Journal of Cell Science SHORT REPORT MAP1S controls microtubule stability throughout the cell cycle in human cells Justus Tegha-Dunghu 1, *, Elena Bausch 1 , Beate Neumann 2 , Annelie Wuensche 3 , Thomas Walter 3,` , Jan Ellenberg 3 and Oliver J. Gruss 1," SUMMARY Understanding the molecular basis for proper cell division requires a detailed functional analysis of microtubule (MT)-associated proteins. MT-associated protein 1S (MAP1S), the most ubiquitously expressed MAP1 family member, is required for accurate cell division. Here, using quantitative analysis of MT plus- end tracking, we show that MAP1S knockdown alters MT dynamics throughout the cell cycle. Surprisingly, MAP1S downregulation results in faster growing, yet short-lived, MTs in all cell cycle stages and in a global loss of MT acetylation. These aberrations correlate with severe defects in the final stages of cell division. In monopolar cytokinesis assays, we demonstrate that MAP1S guides MT-dependent initiation of cytokinesis. Our data underline the key role of MAP1S as a global regulator of MT stability and demonstrate a new primary function of MAP1S to regulate MT dynamics at the onset of cytokinesis. KEY WORDS: Microtubule, Microtubule-associated protein, MAP1S, Microtubule dynamics, Cytokinesis INTRODUCTION Regulating dynamic microtubule (MT) functions involves post- translational tubulin modifications (Sirajuddin et al., 2014; for reviews, see Hammond et al., 2008; Perdiz et al., 2011; Janke and Bulinski, 2011; Magiera and Janke, 2014; Sirajuddin et al., 2014) and changes in the pattern of MT-associated proteins (MAPs) (for reviews, see Manning and Compton, 2008a; Manning and Compton, 2008b; Walczak and Heald, 2008). MAPs of the MAP1 family promote MT stability and modulate MT functions in mammalian cells. The MAP1 family comprises MAP1A, MAP1B and the most recently identified member, MAP1S (Dallol et al., 2007; Halpain and Dehmelt, 2006; Orban-Nemeth et al., 2005). MAP1S binds to MTs in vitro and colocalizes with MTs in cells (Dallol et al., 2007; Orban-Nemeth et al., 2005; Wong et al., 2004). In contrast to MAP1A and MAP1B, MAP1S is found not only in the nervous system, but also in liver, spleen, heart and other organs (Orban-Nemeth et al., 2005) where it seems to fulfil cell-type-specific functions (Eriksson et al., 2010; Eriksson et al., 2007; Xie et al., 2011a; Xie et al., 2011b). The more ubiquitous expression of MAP1S suggests that the protein sustains general MT-dependent functions. The knockdown of MAP1S in HeLa cells compromises spindle assembly, delays metaphase alignment and anaphase entry, and causes genome instability (Dallol et al., 2007). MAP1S was first identified in large-scale screens for spindle proteins (Sauer et al., 2005; Tegha- Dunghu et al., 2008), and has been shown to associate with the spindle in human and murine cells (Dallol et al., 2007; Orban- Nemeth et al., 2005). However, how MAP1S acts on MTs and how it influences the parameters of MT dynamics in different cell cycle stages remained unclear. Here, we show that MAP1S governs MT nucleation and MT dynamics in interphase and during early and late stages of M phase. We demonstrate that MAP1S-deficient cells assemble MTs faster, but that these MTs have a severely reduced lifetime in interphase, mitosis and during mitotic exit, when they are required to coordinate cytokinesis. Together with specific cytokinesis assays, our data underline that the primary function of MAP1S is to maintain balanced MT dynamics during mitosis as well as in cytokinesis. RESULTS AND DISCUSSION To analyze the role of MAP1S in MT organization in human cells, we tested different parameters that mark MT stability. MT stability correlates with acetylation of a-tubulin on lysine 40 (K40 acetylation) (Sirajuddin et al., 2014, for reviews, see Hammond et al., 2008; Perdiz et al., 2011; Janke and Bulinski, 2011; Magiera and Janke, 2014; Sirajuddin et al., 2014). We found that the amount of acetylated tubulin in cells where MAP1S was knocked down by small interfering RNA (siRNA) (Fig. 1A; Fig. 1B, left panel) was reduced by 60% (Fig. 1A; Fig. 1B, right panel). Quantitative indirect immunofluorescence in synchronized cells revealed a significant reduction in MT acetylation, on average by 58% in metaphase, 40% in anaphase, 68% in cytokinesis and 70% in interphase (S and G2) (Fig. 1C,D; see also supplementary material Fig. S3C). In contrast, MAP1S knockdown did not affect glutamylation of tubulin (supplementary material Fig. S1A). The levels of MAP1S itself remained constant during the cell cycle (supplementary material Fig. S1B), as was also indicated by the detection of MAP1S on MTs in mitosis and interphase (supplementary material Fig. S1C). Knockdown of Eml3, which also localizes to MTs in interphase and mitosis (Tegha-Dunghu et al., 2008), as well as the mitotic MT-binding protein TPX2 (Gruss et al., 2002), left MT acetylation unchanged (Fig. 1E; supplementary material Fig. S1D). 1 Zentrum fu ¨ r Molekulare Biologie der Universita ¨ t Heidelberg (ZMBH), DKFZ- ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany. 2 European Molecular Biology Laboratory (EMBL), Advanced Light Microscopy Facility Programme, Meyerhostr.1, 69117 Heidelberg, Germany. 3 European Molecular Biology Laboratory (EMBL), Cell Biology and Biophysics Programme, Meyerhostr.1, 69117 Heidelberg, Germany. *Present address: Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada. ` Present addresses: MINES ParisTech, PSL-Research University, CBIO-Centre for Computational Biology, 35 rue St Honore ´ 77300 Fontainebleau, France; Institut Curie, 75248 Paris CEDEX, France; and INSERM U900, 75248 Paris CEDEX, France " Author for correspondence (o.gruss@zmbh.uni-heidelberg.de) Received 7 June 2013; Accepted 24 September 2014 ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 5007–5013 doi:10.1242/jcs.136457 5007