Elucidating the mechanism of synergy in actin assembly by Spire and Cappuccino
Two actin nucleators, Spire and Cappuccino, collaborate to build a network of actin filaments that connect vesicles and the cortex in the fruit fly oocyte. This actin mesh is built by a conserved mechanism and by homologous proteins (Spire-1/2 and Fmn-2) in mouse oocytes. In each animal, proper regulation of mesh is necessary to establish cell polarity. A direct interaction between Spire and Cappuccino is required to regulate the actin mesh and for in vitro synergistic actin assembly; however, we understand little about why the interaction is necessary. To mimic the geometry of Spire and Cappuccino in vivo, Spire was immobilized on beads and observed by TIRF microscopy. These experiments revealed that increased nucleation is a major part of synergy with Cappuccino and that Spire alone binds both barbed- and pointed-ends of actin filaments. We identified Spire’s barbed-end binding domain (WH2-A) and observed partial rescue of fertility by a loss-of-function mutant, indicating that barbed-end binding is not necessary for Spire’s in vivo function, but that it may play a role under normal circumstances.
In addition, we found that Spire’s four WH2 domains variably participate in nucleation and other functions (e.g. sequestration) and that mutants of each WH2 domain can be modularly combined to tune Spire activities. We also identified new value in an existing, nucleation-incompetent Cappuccino mutant as a tool to reciprocally explore its own contributions to synergy with Spire. We found that this mutant does not nucleate actin but processively protects barbed ends from capping protein, and allows – but does not accelerate – the elongation of filaments in the presence of profilin. Furthermore, preliminary data suggest that this mutant synergizes with Spire – both in pyrene assembly assays and on beads – through a mechanism other than the dimerization of Spire. Finally, we demonstrate that Spire’s amino acids 490-520 – recently identified as containing a MyoV binding site – have an inhibitory effect on nucleation by Spire, resolving some discrepancies in reported actin assembly rates.
Taken together, these insights emphasize the complexity of Spire—Cappuccino synergy, mesh production, and regulation of polarity in the fruit fly oocyte. Our improved understanding of the components of synergy studied herein should permit the development of a more sophisticated, biomimetic mesh assembly system, and a more precise interrogation of actin assembly activities by Spire and Cappuccino.