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

Abstract

The actin nucleation factors Spire and Cappuccino regulate the onset of ooplasmic streaming in Drosophila 1-5 . 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. Here we demonstrate that Cappuccino and Spire have microtubule and microfilament crosslinking activity. The spire locus encodes several distinct protein isoforms (SpireA, SpireC, and SpireD). SpireD was recently shown to nucleate actin, but the activity of the other isoforms has not been addressed. We find that SpireD does not have crosslinking activity, while SpireC is a potent crosslinker. We show that SpireD binds to Cappuccino and inhibits F-actin/microtubule crosslinking, and activated Rho1 abolishes this inhibition, establishing a mechanistic basis for the regulation of Capu and Spire activity. We propose that Rho1, cappuccino and spire are elements of a conserved developmental cassette that is capable of directly mediating crosstalk between microtubules and microfilaments.Cytoskeletal elements must be coordinately regulated for cells to carry out complex functions, such as the cytoplasmic movements required to disperse or localize intracellular components 5 . The formin homology (FH) protein Cappuccino (Capu) and the WASP homology 2 (WH2) domain-containing protein Spire are both required for the proper timing of one such cytoplasmic movement, ooplasmic streaming in Drosophila 1,3 . In wildtype oocytes, vigorous ooplasmic streaming is associated with rapid growth during stages 10b-13, and is never observed prior to this stage. Mutations in capu and spire result in premature ooplasmic streaming, beginning at stage 7/8 and continuing through stage 13. This premature streaming interferes with transport mechanisms required for the localization of early polarity markers, resulting in disruption of dorsal-ventral and anterior-posterior body axes 1,2 . Both the wildtype streaming event and the premature streaming in capu and spire mutants are microtubule-based 1 . Streaming never takes place in oocytes lacking kinesin, and colcemid injection blocks premature streaming in these mutants 5,6 . Recent work suggests that streaming is restrained by the competing effects of dynein and kinesin, and can be initiated by blocking dynein function 7 . Thus, it is somewhat paradoxical that Spire and Capu nucleate actin, but are not known to affect microtubule architecture or dynamics. Interestingly, the premature streaming seen in capu and spire mutant oocytes can be recapitulated by injection of the actindepolymerizing drug cytochalasin D into wildtype oocytes, suggesting that actin assembly may restrict microtubule rearrangements required for ooplasmic streaming 8 . Presumably, microtubule and microfilament dynamics are coordinated in oogenesis by a group of proteins that includes Spire and Capu, as well as one or more upstream signals. However, the signaling events that combine to encode a "switch" from the n...

Role of the C-terminal Extension of Formin 2 in Its Activation by Spire Protein and Processive Assembly of Actin Filaments

“…Recapitulates the Effect of FSI Removal from Fmn2-The FSI targets the KIND domain of Spire (16,17,28,29) by making mainly electrostatic contacts between a basic stretch of FSI and an exposed acidic patch of KIND. We reported that KIND inhibits Fmn2-stimulated spontaneous actin filament assembly from PA (19), but this mechanism was not addressed.…”

Actin cytoskeleton in cell polarity and asymmetric division during mouse oocyte maturation

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A Myc–Groucho complex integrates EGF and Notch signaling to regulate neural development

“…In vitro binding experiments were preformed by using GST-pulldown assays similar to those described (4,34,35).…”