Capzb2 Interacts with β-Tubulin to Regulate Growth Cone Morphology and Neurite Outgrowth

Abstract

An actin regulatory protein unexpectedly also controls microtubule polymerization during the formation and maintenance of cellular outgrowths in neurons. Capping protein (CP) is a heterodimer that regulates actin assembly by binding to the barbed end of F-actin. In cultured nonneuronal cells, each CP subunit plays a critical role in the organization and dynamics of lamellipodia and filopodia. Mutations in either α or β CP subunit result in retinal degeneration in Drosophila. However, the function of CP subunits in mammalian neurons remains unclear. Here, we investigate the role of the β CP subunit expressed in the brain, Capzb2, in growth cone morphology and neurite outgrowth. We found that silencing Capzb2 in hippocampal neurons resulted in short neurites and misshapen growth cones in which microtubules overgrew into the periphery and completely overlapped with F-actin. In searching for the mechanisms underlying these cytoskeletal abnormalities, we identified β-tubulin as a novel binding partner of Capzb2 and demonstrated that Capzb2 decreases the rate and the extent of tubulin polymerization in vitro. We mapped the region of Capzb2 that was required for the subunit to interact with β-tubulin and inhibit microtubule polymerization. A mutant Capzb2 lacking this region was able to bind F-actin and form a CP heterodimer with α2-subunit. However, this mutant was unable to rescue the growth cone and neurite outgrowth phenotypes caused by Capzb2 knockdown. Together, these data suggest that Capzb2 plays an important role in growth cone formation and neurite outgrowth and that the underlying mechanism may involve direct interaction between Capzb2 and microtubules. Author SummaryNeuronal growth, migration, and survival depend on the regulated formation of cellular outgrowths called neurites. Extension of normal neurites requires coordinated interactions between cytoskeletal networks made up of microfilaments (composed of F-actin) and microtubules (formed by tubulin) in structures called growth cones that form at the tips of growing neurites. Capping protein (CP) is a heterodimer that regulates F-actin assembly in a variety of cell types. Surprisingly, the neuronal CP β subunit, Capzb2, not only regulates F-actin assembly, but also inhibits microtubule polymerization by direct interaction with tubulin. We further show that this function of Capzb2 is required for establishment of the normal shape of growth cones and the appropriate length of neurites. Our data thus reveal an unexpected, dual role for CP in the regulation of both microfilaments and microtubules in neurons.