Abstract
Selectively stabilized microtubules (MTs) form in the lamella of fibroblasts and contribute to cell migration. A Rho-mDia-EB1 pathway regulates the formation of stable MTs, yet how selective stabilization of MTs is achieved is unknown. Kinesin activity has been implicated in selective MT stabilization and a number of kinesins regulate MT dynamics both in vitro and in cells. Here, we show that the mammalian homolog of Xenopus XKLP1, Kif4, is both necessary and sufficient for the induction of selective MT stabilization in fibroblasts. Kif4 localized to the ends of stable MTs and participated in the Rho-mDia-EB1 MT stabilization pathway since Kif4 depletion blocked mDia- and EB1-induced selective MT stabilization and EB1 was necessary for Kif4 induction of stable MTs. Kif4 and EB1 interacted in cell extracts, and binding studies revealed that the tail domain of Kif4 interacted directly with the N-terminal domain of EB1. Consistent with its role in regulating formation of stable MTs in interphase cells, Kif4 knockdown inhibited migration of cells into wounded monolayers. These data identify Kif4 as a novel factor in the Rho-mDia-EB1 MT stabilization pathway and cell migration.
Highlights
Rearrangements of microtubules (MTs) play a central role in the establishment of cell polarity in many systems [1]
Throughout this paper we refer to stable MTs with high levels of detyrosinated tubulin as Glu MTs and their dynamic counterparts as Tyr MTs
Green fluorescent protein (GFP)-tagged constructs encoding the motor domain of these kinesins were microinjected into nuclei of starved NIH3T3 fibroblasts bordering an in vitro wound and after 2 hr of expression, levels of Glu MTs were assessed in fixed cells by immunofluorescence
Summary
Rearrangements of microtubules (MTs) play a central role in the establishment of cell polarity in many systems [1]. MTs contribute to the front-back polarity that is essential for directional migration of cells in a variety of environments. MTs regulate myosin contraction in the cell rear in certain migrating cells such as neutrophils and T cells [9,10]. Radial MT arrays are biased toward the front of many migrating cells by the specific orientation of the centrosome toward the leading edge [11]. The oriented centrosome positions the associated Golgi and endocytic recycling compartment to direct vesicular traffic toward the leading edge. The reorientation of the Golgi may reinforce MT asymmetry toward the leading edge as the Golgi itself can nucleate MTs in certain cell types [3]. Factors that interfere with centrosome orientation usually reduce the rate of cell migration [12,13,14], direct laser ablation of the centrosome has modest-to-strong effects on cell migration depending on the cell type [15,16]
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