Cell and Molecular Biology of Microtubule Plus End Tracking Proteins

End Binding Proteins and Their Partners

Susana Montenegro Gouveia; Anna Akhmanova    Department of Cell Biology, Erasmus Medical Center, Rotterdam, The Netherlands

1 Introduction

The cytoskeleton is a scaffold made of proteinaceous fibers that is required for the majority of essential cellular functions, such as maintenance of cellular shape, cell motility, intracellular transport, and cell division. It is a complex and often highly dynamic structure composed of several major components. One of them are microtubules (MTs), which are among the most ubiquitous cytoskeletal elements present in all eukaryotic cells. MTs are hollow asymmetric tubes that are built and broken down from their ends. In this review, we zoom in on a tiny part of the MT—its growing end. This structure, which is 25 nm in diameter and not more than 1 or 2 μm in length, is surprisingly complex: it concentrates a large set of structurally diverse proteins and serves as a site of convergence of numerous cellular processes. Here, we summarize what is known about a peculiar and highly conserved group of proteins that form comet-like accumulations at the growing MT tips, discuss the molecular mechanisms of MT plus end localization, and describe how MT end localization relates to the functions of these proteins.

2 Microtubules (MTs)

2.1 Tubulin

MTs are cylindrical protein filaments found in all eukaryotes. The structural subunit of MTs is tubulin, a heterodimeric protein composed of two polypeptide chains designated α and β tubulin (Krauhs et al., 1981; Ponstingl et al., 1981). The α and β monomers have similar masses of ~ 55 kDa and interact noncovalently to form a very stable heterodimer, the functional form of the protein (Wade, 2009). Individual tubulin heterodimers are 8 nm in length. Each tubulin monomer can be divided into three functional domains: the N-terminal domain containing the GTP-binding site, an intermediate domain containing the taxol-binding site in β tubulin, and the C-terminal domain, which probably forms the binding surface for many MT-associated proteins (MAPs) (Nogales and Wang, 2006b; Nogales et al., 1998).

Each tubulin monomer binds a guanine nucleotide. The GTP bound to α tubulin does not get hydrolyzed during polymerization and is nonexchangeable; the site to which it binds is therefore referred to as the N-site (Desai and Mitchison, 1997). In contrast, GTP bound to β tubulin at the E-site is exposed on the surface of the dimer and can exchange but gets locked in the protofilament as GDP after undergoing hydrolysis during polymerization. When a MT depolymerizes, the GDP-bound β tubulin exchanges GDP to GTP in solution.

In most eukaryotes, the tubulin gene family encodes multiple isoforms or isotypes (Wade, 2007). The expression of multiple genes leads to a variety of slightly different α and β tubulins. Each tubulin isotype differs from the others in its amino acid sequence and/or the temporal, tissue, and subcellular distribution (Luduena, 1998). In addition, both α and β tubulin undergo a variety of posttranslational covalent modifications, including acetylation, phosphorylation, detyrosynation, polyglutamylation, and polyglycylation, many of which target the C-terminal tail of tubulin (Hammond et al., 2008).

2.2 MT organization and structure

MTs within cells can be organized in different types of networks, such as a radial array in interphase fibroblasts, a bipolar spindle in mitosis and meiosis, a parallel array in polarized epithelial cells, and a linear array in neuronal extensions. In addition, MTs form highly stable and precisely organized structures, such as centrioles, basal bodies, and axonemes of cilia and flagella.

MTs are cylinders of 25 nm in diameter but can have variable length. Their basic building blocks, α and β tubulin heterodimers, are arranged in a head-to-tail fashion into linear protofilaments, which associate laterally to form MTs. MTs commonly found in vivo have 13 protofilaments (Ledbetter and Porter, 1964; Tilney et al., 1973), although exceptions have been described, such as 15-protofilament MTs in certain neuronal cells of Caenorhabditis elegans (Bounoutas et al., 2009). In vitro, the protofilament number in MTs spontaneously assembled from mammalian brain tubulin can vary from 9 to 17, even within the same MT (Chretien and Wade, 1991; Chretien et al., 1992). MTs in centrioles and axonemes represent a special case, as they can form doublets or triplets, in which some MTs are incomplete (Unger et al., 1990).

Two distinct MT lattice structures are possible: an A-type and a B-type lattice. In an A-type lattice, α monomers from one protofilament associate with β monomers from the adjacent one, while in a B-type lattice, the α–α and β–β contacts are formed between the adjacent protofilaments (Desai and Mitchison, 1997). Ultrastructural analysis showed that the B-type lattice is the naturally occurring protofilament arrangement (Mandelkow et al., 1986; Song and Mandelkow, 1993). This implies that MTs have a lattice discontinuity, called the seam, where α and β monomers from adjacent protofilaments undergo lateral interactions. In the B-type lattice, the helical pattern is created by α–α and β–β lateral bonds, with α–β bonds present at the seam (Desai and Mitchison, 1997). It was proposed that MTs grow as a sheet and later zipper into a closed tube at the seam (Chretien et al., 1995). Because at the seam the lateral interactions are weaker, it might also play a role in MT disassembly (Wade, 2007). Interestingly, A lattice formation was recently observed in vitro in the presence of a yeast MT end binding protein Mal3 (see below; des Georges et al., 2008). However, careful reexamination of MT lattice structure confirmed earlier findings that 13-protofilament B-lattice MTs with a seam is the predominant form in cultured mammalian cells (McIntosh et al., 2009).

MTs display intrinsic polarity, generated by the head-to-tail assembly of α/β tubulin dimers, with one end growing more rapidly than the other in vitro. The dynamic and faster growing end of a MT is termed the plus end, and the slower growing and less dynamic one the minus end. In vitro experiments showed that the opposite ends of free MTs have different sensitivities to MT depolymerizing agents such as low temperature, Ca2+, or colchicine (Summers and Kirschner, 1979).

MT minus ends are usually anchored at the centrosome or other minus end stabilizing sites (Dammermann et al., 2003), while the plus ends explore the cytoplasmic space and often interact with the cell cortex and other cellular structures. The polarity of MTs is central to the mechanism of action of motor proteins, kinesins, and dyneins, which use ATP hydrolysis to transport various cargos along MTs. Motor proteins are unidirectional: they can move either toward MT plus ends (kinesins), or toward the minus ends (a few kinesins and cytoplasmic dynein) (Mallik and Gross, 2004). Together with asymmetric MT organization, MT-based motors thus contribute to cell polarity and morphogenesis.

2.3 MT dynamics

MTs can assemble spontaneously in solutions of purified tubulin. Observation of a population of fixed MTs led to the discovery of MT dynamic instability, a process whereby MTs interconvert between phases of polymerization and depolymerization (Mitchison and Kirschner, 1984). Transitions between MT growth and shortening are abrupt and stochastic and are defined as catastrophe for the switch from growth to shortening and rescue for the switch from shortening to growth (Desai and Mitchison, 1997). Subsequent extensive studies using dark field and differential interference contrast (DIC) microscopy have established this phenomenon both in vitro and in vivo (Cassimeris et al., 1988; Horio and Hotani, 1986; Hotani and Horio, 1988; Walker et al., 1988). One additional observation was that MTs sometimes neither polymerize nor depolymerize, they just pause; this behavior is much more frequent in vivo but also occurs...