Clasps at a Glance Elizabeth J

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

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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 distinct character to the TOG–tubulin interface CLASP perturbations have revealed paralog-specific functions in cell (Majumdar et al., 2018). Although their contributions to CLASP motility (Bouchet et al., 2016), cell division (Samora et al., 2011) and neurons (Sayas et al., 2019), the CLASP paralogs may be functionally activity and/or function is not as well explored as that of the TOG2, redundant in other settings. More work is needed to define the specific these other CLASP TOGs also show distinctive features compared to roles of different CLASP paralogs or isoforms and the extent to which those in polymerase TOGs, likely indicating specialized αβ-tubulin- they can or cannot compensate for each other. binding properties or other functional specialization (Leano and Slep,

2019; Maki et al., 2015) (see also Box 2). Journal of Cell Science

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Recent reconstitutions of human CLASPs have revealed that microtubule attachments (Maia et al., 2012). However, until now, CLASPs can autonomously recognize growing microtubule ends, the role of CLASPs in forming the initial attachment of kinetochores suppress catastrophe and promote rescue, all without significantly to microtubules has been less well investigated. affecting the rates of microtubule growing or shrinking (Aher et al., Two new studies now add to our understanding of the role of 2018; Lawrence et al., 2018; Lawrence and Zanic, 2019) (see CLASPs at kinetochores. The initial attachment of chromosomes to poster). The anti-catastrophe and rescue activities of human kinetochore microtubules occurs via a lateral interaction between CLASPs are increased in the presence of EB1 family plus-end- the wall of the microtubule and proteins of the kinetochore (Rieder tracking proteins because direct CLASP–EB interactions increase and Alexander, 1990). The lateral interaction is subsequently the amount of CLASP at the growing microtubule end (Aher et al., converted to an end-on attachment via a poorly understood 2018; Lawrence et al., 2018). Reconstitutions of insect CLASP have mechanism. A recent in vitro study reconstituted kinetochore– revealed that they have potent anti-catastrophe activity and suppress microtubule attachment using surface-immobilized beads coated microtubule growth rate (Moriwaki and Goshima, 2016). Whereas with purified kinetochore proteins and stabilized microtubules the polymerase activity of XMAP215 family polymerases strictly (Chakraborty et al., 2019). When beads were coated with CENP-E requires two linked TOG domains (Ayaz et al., 2014; Geyer et al., and CLASP2, microtubules formed stable end-on attachments with 2018; Widlund et al., 2011), isolated human or fungal CLASP TOG the protein-coated beads, with durations lasting up to 14 min. Thus, domains by themselves are sufficient to recapitulate anti-catastrophe CLASP2 promotes robust, long-lived interactions with microtubule and rescue activity (Aher et al., 2018; Majumdar et al., 2018). ends, a process critical for proper chromosome attachment. In Taken together, the new structure and reconstitutions reveal dividing S. cerevisiae, the CLASP homolog Stu1 has been that the operational principles underlying CLASP activity differ identified as a key component of an elegant mechanism by which fundamentally from those that form the basis for polymerase unattached kinetochores promote their own capture (Kolenda et al., activity (Ayaz et al., 2014; Geyer et al., 2018), and they diversify 2018). Here, Stu1 is sequestered at unattached kinetochores in a the understanding of what TOG domains can do. Indeed, manner dependent on the protein kinase Mps1 and the kinetochore the demonstration that a single CLASP TOG can recapitulate the protein Slk19. Sequestration of Stu1 away from the spindle leads to regulatory activity of the intact protein indicates that ‘tethering’ of an turnover of the spindle microtubules, promoting the formation of unpolymerized αβ-tubulin subunit to the lattice is not strictly required new dynamic microtubules that search the nuclear space for for CLASP activity, unlike what is seen for polymerases where unattached kinetochores. Once an attachment is formed, Stu1 tethering is an obligate part of the mechanism (Ayaz et al., 2014; Geyer stabilizes the capturing microtubule and, thus, acts to maintain the et al., 2018). Instead, CLASP TOGs must control the frequencies of microtubule–kinetochore connection (Kolenda et al., 2018). In microtubule catastrophe and rescue by acting directly to enhance summary, CLASPs are involved in modulating microtubule interactions between αβ-tubulin subunits at or very near the bundling, dynamics and kinetochore attachments, all of which microtubule end. The structural basis for how CLASP TOGs bind contribute to proper spindle assembly in dividing cells. αβ-tubulin has not yet been determined, so we can only speculate that the anti-catastrophe and rescue-promoting activities occur as the CLASPs in cell motility result of TOG-mediated stabilization of an intermediate end-specific CLASPs are essential for persistent cell motility. Indeed, a Clasp2- conformation of αβ-tubulin. knockout mouse presents impaired homing of hematopoietic stem cells to the bone marrow niche and disrupted microtubule network CLASPs in cell division stability and organization, indicating a role for CLASPs in regulating Early studies of CLASPs revealed that they have essential and microtubules during stem cell migration (Drabek et al., 2012). conserved functions at the mitotic spindle (Ambrose et al., 2007; In cultured motile cells, CLASPs locally stabilize microtubules at the Hannak and Heald, 2006; Inoue et al., 2000; Lemos et al., 2000; leading edge, and, thus help to polarize the microtubule network Maiato et al., 2002, 2003; Pasqualone and Huffaker, 1994). In towards the direction of migration (Akhmanova et al., 2001; Drabek mammalian cells, loss of CLASP activity causes severe mitotic et al., 2006; Wittmann and Waterman-Storer, 2005). In addition to defects, including metaphase delay, disorganized, mono- or multi- acting at the cortex, CLASPs are recruited to the Golgi by GCC185 polar spindles, misaligned chromosomes and cytokinesis failure (also known as GCC2), where they stabilize nascent Golgi-derived (Maiato et al., 2003; Mimori-Kiyosue et al., 2006; Pereira et al., microtubules (Efimov et al., 2007; Miller et al., 2009). These CLASP- 2006) (see poster). During early mitosis, human CLASPs localize to dependent Golgi-derived microtubules preferentially orient towards the outer kinetochore and spindle poles (Maiato et al., 2003; the leading edge and promote the directional trafficking of vesicles to Mimori-Kiyosue et al., 2006; Pereira et al., 2006). After the the migrating cell front, further contributing to cell polarization. metaphase-anaphase transition, CLASPs re-localize to the spindle CLASPs are also implicated in the turnover of focal adhesions, midzone where they stabilize overlapping microtubules and large multi-protein complexes that link the intracellular cytoskeleton accumulate in the midbody during cytokinesis (Maiato et al., with the extracellular matrix. Previous work has shown that cortical 2003; Mimori-Kiyosue et al., 2006; Pereira et al., 2006). CLASP is recruited near to focal adhesions by direct interaction with Several studies have established a role for CLASPs in regulating LL5β (also known as PHLDB2), a phosphatidylinositol (3,4,5)- kinetochore microtubule dynamics (Maffini et al., 2009; Maiato trisphosphate (PIP3)-binding protein (Basu et al., 2015; Lansbergen et al., 2005). Notably, CLASPs are thought to be important for et al., 2006). Another study in migrating human keratinocytes has promoting poleward flux in spindle microtubules, thus ensuring shown that cortical clusters of CLASPs are essential for efficient focal proper spindle dynamics. CLASPs are targeted to kinetochores by adhesion disassembly (Stehbens et al., 2014). Here, CLASP-tethered direct interaction with centrosome-associated protein E (CENP-E), microtubules provide stable tracks for the delivery, docking and independently of both the microtubule-binding activity of CLASP fusion of exocytic vesicles in the vicinity of focal adhesions. and the motor activity of CENP-E (Maffini et al., 2009; Maiato Additionally, this work revealed that CLASPs are required for et al., 2003). Phosphorylation of CLASP2 by CDK1 and PLK1 has degradation of the extracellular matrix near focal adhesions (Stehbens been implicated in regulating the stability of kinetochore– et al., 2014), placing CLASPs in a local secretion pathway that Journal of Cell Science

3 CELL SCIENCE AT A GLANCE Journal of Cell Science (2020) 133, jcs243097. doi:10.1242/jcs.243097 facilitates focal adhesion turnover. Similarly, CLASP-decorated CLASP2 inhibits, neurite extension, suggesting that the activities of microtubules support directional trafficking in podosomes, actin- the two proteins are finely balanced to achieve proper neuronal based protrusions important for degradation of extracellular matrix differentiation. CLASP2 has also been identified as a cytoskeletal during cell motility and invasion (Efimova et al., 2014). effector in the reelin signaling pathway, which is central to neocortical The localization and activity of CLASPs are spatially modulated development (Dillon et al., 2017). Knockdown of CLASP2 in vivo led in migrating cells by GSK3β-mediated phosphorylation to the mislocalization of neurons and impaired lamination of the (Akhmanova et al., 2001; Kumar et al., 2009; Watanabe et al., developing cortex. These effects are thought to be mediated by 2009; Wittmann and Waterman-Storer, 2005) (see poster). GSK3β GSK3β-dependent phosphorylation of CLASP2, which modulates activity is inhibited in the leading edge of motile cells, stimulating its interaction with the reelin pathway component DAB1 (Dillon CLASP activity. In migrating epithelial cells, CLASPs bind to et al., 2017). Since GSK3β-mediated phosphorylation of CLASP2 microtubule tips in the cell body and along microtubule lattices in modulates the CLASP–microtubule interaction (Akhmanova et al., the lamella (Kumar et al., 2009; Wittmann and Waterman-Storer, 2001; Kumar et al., 2009; Watanabe et al., 2009; Wittmann and 2005). The distinct localization of CLASPs along microtubule Waterman-Storer, 2005), it is possible that GKS3β in the reelin lattices is specified by inhibition of GSK3β in the lamella, and may pathway also regulates CLASP2 localization on microtubules. be important for establishing robust connections of the lamella In addition to the role of CLASPs in growth cone motility, microtubules to the cell cortex. CLASPs also regulate synaptic morphology and protein Interestingly, CLASP1 is implicated in regulating the mechanical composition. Indeed, CLASP2 overexpression in hippocampal properties of microtubules during 3D mesenchymal migration of neurons resulted in an increase in the number and area of synapses cancer cells by promoting persistent microtubule growth in cell and disrupted synaptic protein levels (Beffert et al., 2012). Several protrusions); CLASP1 knockdown completely prevented protrusion recent studies have shed light on the function of CLASPs at formation, 3D motility and invasiveness (Bouchet et al., 2016). neuromuscular junctions. Here, CLASPs capture microtubules at In contrast, CLASP2 knockdown had no effect on motility, the post synaptic membrane, which then serve as stable tracks for the highlighting the potentially different functional roles of CLASP delivery of vesicles containing acetylcholine receptors (Schmidt isoforms. The authors propose that CLASP1 helps microtubules to et al., 2012). CLASP2 is recruited to the post-synaptic membrane by bear compressive forces during cell retraction by suppressing LL5β and is regulated by GSK3β-mediated phosphorylation (Basu microtubule catastrophe, thus promoting protrusion elongation and et al., 2014, 2015). Thus, CLASPs facilitate directed vesicle cell migration (Bouchet et al., 2016). In 3D migrating Drosophila transport by tethering and stabilizing microtubules to the post- macrophages, the CLASP homolog Orbit (also known as MAST) synaptic membrane, in a manner that is reminiscent of CLASP promotes the formation of a microtubule bundle that protrudes into activity near focal adhesions in motile cells (see poster). the lamella and is involved in directed migration and cell–cell contact-mediated repulsion (Stramer et al., 2010). Whether CLASPs Plant CLASP in microtubule cytoskeleton remodeling also modulate microtubule mechanics by promoting microtubule Plants have a single isoform of CLASP that plays important roles in bundling and/or increasing the rigidity of individual microtubules cell and tissue morphogenesis and in cell division (Ambrose et al., during cell invasion has not been investigated. 2007; Kirik et al., 2007; Pietra et al., 2013). In Arabidopsis, cortical microtubules undergo dramatic rearrangements both during growth CLASPs in neurons and upon acute blue-light stimulus (see poster). A recent study CLASPs are crucial regulators of neuronal development and demonstrated that CLASP activity is central to the brassinosteroid regeneration. CLASPs are specifically enriched in neuronal hormone pathway, regulating sustained cell division in Arabidopsis growth cones, where they play a pivotal role in axonal growth root apical meristems (Ruan et al., 2018). The authors found that (Beffert et al., 2012; Hur et al., 2011; Lee et al., 2004; Marx et al., CLASP mediates cortical microtubule re-organization in response 2013). Furthermore, CLASP2 protein levels steadily rise throughout to brassinosteroid signaling. Interestingly, CLASP functions in a neuronal development, suggesting a continued requirement for negative feedback loop in which CLASP-stabilized microtubules CLASP2 activity (Beffert et al., 2012). Paradoxically, while facilitate the delivery of factors that suppress CLASP transcript CLASP2 is required for axon growth, too much CLASP activity levels, thereby downregulating CLASP expression. In addition, inhibits axon growth (Hur et al., 2011; Lee et al., 2004). The CLASP mutants have significant defects in cortical microtubule contradictory effects of CLASP2 can be attributed to GSK3β- organization, exhibiting a delayed timing of array reorganization mediated modulation of CLASP localization (Hur et al., 2011). during growth (Thoms et al., 2018) and complete failure of When CLASP2 activity is elevated, such as when GSK3β is microtubule reorientation in response to blue light (Lindeboom inhibited, CLASP2 shifts from the microtubule tips to lattices, et al., 2019). Earlier studies implicated CLASP in directly linking resulting in microtubule bundling and looping, and preventing axon microtubules to the cortex and locally preventing microtubule extension (Hur et al., 2011; Lee et al., 2004). Therefore, CLASP2 catastrophe, thereby preferentially stabilizing cortical microtubules can differentially regulate growth cone motility depending on its (Ambrose et al., 2007, 2011; Ambrose and Wasteneys, 2008). Now, microtubule-binding activity (see poster). new work provides novel insight into the interplay between CLASP Recent studies have revealed unexpected differences in the and the microtubule-severing enzyme katanin during the process of function of the two CLASP paralogs, as well as GSK3β-dependent microtubule array reorganization. modulation of CLASP activity. Specifically, it has been reported Microtubule severing is a major mechanism for microtubule that the two paralogs have distinct localization in neuroblastoma network remodeling in plants (Lindeboom et al., 2013). Severing a cells, with CLASP2 decorating growing microtubule ends in the single microtubule gives rise to two polymers, which, if stabilized, growth cone, and CLASP1 being enriched towards the cell body can serve as templates for new polymer growth and overall network (Sayas et al., 2019). CLASP1 and CLASP2 were also found to have amplification (Kuo et al., 2019; Vemu et al., 2018). Two recent differential sensitivity to GSk3β-mediated phosphorylation in that studies in plants have shown that CLASP is required to maintain study. Overall, the authors concluded that CLASP1 stimulates, while the population of nascent microtubule plus ends during the Journal of Cell Science

4 CELL SCIENCE AT A GLANCE Journal of Cell Science (2020) 133, jcs243097. doi:10.1242/jcs.243097 array organization. CLASP mutants had ∼30% fewer growing Acknowledgements microtubules and a lower density of longitudinally oriented We thank Jelmer Lindeboom (Carnegie Institution) and Irina Kaverina (Vanderbilt microtubules during cortical array pattern formation (Thoms University) for providing feedback on the manuscript. et al., 2018). CLASP mutants also displayed a significantly Competing interests smaller probability of microtubule regrowth immediately after The authors declare no competing or financial interests. severing (Lindeboom et al., 2019). A possible mechanism for the protective action of CLASP is Funding promoting microtubule rescue at discrete CLASP puncta localized E.J.L. acknowledges the support of the National Institutes of Health IBSTO training grant T32CA119925. M.Z. acknowledges support from the National Institutes of along the lattice of longitudinally oriented microtubules. Health grant R35GM119552, and the Searle Scholars Program. Work on TOG Surprisingly, however, the effects of CLASP mutations on the domain proteins in L.M.R.’s lab has been supported by grants from the National parameters of microtubule dynamics, including rescue frequency, Institutes of Health (R01-GM098543) and from the Robert A. Welch Foundation were mild (Lindeboom et al., 2019; Thoms et al., 2018). (I-1908). Deposited in PMC for release after 12 months. Furthermore, CLASP puncta were also found on transverse microtubules, a population that is not stabilized and is eventually Cell Science at a Glance A high-resolution version of the poster and individual poster panels are available for lost (Lindeboom et al., 2019). Alternatively, CLASP could act downloading at http://jcs.biologists.org/lookup/doi/10.1242/jcs.243097. through the recognition of a lattice ‘defect’. A microtubule supplemental crossover event could introduce defects in the lattice of the newer ‘crossing’ microtubule (Aumeier et al., 2016; de Forges et al., References Aher, A., Kok, M., Sharma, A., Rai, A., Olieric, N., Rodriguez-Garcia, R., 2016); such defects may be recognized by both CLASP and katanin Katrukha, E. A., Weinert, T., Olieric, V., Kapitein, L. C. et al. (2018). CLASP (Díaz-Valencia et al., 2011). Indeed, katanin specifically targets suppresses microtubule catastrophes through a single TOG domain. Dev. Cell 46, microtubule crossovers and preferentially severs the crossing, as 40-58.e8. doi:10.1016/j.devcel.2018.05.032 opposed to the crossed microtubule (Lindeboom et al., 2013; Zhang Aher, A., Rai, D., Schaedel, L., Gaillard, J., John, K., Blanchoin, L., Thery, M. and Akhmanova, A. (2019). CLASP mediates microtubule repair by promoting et al., 2013). However, recruitment of CLASP to lattice defect sites tubulin incorporation into damaged lattices. bioRxiv 15. doi:10.1101/809251 may facilitate incorporation of GTP-bound tubulin for lattice repair, Akhmanova, A. and Steinmetz, M. O. (2015). Control of microtubule organization and enhance the resistance of the microtubule to mechanical and dynamics: two ends in the limelight. Nat. Rev. Mol. Cell Biol. 16, 711-726. doi:10.1038/nrm4084 force (Aher et al., 2019 preprint). Thus, CLASP may compete Akhmanova, A., Hoogenraad, C. C., Drabek, K., Stepanova, T., Dortland, B., with katanin for binding to sites of microtubule defects, as well as Verkerk, T., Vermeulen, W., Burgering, B. M., De Zeeuw, C. I., Grosveld, F. target intermediate lattice structures that are generated during et al. (2001). Clasps are CLIP-115 and -170 associating proteins involved in the severing (Vemu et al., 2018) (see poster). Although enhanced regional regulation of microtubule dynamics in motile fibroblasts. Cell 104, 923-935. doi:10.1016/S0092-8674(01)00288-4 accumulation of CLASP at microtubule crossovers has not been Al-Bassam, J. and Chang, F. (2011). Regulation of microtubule dynamics by TOG- observed (Lindeboom et al., 2019), the localized action of even a domain proteins XMAP215/Dis1 and CLASP. Trends Cell Biol. 21, 604-614. small number of CLASP molecules may be sufficient to stabilize the doi:10.1016/j.tcb.2011.06.007 Al-Bassam, J., van Breugel, M., Harrison, S. C. and Hyman, A. (2006). Stu2p growing microtubule end (Aher et al., 2018, 2019 preprint). binds tubulin and undergoes an open-to-closed conformational change. J. Cell Therefore, CLASP at severing sites may allow immediate Biol. 172, 1009-1022. doi:10.1083/jcb.200511010 continuous growth of nascent microtubule plus ends, while the Al-Bassam, J., Larsen, N. A., Hyman, A. A. and Harrison, S. C. (2007). Crystal minus ends are simultaneously protected by SPIRAL2 (Nakamura structure of a TOG domain: conserved features of XMAP215/Dis1-family TOG domains and implications for tubulin binding. Structure 15, 355-362. doi:10.1016/j. et al., 2018). In this way, the resulting balance between microtubule str.2007.01.012 severing and stabilization facilitates overall network amplification Al-Bassam, J., Kim, H., Brouhard, G., van Oijen, A., Harrison, S. C. and Chang, and reorganization. F. (2010). CLASP promotes microtubule rescue by recruiting tubulin dimers to the microtubule. Dev. Cell 19, 245-258. doi:10.1016/j.devcel.2010.07.016 Ambrose, J. C. and Wasteneys, G. O. (2008). CLASP modulates microtubule- Perspectives cortex interaction during self-organization of acentrosomal microtubules. Mol. CLASP proteins have been subject of significant interest in the Biol. Cell 19, 4730-4737. doi:10.1091/mbc.e08-06-0665 last few years, with studies ranging from structures of individual Ambrose, J. C., Shoji, T., Kotzer, A. M., Pighin, J. A. and Wasteneys, G. O. (2007). The Arabidopsis CLASP gene encodes a microtubule-associated protein CLASP TOG domains to the roles of CLASPs in 3D cell migration involved in cell expansion and division. Plant Cell 19, 2763-2775. doi:10.1105/tpc. and neuronal development. Connecting the findings from a range of 107.053777 scales, from molecular to organismal, and from different contexts Ambrose, C., Allard, J. F., Cytrynbaum, E. N. and Wasteneys, G. O. (2011). A CLASP-modulated cell edge barrier mechanism drives cell-wide cortical presents an exciting challenge for the immediate future. At the microtubule organization in Arabidopsis. Nat. Commun. 2, 430. doi:10.1038/ molecular level, how do distinct features of CLASP TOG domains ncomms1444 ‘encode’ selective association of CLASPs with dynamic lattice and Aumeier, C., Schaedel, L., Gaillard, J., John, K., Blanchoin, L. and Théry, M. (2016). Self-repair promotes microtubule rescue. Nat. 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