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to capuccino (Manseau, 1989 and Theurkauf, 1994) and Capu binds to rho family GTPases (J. Calley and L. Manseau, unpublished, cited in Wellington, 1999).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).
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).
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 otherís 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).
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, our 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).
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, 2007).
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, 2007).
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, 2007 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, 2007).
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, 2007).
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, 2007).
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, 2007).
See the embryonic expression pattern of spir at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
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).
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).
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date revised: 30 May 2008
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