How to control microtubule dynamics: Function and regulation of kinesin -13s in Drosophila S2 cells

Abstract

Regulation of microtubule polymerization and depolymerization is required for proper cell development. Here we report that two proteins of the Drosophila melanogaster Kinesin-13 family, Klp10a and Klp59c, cooperate to drive microtubule depolymerization in interphase cells. Analyses of microtubule dynamics in S2 cells depleted of these proteins indicate that both proteins stimulate depolymerization, but alter distinct parameters of dynamic instability; Klp10a stimulates catastrophe (a switch from growth to shrinkage) whereas Klp59c suppresses rescue (a switch from shrinkage to growth). Moreover, immunofluorescence and live cell analysis of cells expressing tagged kinesins reveal that Klp10a and Klp59c target to polymerizing and depolymerizing microtubule plus ends, respectively. Our data also suggest that Klp10a is deposited on microtubules by the plus-end tracking protein, EB1. Our findings support a model in which these two members of the Kinesin-13 family divide the labor of microtubule depolymerization.;To investigate regulation of Klp10a in vivo, peptide mapping by ESI-MALDI-TOF mass spectrometry was performed. These experiments identified a novel in vivo site of serine phosphorylation (s573) in the motor domain of Klp10a. To assess the role of serine 573 phosphorylation on the cellular activity of Klp10a, double transgenic Drosophila S2 cell lines expressing mRFP tubulin and GFP-Klp10a bearing phospho-mimic (S573E) or dephospho-mimic (S573A) were developed. These studies show that phosphorylated Klp10a is locked in a low depolymerizing activity state compared to the wild type. In vitro biochemical analysis of the mutants suggest that Klp10a phosphorylation directly affects the motor's ability to depolymerize microtubules, while not affecting its ability to efficiently bind microtubules.;To gain structural insights into how phosphorylation affects depolymerization activity, we also performed electron microscopy studies with purified recombinant proteins. In our assays we show that the phospo-mimic form of Klp10a is unable to form ring and spiral structures on the microtubules, while the wild type form can.*.;*This dissertation is a compound document (contains both a paper copy and a CD as part of the dissertation). The CD requires the following system requirements: Windows MediaPlayer or RealPlayer.