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Clasps at a Glance Elizabeth J © 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs243097. doi:10.1242/jcs.243097 CELL SCIENCE AT A GLANCE SUBJECT COLLECTION: MICROTUBULE DYNAMICS CLASPs at a glance Elizabeth J. Lawrence1, Marija Zanic1,2,* and Luke M. Rice3,* ABSTRACT accompanying poster, we will summarize some of these recent CLIP-associating proteins (CLASPs) form an evolutionarily advances, emphasizing how they impact our current understanding of conserved family of regulatory factors that control microtubule CLASP-mediated microtubule regulation. dynamics and the organization of microtubule networks. The KEY WORDS: CLASP, Microtubule dynamics, Mitosis, Motility importance of CLASP activity has been appreciated for some time, but until recently our understanding of the underlying molecular Introduction mechanisms remained basic. Over the past few years, studies of, for The name CLASP, for CLIP-associating protein, was coined in a example, migrating cells, neuronal development, and microtubule foundational study (Akhmanova et al., 2001) that identified proteins reorganization in plants, along with in vitro reconstitutions, have that interacted with cytoplasmic linker protein (CLIP)-170 and provided new insights into the cellular roles and molecular basis of CLIP-115 (also known as CLIP1 and CLIP2, respectively), two CLASP activity. In this Cell Science at a Glance article and the related proteins that associate with growing microtubule ends to regulate their dynamics (see Box 1 for an overview of microtubule dynamics). By virtue of their characteristic domain organization and 1Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, USA. 2Department of Chemical and Biomolecular Engineering, and sequence, CLASP proteins were recognized to be orthologous to Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, USA. gene products that had been previously identified by genetic 3Department of Biophysics and Department of Biochemistry, UT Southwestern screening in Drosophila melanogaster (MAST/Orbit) (Inoue et al., Medical Center, Dallas, TX 75390, USA. 2000; Lemos et al., 2000) and Saccharomyces cerevisiae (Stu1) *Authors for correspondence ([email protected]; Luke.Rice@ (Pasqualone and Huffaker, 1994). CLASP orthologs were also UTSouthwestern.edu) identified in Caernorhabditis elegans (Hannak and Heald, 2006) E.J.L., 0000-0001-9543-8678; M.Z., 0000-0002-5127-5819; L.M.R., 0000-0001- and in Arabidopsis thaliana (Ambrose et al., 2007). The first studies 6551-3307 on human CLASP paralogs (see Box 2) reported that CLASP1 is Journal of Cell Science 1 CELL SCIENCE AT A GLANCE Journal of Cell Science (2020) 133, jcs243097. doi:10.1242/jcs.243097 microtubule density (Efimov et al., 2007; Miller et al., 2009; Mimori- Box 1. Microtubule dynamic instability Kiyosue et al., 2005). Overall, a common theme that emerged from Microtubule dynamic instability is the hallmark behavior of microtubule these early studies was that CLASP activity is important for proper polymers (Mitchison and Kirschner, 1984), in which individual formation of specific networks or subpopulations of microtubules. microtubule ends stochastically switch between periods of growth and CLASPs can bind directly to microtubules and to other ‘ ’ shrinkage. A growing microtubule end maintains a cap of GTP-bound microtubule-associated proteins including EB1 (also known as tubulin that protects against depolymerization. ‘Catastrophe’, the sudden transition from growth to shrinkage, occurs when this cap is lost. MAPRE1), as well as to other binding partners that are not A shrinking microtubule can revert to growth, a transition known as associated with microtubules (Akhmanova et al., 2001; Bratman ‘rescue’. CLASPs and several other microtubule-associated proteins can and Chang, 2007; Cheeseman et al., 2005; Efimov et al., 2007; selectively localize to the dynamic microtubule ends, where they regulate Lansbergen et al., 2006; Maffini et al., 2009; Manning et al., 2010; polymerization dynamics by modulating the structure and/or biochemical Mimori-Kiyosue et al., 2005; Wittmann and Waterman-Storer, 2005). properties of the microtubule end (Akhmanova and Steinmetz, 2015; CLASP proteins were also recognized to contain a tubulin-binding Brouhard and Rice, 2018). TOG domain homologous to those found in otherwise unrelated microtubule polymerases of the XMAP215/Stu2/chTOG/CKAP5 family (Akhmanova et al., 2001; reviewed in Al-Bassam and Chang, ubiquitously expressed whereas CLASP2 is enriched in brain tissue 2011; Slep, 2009), providing a potential clue about their mechanism. (Akhmanova et al., 2001). However, deeper insights into the molecular mechanism were limited Early functional observations on CLASPs indicated that CLASP by a lack of in vitro reconstitutions. Although the first in vitro activity stabilized microtubules (Akhmanova et al., 2001; Bratman reconstitution of CLASP activity was reported in 2007 using the and Chang, 2007; Drabek et al., 2006; Mimori-Kiyosue et al., 2005; Schizosaccharomyces pombe CLASP (Al-Bassam et al., 2010), a Sousa et al., 2007), and revealed roles in cell polarization second reconstitution only followed in 2016 (Moriwaki and Goshima, (Akhmanova et al., 2001; Efimov et al., 2007; Miller et al., 2009; 2016) using CLASP from Drosophila. More recent reconstitutions are Mimori-Kiyosue et al., 2005; Wittmann and Waterman-Storer, discussed below. 2005) and cell division (Maiato et al., 2002, 2003, 2005; Mimori- In this Cell Science at a Glance article and the accompanying Kiyosue et al., 2006; Ortiz et al., 2009; Pereira et al., 2006). Indeed, poster, we will summarize recent developments that provide insight in multicellular organisms, CLASPs were observed to associate into the cellular function or molecular mechanism of CLASP activity, with growing microtubules at the leading edge of motile cells drawing on studies of migrating cells, neuronal development, (Akhmanova et al., 2001; Mimori-Kiyosue et al., 2005; Wittmann microtubule reorganization in plants, in vitro reconstitution, and more. and Waterman-Storer, 2005), and to also localize at kinetochores and the mitotic spindle during mitosis (Akhmanova et al., 2001; Structural and mechanistic insights Hannak and Heald, 2006; Inoue et al., 2000; Lemos et al., 2000; The first structures of CLASP family TOG domains and new Maiato et al., 2002, 2003; Mimori-Kiyosue et al., 2006). In yeasts, reconstitutions of human, insect and fungal CLASP activity have CLASPs were similarly found to bind microtubules and to have reshaped our thinking about the mechanisms by which CLASPs roles in spindle assembly (Bratman and Chang, 2007; Funk et al., control microtubule dynamics. 2014; Ortiz et al., 2009; Pasqualone and Huffaker, 1994; Yin et al., TOG domains are helical repeat modules that bind the α-tubulin–β- 2002). In addition to demonstrating that CLASPs could regulate tubulin heterodimers (αβ-tubulin) (Al-Bassam et al., 2006, 2007; Slep microtubule dynamics, CLASPs were also shown to localize to the and Vale, 2007), and that can interact selectively with specific major microtubule-nucleating centers, including centrosomes and conformations of αβ-tubulin (Ayaz et al., 2012, 2014) (see poster). the Golgi, where they promote microtubule nucleation and increase CLASP proteins can bind to unpolymerized αβ-tubulin, and early analyses of CLASP sequences identified one TOG domain that is homologous to those found in XMAP215 family polymerases Box 2. Human CLASP proteins (Akhmanova et al., 2001). Subsequent sequence analysis uncovered There are two paralogs of mammalian CLASPs, CLASP1 (also known as additional, divergent TOG domains that retained tubulin-binding CLASP1α) and CLASP2, with three isoforms of CLASP2 (α, β and γ) elements that have been identified in studies of polymerase TOGs (Slep (Akhmanova et al., 2001). CLASP1 is broadly expressed, whereas and Vale, 2007). Structures of CLASP-specific TOG2 domains CLASP2 expression is primarily limited to the brain. CLASP1 and CLASP2α represent the longest isoforms, each containing three TOG revealed differences compared to that seen for polymerase TOGs. First, domains, but the N-terminal TOG domain has apparently lost the ability the putative tubulin-binding residues are arranged differently to those in to interact with αβ-tubulin or microtubules (Aher et al., 2018; De la Mora- polymerase TOGs, possibly indicating that CLASP TOG2 recognizes Rey et al., 2013) and may instead mediate autoregulatory interactions a different conformation of αβ-tubulin from that recognized by with other regions of the protein (Aher et al., 2018). CLASP2β polymerase TOGs (Leano et al., 2013; Majumdar et al., 2018; Maki and CLASP2γ each lack the N-terminal TOG domain present in α β et al., 2015). Second, a portion of the primary sequence preceding CLASP1 and CLASP2 . CLASP2 contains a palmitoylation motif α near the N-terminus that presumably directs membrane localization, but TOG2 folds into an -helix that docks onto one face of the TOG, the functional impact of membrane targeting is not known because this suggesting that the linker between CLASP TOGs is less flexible than isoform is relatively understudied. Notably, the Clasp2-knockout mouse observed in polymerases (Leano et al., 2013; Majumdar et al., 2018; has a very mild phenotype (Drabek et al., 2012), whereas the double Maki et al., 2015). Third, a CLASP-specific conserved arginine residue knockout (to our knowledge) is embryonic lethal. Therefore, although likely confers a
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