InteractiveFly: GeneBrief

cappuccino: Biological Overview | References

Gene name - cappuccino

Synonyms -

Cytological map position - 24C8-24C9

Function - signaling

Keywords - actin filament capping and nucleation, suppression of kinesin motility, oogenesis, dorsal group, Posterior group, cytoskeleton

Symbol - capu

FlyBase ID: FBgn0000256

Genetic map position - 2L: 3,872,658..3,902,860 [-]

Classification - Formin Homology 2 Domain

Cellular location - cytoplasmic

NCBI links: Precomputed BLAST | EntrezGene

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 (St Johnston, 2005). 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 (Theurkauf, 1994), 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 (Emmons, 1995; Kovar, 2004; Pruyne, 2002; Sagot, 2002; 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 (Doerflinger, 2006; Rosales-Nieves, 2006). 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 (Evangelista, 2002). 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 (Rosales-Nieves, 2006). 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 (Rosales-Nieves, 2006). 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 (Theurkauf, 1994) 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 (Evangelista, 2002; Martin, 2005; Palazzo, 2001). 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 (Rosales-Nieves, 2006). 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 (Rosales-Nieves, 2006). 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 the 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 (Rosales-Nieves, 2006). 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 (Magie, 1999; Rosales-Nieves, 2006). 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 (Goode, 2007). 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 (Emmons, 1995; Manseau, 1996; Quinlan, 2005). 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 (Quinlan, 2005). 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 (Kerkhoff, 2001; Otto, 2000). 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 (Rosales-Nieves, 2006; Wellington, 1999). 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).

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).

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 (Theurkauf, 1994) 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 (Serbus, 2005; 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 (Goode, 2007). 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 (Magie, 1999; Wellington et al., 1999). 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).

Regulatory interactions between two actin nucleators, Spire and Cappuccino

Spire and Cappuccino are actin nucleation factors that are required to establish the polarity of Drosophila oocytes. Their mutant phenotypes are nearly identical, and the proteins interact biochemically. This study found that interaction between Spire and Cappuccino family proteins is conserved across metazoan phyla and is mediated by binding of the formin homology 2 (FH2) domain from Cappuccino (or its mammalian homologue formin-2) to the kinase noncatalytic C-lobe domain (KIND) from Spire. In vitro, the KIND domain is a monomeric folded domain. Two KIND monomers bind each FH2 dimer with nanomolar affinity and strongly inhibit actin nucleation by the FH2 domain. In contrast, formation of the Spire-Cappuccino complex enhances actin nucleation by Spire. In Drosophila oocytes, Spire localizes to the cortex early in oogenesis and disappears around stage 10b, coincident with the onset of cytoplasmic streaming (Quinlan, 2007).

The spire and cappuccino genes have been linked since their discovery in a genetic screen 17 yr ago (Manseau, 1989). The KIND domain of Spir binds with high affinity to the Capu-FH2 domain at a stoichiometry of 2:2 (two KIND monomers to one FH2 dimer). The WH2 cluster of Spir interacts with Capu-FH2 but that this interaction is three orders of magnitude weaker than that between the Capu-FH2 and the KIND domain. Although binding is detected between the two domains, the Capu-FH2 domain has no direct effect on actin nucleation by the Spir-WH2 cluster. However, if the KIND domain is present and correctly folded, binding of the FH2 dimer increased nucleation activity of the WH2 cluster. In contrast, the KIND domain potently inhibits actin nucleation by the Capu-FH2 domain. Constructs containing both the KIND and WH2 cluster do not enhance the inhibition of Capu-FH2-mediated actin nucleation or microtubule bundling over that observed for the KIND domain alone. For these reasons, it is proposed that the KIND-FH2 interaction is more physiologically relevant than the WH2-FH2 interaction. Additional structural and functional studies of the KIND domain are required to determine how many KIND domains are required to inhibit actin nucleation and to compete for actin and microtubule binding (Quinlan, 2007).

The KIND module was initially identified as a conserved region in the N-terminal half of Spir proteins (Ciccarelli, 2003), and the region was named based on its sequence similarity to the C-lobe of the protein kinase fold (Ciccarelli, 2003). The KIND domain is found only in metazoa, and its consensus sequence lacks catalytic residues required for kinase activity. Because the substrates of protein kinases interact with α-helical regions in the C-lobe, it was hypothesized that the KIND domain evolved from a functional kinase into a protein-protein interaction domain. The discovery that the Spir KIND domains bind specifically to Capu family FH2 domains supports this hypothesis (Quinlan, 2007).

What role do Spir and Capu play in oogenesis? Spir disappears from the oocyte cortex at stage 10, when rapid streaming normally begins and its absence in spire mutant flies leads to premature streaming. This strongly suggests that Spir plays an inhibitory role in rapid streaming. It is not yet known whether endogenous Capu has the same restricted temporal pattern observed for Spir. This information will be essential to understanding the nature of the Spir-Capu complex and its role during oogenesis. Spir and Capu interact in the oocyte, and Rosales-Nieves (2006) found that GFP fusions of these proteins both exist at the oocyte cortex, placing them in an ideal location to coordinate actin and possibly anchor microtubules. Rapid streaming is, in part, characterized by bundling and movement of microtubules. Capu bundles microtubules, which is an activity regulated by Spir. If Spir is removed at stage 10 but Capu remains, Capu could play a role in reorganizing the microtubule cytoskeleton and possibly coordinating it with the actin cytoskeleton. A complete understanding of how Spir and Capu achieve this coordination depends on knowing when and how the Spir-Capu complex is regulated (Quinlan, 2007).

Capu and other members of the formin family nucleate de novo actin filament assembly and remain associated with elongating barbed ends of newly formed filaments (Pring, 2003; Quinlan, 2005). The activity of most formin family proteins is regulated by an autoinhibitory interaction between an N-terminal sequence (the Diaphanous inhibitory domain [DID]) and a C-terminal sequence (the Diaphanous autoinhibitory domain [DAD]). Small G proteins of the Rho family stimulate nucleation activity by binding to the DID domain and disrupting its interaction with DAD. However, Capu family formins lack both DID and DAD domains (Higgs, 2005). In fact, Rosales-Nieves (2006) did not observe autoinhibition when combining the N terminus of Capu with the FH2 domain, as has been observed for mDia1 (Li 2003). The results argue strongly that Capu activity is regulated in trans by interaction with Spir (Quinlan, 2007).

The mechanism of actin nucleation by Spir is very different from that of formins like Capu. Spir binds four actin monomers using four closely apposed binding sites and then assembles them into a filament nucleus. After nucleation, Spir proteins remain associated with the slow-growing pointed end of the new filament. If Spir and Capu always function together as a single filament-forming complex, it is suggested that their activities might synergize. One intriguing possibility is that Spir nucleates filaments whose free barbed ends are then handed off to Capu. Such a mechanism would enable the independent control of filament nucleation and barbed end binding. The tight binding that was measured suggests that Spir and Capu may not dissociate upon nucleation but that actin and microtubules do bind competitively. This idea begs two important questions: (1) Does the activation of Capu require the complete dissociation of Spir, or can the two proteins function together as a single filament-forming unit? (2) How is the Spir-Capu interaction modulated by upstream signaling systems? Recent data implicate the GTPase Rho as a regulator of Spir-Capu interaction in Drosophila (Rosales-Nieves, 2006). The Spir-Capu interaction is evolutionally conserved, but whether or not this mode of regulation is conserved remains to be tested (Quinlan, 2007).

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).

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).

Interaction between microtubules and the Drosophila formin Cappuccino and its effect on actin assembly

Formin family actin nucleators are potential coordinators of the actin and microtubule cytoskeletons, as they can both nucleate actin filaments and bind microtubules in vitro. To gain a more detailed mechanistic understanding of formin-microtubule interactions and formin-mediated actin-microtubule crosstalk, microtubule binding by Cappuccino (Capu), a formin involved in regulating actin and microtubule organization was studied during Drosophila oogenesis. Two distinct domains were found within Capu; FH2 and tail, work together to promote high-affinity microtubule binding. The tail domain appears to bind microtubules through non-specific charge-based interactions. In contrast, distinct residues within the FH2 domain are important for microtubule binding. The first visualization is reported of a formin polymerizing actin filaments in the presence of microtubules. Interestingly, microtubules are potent inhibitors of Capu's actin nucleation activity but appear to have little effect on Capu once it is bound to the barbed end of an elongating filament. Because Capu does not simultaneously bind microtubules and assemble actin filaments in vitro, its actin assembly and microtubule binding activities likely require spatial and/or temporal regulation within the Drosophila oocyte (Roth-Johnson, 2013).

Based on structural information and the current experimental findings, a simple model is proposed to describe the mechanism of Capu-microtubule binding. A homology model of Capu based on the crystal structure of hDAAM1 reveals that residues within the FH2 domain that affect microtubule binding (K851, K853, K856) are clustered into a positively charged patch near the base of Capu's tail domain, suggesting that these regions form a continuous binding surface. Negatively charged residues in the patch (D854) or positively charged residues located away from the patch (K858) have little or no effect on microtubule binding. Capu binds microtubules through a seemingly synergistic interaction involving both its tail and FH2 domains. The FH2 domain alone is insufficient to measurably bind microtubules in vitro, but the highly charged tail domain could act as an electrostatic tether that promotes FH2 binding by increasing the local FH2 concentration at the microtubule surface (Roth-Johnson, 2013).

It is further proposed that the differences in microtubule binding density that were observed reflect the size of the FH2 binding footprint along the microtubule lattice: loss (GST-tail) or reduction (K856A mutation) of FH2 domain binding results in a smaller binding footprint and a corresponding increase in binding density. This model could also explain the different microtubule binding densities seen among different formins. An emerging trend shows that formins whose tails can bind microtubules (Capu and mDia2) have higher binding densities than formins whose tails do not bind microtubules (mDia1 and hINF2). In the absence of tail binding, formins may rely on more extensive FH2 contacts with the microtubule lattice and thus have a larger binding footprint (Roth-Johnson, 2013).

The data also support a model in which Capu does not simultaneously assemble actin filaments and bind microtubules. Microtubules potently inhibit Capu's actin nucleation activity in bulk assays but had little effect on Capu once it was bound to the end of an elongating actin filament. The location of residues K851, K853, and K856 on the homology model suggests that the FH2 domain binds both microtubules and the actin barbed end through similar or overlapping surfaces, further supporting the model that microtubules and actin barbed ends directly compete for Capu binding. When Capu binds microtubules, its tail domain becomes unavailable for actin monomer binding and the inner FH2 domain becomes sterically occluded and inaccessible to newly formed actin barbed ends. Conversely, the microtubule binding surface in the FH2 domain is inaccessible when Capu is associated with an actin barbed end, allowing Capu to elongate actin filaments in the presence of microtubules (Roth-Johnson, 2013).

Total internal reflection fluorescence (TIRF) microscopy assays provide additional insight into important questions surrounding formin-mediated actin-microtubule crosstalk. Microtubules did not anchor elongating actin filaments nor act as scaffolds for actin nucleation. Although actin filaments have been observed tracking along microtubules, this behavior was essentially the same in the presence or absence of CapuCT, suggesting that low concentrations of Capu do not actively align or bundle microtubules and actin filaments. It was previously shown that Capu can crosslink actin filaments and microtubules, mostly likely through side binding of both microtubules and actin filaments. However, at the very low CapuCT concentrations used in the current TIRF assays, much of the CapuCT is expected to be bound to the barbed end of elongating filaments and unavailable for binding the sides of actin filaments. Because of this, the relationship between microtubule and barbed end binding was specifically tested rather than filament side binding (Roth-Johnson, 2013).

There is strong evidence that Capu helps build a cytoplasmic actin mesh in the mid-stage Drosophila oocyte. This discussion specifically considers Capu's role as both a microtubule binding protein and actin assembly factor in the ooctye and how these functions might be regulated. The observation that actin barbed ends and microtubules compete for Capu binding suggests that Capu does not simultaneously bind microtubules and assemble actin filaments within the oocyte. Acting as a microtubule binding protein, Capu could directly regulate the microtubule cytoskeleton without invoking its actin assembly activity. It could crosslink microtubules to each other and/or to pre-existing actin filaments. Conversely, when assembling actin filaments and not binding microtubules, Capu could still indirectly influence microtubule organization through the actin cytoskeleton. Spatial and/or temporal regulation of Capu could control when Capu is associated with microtubules versus actin filaments and could be achieved through additional binding partners, post-translational modification, or some combination thereof. For instance, Capu binds the actin nucleator Spir through its tail domain. Spir competes directly with microtubules for Capu binding and forms a functional nucleation unit when bound to Capu. This Spir-Capu nucleation unit may be much more efficient at nucleating actin in the microtubule-rich oocyte than Capu would be alone. Additional unidentified binding partners may also play a role in regulating Capu association with microtubules. Several mammalian Diaphanous family formins bind and/or colocalize with microtubule-associated proteins such as EB1, APC, and CLIP-170. Future work will determine whether Capu has similar binding partners within the Drosophila oocyte (Roth-Johnson, 2013).

Post-translational modifications, especially phosphorylation, are commonly used to regulate microtubule binding by a variety of microtubule-associated proteins. With respect to formins, mDia3 association with microtubules has been shown to be mediated by phosphorylation by the AuroraB kinase. Notably, one mDia3 phosphorylation site is conserved across many formins and is within 10 amino acids of Capu's conserved K856 residue. Moreover, lysine-to-alanine mutations in this region of mDia1 have been shown to disrupt actin-microtubule coordination in Hela cells, suggesting that this region within the FH2 domain could be a hotspot for microtubule binding among formins. Additionally, it is anticipated that post-translational modification within Capu's tail will be important for regulating Capu function in the Drosophila oocyte. Though only approximately 30 amino acids long, the tail domain is involved in Spir binding, microtubule binding, actin nucleation, and Capu autoinhibition. Such a promiscuous domain will likely require careful regulation in vivo (Roth-Johnson, 2013).

Finally, it is possible that microtubules themselves are a means of regulating Capu activity in the oocyte. Throughout mid- and late-oogenesis, microtubules are nucleated from all regions of the oocyte cortex except the posterior pole, and it was recently shown that Capu-dependent actin projections emanate specifically from the posterior cortex of the oocyte. Could microtubule organization restrict the location of these Capu-dependent actin projections to the oocyte posterior? Similarly, microtubules could tune the processivity of Capu as it elongates actin filaments within the oocyte. The TIRF experiments show that microtubules do not effectively compete Capu away from the barbed end of actin filaments, causing barbed end dissociation in only 2% of all microtubule encounters. However, this relatively low probability of dissociation could have a much more substantial effect over the span of the entire oocyte where a single barbed end may encounter hundreds of microtubules. Together with Capu's own autoinhibitory activity, microtubules could also prevent Capu from nucleating new actin filaments after falling off the end of an elongating filament (Roth-Johnson, 2013).

These results provide mechanistic details of Capu-microtubule binding and the interplay of microtubule binding and actin assembly in vitro. Beyond providing valuable insight into Capu's role as a cytoskeletal regulator in the Drosophila oocyte, these findings may help advance understanding of Capu's mammalian homologs, Fmn-1 and Fmn-2. Capu FH2 residues 851-856 are perfectly conserved in Fmn-1 and Fmn-2, and both formins contain well conserved, short, basic C-terminal tail domains, suggesting a conserved mechanism for microtubule binding. These formins have been implicated in a number of processes in a wide variety of cell types, including intercellular adhesion and cell spreading, as well as spindle positioning and cytokinesis during mammalian oogenesis. How these findings relate to the broader class of formin proteins remains to be seen. Although the microtubule binding FH2 residues identified in this study are well conserved across several formin groups, the C-terminal tail domains are more variable. It has been recently shown that INF2, mDia1, and mDia2 have distinct microtubule interaction properties. Notably, mDia2 has the most basic tail of the three formins and behaves the most like Capu in vitro. Future work will reveal whether the current model for Capu-microtubule binding can be generalized to other formins such as mDia2 (Roth-Johnson, 2013).


Search PubMed for articles about Drosophila Cappuccino

Ciccarelli, F. D., Bork, P. and Kerkhoff, E. (2003). The KIND module: a putative signalling domain evolved from the C lobe of the protein kinase fold. Trends Biochem. Sci. 28: 349-352. PubMed ID: 12877999

Dahlgaard, K., Raposo, A. A., Niccoli, T. and St Johnston, D. (2007). Capu and Spire assemble a cytoplasmic actin mesh that maintains microtubule organization in the Drosophila oocyte. Dev. Cell 13(4): 539-53. PubMed ID: 17925229

Doerflinger H., Benton R., Torres I. L., Zwart M. F. and St Johnston D. (2006). Drosophila anterior-posterior polarity requires actin-dependent PAR-1 recruitment to the oocyte posterior. Curr. Biol. 16: 1090-1095. PubMed ID: 16753562

Emmons S., et al. (1995). cappuccino, a Drosophila maternal effect gene required for polarity of the egg and embryo, is related to the vertebrate limb deformity locus. Genes Dev. 9: 2482-2494. PubMed ID: 7590229

Evangelista, M., et al. (1997). Bni1p, a yeast formin linking cdc42p and the actin cytoskeleton during polarized morphogenesis. Science 276: 118-122. PubMed ID: 9082982

Goode, B. L. and Eck, M. J. (2007). Mechanism and function of formins in the control of actin assembly. Annu. Rev. Biochem. 76: 593-627. PubMed ID: 17373907

Higgs, H. N. and Peterson, K.J. (2005). Phylogenetic analysis of the formin homology 2 domain. Mol. Biol. Cell. 16: 1-13. PubMed ID: 15509653

Kanamori T., et al. (2008). β-Catenin asymmetry is regulated by PLA1 and retrograde traffic in C. elegans stem cell divisions. EMBO J. 27: 1647-1657. PubMed ID: 18497747

Kerkhoff, E., et al. (2001). The Spir actin organizers are involved in vesicle transport processes. Curr. Biol. 11: 1963-1968. PubMed ID: 11747823

Kovar, D. R. and Pollard, T. D. (2004). Insertional assembly of actin filament barbed ends in association with formins produces piconewton forces. Proc. Natl. Acad. Sci. 101: 14725-14730. PubMed ID: 15377785

Li, F. and Higgs, H. N. (2003). The mouse Formin mDia1 is a potent actin nucleation factor regulated by autoinhibition. Curr. Biol. 13: 1335-1340. PubMed ID: 12906795

Liu, R., Abreu-Blanco, M. T., Barry, K. C., Linardopoulou, E. V., Osborn, G. E. and Parkhurst, S. M. (2008). Wash functions downstream of Rho and links linear and branched actin nucleation factors. Development 136(16): 2849-60. PubMed ID: 19633175

Magie, C. R., Meyer, M. R., Gorsuch, M. S. and Parkhurst, S. M. (1999). Mutations in the Rho1 small GTPase disrupt morphogenesis and segmentation during early Drosophila development. Development 126: 5353-5364. PubMed ID: 10556060

Manseau, L. J. and Schupbach, T. (1989). cappuccino and spire: two unique maternal-effect loci required for both the anteroposterior and dorsoventral patterns of the Drosophila embryo. Genes Dev. 3: 1437-52. PubMed ID: 2514120

Martin, S. G., McDonald, W. H., Yates, J. R. and Chang F. (2005). Tea4p links microtubule plus ends with the formin For3p in the establishment of cell polarity. Dev. Cell. 8: 479-491. PubMed ID: 15809031

Otto, I. M., et al. (2000). The p150Spir protein provides a link between c-Jun N-terminal kinase function and actin reorganization. Curr. Biol. 10: 345-348. PubMed ID: 10744979

Palazzo, A. F., Cook, T. A., Alberts, A. S. and Gundersen, G. G. (2001). mDia mediates Rho-regulated formation and orientation of stable microtubules. Nat. Cell Biol. 3: 723-729. PubMed ID: 11483957

Pring, M., et al. (2003). Mechanism of formin-induced nucleation of actin filaments. Biochemistry 42: 486-496. PubMed ID: 12525176

Pruyne, D., et al. (2002). Role of formins in actin assembly: nucleation and barbed-end association. Science 297: 612-615. PubMed ID: 12052901

Qualmann, B. and Kessels, M. M. (2009). New players in actin polymerization - WH2-domain-containing actin nucleators. Trends Cell Biol. 19: 276-285. PubMed ID: 19406642

Quinlan, M. E., Heuser, J. E., Kerkhoff, E. and Mullins, R. D. (2005). Drosophila Spire is an actin nucleation factor. Nature 433(7024): 382-8. PubMed ID: 15674283

Quinlan, M. E., et al. (2007). Regulatory interactions between two actin nucleators, Spire and Cappuccino. J. Cell Biol. 179(1): 117-28. PubMed ID: 17923532

Rosales-Nieves, A. E., et al. (2006). Coordination of microtubule and microfilament dynamics by Drosophila Rho1, Spire and Cappuccino. Nat. Cell Biol. 8(4): 367-76. PubMed ID: 16518391

Roth-Johnson, E. A., Vizcarra, C. L., Bois, J. S. and Quinlan, M. E. (2013). Interaction between microtubules and the Drosophila formin Cappuccino and its effect on actin assembly. J Biol Chem 289(7):4395-404. PubMed ID: 24362037

Sagot, I., et al. (2002). An actin nucleation mechanism mediated by Bni1 and profilin. Nat. Cell Biol. 4: 626-631. PubMed ID: 12134165

Serbus, L. R., Cha, B. J. Theurkauf, W. E. and Saxton, W. M. (2005). Dynein and the actin cytoskeleton control kinesin-driven cytoplasmic streaming in Drosophila oocytes. Development 132: 3743-3752. PubMed ID: 16077093

St Johnston, D. (2005). Moving messages: the intracellular localization of mRNAs. Nat. Rev. Mol. Cell Biol. 6: 363-375. PubMed ID: 15852043

Tanaka, T. and Nakamura, A. (2008). The endocytic pathway acts downstream of Oskar in Drosophila germ plasm assembly. Development 135: 1107-1117. PubMed ID: 18272590

Tanaka, T., et al. (2011). Drosophila Mon2 couples Oskar-induced endocytosis with actin remodeling for cortical anchorage of the germ plasm. Development 138(12): 2523-32. PubMed ID: 21610029

Theurkauf, W. E. (1994). Premature microtubule-dependent cytoplasmic streaming in cappuccino and spire mutant oocytes. Science 265: 2093-6. PubMed ID: 8091233

Wang, Y. and Riechmann, V. (2008). Microtubule anchoring by cortical actin bundles prevents streaming of the oocyte cytoplasm. Mech. Dev. 125(1-2): 142-52. PubMed ID: 18053693

Wellington, A., et al. (1999). Spire contains actin binding domains and is related to ascidian posterior end mark-5. Development 126: 5267-5274. PubMed ID: 10556052

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date revised: 3 July 2014

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