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The KASH Protein Kms2 Coordinates Mitotic Remodeling of the Spindle

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The KASH Protein Kms2 Coordinates Mitotic Remodeling of the Spindle ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 3625–3640 doi:10.1242/jcs.154997 RESEARCH ARTICLE The KASH protein Kms2 coordinates mitotic remodeling of the spindle pole body Sarah Wa¨lde and Megan C. King* ABSTRACT The centrosome is composed of a pair of centrioles and a surrounding cloud of electron-dense pericentriolar material Defects in the biogenesis of the spindle pole body (SPB), the yeast (PCM; Bettencourt-Dias and Glover, 2007). The PCM anchors centrosome equivalent, can lead to monopolar spindles and mitotic and nucleates interphase and spindle microtubules (Mennella catastrophe. The KASH domain protein Kms2 and the SUN domain et al., 2012; Moritz et al., 1995; Ou and Rattner, 2004). During protein Sad1 colocalize within the nuclear envelope at the site of centrosome maturation in late G2, the PCM expands in size and SPB attachment during interphase and at the spindle poles during recruits additional c-tubulin ring complex (cTuRC) to support mitosis in Schizosaccharomyces pombe. We show that Kms2 robust mitotic spindle assembly (Decker et al., 2011; Palazzo interacts with the essential SPB components Cut12 and Pcp1 and et al., 2000). The recruitment and incorporation of cTuRC is the Polo kinase Plo1. Depletion of Kms2 delays mitotic entry and achieved by several PCM proteins, among them the large coiled- leads to defects in the insertion of the SPB into the nuclear coil protein pericentrin (Dictenberg et al., 1998; Doxsey et al., envelope, disrupting stable bipolar spindle formation. These effects 1994). Pericentrin is required for centrosome maturation, as its are mediated in part by a delay in the recruitment of Plo1 to the SPB depletion leads to monopolar spindles and perturbed PCM at mitotic entry. Plo1 activity supports mitotic SPB remodeling by assembly (Zimmerman et al., 2004). Phosphorylation of PCM driving a burst of incorporation of Cut12 and Pcp1. Thus, a fission components occurs concomitant with centrosome maturation, thus yeast SUN–KASH complex plays an important role in supporting the coupling changes in its physical state to cell cycle kinase activity remodeling of the SPB at mitotic entry. (Barr et al., 2004; Glover et al., 1998). Quantitative proteomics of isolated mitotic spindles has identified pericentrin and other PCM KEY WORDS: LINC complex, Nuclear envelope, Spindle pole body components as Plk1 substrates; phosphorylation of pericentrin by Plk1 is required for proper spindle formation and centrosomal microtubule nucleation in HeLa cells (Lee and Rhee, 2011; INTRODUCTION Santamaria et al., 2011). In eukaryotic cells, the activity of the cyclin-dependent kinase In S. pombe, the electron-dense material equivalent to the Cdk1 (also known as Cdc2, Cdc28 or mitosis-promoting factor) PCM, including the pericentrin homolog, Pcp1 (Flory et al., determines the timing of mitotic entry and cell division (Nurse, 2002), resides close to the outer nuclear membrane, with 1990). Cdk1 activity and the transition from G2 to M phase are an additional enrichment of nuclear c-tubulin underneath the tightly controlled by the antagonistic activities of the Wee1 apposed inner nuclear membrane (Ding et al., 1997). Late in G2, kinase and Cdc25 phosphatase (Nurse, 1990). In response to the sole Polo-like kinase, Plo1, is recruited to the SPB, in part physiological cues, such as appropriate cell mass and nutrient through its interaction with Pcp1 and the SPB component Cut12, availability, the inhibitory phosphorylation of Cdk1 (catalyzed by where it helps to drive mitotic entry (Ba¨hler et al., 1998a; Fong Wee1) is removed by Cdc25; Cdc25 is further activated by Cdk1 et al., 2010; Grallert et al., 2013b; MacIver et al., 2003; Mulvihill phosphorylation (Decottignies et al., 2001; Gould et al., 1991; et al., 1999). As the cell enters ‘closed’ mitosis, cytoplasmic Gould and Nurse, 1989; Russell and Nurse, 1986; Strausfeld microtubules depolymerize and the nuclear envelope opens to et al., 1991). The highly conserved serine/threonine Polo-like form fenestrae to allow for the insertion of the mother and kinases (Plks) reinforce this positive-feedback loop by activating daughter SPBs (Ding et al., 1997). Pcp1, Cut12 and Plo1 are Cdc25 and inhibiting Wee1 (Barr et al., 2004; Llamazares et al., required for bipolar spindle formation and mitotic progression 1991; Lowery et al., 2005). This robust signaling network is (Bridge et al., 1998; Flory et al., 2002; Ohkura et al., 1995). coordinated in space as well as time, with the Cdk1 feedback When mitosis is complete, the SPBs are extruded back into the mechanism occurring primarily at the centrosome (Jackman et al., cytoplasm (Ding et al., 1997). 2003). In Schizosaccharomyces pombe, regulation of mitotic Little is known about how Pcp1 and other components of the entry is also linked to components of the centrosome equivalent, SPB are tethered to (and coordinated across) the nuclear envelope the spindle pole body (SPB) (Bridge et al., 1998; Grallert et al., in S. pombe. In other model systems, the centrosome is tethered 2013a; Grallert et al., 2013b; MacIver et al., 2003; Mulvihill to the nuclear envelope by KASH (Klarsicht, Anc1, SYNE1 et al., 1999; Tallada et al., 2007; Tallada et al., 2009). homology) domain proteins integrated into the outer nuclear membrane either directly or indirectly through microtubules and the associated motors dynein and kinesin (Fan and Ready, 1997; Department of Cell Biology, Yale School of Medicine, 333 Cedar Street, New Malone et al., 2003; Minn et al., 2009; Roux et al., 2009; Whited Haven, CT 06520, USA. et al., 2004; Zhang et al., 2009). KASH proteins bind to well- *Author for correspondence ([email protected]) conserved integral inner nuclear membrane SUN (Sad1/Unc84) proteins within the nuclear envelope lumen (Razafsky and Received 21 April 2014; Accepted 9 June 2014 Hodzic, 2009; Sosa et al., 2012; Tzur et al., 2006). The S. Journal of Cell Science 3625 RESEARCH ARTICLE Journal of Cell Science (2014) 127, 3625–3640 doi:10.1242/jcs.154997 pombe genome encodes two KASH proteins, Kms1 and Kms2, Combining the kms2 DAmP allele with the cdc25-22 allele (Nurse and one SUN protein, Sad1 (Hagan and Yanagida, 1995; Miki et al., 1976), which disrupts the dephosphorylation and activation et al., 2004; Shimanuki et al., 1997). Kms1 and Kms2 are strong of Cdc2/Cdk1, caused a significant increase in cell length candidates for mediating the association of the SPB with the outer compared with that observed in cells carrying single-mutant nuclear membrane, thereby coupling the SPB to the nuclear alleles (Fig. 1F). Strikingly, the combination of the kms2 DAmP interior through interactions with Sad1. Sad1 is also required for and cdc25-22 alleles was synthetically lethal at 34˚C (Fig. 1G). normal SPB function in mitosis, although its molecular function These observations suggest a role for Kms2 in promoting mitotic is not explicitly known (Hagan and Yanagida, 1995); alleles of entry. the budding yeast SUN protein Mps3 lead to defects in SPB duplication or insertion of the SPB into the nuclear envelope Kms2 contributes to stable bipolar spindle formation (Friederichs et al., 2011; Jaspersen et al., 2002). Mitotic entry is linked to the activation and insertion of the SPBs Here, we investigate the contribution of the KASH protein into the nuclear envelope to support spindle formation. To test Kms2 to SPB function. We find that Kms2 supports timely whether spindle formation is affected by the kms2 DAmP allele, mitotic onset and stable formation of the bipolar spindle, in part we monitored SPB position (marked by Sad1–mCherry) and by supporting insertion of the SPBs into the nuclear envelope. mitotic spindle formation and stabilization (marked by a2- Furthermore, Kms2 interacts with Pcp1, Cut12 and Plo1. Plo1 is tubulin, GFP–Atb2) in live cells progressing through mitosis. required for the phosphorylation of Pcp1 at mitotic onset and its Wild-type cells formed bipolar spindles that grew to the cell poles daughter-specific incorporation into the SPB. Depletion of Kms2 after the anaphase A to anaphase B transition (Fig. 2A,B). By affects the efficiency of SPB remodeling at mitotic entry, contrast, kms2 DAmP cells often displayed spindle stabilization suggesting that Kms2 helps to coordinate SPB remodeling with problems (SSP; Fig. 2A,B), which were sometimes so severe as the cell cycle. to lead to persistent monopolar spindles (MPS; Fig. 2A,B; supplementary material Fig. S2B). In order to further RESULTS understand the events leading to the spindle defects, we Kms2 colocalizes with Sad1 and the SPB throughout the cell investigated the timing of SPB separation, which coincides with cycle the initiation of bipolar spindle formation. In normal mitoses, As observed previously, GFP–Kms2 colocalizes with the SUN spindle assembly follows immediately after the depolymerization domain protein Sad1 during interphase, oscillating along the of the cytoplasmic microtubule array. As in cells harboring a nuclear envelope in concert with the SPB as it is pushed by temperature-sensitive allele of Cut12 (cut12.1) or Cut11 microtubules (Fig. 1A; King et al., 2008). We also find that Sad1 (cut11.1), we see a ‘nucleation delay’ between the loss of and Kms2 remain constitutively associated with the SPB during the cytoplasmic microtubules and SPB separation (Fig. 2C; closed mitosis of S. pombe; at each mitosis the SPB goes through a supplementary material Fig. S2C; Tallada et al., 2009). This cycle of insertion at mitotic entry and extrusion during anaphase B, delay also corresponds to a period in which a-tubulin appears (Fig. 1A; supplementary material Fig. S1; Ding et al., 1997). diffusely nucleoplasmic (supplementary material Fig. S2C, kms2 DAmP panel, time-points 0–4). These results suggest a possible Kms2 is essential after germination delay in the insertion and/or activation of the SPBs. Kms2 is reported to be an essential gene (Kim et al., 2010). Thus, In kms2 DAmP cells that successfully separated SPBs, in order to better understand the impact of loss of Kms2 additional defects in spindle stabilization arose.
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