The actin nucleating Arp2/3 complex contributes to the formation of axonal filopodia and branches through the regulation of actin patch precursors to filopodia.

The emergence of axonal filopodia is the first step in the formation of axon collateral branches. In vitro, axonal filopodia emerge from precursor cytoskeletal structures termed actin patches. However, nothing is known about the cytoskeletal dynamics of the axon leading to the formation of filopodia in the relevant tissue environment. In this study we investigated the role of the actin nucleating Arp2/3 complex in the formation of sensory axon actin patches, filopodia, and branches. By combining in ovo chicken embryo electroporation mediated gene delivery with a novel acute ex vivo spinal cord preparation, we demonstrate that actin patches form along sensory axons and give rise to filopodia in situ. Inhibition of Arp2/3 complex function in vitro and in vivo decreases the number of axonal filopodia. In vitro, Arp2/3 complex subunits and upstream regulators localize to actin patches. Analysis of the organization of actin filaments in actin patches using platinum replica electron microscopy reveals that patches consist of networks of actin filaments, and filaments in axonal filopodia exhibit an organization consistent with the Arp2/3-based convergent elongation mechanism. Nerve growth factor (NGF) promotes formation of axonal filopodia and branches through phosphoinositide 3-kinase (PI3K). Inhibition of the Arp2/3 complex impairs NGF/PI3K-induced formation of axonal actin patches, filopodia, and the formation of collateral branches. Collectively, these data reveal that the Arp2/3 complex contributes to the formation of axon collateral branches through its involvement in the formation of actin patches leading to the emergence of axonal filopodia.

The small GTPase Cdc42 promotes membrane protrusion during polar body emission via ARP2-nucleated actin polymerization.

Polar body emission is a specialized cell division throughout the animal kingdom, serving to reduce chromosome ploidy while preserving the egg cytoplasm. Critical to polar body emission are the asymmetric positioning of the meiotic spindle prior to anaphase, with one pole attached to the oocyte cortex, and the simultaneous membrane protrusion during subsequent cytokinesis. We have shown that, during Xenopus oocyte maturation, the small GTPase Cdc42 promotes membrane protrusion while a classical RhoA contractile ring forms and constricts at the base of the protrusion. We report here that treating oocytes with low concentrations of nocodazole diminished the size of metaphase I spindles and prevented polar body emission, and yet an active Cdc42 cap of correspondingly diminished size still developed, on time, atop of the spindle pole. Conversely, treating oocytes with low concentrations of taxol resulted in a spindle with multiple poles attached to the cortex, but still each of these poles were associated with activated cortical Cdc42 at the appropriate time. Therefore, the asymmetric positioning of the meiotic spindle with one pole anchored to the cortex is a prerequisite for Cdc42 activation. Furthermore, we demonstrated that the Cdc42-regulated F-actin nucleator ARP2/3 complex was similarly localized at the cortex of the protruding polar body membrane, suggesting that Cdc42 promotes membrane protrusion through an F-actin meshwork mechanism. Finally, we demonstrated that Cdc42 and RhoA formed similarly complementary activity zones during egg activation and that inhibition of Cdc42 prevented second polar body emission. Therefore, Cdc42 activation likely promotes membrane protrusion during polar body emission in widespread systems.

Characterization of two classes of small molecule inhibitors of Arp2/3 complex.

Polymerization of actin filaments directed by the actin-related protein (Arp)2/3 complex supports many types of cellular movements. However, questions remain regarding the relative contributions of Arp2/3 complex versus other mechanisms of actin filament nucleation to processes such as path finding by neuronal growth cones; this is because of the lack of simple methods to inhibit Arp2/3 complex reversibly in living cells. Here we describe two classes of small molecules that bind to different sites on the Arp2/3 complex and inhibit its ability to nucleate actin filaments. CK-0944636 binds between Arp2 and Arp3, where it appears to block movement of Arp2 and Arp3 into their active conformation. CK-0993548 inserts into the hydrophobic core of Arp3 and alters its conformation. Both classes of compounds inhibit formation of actin filament comet tails by Listeria and podosomes by monocytes. Two inhibitors with different mechanisms of action provide a powerful approach for studying the Arp2/3 complex in living cells.