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Microtubule binding by the formin Cappuccino and its implications for Drosophila oogenesis

  • Author(s): Roth-Johnson, Elizabeth Anne
  • Advisor(s): Quinlan, Margot E
  • et al.
Abstract

Coordination of actin and microtubule cytoskeletal networks is required for a number of fundamental cellular processes. Formin family actin nucleators are emerging 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, we studied microtubule binding by Cappuccino (Capu), a formin involved in regulating actin and microtubule organization during Drosophila oogenesis. We report that two distinct domains within Capu, FH2 and tail, work together to promote high-affinity microtubule binding. Microtubules potently inhibit Capu's actin nucleation activity but have little effect on Capu once bound to the barbed end of an elongating actin filament, supporting a model in which Capu does not simultaneously bind microtubules and assemble actin in vivo. After characterizing Capu-microtubule binding in vitro, we examined the localization of Capu and microtubules in S2 cells. We did not observe any obvious colocalization and conclude that S2 cells are not an ideal system for visualizing in vivo formin-microtubule interactions. To better understand Capu's mechanisms of action during oogenesis, we then systematically characterized several classical capu alleles. While all of these capu mutants exhibit severe developmental defects, they variably affect Capu's actin assembly activity in vitro. Together with our microtubule binding data, this indicates that Capu's physiological role extends beyond its ability to nucleate actin filaments. We therefore performed tandem affinity purification experiments to identify novel Capu binding partners and gain additional insight into Capu's role during oogenesis. We report several candidate binding partners involved in such diverse cellular processes as mitochondrial fission, endocytosis, and nuclear import. Though preliminary, these results suggest that Capu has additional uncharacterized functions in development; further investigation may help us uncover the physiological role of Capu-microtubule binding. Ultimately, our findings expand our understanding of Capu's role in Drosophila oogenesis and provide mechanistic insight into formins as coordinators of the actin and microtubule cytoskeletons.

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