Building the Microtubule Cytoskeleton Piece by Piece

Abstract

The microtubule (MT) cytoskeleton gives cells their shape, organizes the cellular interior, and segregates chromosomes. These functions rely on the precise arrangement of MTs, which is achieved by the coordinated action of MT-associated proteins (MAPs). We highlight the first and most important examples of how different MAP activities are combined in vitro to create an ensemble function that exceeds the simple addition of their individual activities, and how the Xenopus laevis egg extract system has been utilized as a powerful intermediate between cellular and purified systems to uncover the design principles of selforganized MT networks in the cell. The microtubule (MT)2 cytoskeleton forms the skeletal framework that gives eukaryotic cells their shape and organizes their cytoplasm by positioning organelles, providing tracks for transport, and establishing cell polarity. In an interphase cell, the MT cytoskeleton is also critical for cell motility and a key constituent of cilia and flagella. During cell division, the MT cytoskeleton gets remodeled into a spindle structure that segregates chromosomes. Each of these functions relies on a specific MT architecture, which must be capable of rapid and prolonged change followed by an eventual resumption of a steady state to respond to the cellular environment and morphology changes during growth and differentiation.MTs are made of ␣/␤-tubulin heterodimers, which assemble into a polar, cylindrical structure in the presence of GTP and above the so-called critical concentration in vitro. MT growth phases alternate with swift shrinkage phases (dynamic instability), and their transitions are referred to as catastrophe (switching from growth to shrinkage) and rescue (switching from shrinkage to growth) (1). In cells, a plethora of different MT-associated proteins (MAPs) regulate the MT-inherent abilities of MT nucleation and dynamics ( Fig. 1A) (2). In addition, MT cross-linking proteins connect MTs into networks and molecular motors use MTs as tracks for cargo transport or transport MTs themselves (Fig. 1A). Altogether, different combinations of these four basic groups of MAP activities drive the self-organization of the MT cytoskeleton into discrete three-dimensional patterns (Fig. 1B) (3). Thus, they establish, maintain, and disassemble functional MT structures that are observed on the cellular level.Traditionally, individual MAPs were identified by loss-offunction experiments in cells followed by their detailed in vivo and in vitro characterization. During the past decade, highthroughput genomic and proteomic screens accelerated MAP discovery by cataloging RNAi phenotypes and identifying novel microtubule binders, resulting in comprehensive lists of candidates involved in organizing the MT cytoskeleton in various cell states (4 -7). Now, the challenge is to understand how these MAPs work together to establish the physiological MT architecture of the cell. What specific MAP building blocks can generate the MT networks that shape a dendrite or a polarized epithelial cell (...

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