracting changes in different cells. Nonetheless, it remains that a genome-wide analysis failed to identify any single major-effect lola target that would account for the lola axonal phenotypes. It is also true that there is a substantial maternal contribution of Lola to the embryo, and this may limit the measured effect of the mutation on downstream targets. It is noted, however, that it is the zygotic mutant phenotype of lola that this study seeks to explain, and it is therefore the quantitative effect of that zygotic mutant that is the relevant measurement for investigating the phenotype (Gates, 2011).

Microarray analysis has been widely used to identify genes associated with, or responsible for, many developmental and physiological processes. Typical analyses of expression microarray data emphasize genes whose level is strongly altered by the biological manipulation, often setting numerical cutoffs for change in expression level, together with statistical criteria, to identify true positives. In these experiments, it was necessary to eschew the use of a quantitative cutoff in fold change; for example, a commonly used criterion of a two-fold minimum change would have excluded from analysis all but 26 single-copy genes in the genome. Rather, the nature of the biological process studied, and the nature of lola, required that the biological and technical variance be minimized to achieve exceptionally tight statistics. In the end, qRT-PCR validation of expression changes from 1.2-fold (genderblind) to 2.5-fold (spire) provided support for 50% of the putative downstream effects of lola. It is noteed that this is likely to be an underestimate of the reliability of the array results since these small fold differences were at or beyond the usual sensitivity of RT-PCR itself, and it is as likely that RT-PCR was reporting false negatives as the microarrays were reporting false positives. Validity of the results was also supported more globally by independent expression profiling of another lola allele. Thus, these data underscore the efficacy of microarray analysis for detecting reliably even quite small changes in expression level. Known genes whose expression was altered in the lola mutant shed light on many lola-dependent processes. Previous experiments had led to the notion that lola likely coregulates a suite of interacting genes that are important for particular axon guidance decisions, and indeed, it was found that expression of a number of well-characterized guidance receptors is altered in lola. frazzled, which was identified as a downstream target of lola, is on its own known to be required for three lola-dependent axonal processes: ISNb development in the periphery, and both longitudinal and commissural axon extension in the CNS. Among other factors downstream of lola are midline fasciclin (longitudinal and commissural axons), fasciclin 3 and capricious (ISNb) and neural lazarillo (thought to be involved in both longitudinal and commissural axon guidance). Also identified were genes for a number of ligands, receptors and receptor-modifying proteins not previously associated with lola-dependent processes, such as sugarless, dallylike, wnt4a and PVF-1. It now becomes interesting to investigate the potential role of these genes in axon patterning, and in migration and orientation of sensory neurons. Aside from cell surface and extracellular proteins, expression of genes encoding a number of intracellular signaling proteins was found to be altered, including prospero (which in hypomorphic alleles produces a phenotype very similar to that of lola), as well as moesin, Rac2, and a calmodulin-dependent protein kinase (CAKI). An unexpected cluster of downstream effects comprised genes for proteins modulating microtubule structure and function, including katanin, stathmin, NudC and KLP-59C. lola also interacts genetically with the axon patterning function and other activities of the receptor Notch, and a cluster of affected genes was found that modulate Notch action, including sca, Nak, Dap-160 and O-fut1. In addition to these known genes, Gene Ontology analysis identifies a large number of lola-dependent loci that have not yet been characterized in the fly, but whose annotations cluster with lola-dependent genes of known function. This provides a substantial list of excellent candidates for additional contributors to lola-dependent processes. Unfortunately, the large number of Lola isoforms, and their heteromeric combinations, makes it impossible to extract Lola binding site consensus sequences from these candidates using standard computational approaches. Extensive molecular experiments will be necessary in the future to identify response elements for individual heteromeric forms of Lola (Gates, 2011).

lola has several characterized functions outside of axon patterning. For example, it affects cell fates in the eye, and indeed, there is a substantial group of eye patterning genes included in the list of lola-affected loci (sickle, charlatan, asense, rap, roughex, Lobe, target of eyeless and fat facets). Additionally, consistent with the role of lola in controlling programmed cell death during oogenesis, grim, scylla, charybde, bunched and Nedd2-like caspase were found among the downstream effects. It should be noted that since the microarray analysis was performed only with mid-stage embryos, it cannot be distinguished whether the effects of lola on these postembryonic processes are mediated by the same downstream targets that were see affected during embryogenesis. Seeing that these genes can be modulated by lola at one stage of the lifecycle, however, makes them more attractive candidates for analysis at other stages. Finally, in addition to genes affecting known lola-dependent processes, the set of genes altered in lola mutants identifies clusters associated with new processes that would be worth investigating for a role of lola. These include aging, oxidative stress, hormonal regulation of development, tracheal development and maintenance, cell polarity and olfactory learning, among others (Gates, 2011).

One of the most robust putative downstream effects identified for lola was downregulation of the actin nucleation factor Spire. This was immediately striking since spire is known to be a critical regulator of the oocyte cytoskeleton during Drosophila oogenesis. spire is required for both anteroposterior and dorsoventral patterning of the developing oocyte. By modulating actin structure, Spire restrains bundling of oocyte microtubules, thereby blocking cytoplasmic streaming in the oocyte until critical anteroposterior and dorso-ventral polarity cues become stably bound to cortical anchoring sites or initiate irreversible signaling cascades. At the biochemical level, Spire nucleates actin filaments by bringing together actin monomers to assemble a filament nucleus, and it may then transfer this nucleus to the associated formin, Cappuccino, which stimulates filament growth. While the developmental function of spire has been studied most thoroughly in the oocyte, strong mutations in this gene are largely lethal, with only small numbers of escapers surviving to adulthood, and this suggested the existence of as yet uncharacterized zygotic functions of spire. Moreover, a mouse ortholog of spire is expressed in the developing and adult brain. This study found that spire is required for a well-characterized lola-dependent neuronal process, extension of the ISNb motonerve. ISNb was an ideal candidate for the sort of function that had previously been hypothesized for lola, since it is known to depend on the summed, quantitative effects of a large collection of regulators. Therefore ISNb was exploited to examine more carefully the potential interaction of lola and spire, and it was found that genetic reduction of spire suppressed the ISNb mutant phenotype of lola, consistent with the upregulation of spire in a lola mutant making a significant contribution to ISNb axon stalling in lola. By itself, expression analysis cannot distinguish whether spire is a direct target of Lola or whether the upregulation of spire message is a downstream consequence of other changes set in motion by lola. Further biochemical studies of the DNA binding properties of Lolaisoforms will be necessary to assess this. Finally, lola has other phenotypes that are not suppressed by reduction of spire. These may reflect, for example, roles of lola-dependent guidance molecules that are themselves spire -independent, or the action of Spire-independent aspects of growth cone signaling (Gates, 2011).

Efforts to mimic the lola ISNb phenotype by overexpression of spire were not successful. There are several possible reasons for this. First, there are thought to be at least eight Spire protein isoforms, based on cDNA and expressed sequence tag data, and it may be that a particular combination of isoforms, or a specific ratio of expression levels of different isoforms, is necessary to give the ISNb stalling phenotype. Alternatively, it may be that this phenotype is produced only when spire upregulation occurs in the context of some other downstream effect(s) of lola. Additional experiments will be necessary to discriminate among these models (Gates, 2011).

Superficially, it seems remarkable that complete loss of spire causes stalling of ISNb axons, yet the upregulation of spire that occurs in a lola mutant also contributes to ISNb stalling. Evidently, excessive nucleation of actin filaments from spire overexpression is as detrimental to growth cone motility as is the failure of actin nucleation from absence of the protein. Similar non-linearity has been observed in the effects of a number of signaling and cytoskeletal regulatory proteins in other axon guidance paradigms, and it appears to be a common feature of the relationship of signaling to morphogenesis. Thus, for example, even though Abl tyrosine kinase pathway signaling appears to be essential for most axon growth, extension of longitudinal pioneer axons of the fly CNS requires suppression of Abl signaling to achieve the proper balance in the steps of actin dynamics. Similarly, for the Rac GTPases, expression of dominant negative and dominant constitutive forms of the protein are equally effective for inhibiting axon motility, but in one case from excessive stabilization of actin filaments and in the other from insufficient stabilization. Spire now provides another example of this generalization, and underscores the need for signaling networks to evoke a balance in the steps in actin dynamics, thus optimizing throughput through the mechanical cycle of growth cone motility (Gates, 2011).

lola mutants have profound effects on axon patterning, even though systematic molecular analysis reveals only subtle modulation of downstream target gene expression. This observation highlights the exquisite sensitivity of motility and guidance to the balance among cell signaling networks, and thus also to the gene expression mechanisms that set the boundaries of that balance (Gates, 2011).

This study has used a genome-wide analysis to identify the suite of genes whose expression is altered in embryos lacking the Drosophila transcription factor Lola. Gene Ontology analysis sheds light on the regulation of several characterized lola functions, including axon guidance, synapse formation, eye development and oogenesis, by revealing the loladependence of genes known to be involved in those processes, and also by identifying a large number of previously uncharacterized lola-dependent genes that are likely to contribute to these processes. Additionally, these results identify novel processes that are likely to be regulated by lola. Regarding axon patterning, this analysis reveals that Lola suppresses expression of the actin nucleation factor Spire, and this is crucial for its ability to promote growth of motor axons in vivo. These data underscore the critical importance of ensuring the correct levels of actin regulatory proteins in a cell to promote motility effectively (Gates, 2011).

Wash functions downstream of Rho and links linear and branched actin nucleation factors

Wiskott-Aldrich Syndrome (WAS) family proteins are Arp2/3 activators that mediate the branched-actin network formation required for cytoskeletal remodeling, intracellular transport and cell locomotion. Wasp and Scar/WAVE, the two founding members of the family, are regulated by the GTPases Cdc42 and Rac, respectively. By contrast, linear actin nucleators, such as Spire and formins, are regulated by the GTPase Rho. A third WAS family member, called Washout (Wash), has Arp2/3-mediated actin nucleation activity. This study shows that Drosophila Wash interacts genetically with Arp2/3, and also functions downstream of Rho1 with Spire and the formin Cappuccino to control actin and microtubule dynamics during Drosophila oogenesis. Wash bundles and crosslinks F-actin and microtubules, is regulated by Rho1, Spire and Arp2/3, and is essential for actin cytoskeleton organization in the egg chamber. These results establish Wash and Rho as regulators of both linear- and branched-actin networks, and suggest an Arp2/3-mediated mechanism for how cells might coordinately regulate these structures (Liu, 2009).

The actin cytoskeleton consists of linear and branched filament networks required for processes ranging from cell division to migration. How these two networks function and are coordinated is of major interest, as their misregulation results in infertility, immunodeficiency, and tumor metastasis in humans. Linear actin filament networks, required for cytokinesis and filopodia formation, are regulated by nucleators and bundling proteins, which enhance filament formation rates and control filament organization, respectively. Examples include Spire and the formin Cappuccino (Capu), which exhibit both nucleation and bundling activities and are essential for oocyte development during Drosophila oogenesis. Both Spire and Capu are regulated by the GTPase Rho1 of the Rho family of small GTPases, which is upstream of other linear nucleators, such as Diaphanous, and is considered a key regulator of linear filament formation (Liu, 2009).

Branched or dendritic actin filament networks, which are required for phagocytosis and lamellipodia formation, are primarily regulated by the Arp2/3 complex and by nucleation-promoting factors that associate with Arp2/3 and actin monomers to nucleate daughter filaments off of existing mother filaments. Like Spire and Capu, Arp2/3 is essential for Drosophila oogenesis, specifically for maintaining proper nurse cell cyto-architecture and function. One family of Arp2/3 activators, the Wiskott-Aldrich Syndrome (WAS) protein family, has been shown to function downstream of Rho GTPases to mediate the branched-actin network formation required for cytoskeletal remodeling, intracellular transport and cell locomotion. WASP and SCAR/WAVE, the two founding subclasses of the family, are activated by the GTPases Cdc42 and Rac, respectively. Two new WAS subclasses, WASH and WHAMM, have recently been reported and have been shown to exhibit Arp2/3-mediated branched nucleation activity. Which GTPases might regulate them, however, is not known (Liu, 2009).

This study reports that Drosophila Wash functions downstream of Rho1 and interacts with Spire and Capu to regulate actin and microtubule organization during Drosophila oogenesis. Wash nucleates actin in an Arp2/3-dependent manner, and exhibits F-actin and microtubule bundling and crosslinking activity that is regulated by a pathway involving Rho1, Spire and Arp2/3. Wash genetically interacts with Rho1, Capu, Spire and Arp2/3, and is essential for actin cytoskeleton organization during oogenesis. These results establish Wash and Rho as regulators of both linear- and branched-actin networks, and suggest an Arp2/3-mediated mechanism of cytoskeletal control through which cells might coordinately regulate linear and branched architectures (Liu, 2009).

It has been suggested that Rho1 regulates the timing of ooplasmic streaming by regulating the MT/microfilament crosslinking that occurs at the oocyte cortex. In this model, crosslinking antagonizes the formation of the dynamic subcortical MT arrays that are required for ooplasm streaming, but does not require the actin-nucleation activity of these proteins. The current model depends on the presence of SpirC and the cortical localization of Rho1, Capu, the Spire isoforms, and now Wash during late-stage oocytes. Support for this model comes from a recent study demonstrating that chickadee, encoding fly Profilin, is required for the formation of cortical actin bundles in the oocyte, and that Capu and Spire anchor the minus ends of MTs to a scaffold made from these cortical actin bundles. These results suggest dual or multifaceted biochemical roles for these proteins in regulating developmental processes. Consistent with this concept, non-actin-nucleating roles for other formins (i.e. actin severing/depolymerization, MT stabilization, signaling, and transcriptional regulation) are beginning to be reported (Liu, 2009).

St Johnston and colleagues have recently proposed an alternative model in which Capu and Spire are required to organize an isotropic mesh of actin filaments in the oocyte cytoplasm that suppresses the motility of kinesin, a plus-end directed MT motor protein that is required for ooplasmic streaming (Dahlgaard, 2007). Their model was formulated with the assumptions that the SpirC isoform does not exist, that spirRP is a null allele, and that the cortical localization of Capu and Spire is lost in late-stage oocytes. This study found these assumptions not to be the case. mRNA and protein evidence is provided for the existence of the SpirC isoform. The existence of SpirC is also supported by ESTs from the Drosophila Genome Project. The spirRP allele affects only the SpirA and SpirD isoforms; it does not affect the SpirC isoform because this isoform has a unique 5' end. Ectopic SpirC expression would not be expected to rescue spirRP because it is already being expressed. The cortical localization of Capu and the Spire proteins during the late stages is masked by intense yolk auto-fluorescence in the green channel when using live imaging of GFP fusions, but can be observed by fixing, by antibody staining, or by the use of ChFP ('cherry' fusion protein). In addition, a subsequent study has shown that kinesin is not required for this cytoskeletal reorganization, suggesting that Capu and Spire might not act as indirect kinesin regulators, but as direct modulators of the MT cytoskeleton). One possibility is that Capu and Spire are bundling and crosslinking MTs to Profilin-dependent F-actin at the oocyte cortex, as has been demonstrated in vitro (Liu, 2009).

Since the discovery of Arp2/3 activators and other actin-nucleation promoting factors, much of the work examining the functions of these proteins has been focused on the properties of their nucleation activities. Recent studies, however, have begun reporting novel biochemical activities for actin nucleators, including MT stabilization activity by mammalian Diaphanous, filopodia inhibition by WAVE/Arp2/3, and F-actin and MT bundling and crosslinking by Spire and Capu (Rosales-Nieves, 2006). Consistent with this, not all disease-associated WASP mutations are predicted to affect its actin-nucleation activity (Notarangelo, 2008). The current results contribute to this growing list of actin nucleators with significant non-nucleation activities, since this study shows that Wash is both an Arp2/3 activator and a crosslinker/bundler of F-actin and microtubules. What is unique about Wash, however, is that its combination of biochemical activities suggests that it is an important intermediary molecule functioning at the intersection of linear and branched actin architectures, with Spire, Rho and Arp2/3 acting as the factors that direct these dual functions of Wash. Based on these findings, the following model is proposed for Wash function in the context of Drosophila oogenesis. In the nucleation pathway, upstream signals and factors, possibly Rho, induce Wash activation, which acts with Arp2/3 to promote branched filament formation and cytoskeletal integrity in nurse cells. In the crosslinking/bundling pathway, Wash bundles and crosslinks filaments of actin and MTs, under the control of Rho and SpirD, to maintain cortical bundle stability in the oocyte and to prevent premature ooplasmic streaming. Together with Capu and Spire (Rosales-Nieves, 2006), Wash maintains the correct timing of ooplasmic streaming by preventing the formation of the microtubule tracks required for motor proteins to drive cytoplasmic flow. The dual functions of Wash might also be regulated spatially by Arp2/3 and depend on the availability or concentration of Arp2/3. Since the nucleation activity of Wash is Arp2/3 dependent, Wash-mediated actin nucleation might require some threshold concentration of locally available Arp2/3; for example, at the ring canals. Spatiotemporal regulation is also possible through the changing levels of Arp2/3 during oogenesis. Arp2/3, for example, might transiently accumulate at the oocyte cortex during the onset of streaming to disrupt Wash bundling activity (Liu, 2009).

These findings contribute to previous studies examining the functions of Wasp and Scar in Drosophila, and together describe a spectrum of phenotypes that illustrate the multiple functions exerted by WAS family members in development. Scar has been shown to be required for axon development, egg chamber structure, adult eye morphology and myoblast fusion; Wasp has been demonstrated to be required for Notch-mediated cell-fate decisions, rhabdomere microvilli formation, bristle development and myoblast fusion; and Wash is required for pupal development and oogenesis, as described in this study. Mutants in various subunits of Arp2/3 have also been described, offering additional insight into how Wash, Wasp and Scar shape the cytoskeleton during development. Interestingly, the spectrum of Arp2/3 mutant phenotypes reported does not completely overlap with all of the phenotypes associated with these WAS family mutants. This might be because Arp2/3 has not been examined in all of the processes in which WAS members play a role, or it might be an indication that WAS members have additional, Arp2/3-independent functions, which is the case for Wash. The current observations support the idea that these and other actin nucleators, such as Capu and Spire, are required at different times or locations during development, and are thus tightly regulated spatiotemporally by Rho GTPases and other factors (Liu, 2009).

The data indicate that Wash acts as a downstream effector of Rho. Indeed, Rho is shown to regulates the bundling/crosslinking activity of Wash through the relief of SpirD inhibition. However, Rho does not enhance the ability of Wash to induce Arp2/3-mediated actin nucleation, raising the question of how or whether Rho might regulate the Arp2/3-associated functions of Wash. Interestingly, the results are consistent with studies examining the Cdc42 regulation of Wasp in Drosophila, which conclude that Cdc42 activation of Wasp is not required for Wasp function in myoblast fusion or bristle development. Although Wasp exhibits a strong and specific interaction with active Cdc42GTP in vitro, these studies provide strong evidence that, at least for the subset of developmental processes examined, Wasp is not regulated upstream by Cdc42GTP. As previously noted, Drosophila Wasp differs from mammalian homologs in that it is not auto-inhibited; Cdc42, therefore, might not be required for the activation of its actin nucleation-promoting functions. This might also be the case for Wash, as it too appears to be constitutively active, and might act as a downstream effector of Rho only where its bundling/crosslinking activities are concerned. The data, however, do not rule out the possibility that the nucleation activity of Wash is regulated by a complex in vivo. In fact, recent reports have shown that two proteins originally associated with Scar regulation, Abi and Kette, control Wasp function in Drosophila as well. It remains to be determined whether Abi and Kette also regulate Wash function, and whether Rho might play a role in mediating these interactions (Liu, 2009).

Wash requires Arp2/3 for actin nucleation, but, interestingly, this association appears to disrupt the ability of Wash to bundle and crosslink F-actin and microtubules, as a loss of F-actin/MT bundling favored branching actin filaments. This suggests that Arp2/3 might act as a molecular switch that shifts Wash function from bundling to nucleation and, in terms of cytoskeletal remodeling, supports the hypothesis that Arp2/3 regulates the balance between linear and branched actin architectures in the cell. This is predicated on the assumption that the Wash bundling/crosslinking and nucleation-inducing activities are mutually exclusive, and would represent a previously uncharacterized function of Arp2/3. However, scenarios cannot be ruled out in which nucleation and bundling might coexist. F-actin bundling might be preserved if the branched-actin structures created by Wash and Arp2/3 in vitro are bundled by Wash in parallel (form angled, branching bundles rather than the tortuous bundles observed under non-Arp2/3 conditions), or if filaments emanating from vertices are clamped together by Wash at the branching point to form angled bundles that branch from these vertices. An example of this latter case has been reported in a recent study examining the concerted actions of N-Wasp and Hsp90 to nucleate branched actin filaments (via N-Wasp activation of Arp2/3) and clamp the angled filaments to form a linear bundle (mediated by Hsp90). Wash therefore, in having both nucleation and bundling activities, might perform both functions simultaneously in the presence of Arp2/3. At the very least, Arp2/3 abolishes the ability of Wash to bundle MTs and crosslink them to actin, and so might contribute to regulating crosstalk between the actin and microtubule cytoskeletons. Further studies examining the molecular interactions of WAS family members and Arp2/3 will be invaluable for understanding the full range of cytoskeletal regulation in the cell (Liu, 2009).

In motile cells the actin cytoskeleton can be represented as a dynamic sum of two general geometries - strands or bundles of linear actin filaments, and broad dendritic networks of branched filaments. The mechanisms by which these two networks are remodeled and coordinated are areas of intense investigation and are important for understanding how processes such as lamellipodia and filopodia formation occur. It is intriguing to note that, in the latter case, the biochemical properties of Wash suggest that it might play a role in the convergent extension model of filopodia formation, whereby uncapped actin filaments nucleated from a dendritic branched-actin array are captured at the cell periphery and bundled to form long extensions (Mattila, 2008). Wash, as both an Arp2/3 activator and an F-actin bundling protein, is in an ideal position in which to carry out both the nucleation and the bundling functions, and might thus be an important regulator of filopodia formation alongside previously discovered molecules (Mattila, 2008). The presence of Spire and Arp2/3 at the dendritic bed and active Rho at the cell membrane could form two zones of differential activity to switch Wash function from nucleation to bundling and crosslinking. This form of spatial regulation is analogous to how Rho, Cdc42 and Rac define regions of differential activity during wound healing and cell adhesion. Further investigation into the role of Wash in filopodia and lamellipodia formation will be important, as these protrusions play essential roles in wound healing, substrate adhesion and neurite outgrowth (Liu, 2009).

In humans, the misregulation of WAS members results in disorders such as Wiskott-Aldrich Syndrome, and cancer metastasis. As a new member of the WAS family, human WASH appears to also be clinically relevant. WASH has been reported to be overexpressed in a breast cancer cell line and might, like the overexpression of N-WASP and Scar/WAVEs, contribute to metastasis (Leirdal, 2004). Moreover, the subtelomeric location of human WASH places it at high risk for deletion and rearrangement, as subtelomeres are hotspots of meiotic interchromosomal sequence transfers. The data presented in this study demonstrate that Wash is essential for development in Drosophila, and suggest that Wash might function in actin organization in other contexts. Further work will be required to understand how Wash and other WAS family members coordinate linear- and branched-actin networks during oogenesis and other cellular processes, and how the misregulation of these processes results in disease (Liu, 2009).

Drosophila Mon2 couples Oskar-induced endocytosis with actin remodeling for cortical anchorage of the germ plasm

Drosophila pole (germ) plasm contains germline and abdominal determinants. Its assembly begins with the localization and translation of oskar (osk) RNA at the oocyte posterior, to which the pole plasm must be restricted for proper embryonic development. Osk stimulates endocytosis, which in turn promotes actin remodeling to form long F-actin projections at the oocyte posterior pole. Although the endocytosis-coupled actin remodeling appears to be crucial for the pole plasm anchoring, the mechanism linking Osk-induced endocytic activity and actin remodeling is unknown. This study reports that a Golgi-endosomal protein, Mon2, acts downstream of Osk to remodel cortical actin and to anchor the pole plasm. Mon2 interacts with two actin nucleators known to be involved in osk RNA localization in the oocyte, Cappuccino (Capu) and Spire (Spir), and promotes the accumulation of the small GTPase Rho1 at the oocyte posterior. This study also found that these actin regulators are required for Osk-dependent formation of long F-actin projections and cortical anchoring of pole plasm components. It is proposed that, in response to the Osk-mediated endocytic activation, vesicle-localized Mon2 acts as a scaffold that instructs the actin-remodeling complex to form long F-actin projections. This Mon2-mediated coupling event is crucial to restrict the pole plasm to the oocyte posterior cortex (Tanaka, 2011).

In many cell types, asymmetric localization of specific RNAs and proteins is essential for exhibiting proper structure and function. These macromolecules are transported to their final destinations and anchored there. This latter step is particularly important for the long-term maintenance of cell asymmetry. A genetically tractable model for studying intracellular RNA and protein localization is the assembly of the pole (germ) plasm in Drosophila oocytes and embryos. The pole plasm is a specialized cytoplasm that contains maternal RNAs and proteins essential for germline and abdominal development. It is assembled at the posterior pole of the oocyte during oogenesis. Drosophila oogenesis is subdivided into 14 stages, with pole plasm assembly starting at stage 8. The functional pole plasm is assembled by stage 13, stably anchored at the posterior cortex of the oocyte and later inherited by the germline progenitors (pole cells) during embryogenesis (Tanaka, 2011).

Pole plasm assembly begins with the transport of oskar (osk) RNA along microtubules to the posterior pole of the oocyte. There, the osk RNA is translated, producing two isoforms, long and short Osk, by the alternate use of two in-frame translation start sites. Although short Osk shares its entire sequence with long Osk, the isoforms have distinct functions in pole plasm assembly. Downstream, short Osk recruits other pole plasm components, such as Vasa (Vas), to the oocyte posterior, presumably through direct interactions. By contrast, long Osk prevents pole plasm components from diffusing back into the cytoplasm. Intriguingly, embryonic patterning defects are caused by either the ectopic assembly of pole plasm [elicited by Osk translation at the oocyte anterior directed by the osk-bicoid (bcd) 3'UTR] or the leakage of pole plasm activity into the bulk cytoplasm (induced by overexpressing osk). Thus, the pole plasm must be anchored at the posterior cortex for proper embryonic development (Tanaka, 2011).

Short and long Osk also differ in their subcellular distributions. Short Osk is located on polar granules, specialized ribonucleoprotein aggregates in the pole plasm, and long Osk is associated with endosome surfaces. Intriguingly, the oocyte posterior, where endocytosis is increased, is highly enriched with markers of early, late and recycling endosomes (Rab5, Rab7 and Rab11, respectively). osk oocytes, however, do not maintain either the accumulation of endosomal proteins or the increased endocytic activity at the posterior. Furthermore, the ectopic expression of long Osk at the anterior pole of the oocyte results in the anterior accumulation of endosomal proteins along with increased endocytosis. Thus, long Osk regulates endocytic activity spatially within the oocyte (Tanaka, 2011).

The endocytic pathway has two separate roles in pole plasm assembly (see Tanaka, 2008). First, it is required for the sustained transport of osk RNA by maintaining microtubule alignment. For example, in oocytes lacking Rabenosyn-5 (Rbsn-5), a Rab5 effector protein essential for endocytosis, the polarity of the microtubule array is not maintained, disrupting osk RNA localization. A similar defect occurs in hypomorphic rab11 oocytes. Second, the endocytic pathway acts downstream of Osk to anchor the pole plasm components. In rbsn-5 oocytes aberrantly expressing osk at the anterior, Osk and other pole plasm components diffuse from the anterior cortex into the ooplasm, indicating that endocytic activity is essential for stably anchoring them to the cortex (Tanaka, 2011).

The endocytic pathway is thought to anchor pole plasm components by remodeling the cortical actin cytoskeleton in response to Osk. Pole plasm anchoring is sensitive to cytochalasin D, which disrupts actin dynamics, and requires several actin-binding proteins, such as Moesin, Bifocal and Homer. Osk induces long F-actin projections emanating from cortical F-actin bundles at the posterior pole of the oocyte. Ectopic F-actin projections are also induced at the anterior pole when long Osk is misexpressed at the oocyte anterior (Tanaka, 2008). However, when the endocytic pathway is disrupted, F-actin forms aggregates and diffuses into the ooplasm, along with pole plasm components (Tanaka, 2008). These observations led to the hypothesis that Osk stimulates endocytosis, which promotes actin remodeling, which in turn anchors the pole plasm components at the posterior oocyte cortex. However, the molecular mechanism linking Osk, the endocytic pathway and actin remodeling is still unknown (Tanaka, 2011).

This study has identified Mon2, a conserved Golgi/endosomal protein, as an essential factor in anchoring pole plasm components at the oocyte posterior cortex. Oocytes lacking Mon2 did not form F-actin projections in response to Osk, but neither did they exhibit obvious defects in microtubule alignment or endocytosis. It was also shown that two actin nucleators that function in osk RNA localization in the oocyte, Cappuccino (Capu) and Spire (Spir), play an essential role in a second aspect of pole plasm assembly: the Osk-dependent formation of long F-actin projections and cortical anchoring of pole plasm components. Finally, it was found that Mon2 interacts with Capu and Spir, and promotes the accumulation of the small GTPase Rho1 at the oocyte posterior. These data support a model in which Mon2 acts as a scaffold, linking Osk-induced vesicles with these actin regulators to anchor the pole plasm to the oocyte cortex (Tanaka, 2011).

To learn more about how the pole plasm is assembled and anchored during Drosophila oogenesis, a germline clone (GLC) screen was conducted for ethyl methanesulfonate-induced mutations showing the abnormal localization of GFP-Vas, a fluorescent pole plasm marker (Tanaka, 2008). In a screen targeting chromosomal arm 2L, six mutants were identified that mapped into a single lethal complementation group, which was named no anchor (noan). In wild-type oocytes, GFP-Vas was first detectable at the posterior pole at stage 9, where it remained tightly anchored, with a progressive accumulation of protein until the end of oogenesis. In the noan GLC oocyte, GFP-Vas initially localized to the oocyte posterior during stages 9-10a, but its level gradually decreased, becoming undetectable in the mature oocyte. Similarly, the localization of Staufen (Stau) and Osk at the posterior pole, which occurs prior to that of Vas, was not maintained in the noan oocytes. Although the noan oocytes developed into normal-looking mature oocytes, the eggs were fragile and did not develop. Therefore, it was not possible to analyze the effects of the loss of maternal noan activity on the formation of abdomen or germ cells in embryos. Nevertheless, these results indicated that noan mutations cause defective anchoring of pole plasm components to the posterior pole of the oocyte (Tanaka, 2011).

The genetic mapping and subsequent DNA sequencing of the noan locus revealed that all the noan alleles had a nonsense mutation in CG8683, which encodes a homolog of a budding yeast protein, Mon2p, also termed Ysl2p. noan is referred to as mon2. All the mon2 alleles showed identical defects in the posterior localization of GFP-Vas with full penetrance. As the mutation in the mon2K388 allele was the most proximal to the translational initiation site among the six alleles identified, mon2K388 was primarily used to characterize the mon2 phenotype (Tanaka, 2011).

em>Drosophila Mon2 consists of 1684 amino acids and represents a highly conserved protein among eukaryotes. It has two Armadillo (ARM) repeat domains, which are likely to mediate protein-protein interactions, and a DUF1981 domain, which is functionally uncharacterized. In budding yeasts, mon2 (ysl2) was identified as a gene whose mutation increases sensitivity to the Na+/H+ ionophore monensin, and is synthetically lethal with a mutation in ypt51, which encodes a Rab5 homolo. Yeast Mon2p (Ysl2p) forms a large protein complex on the surface of the trans-Golgi network and early endosomes, and it is proposed to act as a scaffold to regulate antero- and retrograde trafficking between the Golgi, endosomes and vacuoles (Tanaka, 2011 and references therein).

This study found that Capu and Spir act together to form long F-actin projections and to anchor pole plasm components at the oocyte cortex, and that Mon2 is essential to these processes. Capu and Spir also regulate the timing for initiating ooplasmic streaming and microtubule array polarization in the oocyte (Qualmann, 2009). However, the polarity of microtubule arrays was not affected in mon2 oocytes. Therefore, Mon2 is not always required for Capu and Spir to function. Rather, it appears to regulate specifically these actin nucleators through the Osk-induced endocytic pathway (Tanaka, 2011).

Mon2 is required for the formation of Osk-induced long F-actin projections at the oocyte posterior. Interestingly, ectopic overexpression of Osk at the anterior pole in the mon2 oocyte induced granular, albeit faint, F-actin structures, indicating that Osk-induced actin remodeling does not totally cease in the mon2 oocyte. Ectopic Osk at the anterior of capu spir double-mutant oocytes also induced faint F-actin granules in the cytoplasm. Thus, additional, as yet uncharacterized, actin regulators appear to function in response to Osk. Notably, two actin-binding proteins, Bifocal and Homer, play redundant roles in anchoring Osk to the cortex. Although the precise roles of Bifocal and Homer in this process remain elusive, they might function independently of Mon2 (Tanaka, 2011).

Oocytes lacking Rab5 showed disrupted posterior cortical F-actin bundles, which was suppressed by the simultaneous loss of Osk. These results reconfirm that the endocytic pathway needs intact Osk function for actin remodeling (Tanaka, 2008). This study also found that the F-actin disorganization in rab5 oocytes is Mon2-dependent. Therefore, Mon2 can facilitate actin remodeling even when Rab5 is absent, but endosomal trafficking, in which Rab5 is involved, is crucial for regulating Mon2. Mammalian Rab5 is also involved in actin remodeling. For example, Rac1 GTPase, a regulator of F-actin dynamics, is activated by Rab5-dependent endocytosis, and the local activation of Rac1 on early endosomes and its subsequent recycling to the plasma membrane spatially regulate actin remodeling. Thus, local endocytic cycling provides a specific platform for actin remodeling in a wide range of cell types (Tanaka, 2011).

There is growing evidence that endosomes act as multifunctional platforms for many types of molecular machinery. Intriguingly, Mon2 is located on the Golgi and endosomes, without entirely accumulating at the oocyte posterior. It is therefore proposed that the Osk-induced stimulation of endocytic cycling at the oocyte posterior leads to the formation of specialized vesicles, which instruct a fraction of Mon2 to regulate the activity of Capu, Spir and Rho1 to form long F-actin projections from the cortex. Although the functional property of Osk-induced endocytic vesicles has yet to be ascertained, long Osk is known to associate with the surface of endosomes. Therefore, long Osk might modify endosome specificity to recruit and/or stabilize the machineries responsible for actin remodeling (Tanaka, 2011).

Oocytes lacking Mon2 can mature without morphological abnormalities, but their eggs are nonviable. Furthermore, Drosophila mon2 mutations show recessive lethality, indicating that Mon2 has additional functions in somatic cell development. It might function in regulating vesicle trafficking or protein targeting, as reported in yeasts. As vesicle trafficking is often linked with establishing and maintaining cell polarity, it is an attractive idea that Mon2 might regulate the polarity protein localization and/or mediate the signal transduction for cell polarization in somatic cells, as well as in germ cells. Supporting this idea, a Mon2 homolog in C. elegans has been implicated in the asymmetric division of epithelial stem cells (Kanamori, 2008; Tanaka, 2011 and references therein).

It has been proposed that long Osk localizes to the endosomal membrane and generates a positive-feedback loop for cortical anchoring of pole plasm components. Osk is also thought to generate another positive-feedback loop to maintain the polarity of microtubule arrays, and the process appears to be endosomal protein-dependent. Although Rbsn-5 is required for both feedback loops, Mon2 acts specifically in the loop regulating actin remodeling for pole plasm anchoring, indicating that the two feedback loops are regulated by distinct mechanisms. The endocytic pathway consists of multiple vesicle trafficking steps, including endocytosis, endosomal recycling, late-endosomal sorting and endosome-to-Golgi trafficking. Therefore, determining which steps in the endocytic pathway are used by the two Osk-dependent positive-feedback loops is an important aim for future exploration (Tanaka, 2011).

Protein Interactions

Since Spire contains sequence similarity to WH2 domains that bind directly to actin in vitro, Spire was tested for its ability to bind actin in the yeast interaction trap system. Co-expression of Spire::lexA with Drosophila actin 5C fused to a transcriptional activation domain results in growth on galactose medium lacking leucine. This indicates that Spire interacts with actin to stimulate expression of the leucine reporter gene. To identify the region of Spire responsible for the interaction with actin, smaller fragments of Spire were tested. The actin binding region is contained in a fragment of Spire (aa 296-585) that contains both WH2 domains (aa 398-416 and aa 462-479). Smaller fragments within this region of Spire have only weak or no interactions with actin. A construct of the actin binding region of Spire containing mutations in the second WH2 domain fail to interact with actin, confirming that the WH2 domains are responsible for Spire's actin binding capability. In vitro binding assays between in vitro translated Spire and G-actin isolated from chicken muscle demonstrate that Spire binds directly to purified G-actin (Wellington, 1999).

The actin-binding WH2 domains of WASP and SCAR1 are followed by domains that interact with the p21 Arc of the Arp2/3 complex. Together these domains affect polymerization of actin. Spire does not appear to contain these domains and no interaction between Spire and p21 Arc could be detected in the interaction trap system (Wellington, 1999).

Spire also interacts with rho family GTPases. spire has similar phenotypes to capuccino (Manseau, 1989 and Theurkauf, 1994) and Capu binds to rho family GTPases (J. Calley and L. Manseau, unpublished, cited in Wellington, 1999). For these reasons, a test was performed to see whether Spire interacts with rho family GTPases in the interaction trap system. Spire interacts with wild-type and dominant negative mutants of RHOA, RAC1 and CDC42, but not with RHOL. Attempts were also made to test the constitutively active forms of the rho family members, but it was found that they self-activate the reporters, making it difficult to assess whether Spire interacts with the constitutively active forms. Deletion analysis localizes the rho binding domain of Spire to the first 100 amino acids (Wellington, 1999).

Drosophila Spire is an actin nucleation factor

The actin cytoskeleton is essential for many cellular functions including shape determination, intracellular transport and locomotion. Two factors -- the Arp2/3 complex and the formin family of proteins -- have been identified that nucleate new actin filaments via different mechanisms. This study shows that the Drosophila protein Spire represents a third class of actin nucleation factor. In vitro, Spire nucleates new filaments at a rate similar to that of the formin family of proteins but slower than in the activated Arp2/3 complex, and it remains associated with the slow-growing pointed end of the new filament. Spire contains a cluster of four WASP homology 2 (WH2) domains, each of which binds an actin monomer. Maximal nucleation activity requires all four WH2 domains along with an additional actin-binding motif, conserved among Spire proteins. Spire itself is conserved among metazoans and, together with the formin Cappuccino, is required for axis specification in oocytes and embryos, suggesting that multiple actin nucleation factors collaborate to construct essential cytoskeletal structures (Quinlan, 2005).

The results indicate that the C-terminal half of the Spir WH2 cluster (composed of WH2-C, the linker region L-3 and WH2-D) is the functional core of the protein. The main kinetic barrier to nucleation is formation of an actin dimer. It is proposed that Spir assembles an actin dimer when WH2-C and WH2-D each bind an actin monomer and L-3 coordinates their interaction. Similar to the first dimer formed during spontaneous nucleation, it is expected that this dimer lies along one strand of the long-pitch actin helix. On the basis of their effects on nucleation and electron microscopy data, it is proposed that WH2-B and WH2-A add a third and fourth monomer to the initial dimer (Quinlan, 2005).

In addition to electron microscopy, spatial constraints imposed by the Spir sequence and the atomic structure of the WH2-like domain of Drosophila Ciboulot, a protein that participates in actin filament assembly, suggest that Spir stacks monomers along one strand of the long-pitch filament helix. The N-terminal portion of a WH2 domain would block addition of the next monomer at the barbed end. It is proposed that, similar to Ciboulot and the WASP-family WH2 domains, the N-terminal portion of the Spir WH2 domains dissociates rapidly upon incorporation into the nascent nucleus. Consistent with this idea, residues in the Ciboulot actin-binding site important for dissociation and promotion of actin filament assembly are conserved in Spir. The only structure consistent with the lengths of the linker sequences is a linear arrangement of the four WH2-bound monomers, with WH2-D at the pointed end and WH2-A at the barbed end. Binding of an additional monomer to the interface between any of the WH2-bound monomers would result in formation of a stable nucleus and rapid filament elongation (Quinlan, 2005).

The fact that Spir assembles nuclei even when all three linker sequences are mutated [(NT)Spir(gs123)] suggests that tethering multiple, WH2-bound actin monomers in close proximity may be sufficient to promote filament formation. The only other known tandem repeats of competent WH2 domains are found in N-WASP and tetra-thymosin-ß. By themselves, N-WASP and tetra-thymosin-ß allow elongation of the barbed ends of filaments, but strongly inhibit spontaneous nucleation. Further work is required to determine whether WH2 domains in Spir are specially adapted to promote nucleation, or whether sequences in other WH2-containing proteins are specially adapted to prevent nucleation, as in the case of thymosin-ß4 (Quinlan, 2005).

After the Arp2/3 complex and the formins, Spir is the third actin nucleation factor to be discovered. Why do cells require more than one mechanism for constructing actin filaments? The answer probably lies in the diversity of functions performed by the actin cytoskeleton. Different functions require actin networks with different architectures, and the architecture of an actin network is determined in part by the mechanism of filament nucleation. Dendritic nucleation by the Arp2/3 complex, for example, produces space-filling actin networks capable of resisting mechanical deformation. This activity is required for amoeboid motility, phagocytosis and intracellular motility of endosomal vesicles and some pathogens. The formins do not crosslink new filaments into branched arrays but remain attached to their growing ends and probably tether them to specific locations. Unbranched filaments generated by formins are essential for construction of actin cables in budding yeast, and stress fibres and contractile rings in mammalian cells (Quinlan, 2005).

The regulation and expression patterns of Spir differ from those of the other nucleators. Unlike the Arp2/3 complex and the formins, Spir has no obvious orthologues in any sequenced protozoan genome; however, it is highly conserved across metazoan species. Two mammalian isoforms, Spir-1 and Spir-2, are widely expressed in embryonic tissues but limited primarily to the central nervous system of adults. The Arp2/3 complex and the Diaphanous-related formins are downstream of Rho-family G proteins, whereas Spir and the mammalian homologue of Capu, formin-1, are MAP kinase substrates. Spir proteins are targeted to intracellular membranes by a C-terminal-modified FYVE zinc finger motif, and co-localize with the GTPase Rab11, which is involved in vesicle transport processes. As with spir and capu, Drosophila Rab11 belongs to the posterior group of genes. In addition, Rab11, spir and capu mutants have similar defects in microtubule plus-end orientation during oogenesis. These data suggest that Spir has evolved specifically to construct actin-based structures required for polarization of multicellular organisms (Quinlan, 2005).

Coordination of microtubule and microfilament dynamics by Drosophila Rho1, Spire and Cappuccino

The actin-nucleation factors Spire and Cappuccino (Capu) regulate the onset of ooplasmic streaming in Drosophila melanogaster. Although this streaming event is microtubule-based, actin assembly is required for its timing. It is not understood how the interaction of microtubules and microfilaments is mediated in this context. This study demonstrates that Capu and Spire have microtubule and microfilament crosslinking activity. The spire locus encodes several distinct protein isoforms (SpireA, SpireC and SpireD). SpireD nucleates actin, but the activity of the other isoforms has not been addressed. This study finds that SpireD does not have crosslinking activity, whereas SpireC is a potent crosslinker. SpireD binds to Capu and inhibits F-actin/microtubule crosslinking, and activated Rho1 abolishes this inhibition, establishing a mechanistic basis for the regulation of Capu and Spire activity. It is proposed that Rho1, Cappuccino and Spire are elements of a conserved developmental cassette that is capable of directly mediating crosstalk between microtubules and microfilaments (Rosales-Nieves, 2006).

The results indicate that Rho1 regulates the timing of ooplasmic streaming by regulating the microtubule/microfilament crosslinking that occurs at the oocyte cortex. In this model, crosslinking antagonizes the formation of the dynamic subcortical microtubule arrays that are required for ooplasmic streaming. It is proposed that activated Rho1 transduces a signal during stages 8-10b that promotes the crosslinking activity of Capu and SpireC by preventing binding of SpireD to both Capu and SpireC. Rho1 then becomes inactivated at stage 10b, presumably by a signalling event, allowing SpireD to bind to Capu and SpireC, thereby inhibiting microtubule/microfilament crosslinking. When signalling through this pathway or the level of Capu and/or Spire protein is reduced through mutation, ooplasmic streaming occurs constitutively from stage 8 up to and through stage 13, resulting in the severe patterning defects that are observed in these mutants. That SpireD also inhibits the crosslinking activity of SpireC indicates that a parallel regulatory mechanism exists for SpireC-mediated crosslinking. Although a role for Rho1 in regulating actin nucleation by Capu and Spire cannot be ruled out, the mechanism established in this study by which Spire and Rho1 regulate the crosslinking activity of Capu does not seem pertinent to actin nucleation. Viewed in light of the fact that the P597T mutation in the FH2 domain, which is encoded by the capu2F allele, does not affect actin-nucleation activity but is less efficient at crosslinking microtubules and microfilaments, the crosslinking activity describe in this study seems to be an important aspect of how ooplasmic streaming is regulated in vivo (Rosales-Nieves, 2006).

The data have several broader implications. The finding that Capu and Spire regulate each other’s activity indicates an explanation for the conserved co-expression of these two de novo actin-nucleation factors, both of which create linear actin filaments and are required to mediate the same developmental events. Moreover, this work establishes Rho1 as a direct regulator of a broader group of actin-nucleating proteins, and is the first evidence for how the activities of Spire and Capu are regulated to coordinate the ooplasmic streaming events in vivo. The direct interaction between Rho1 and Capu indicates an additional level of complexity to this mechanism. It is, therefore, possible that Rho1 may simultaneously regulate the nucleation and crosslinking activities of Capu through an, as yet unclear, mechanism. Further investigation of this will require the expression of full-length Capu constructs that contain the relevant binding site (Rosales-Nieves, 2006).

To date, much work has been devoted to understanding the role of formins, and more recently Spire, in controlling actin dynamics and nucleation. However, diaphanous-related formin proteins also have profound effects on microtubule dynamics and stability, with recent evidence indicating that these effects are, at least in some cases, independent of the actin-nucleation function. The data presented in this study indicate that direct regulation of microtubule architecture may be a property that is common to a larger subset of formins, as well as to at least one of the Spire protein isoforms. The distinct mechanism by which Spire and Capu regulate microtubule/microfilament crosstalk is consistent with the highly specialized function of these proteins in regulating germline development in Drosophila. Indeed, the mammalian homologue of Capu, formin-2, is also required only in the female germline, where it regulates proper chromosome segregation, which is another process that involves intimate coordination of microtubule and microfilament dynamics. Recently, a mutation at the formin-2 locus has been implicated in unexplained female infertility in humans. Therefore, Capu and Spire seem to be elements of a highly conserved cassette that is required for the earliest stages of metazoan development. Precisely how the activity of these proteins is coordinated with developmental signalling circuits to allow for the proper regulation of ooplasmic streaming or chromosome segregation will certainly provide interesting areas for future work (Rosales-Nieves, 2006).

Capu and Spire assemble a cytoplasmic actin mesh that maintains microtubule organization in the Drosophila oocyte

Mutants in the actin nucleators Cappuccino and Spire disrupt the polarized microtubule network in the Drosophila oocyte that defines the anterior-posterior axis, suggesting that microtubule organization depends on actin. Cappuccino and Spire organize an isotropic mesh of actin filaments in the oocyte cytoplasm. capu and spire mutants lack this mesh, whereas overexpressed truncated Cappuccino stabilizes the mesh in the presence of Latrunculin A and partially rescues spire mutants. Spire overexpression cannot rescue capu mutants, but prevents actin mesh disassembly at stage 10B and blocks late cytoplasmic streaming. This study also shows that the actin mesh regulates microtubules indirectly, by inhibiting kinesin-dependent cytoplasmic flows. Thus, the Capu pathway controls alternative states of the oocyte cytoplasm: when active, it assembles an actin mesh that suppresses kinesin motility to maintain a polarized microtubule cytoskeleton. When inactive, unrestrained kinesin movement generates flows that wash microtubules to the cortex (Dahlgaard, 2007).

The main body axes of Drosophila are established during stages 7-9 of oogenesis when the oocyte microtubule (MT) cytoskeleton is reorganized to direct the asymmetric localization of bicoid (bcd), oskar (osk), and gurken mRNAs. At stage 7 of oogenesis, an unknown signal from the posterior follicle cells induces the disassembly of a microtubule-organizing center at the posterior of the oocyte, while new MTs nucleate from the anterior-lateral cortex with their plus ends extending toward the posterior pole. This results in the formation of an anterior-to-posterior gradient of MTs that directs the localization of bcd and osk mRNAs to the anterior and posterior poles of the oocyte, respectively, where they act to determine the anterior-posterior axis of the embryo. The polarized MT cytoskeleton is also required for the migration of the oocyte nucleus from the posterior of the oocyte to a point at the anterior margin, and this defines the dorsal-ventral axis by directing the localization of gurken mRNA to one side of the nucleus, where Gurken protein is secreted to induce dorsal follicle cell fates (Dahlgaard, 2007 and references therein).

The organization of the MTs changes during stage 10B, and they form parallel arrays around the cortex of the oocyte that drive a fast unidirectional movement of the oocyte cytoplasm, called ooplasmic streaming. Ooplasmic streaming requires the plus-end-directed MT motor, Kinesin, suggesting that the flows are generated by kinesin-dependent transport of organelles or vesicles. The cytoplasm is also in motion in oocytes from stages 8-10A, but these movements are slower and uncoordinated and have been named ooplasmic seething (Dahlgaard, 2007).

The polarized organization of the MTs at mid-oogenesis requires the function of par-1 and capu groups of genes. In mutants in the former group, which comprises par-1, lkb-1, and 14-3-3epsilon, the MTs appear to be nucleated all around the oocyte cortex, with their plus ends in the center. As a consequence, osk mRNA is mislocalized to a dot in the center of the oocyte, while bcd mRNA spreads from the anterior around most of the cortex. However, the localization of gurken mRNA is wild-type in these mutants. The polarity signal from the follicle cells induces the actin-dependent localization of PAR-1 to the posterior cortex of the oocyte, suggesting that asymmetric PAR-1 activity plays a key role in the polarization of the oocyte MT cytoskeleton (Dahlgaard, 2007).

Mutants in cappuccino (capu), chickadee (chic), and spire produce a distinct phenotype, in which the MTs form prominent arrays around the oocyte cortex during stages 8-10 and MT plus-end markers no longer localize to the posterior pole. These mutants also cause premature streaming of the oocyte cytoplasm, which resembles the cytoplasmic streaming seen in wild-type oocytes after stage 10B. As a result, both osk and gurken mRNAs are mislocalized, leading to abdominal defects in the embryo and ventralized eggs, although the localization of bcd mRNA is unaffected (Dahlgaard, 2007).

Actin-depolymerizing drugs produce identical MT and premature cytoplasmic streaming phenotypes to capu, chic, and spire mutants, indicating that actin is required for the correct organization of the MT cytoskeleton. Consistent with this, all three genes encode regulators of the actin cytoskeleton. Chickadee is Drosophila Profilin, which binds free G-actin protein to regulate actin dynamics; Spire is the founding member of a new family of actin nucleation factors that nucleate filaments from their pointed ends; Capu is a member of the Formin family of proteins, which also nucleate actin filaments, but in this case from their barbed ends (Dahlgaard, 2007 and references therein).

Although effects of actin depolymerization strongly suggest that actin plays a key role in the organization of the oocyte MT cytoskeleton, it is not clear which population of F-actin in the oocyte is responsible for this effect, or how Capu, Spire, and Profilin participate in the interaction between actin and MTs. One possibility is that Capu, Profilin, and Spire regulate MTs by directing the posterior recruitment of PAR-1, since they have been proposed to play a role in the organization of cortical actin, which is required for PAR-1 localization. This cannot account for all of the effects of the capu group mutants, however, since they produce a different phenotype from par-1 mutants. An alternative possibility is suggested by experiments showing that formin-related proteins can control the positioning or stability of MT plus ends. Bni1p is required for spindle positioning during early metaphase in budding yeast, through the recruitment of the plus ends of astral MTs to the bud tip. Bni1p localizes to the emerging bud tip and nucleates unbranched actin filaments. The myosin, Myo2p, then transports the MT plus ends along these actin cables to the bud tip, through its linkage to the plus-end-binding protein, Kar9p. In contrast, the mouse formin mDia1 acts independently of actin to stabilize MT plus ends at the leading edge of migrating NIH 3T3 cells, through a pathway that involves the inhibition of GSK3β and the plus-end-binding proteins, EB1 and APC. Thus, Capu may function in a similar way to either Bni1 or mDia to recruit or stabilize MT plus ends at the posterior of the oocyte (Dahlgaard, 2007).

A different model has been proposed for the function of Capu and Spire, in which they act not as actin nucleators but as crosslinkers between MTs and cortical actin. The spire locus encodes multiple isoforms, including two short forms, Spire D and Spire C, that encompass the N-terminal and C-terminal halves of the longest isoform, respectively. Spire D contains the KIND domain and the 4 WH2 domains that have been shown to nucleate actin in vitro and when transiently expressed in mouse fibroblasts, whereas Spire C includes an mFYVE domain and a JNK-binding site. In binding studies with tubulin and actin in vitro, both Capu and Spire C induced the bundling of actin with MTs. In contrast, Spire D nucleated F-actin in vitro but did not interact with MTs and inhibited the actin/MT crosslinking activity of Capu and Spire C. This has led to the proposal that Capu and Spire C repress the cortical bundling of MTs and premature cytoplasmic streaming by crosslinking the MTs to the cortical actin, whereas Spire D is a negative regulator of this process (Dahlgaard, 2007).

To distinguish between the different models for the function of Capu and Spire, various domains of each protein were expressed in wild-type and mutant egg chambers in order to analyze their subcellular localizations and their effects on actin, MTs, and cytoplasmic streaming in vivo. The results indicate that neither the cortical localization nor the MT-binding activity of Capu and Spire is required for their function. Instead, Capu and Spire are shown to act to assemble a dynamic actin mesh in the oocyte cytoplasm (Dahlgaard, 2007).

Formin-related proteins play a key role in cell polarity in a number of systems and usually show a highly polarized distribution to one end of the cell. For example, Bni1p and For3p localize to the poles of budding and fission yeast, respectively, where they nucleate actin cables that are required for polarized growth, while mDia stabilizes MT plus ends at the leading edge of migrating fibroblasts. Although the Drosophila forming-related protein, Capu, is similarly required for the polarization of the oocyte MT cytoskeleton and for the formation of both the anterior-posterior and dorsal-ventral axes, the results reported in this study demonstrate that Capu regulates MTs by a very different mechanism from these other formins. Neither Capu nor its partner Spire shows a polarized distribution within the oocyte, nor do they play a direct role in MT organization in a particular region of the cell. Instead, they act together with Profilin to assemble an isotropic actin mesh in the oocyte cytoplasm, which maintains the polarized arrangement of MTs by suppressing kinesin-dependent cytoplasmic streaming (Dahlgaard, 2007).

This function for Capu and Spire contrasts with the recent proposal that they act at the oocyte cortex to regulate cortical polarity and to crosslink the actin and MT cytoskeletons. The results argue against this model for several reasons. First, cortical polarity appears to be unaffected in capu and spire mutant egg chambers. PAR-1 still localizes normally to the posterior cortex, and osk mRNA is specifically anchored at the posterior in spire mutant egg chambers, indicating that this region of the cortex is different from the anterior and lateral domains. Furthermore, the MTs show a normal association with the anterior and lateral cortex in capu and spire mutants, as is most clearly demonstrated by the wild-type MT arrangement in capu mutants in which kinesin function is impaired (Dahlgaard, 2007).

Second, although Capu and Spire interact with MTs in vitro, this activity does not appear to be required for their function in vivo. Spire D, which lacks the MT-binding domain, completely suppresses cytoplasmic streaming at all stages, whereas Spire C, which contains the domain, has no effect on the spire mutant phenotype. Thus, the MT-binding activity of Spire is not required for its in vivo activity. A similar argument can made for the MT-binding activity of Capu. Capu binds MT in vitro through its FH2 domain, and a P597T substitution in the capu2F allele blocks this activity. Despite this loss of MT binding, capu2F has the weakest phenotype of all capu alleles examined, indicating that the inability to interact with MT has little effect on Capu's in vivo activity. Furthermore, the weak phenotype of capu2F is more easily explained by an effect on actin nucleation, since a clear reduction in the actin mesh in was observed capu2F homozygous oocytes, although the P597T mutation was reported to have minimal effect on actin nucleation in vitro (Dahlgaard, 2007).

The localization of Capu and Spire also argues against a model in which they act exclusively to anchor MTs to the cortex. Neither GFP-tagged Capu nor Spire D is enriched at the oocyte cortex when visualized in living oocytes, even though these fusion proteins are functional, since they rescue the strongest alleles of capu and spire, respectively. This contrasts with a previous study in which both proteins were reported to localize to the oocyte cortex, and may reflect the fact that the latter examined their distribution in detergent-extracted and fixed samples. It therefore seems unlikely that the direct crosslinking of actin and MTs by Capu or Spire at the cortex plays a significant role in their function in the oocyte (Dahlgaard, 2007).

Instead, the results indicate that the Capu pathway functions to organize a dynamic network of actin filaments throughout the oocyte cytoplasm. This actin mesh is lost in capu, spire, and chic mutants, indicating that Capu, Spire, and Profilin are necessary for its formation. Furthermore, overexpression of Capu or Spire D induces an ectopic mesh in the nurse cells, while Spire D induces an ectopic mesh in late oocytes, strongly suggesting that both proteins play a direct role in its assembly. Indeed, the role of Capu in the formation of the cytoplasmic actin mesh may explain the seemingly paradoxical observation that capu mutants cause an increase in the amount of cortical actin in the oocyte. The failure to form the actin mesh in capu mutants should lead to a rise in the concentration of free G-actin in the oocyte, which may promote excess actin polymerization at the oocyte cortex by a Capu- and Spire-independent mechanism (Dahlgaard, 2007).

The presence of the ooplasmic actin mesh correlates perfectly with the polarized arrangement of the MTs in the oocyte. The mesh is present from stage 5 to stage 10A of oogenesis, which is the period during which the anterior-posterior gradient of MT persists, and the disappearance of the mesh at stage 10B coincides with the onset of fast cytoplasmic streaming and the rearrangement of the MT into parallel cortical arrays. Furthermore, the loss of the mesh in capu, spire, and chic mutants leads to premature streaming and the precocious formation of cortical MT arrays, whereas the overexpression of Spire D maintains the mesh during stage 11 and suppresses the normal rearrangement of the MT and streaming at this stage. Indeed, the density of the mesh correlates with the severity of the mutant phenotype, since the weakest alleles of capu and chic cause a reduction in the mesh without abolishing it entirely (Dahlgaard, 2007).

This revised view of the function of Capu and Spire is consistent with the known biochemical properties of the other formin-related proteins and Spire. In vitro studies have shown that formin-related proteins nucleate actin filaments through their FH2 domains and then remain associated with the barbed end, which they protect from actin-capping proteins, while increasing the rate of elongation through the interaction of the FH1 domain with Profilin/Actin complexes. Although Capu is not a typical formin, it contains well-conserved FH1 and FH2 domains, nucleates actin in vitro, and has been shown to interact with Profilin in yeast two-hybrid assays. Furthermore, the protection of the actin mesh from Latrunculin A-induced depolymerization by CapuΔN is consistent with a model in which the protein remains associated with the barbed ends and prevents their disassembly. Spire, on the other hand, nucleates actin filaments from their pointed ends and caps this end of the filament as it grows. Thus, both Capu and Spire have the capacity to nucleate and stabilize actin filaments, raising the possibility that each protein independently nucleates and stabilizes actin filaments in the mesh. This is consistent with the observation that overexpression of Capu can induce the formation of an actin mesh in the absence of Spire. The mesh induced by overexpressed Capu alone is significantly weaker than normal, however, and persists for a shorter time, while Spire D cannot form a mesh in the absence of Capu. Furthermore, the ability of GFP-CapuΔN to stabilize the actin mesh in the presence of Latrunculin depends on endogenous Spire activity. Thus, the two proteins must cooperate to form a normal mesh, and one possibility is that they assist each other by capping the opposite ends of filaments nucleated by the other. Since Spire D associates with Capu in vitro, it is even possible that they collaborate to nucleate the same filament and remain attached to opposite ends as it grows (Dahlgaard, 2007).

Although the mesh is essential for the polarized arrangement of the MTs in the oocyte, it appears to play a permissive rather than an instructive role, because the defects in MT organization and osk mRNA localization caused by its loss can be rescued by slowing the speed of kinesin. This suggests that the mesh normally serves to restrain kinesin-dependent motility and that the rearrangement of the MTs and premature streaming are a consequence of unrestricted kinesin activity. Kinesin is required both for the slow disorganized cytoplasmic movements during stage 9, called seething, and for the rapid directional streaming at stage 11, leading to the proposal that the motor generates ooplasmic flows by moving large organelles or vesicles along MTs. This suggests the following model for how loss of the actin mesh and unrestrained kinesin motility cause the rearrangement of the MT. In the absence of the actin mesh, there is an increase in the speed or frequency of kinesin-dependent organelle transport, resulting in a concomitant increase in the strength of the cytoplasmic flows that these movements generate. Since the MTs move with the cytoplasmic flows, the stronger flows will start to wash the MTs into alignment, thereby aligning the kinesin-dependent organelle movements, which will amplify the cytoplasmic flows still further. This positive-feedback loop then continues to coordinate and increase the flows until all MTs have been washed to the oocyte cortex, with the oocyte cytoplasm rapidly rotating inside (Dahlgaard, 2007).

This model raises the question of how the actin mesh restrains the kinesin-dependent cytoplasmic flows to prevent their amplification into cytoplasmic streaming. This could be an entirely passive process, in which the actin mesh increases the viscosity of the oocyte cytoplasm, thereby increasing the drag on kinesin-dependent transport. However, a model is favored in which the mesh plays a more direct role in the inhibition of kinesin-mediated movement of the cytoplasm, and one attractive possibility is that it tethers the cargoes of kinesin that generate the flows, thereby limiting their movement. One way that the organelles might be tethered to the actin mesh is by binding to either Capu or Spire, and it is interesting to note that Spire-D shows a punctate distribution that is consistent with an association with a population of organelles or vesicles. In addition, full-length Spire contains an mFYVE domain that is predicted to target it to endosomal membranes, and has been shown to colocalize with Rab11 on vesicular structures when expressed in tissue culture cells. This tethering mechanism is very similar to the function of mDia in the anchoring of endosomes to actin at the cell periphery, which inhibits their movement along MT, and also resembles the tethering of mitochondria in neuronal cells, where mDia nucleates actin filaments that anchor the mitochondria, without affecting the motility of lysosomes or peroxisomes (Dahlgaard, 2007).

A third possibility is that the mesh anchors the MTs within the cytoplasm and prevents them from being washed into alignment at the cortex by the cytoplasmic flows. It seems unlikely, however, that direct crosslinking of actin and MT by Capu and Spire is important in vivo, but some other protein may anchor the MT to actin. Alternatively, the actin and MTs could be crosslinked indirectly. For example, Capu or Spire could interact with a vesicle or organelle that is associated with MT, thereby linking the two cytoskeletons (Dahlgaard, 2007).

The formation of the actin mesh must be tightly regulated both spatially and temporally, since the mesh normally forms only in the oocyte and not the nurse cells and is disassembled during stage 10B to allow the onset of rapid streaming. Both Capu and Spire bind Rho-GTP, raising the possibility that one or both proteins are regulated by Rho). Indeed, the GFP-CapuΔN construct was generated to test if deletion of its Rho-binding domain would lead to a constitutively active form of the protein. However, overexpression of GFP-Capu or of full-length untagged Capu produces very similar effects to GFP-CapuΔN. The only obvious difference between the three constructs is the ability of CapuΔN to protect the actin mesh from Latrunculin A-induced depolymerization, but it is unclear whether this is due to constitutive activation of Capu or some other alteration to its activity. More importantly, these data suggest that the regulation of Capu activity is unlikely to determine where and when the mesh forms. Although overexpressed Capu can assemble a mesh in both the oocyte and the nurse cells in the absence of Spire until stage 10A, Spire D cannot induce the formation of an actin mesh in the absence of Capu. The ability of Spire to form an ectopic mesh in the nurse cells and in late oocytes therefore implies that endogenous Capu must be active in the nurse cells and during the late stages of oogenesis. This suggests that the regulation of Spire determines the temporal and spatial control of actin mesh formation and disassembly (Dahlgaard, 2007).

In summary, these results suggest that the Capu pathway controls the formation of an actin mesh, which acts as a switch between two alternative states of the oocyte cytoplasm, both of which are essential for the formation of a viable egg. During stages 5–10A, the mesh inhibits kinesin-dependent motility to allow the formation of the anterior-posterior MT array that directs the localization of oskar and gurken mRNAs, and this establishes the polarity of both body axes. Once oskar mRNA has been localized and anchored to the oocyte cortex and Gurken has signaled to polarize the dorsal-ventral axis, the actin mesh is disassembled. This relieves the inhibition of kinesin-dependent organelle movement and switches on fast ooplasmic streaming. As a result, the oocyte cytoplasm becomes thoroughly mixed with the cytoplasm that enters from the nurse cells during nurse-cell dumping, leading to a uniform distribution of maternal proteins and mRNAs throughout the egg. This is important for subsequent development, because most housekeeping functions in the embryo depend on maternal gene products, which must be evenly distributed in the egg, so that they are equally partitioned into all cells (Dahlgaard, 2007).

Microtubule anchoring by cortical actin bundles prevents streaming of the oocyte cytoplasm

The localisation of the determinants of the body axis during Drosophila oogenesis is dependent on the microtubule (MT) cytoskeleton. Mutations in the actin binding proteins Profilin, Cappuccino (Capu) and Spire result in premature streaming of the cytoplasm and a reorganisation of the oocyte MT network. As a consequence, the localisation of axis determinants is abolished in these mutants. It is unclear how actin regulates the organisation of the MTs, or what the spatial relationship between these two cytoskeletal elements is. This study reports a careful analysis of the oocyte cytoskeleton. Thick actin bundles are identified at the oocyte cortex, in which the minus ends of the MTs are embedded. Disruption of these bundles results in cortical release of the MT minus ends, and premature onset of cytoplasmic streaming. Thus, the data indicate that the actin bundles anchor the MTs minus ends at the oocyte cortex, and thereby prevent streaming of the cytoplasm. Actin bundle formation requires Profilin but not Capu and Spire. Thus, these results support a model in which Profilin acts in actin bundle nucleation, while Capu and Spire link the bundles to MTs. Finally, these data indicate how cytoplasmic streaming contributes to the reorganisation of the MT cytoskeleton. The release of the MT minus ends from the cortex occurs independently of streaming, while the formation of MT bundles is streaming dependent (Wang, 2008).

This study reports the existence of actin bundles at the cortex of the oocyte which are involved in the cortical localisation of γTubulin. γTubulin is part of the γTubulin ring complex that is stabilising the minus ends of MTs. The presence of γTubulin alone does not allow distinguishing whether the protein is part of a microtubule organising centre (MTOC) that nucleates MTs or whether it only protects existing MTs from depolymerisation. Here, γTubulin was used solely as a maker for the MT minus ends, and it was shown that these are embedded within the cortical actin bundles before stage 10b (Wang, 2008).

The cytoskeletal rearrangements at stage 10b include the disassembly of the cortical actin bundles, the redistribution of the MT minus ends from the cortex to subcortical regions and the formation of MT arrays parallel to the oocyte cortex. Concomitantly with these cytoskeletal changes, the transition from slow to fast cytoplasmic streaming is triggered. What is the causal relationship between these events? The finding that interfering with actin bundle formation by drug treatment and GFPactin5c overexpression results in MT minus ends redistribution, MT array formation and premature fast streaming indicates that actin bundling acts upstream of MT reorganisation and streaming. The analysis of Khc mutants allows to further dissect the subsequent steps reorganising the MT cytoskeleton. In the absence of streaming, caused by the loss of Khc function, the redistribution of MT minus ends occurs normally, while the formation of MT arrays is abolished. Thus, minus end redistribution is upstream of streaming, and array formation is downstream. It is therefore proposed that streaming is initiated by the disassembly of the cortical actin bundles resulting in loss of cortical MT minus end anchoring. It is further proposed that the redistribution of the minus ends to subcortical regions is important for the reorganisation of the MT cytoskeleton into arrays that run parallel to the oocyte cortex. At this step a previously suggested self amplifying loop could be initiated, in which MT array formation and Kinesin movement enhance each other. In this loop the Kinesin driven streaming helps to sweep MTs into parallel arrays, which in turn allow more robust currents in the cytoplasm (Wang, 2008 and references therein).

How do the actin binding proteins Capu, Spire and Profilin act on the oocyte cytoskeleton to prevent premature cytoplasmic streaming? chic/Profilin mutants and latrunculin A treatment both interfere with bundle formation. Latrunculin A treatment inhibits actin polymerisation by binding to and sequestering actin monomers. Profilin is involved in actin polymerisation by delivering actin monomers to the growing ends of actin filaments. Thus, latrunculin A and Profilin mutants appear to interfere with bundling by limiting the pool of monomers that can be added to growing actin filaments. In contrast, capu and spire mutants are not required for the formation of actin bundles. It is proposed that Capu and Spire anchor the MT minus ends in a scaffold provided by the cortical actin bundles. The lack of Capu and Spire activity in the mutants prevents cortical MT anchoring and allows streaming in the presence of actin bundles. This model is supported by the work of Rosales-Nieves (2006) who have shown that Capu and Spire proteins are able to crosslink F-actin and MTs in vitro, and that both proteins localise to the oocyte cortex (Wang, 2008).

The regulation of fast ooplasmic streaming could be controlled at the level of the cortical localisation of Capu and Spire. The displacement of the two proteins from the cortex at stage 10b might result in loss of MT minus end anchoring, and thereby induce fast streaming. To test this, the localisation of GFP-Capu and GFP-Spire was analysed in cross sections of oocytes. However, no difference were in the localisation of the two proteins before and after onset of fast streaming. In addition, no displacement of GFP-Capu and GFP-Spire was detected after induction of premature streaming by latrunculin A treatment. Thus, Capu and Spire activities are not regulated at the level of their localisation (Wang, 2008).

A different mode of Capu and Spire regulation is suggested by their genetic and biochemical interaction with Rho1. This interaction led to a model in which Rho1 initiates fast streaming by regulating the crosslinking activities of Capu and Spire (Rosales-Nieves, 2006). The prevention of streaming requires not only capu and spire but also the presence of actin bundles. The formation of these bundles occurs, however, independently of capu and spire. This suggests that the onset of fast streaming is not only controlled by regulating Capu and Spire activities, but also by disassembly of the actin bundles (Wang, 2008).

Genes were also tested that are involved in actin regulation in the oocyte but do not induce premature streaming. For capulet, swallow and moesin mutants the formation of ectopic actin clumps has been reported reflecting defects in the organisation of the oocyte actin cytoskeleton. The presence was confirmed of ectopic F-actin in the oocyte cytoplasm in these mutants, but nevertheless the formation cortical actin bundles was detected. Thus, actin defects in the oocyte do not necessarily affect cortical actin bundling (Wang, 2008).

Molecular architecture of the Spire-actin nucleus and its implication for actin filament assembly

The Spire protein is a multifunctional regulator of actin assembly. The structures and properties of Spire-actin complexes have been studied by X-ray scattering, X-ray crystallography, total internal reflection fluorescence microscopy, and actin polymerization assays. Spire-actin complexes in solution assume a unique, longitudinal-like shape, in which Wiskott-Aldrich syndrome protein homology 2 domains (WH2), in an extended configuration, line up actins along the long axis of the core of the Spire-actin particle. In the complex, the kinase noncatalytic C-lobe domain is positioned at the side of the first N-terminal Spire-actin module. In addition, preformed, isolated Spire-actin complexes were found to be very efficient nucleators of polymerization and they afterward dissociate from the growing filament. However, under certain conditions, all Spire constructs -- even a single WH2 repeat -- sequester actin and disrupt existing filaments. This molecular and structural mechanism of actin polymerization by Spire should apply to other actin-binding proteins that contain WH2 domains in tandem (Sitar, 2011).

Small-angle X-ray scattering was applied to study the architecture of several Spire-actin complexes including the most native N-terminal part of Spire (SpireNT) and other truncated but fully functional constructs. The SAXS (small-angle X-ray scattering) ab initio reconstruction directly visualized the overall shape of complexes in solution and the rigid body refinement provided distribution of Spire-actin conformers. Spire forms stable longitudinal-like complexes with actin loosely positioned along the stretch of WH2 repeats. Interestingly, Spire forms identical complexes with Drosophila cytoskeletal 5C actin, native rabbit skeletal actin, and even when the latter one is inhibited by latrunculin B, suggesting that actins in the Spire-actin complexes do not form F-actin-like longitudinal bonds in the polymerization initiation complex (Sitar, 2011).

Combining the high-resolution crystallographic models with the SAXS data clearly singles out the straight-longitudinal configuration for crystals and solution. The rigid body fitting of each complex to the SAXS data indicates small shifts from the crystal structures and shows arrangements that are predominantly straight-longitudinal with the admixture of the elongated side-to-side structures. The X-ray data suggest that actin molecules and parts of the WH2 domains, which consist of the N-terminal helix and C-terminal tail, constitute rigid units of the complexes. These are linked by unstructured and flexible linkers. The variability of the 'around linker' regions is observed not only between actin/WH2 units of different constructs but also within one complex. This finding is in agreement with the rigid body refinement of the SAXS data, where for each construct only a few models with similar chi squared f values are produced with slightly different actin/WH2 modules orientation. The different solution conformations derived from SAXS measurements underline the dynamic character of the Spire-actin nucleus and explain the importance of the length and flexibility of Spire's linkers (Sitar, 2011).

ITC (Isothermal Titration Calorimetry) investigations show that WH2 domains -A, -B, -C, and -D bind monomeric G-actin with comparable affinities, and the presence of L3 linker does not significantly influence the Kd of either L3-WH2D or WH2C-L3. The L3 linker alone also does not interact with actin in these experiments. These results do not confirm a previously described role of the L3 linker, which was also designated as a monomer binding linker. ITC data cannot exclude sequential or cooperative binding but rather suggest equal binding sites mode of interaction, at least at the stage of primary nucleus formation (Sitar, 2011).

Polymerization assays clearly show that the prepurified Spire-actin complexes are the intermediates that lead to enhanced nucleation. Actin is not solely sequestered in the dead-end Spire-actin complex, but forms a dynamic Spire-actin nucleus that triggers fast polymerization. The shortest nucleating construct of Spire must have two WH2 domains; a single WH2 domain or the WH2 domain with a linker do not show any nucleation activity. In contrast, sequestering activity as well as the disruption of actin filaments are the attributes of all constructs, including a single 20 amino acid long WH2 domain. These results suggest that the disintegration of an actin filament by Spire does not require a concerted action of two actin-binding sites as is typical for F-actin severing proteins from the gelsolin protein family. Thus, a single Spire WH2 domain can remove an actin monomer from the filament despite its F-actin conformation. Consequently, the filament will break at this position and the recently described enhanced depolymerization in dilution-induced depolymerization assays is just because of the rapid increase of free barbed ends. An explanation for the filament dissociating activity of a single WH2 domain must be sought in conformational differences of the actin monomers in F-actin and WH2 complexes and mutual incompatibility of these conformers. Indeed, the crystal structure studies described earlier and referred to in detail in this work offer strong evidence for this view (Sitar, 2011).


Rab11 functions during oogenesis and during cellularization of Drosophila embryos. The Nuclear fallout and Rab11 function in membrane trafficking and actin remodeling during the initial stages of furrow formation during cellularization. Membrane addition is mediated via endosomal-mediated membrane delivery to the site of furrow formation. Thus Rab11 regulates endosomes as key trafficking intermediates that control vesicle exocytosis and membrane growth during cellularization. Rab11 is required in endocytic recycling and in the organization of posterior membrane compartments during oogenesis. Rab11 is also required in the organization of microtubule plus ends and osk mRNA localization and translation at the posterior pole. It is proposed that microtubule plus ends and, possibly, translation factors for osk mRNA are anchored to posterior membrane compartments that are defined by Rab11-mediated trafficking and reinforced by Rab11-Osk interactions (Dollar, 2002).

The p150-Spire protein, which was discovered as a phosphorylation target of the Jun N-terminal kinase, is an essential regulator of the polarization of the Drosophila oocyte. Spire proteins are highly conserved between species and belong to the family of Wiskott-Aldrich homology region 2 (WH2) proteins involved in actin organization. The C-terminal region of Spire encodes a zinc finger structure highly homologous to FYVE motifs. A region with high homology between the Spire family proteins is located adjacent (N-terminal) to the modified FYVE domain and is designated as 'Spir-box'. The Spir-box has sequence similarity to a region of rabphilin-3A, which mediates interaction with the small GTPase Rab3A. Coexpression of Drosophila p150-Spire and green fluorescent protein-tagged Rab GTPases in NIH 3T3 cells revealed that the Spire protein colocalizes specifically with the Rab11 GTPase, which is localized at the trans-Golgi network (TGN), post-Golgi vesicles, and the recycling endosome. The distinct Spire localization pattern is dependent on the integrity of the modified FYVE finger motif and the Spir-box. Overexpression of a mouse Spir-1 dominant interfering mutant strongly inhibits the transport of the vesicular stomatitis virus G (VSV G) protein to the plasma membrane. The viral protein arrests in membrane structures, largely colocalizing with the TGN marker TGN46. The findings that the Spire actin organizer is targeted to intracellular membrane structures by its modified FYVE zinc finger and is involved in vesicle transport processes provide a novel link between actin organization and intracellular transport (Kerkhoff, 2001).

Effects of Mutation or Deletion

cappuccino and spire are unique Drosophila maternal-effect loci that participate in pattern formation in both the anteroposterior and dorsoventral axes of the early embryo. Mutant females produce embryos lacking pole cells, polar granules, and normal abdominal segmentation. They share these defects with the posterior group of maternal-effect genes. Although embryos are defective in abdominal segmentation, in double mutant combinations with Bicaudal D, abdominal segments can be formed in the anterior half of the egg. This indicates that embryos produced by mutant females contain the 'posterior determinant' required for abdominal segmentation and suggests that the wild-type gene products are not required for production of the posterior determinant but, rather, for its localization or stabilization. The Vasa protein, a component of polar granules, is not localized at the posterior pole of mutant egg chambers or embryos, providing additional support for the hypothesis that localization to or stabilization of substances at the posterior pole of the egg chamber is defective in mutant females. Females mutant for the strongest alleles also produce dorsalized embryos. Phenotypic analysis reveals that these dorsalized embryos also have abdominal segmentation defects. The mutant phenotypes can be ordered in a series of increasing severity. Pole cell formation is most sensitive to loss of functional gene products, followed by abdominal segmentation, whereas normal dorsoventral patterning is the least sensitive to loss of functional gene products. In addition, mutant females contain egg chambers that appear to be dorsalized, resulting in the production of eggs with dorsalized eggshells. Germ-line mosaics indicate that cappuccino and spire are required in the oocyte-nurse cell complex. This suggests that the eggshell phenotype results from altered pattern in the underlying germ cell. Also, the epistatic relationships between several early patterning loci, are defined on the basis of an analysis of the eggs and embryos produced by females doubly mutant for cappuccino or spire and other loci that affect the pattern of both the egg and the embryo (Manseau, 1989).

Staufen is required to localize Oskar mRNA. However, oskar function is required to stabilize the posterior localization of Staufen protein. In oskar mutants, Staufen accumulates only transiently at the posterior pole. It thus seems that Oskar protein is required to keep Oskar mRNA and Staufen protein at the posterior pole. Mutations in the posterior group genes nanos, pumilio, tudor, valois and vasa have no effect on the localization of Staufen to the pole plasm of freshly laid embryos. In contrast, all cappuccino and spire mutations have dramatic effects on this localization (St Johnston, 1991).

Mutations in vasa, pumilio and nanos display no effect on Oskar mRNA localization, while capuccino, spire and staufen all show defects in Oskar mRNA localization (Kim-Ha, 1991).

Maternally synthesized Hsp83 transcripts are localized to the posterior pole of the early Drosophila embryo by a novel mechanism involving a combination of generalized RNA degradation and local protection at the posterior. Hsp83 RNA is not protected at the posterior pole of embryos produced by females carrying maternal mutations that disrupt the posterior polar plasm and the polar granules -- cappuccino, oskar, spire, staufen, tudor, valois, and vasa (Ding, 1993).

Embryonic axis specification in Drosophila melanogaster is achieved through the asymmetric subcellular localization of morphogenetic molecules within the oocyte. The cappuccino and spire loci are required for both posterior and dorsoventral patterning. Time-lapse confocal microscopic analyses of living egg chambers have demonstrated that these mutations induce microtubule reorganization and the premature initiation of microtubule-dependent ooplasmic streaming. As a result, microtubule organization is altered and bulk ooplasm rapidly streams during the developmental stages in which morphogens are normally localized. These changes in oocyte cytoarchitecture and dynamics appear to disrupt axial patterning of the embryo (Theurkauf, 1994).

Posterior localization of Vasa protein depends upon the functions of four genes: cappuccino, spire, oskar and staufen. Localization of Vasa to the perinuclear nuage (fibrous bodies making electron-dense clumps on the cytoplasmic face of the nuclear envelop of germ line and nurse cells) is abolished in most vas alleles, but is unaffected by mutations in the four genes required upstream for Vasa's pole plasm localization. Thus localization of Vasa to the nuage particles in the perinuclear region of the oocyte is independent of the pole plasm assembly pathway. Proteins from two mutant alleles that retain the capacity to localize to the posterior pole of the oocyte are both severely reduced in RNA-binding and -unwinding activity, as compared to the wild-type protein on a variety of RNA substrates, including in vitro synthesized pole plasm RNAs. Thus the RNA helicase function is not required for localization to the pole plasm. Initial recruitment of Vasa to the pole plasm must consequently depend upon protein-protein interactions but once localized Vasa must bind to RNA to mediate germ cell formation (Liang, 1994).

Three distinct segments of OSK mRNA, termed the A, B and C regions, contain Bruno-binding sites. The A and B regions are adjacent to one another near the beginning of the 3' UTR, whereas the C region is located downstream of the AB region, close to the polyadenylation site. The Bruno binding sites contain the consensus sequence UU(G/A)U(A.G)U(G/A)U. When this sequence is modified by mutation, Bru fails to bind. Oskar transgenes lacking Bru binding sites produce embryos that display substantial patterning defects. These defects indicate a posteriorization of the embryo and can be attributed to excess or mislocalized osk activity. These results suggest that Bruno normally acts to restrict OSK activity. BRE mutations have no effect on OSK mRNA localization; rather, they affect the level of translation. The posterior group genes cappuccino, spire, mago nashi, staufen and oo18, each of which are required for the localization of OSK mRNA to the posterior pole of the oocyte, are still required for OSK translation when Bruno-mediated translational repression is missing, due to deleted BREs (Kim-Ha, 1995).

Shortly after fertilization in Drosophila embryos, the G-protein alpha subunit, Gi alpha, undergoes a dramatic redistribution. Initially granules containing Gi alpha are present throughout the embryonic cortex but during nuclear cleavage they become concentrated at the posterior pole and are lost by the blastoderm stage. Mutations that eliminate anterior structures (bicoid, swallow, and exuperantia) do not prevent the posterior accumulation of Gi alpha. Likewise, embryos from mothers with dominant gain of function mutations in the Bicaudal D gene show normal polarization of Gi alpha granules. By contrast, a subset of mutations that eliminate posterior structures (cappuccino, spire, staufen, mago nashi, valois, and oskar) prevent the posterior accumulation of Gi alpha. It is important to note that mutations in posterior genes that are found lower in the putative hierarchy (vasa, tudor, nanos, and pumilio) do not affect Gi alpha redistribution. From these results it is concluded that Gi alpha redistribution to the posterior pole depends on maternal factors involved in the localization of the posterior morphogen Nanos (Wolfgang, 1995).

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 because 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).

The phenotypes of eggs laid by squid1 and fs(1)K10 females are similar. In both cases, GRK mRNA is mislocalized and Grk protein is produced around the entire anterior circumference of the oocyte. These two female sterile mutations, although similar, are also unique with respect to other known female sterile mutants. In most other cases in which grk mRNA is mislocalized, for example, in orb and spindleB mutant egg chambers, the unlocalized RNA is not translated efficiently. Mutations in cappuccino and spire also result in a mislocalization of grk RNA and translation of the mislocalized message, but these two mutations have more generalized effects on oocyte patterning and do not seem as specific for grk function as K10 or the germ-line forms of Sqd. In addition, early in oogenesis Grk is necessary for the establishment of anteroposterior patterning. However, eggs laid by both sqd1 and K10 mutant mothers display no anteroposterior defects, but are abnormal along only the D/V axis. When the expression of K10 protein was analyzed in sqd1 mutant ovaries, it was found that the distribution of K10 in sqd1 mutants is unaffected and K10 protein is detected in the oocyte nucleus of late stage egg chambers. Conversely, however, the expression of Sqd protein is affected by the K10 mutation. In K10 mutant ovaries, Sqd is present in the nurse cell nuclei but absent from the oocyte nucleus. In addition, in wild-type egg chambers Sqd protein is detected at the posterior pole at late stages and this cytoplasmic Sqd localization is unaffected in K10 egg chambers. These data indicate that Sqd protein is, in fact, expressed in K10 mutant egg chambers, but that its accumulation in the oocyte nucleus is specifically lost in the absence of K10 function (Norvell, 1999).

To determine the molecular nature of the mutant lesions in spire, reverse transcription-PCR was used to identify the molecular lesions. The lesions in spir2F, spirRP and spirPJ are nonsense mutations, creating premature termination codons, while the lesion in spirQF alters a splice junction, leading to premature termination within the intron. SpirI83, a hybrid dysgenesis-induced allele of spire, contains an insertion of a roo element in the region specific to the long form of spire (Wellington, 1999).

Rhodamine-phalloidin staining of the actin cytoskeleton in spirEC and spir2F appears normal after analysis of the actin cytoskeleton in spire mutants. (Wellington, 1999).

Spire, an actin nucleation factor, regulates cell division during Drosophila heart development

The Drosophila dorsal vessel is a beneficial model system for studying the regulation of early heart development. Spire (Spir), an actin-nucleation factor, regulates actin dynamics in many developmental processes, such as cell shape determination, intracellular transport, and locomotion. Through protein expression pattern analysis, this study demonstrates that the absence of spir function affects cell division in Myocyte enhancer factor 2-, Tinman (Tin)-, Even-skipped- and Seven up (Svp)-positive heart cells. In addition, genetic interaction analysis shows that spir functionally interacts with Dorsocross, tin, and pannier to properly specify the cardiac fate. Furthermore, through visualization of double heterozygous embryos, it was determined that spir cooperates with CycA for heart cell specification and division. Finally, when comparing the spir mutant phenotype with that of a CycA mutant, the results suggest that most Svp-positive progenitors in spir mutant embryos cannot undergo full cell division at cell cycle 15, and that Tin-positive progenitors are arrested at cell cycle 16 as double-nucleated cells. It is concluded that Spir plays a crucial role in controlling dorsal vessel formation and has a function in cell division during heart tube morphogenesis (Xu, 2012).

Proper dorsal vessel morphogenesis is critically dependent upon intercellular signaling and the regulation of gene expression. Although great progress has been made in the study of heart development, it is not known exactly how many genes and pathways are involved in this cardiogenic process or how many of these factors cooperate together. Previous genetic screens have identified genes that play roles in the specification, morphogenesis, and differentiation of the heart, including mastermind and tup. The current sensitized screen has also proved to be an efficient method to find additional factors in this process, suggesting that much remains to be learned about the molecular components involved in correct dorsal vessel formation (Xu, 2012).

Spir is required for the proper timing of cytoplasmic streaming in Drosophila, and loss of spir leads to premature microtubule-dependent fast cytoplasmic streaming during oogenesis, the loss of oocyte polarity, and female sterility. Even though it is known that spir is an important actin filament nucleation factor, the findings are the first report to describe a function of spir for cell division. Through phenotypic analysis of the spir mutant phenotype, it was found that many cardioblast nuclei are partially or completely divided. However, the cytoplasm is not divided in the absence of spir, which is consistent with the function of spir in cytoplasmic movement. Thirteen rapid nuclear division cycles without cell division initiate Drosophila embryo development, followed by three waves of cell division. The first wave of cell division occurs in the mesoderm at cell cycle 14. After this initial division, cells migrate, spread dorsally and undergo a second round of cell division at cell cycle 15. The third wave of cell division in the mesoderm occurs at the end of germband extension during cell cycle 16. There are two different types of cardioblast precursor cells: one type divides into two identical Tin-positive cardioblasts (TC), and the other type divides into one Svp-positive cardioblast (SC) and one Svp-positive pericardial cell (SPC). Based on the comparison of CycA and spir mutant phenotypes, a tentative cell division model is proposed to demonstrate spir function in determining cardiac cell fate (see A cell division model of spir function during heart development). In a wild-type background, one Svp-positive super progenitor (SSP) divides into two Svp-positive progenitors (SP), then each of these cells divides into one SPC and one SC. For Tin-positive super progenitors (TSP), after each divides into two Tin-positive progenitors (TP), each TP further divides into two identical TCs. In the current model, division from the super progenitor to progenitors takes place at cell cycle 15, and division from progenitors to full differentiated heart cells occurs at cell cycle 16. In CycA mutants, mitosis 16 is blocked such that both SPs and TPs stop cell division. This results in the two SPs assuming a myocardial fate. Thus the number of SCs remains normal, but half of the TCs are missing in the CycA mutants. The data suggest that in spir mutant embryos, most of the SPs fail to undergo full cell division at cycle 15 resulting in a SPC fate with paired nuclei. A subset of these cells are able to undergo the 15th cell division but are arrested at cycle 16 as double-nucleated cells which exhibit both Svp and Mef2 staining, characteristic of the SCs seen in the CycA mutants. Similarly, for TPs, cycle 16 was also blocked such that it resulted in two double-nucleated cells. In summary, Spir affects mitosis 16 for Tin-positive cell division and both mitosis 15 and 16 for Svp-positive cell division (Xu, 2012).

Antibody staining suggests that Spir is expressed ubiquitously before stages 12-13 and is located in both nuclei and cytoplasm. After cell cycle 16 cell division stops, occurring during stage 10-11. The expression of Spir in the cytoplasm then decreases gradually. At stage 15, the staining pattern shows mostly nucleus expression with some cytoplasmic expression and by stage 16 the nuclei become distinct indicating nucleus staining only. It is hypothesized that expression of Spir decreases in the cytoplasm but remains constant in the nuclei when cell division halts (Xu, 2012).

The genetic analysis of spir, Doc, pnr and tin suggests that these factors may regulate each other during dorsal vessel formation, and especially significant is the interaction between spir and pnr. Pnr is a GATA class transcription factor expressed in both the dorsal ectoderm and dorsal mesoderm, where it is required for cardiac cell specification. Proper dorsal vessel formation is inhibited in pnr loss-of-function embryos due to failure in the specification of the cardiac progenitors. In spir mutants, the expression pattern of Pnr remains normal. However, Spir is over-expressed in the cardiac mesoderm in pnr mutants, suggesting that Pnr may repress the expression of the spir (Xu, 2012).

In conclusion, Spir is a newly-identified factor functioning in cell division during dorsal vessel formation. Tin-, Eve- and Svp-positive heart cells are all affected in the absence of spir. Also, spir expression depends on the transcription factors Doc, tin and pnr. Genetic interaction data also show that spir cooperates with CycA in heart cell division (Xu, 2012).


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spire: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 17 December 2021

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