Activated Ezrin Controls MISP Levels to Ensure Correct Numa Polarization and Spindle Orientation Yvonne T

© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs214544. doi:10.1242/jcs.214544 RESEARCH ARTICLE Activated ezrin controls MISP levels to ensure correct NuMA polarization and spindle orientation Yvonne T. Kschonsak1,2 and Ingrid Hoffmann1,* ABSTRACT misregulation in spindle orientation can result in disorganized tissue Correct spindle orientation is achieved through signaling pathways that morphology due to cell multi-layering, which could be associated provide a molecular link between the cell cortex and spindle with the earliest cancer developments (McCaffrey and Macara, microtubules in an F-actin-dependent manner. A conserved cortical 2011; Pease and Tirnauer, 2011). protein complex, composed of LGN (also known as GPSM2), NuMA The precise spindle position and orientation in the cell is achieved (also known as NUMA1) and dynein–dynactin, plays a key role in by signaling pathways generating pulling and pushing forces on the establishing proper spindle orientation. It has also been shown that the spindle, both externally or internally (Gönczy, 2002; Grill and actin-binding protein MISP and the ERM family, which are activated by Hyman, 2005; Théry et al., 2005; Fink et al., 2011). The longest lymphocyte-oriented kinase (LOK, also known as STK10) and Ste20- established player in spindle orientation is the conserved ternary α like kinase (SLK) (hereafter, SLK/LOK) in mitosis, regulate spindle complex, composed of G i, Leu-Gly-Asn repeat-enriched protein orientation. Here, we report that MISP functions downstream of the (LGN, also known as GPSM2) and nuclear mitotic apparatus ERM family member ezrin and upstream of NuMA to allow optimal (NuMA, also known as NUMA1) (Du et al., 2001; Du and Macara, α spindle positioning. We show that MISP directly interacts with ezrin and 2004). Whereas G i localizes at the whole cortex, LGN and NuMA that SLK/LOK-activated ezrin ensures appropriate cortical MISP levels are found in a crescent-shaped localization at the cortex, facing one in mitosis by competing with MISP for actin-binding sites at the cell or both spindle poles. Thereby, the crescent localization of LGN and cortex. Furthermore, we found that regulation of the correct cortical NuMA determines the position of force concentration. Through MISP levels, by preventing its excessive accumulation, is essential direct binding, NuMA recruits the force generator minus-end- for crescent-like polarized NuMA localization at the cortex and, as a directed motor protein complex of dynein and dynactin to the consequence, leads to highly dynamic astral microtubules. Our results cortex, where it generates pulling forces by binding to astral uncover how appropriate MISP levels at the cortex are required for microtubules (MTs) (Toyoshima and Nishida, 2007; di Pietro et al., proper NuMA polarization and, therefore, an optimal placement of the 2016). Through the polarized localization of LGN, NuMA and – mitotic spindle within the cell. dynein dynactin, the forces are generated at opposite sides of the cell ensuring optimal spindle orientation/positioning and equal cell This article has an associated First Person interview with the first division. In epithelial cells, LGN and NuMA specifically localize to author of the paper. the lateral sides, allowing planar cell division (Zheng et al., 2010; Peyre et al., 2011). Besides a few recently identified proteins that KEY WORDS: MISP, ERM, NuMA, Spindle orientation, Astral regulate polarized distribution of LGN and NuMA, like Afadin and microtubule aPKC, the regulatory mechanism that restricts LGN and NuMA localization still has to be identified (Hao et al., 2010; Zheng et al., INTRODUCTION 2010; Carminati et al., 2016). Correct positioning of the mitotic spindle axis within the cell is a Recently published data reveal that the ezrin/radixin/moesin fundamental process in development and stem cell division (ERM) protein family acts as an additional factor in mitotic spindle (Gönczy, 2002; Siller and Doe, 2009). In symmetrically dividing orientation and positioning (Machicoane et al., 2014). More cells, precise spindle orientation and positioning ensures equal precisely, activated cortical ERM proteins in mitosis are involved distribution of cellular components. In contrast, in asymmetrically in promoting polarized, cortical association of LGN and NuMA. dividing cells, accurate orientation and placement of the mitotic However, the direct molecular link between ERM and the ternary spindle away from the center of the cell results in cell fate diversity complex is not known. The ERM proteins, namely ezrin, radixin (Horvitz and Herskowitz, 1992; Gönczy, 2002; Ahringer, 2003; and moesin, are evolutionarily highly conserved proteins, and are Grill and Hyman, 2005). In most epithelia, cells divide expressed differently in various tissues (Fehon et al., 2010). They symmetrically and orient their mitotic spindle parallel to the exert crucial functions in cell migration, as well as cell invasion, by apical–basal surface, ensuring expansion of the epithelial sheet with directly linking the actin cytoskeleton to the plasma membrane side-by-side growing daughter cells (Fleming et al., 2007). Any (Algrain et al., 1993; Clucas and Valderrama, 2014). ERM proteins exist in two conformational states, an inactive, closed conformation with the FERM and C-terminal tail domain (C-ERMAD) forming 1Cell Cycle Control and Carcinogenesis, F045, German Cancer Research Center, an intramolecular interaction, and an active, open confirmation DKFZ, 69120 Heidelberg, Germany. 2Faculty of Biosciences, Heidelberg University, 69120 Heidelberg, Germany. where the two domains are dissociated (Bretscher et al., 1995, 1997; Gary and Bretscher, 1995). The active form is produced by *Author for correspondence ([email protected]) phosphatidylinositol-4,5-bisphosphate (PIP2) binding to the FERM I.H., 0000-0002-8894-8597 domain and a subsequent membrane-dependent phosphorylation of the highly conserved C-terminally located threonine residue at Received 19 December 2017; Accepted 11 April 2018 position 567, 564 and 558 for ezrin, radixin and moesin, Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs214544. doi:10.1242/jcs.214544 respectively (Nakamura et al., 1995; Niggli et al., 1995; Hirao et al., polarization of NuMA and impairs astral MT dynamics, which in 1996; Matsui et al., 1998; Simons et al., 1998; Bretscher et al., 2002; turn results in spindle misorientation. Our data suggest that the Fievet et al., 2004; Pelaseyed et al., 2017). This phosphorylation in protein levels of MISP at the cortex need to be tightly controlled to epithelial cells is mainly catalyzed by lymphocyte-oriented kinase ensure NuMA polarization and proper spindle orientation. Thus, we (LOK, also known as STK10) and Ste20-like kinase (SLK) provide novel mechanistic insights into how ezrin governs NuMA- (hereafter SLK/LOK) (Kunda et al., 2008; Machicoane et al., 2014). regulated spindle orientation by monitoring cortical levels of the The recently identified unstructured protein mitotic interactor and actin-binding protein MISP. substrate of Plk1 (MISP) is strongly associated with actin and is highly phosphorylated during mitosis by Cdk1 and Plk1 (Maier RESULTS et al., 2013; Zhu et al., 2013; Kumeta et al., 2014). MISP localizes at Activated ezrin interacts with MISP at the cell cortex the cell cortex throughout the cell cycle and contributes to proper To understand the mechanism by which MISP regulates spindle cortical distribution of p150glued (also known as DCTN1), astral MT orientation, we aimed at identifying MISP-interacting proteins in stability and correct mitotic spindle placement (Zhu et al., 2013). both asynchronous and mitotic cells. By using several approaches, However, the exact mechanism of how cortical proteins collectively including co-immunoprecipitations (co-IPs) and BioID (Roux et al., contribute to the regulation of mitotic spindle orientation is not fully 2012), we identified the human ERM protein family, consisting of understood. ezrin, radixin and moesin, as such interacting proteins (Fig. S1A). Here, we report that the ERM family member ezrin acts upstream We verified the interaction between MISP and the ERM proteins of MISP in controlling mitotic spindle orientation and positioning. ezrin, radixin and moesin, respectively, after co-expression in Our results indicate that MISP is a direct binding partner of ezrin in HEK293T cells (Fig. 1A; Fig. S1B). Since ezrin is the most its open active state. We uncover that, in mitotic cells, SLK/LOK- ubiquitous ERM protein in epithelial cells, we focused on the activated ezrin is required to prevent excessive accumulation of interplay between MISP and ezrin, and could observe that MISP MISP at the cell cortex. Aberrant MISP levels abolish cortical also interacted with ezrin in endogenous co-IPs from HeLa cells Fig. 1. Ezrin in its open, activated conformation binds and colocalizes with MISP at the cell cortex. (A) Flag–MISP and GFP–ezrin were co-expressed in HEK293T cells. Complexes were immunoprecipitated with Flag-M2 antibody (Flag-IP) or anti-GFP antibody (GFP-IP), and analyzed by western blotting using the indicated antibodies. (B) Co-IP of endogenous ezrin from HeLa cells as analyzed by western blotting using anti-MISP or -ezrin antibodies. IgG, control immunoglobulin. (C) Domain structure of full-length ezrin (comprising the N-terminal located FERM domain, the α-tail and the C-ERMAD domain. Indicated residue T567 gets phosphorylated,

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