A Protein Interaction Map of the Mitotic Spindle

Wong, Jonathan; Nakajima, Yuko; Westermann, Stefan; Shang, Ching; Kang, Jungseog; Goodner, Crystal; Houshmand, Pantea; Fields, Stanley; Chan, Chung Chow; Drubin, David G.; Barnes, Georjana; Hazbun, Tony R.

doi:10.1091/mbc.e07-06-0536

Cited by 90 publications

(116 citation statements)

References 55 publications

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“…All Cnn1 mutants were expressed as fusion proteins with the Gal4 activation domain. Yeast two-hybrid was performed as previously described to analyze for the ability to grow as a consequence of HIS3 transcription using medium lacking tryptophan, leucine and histidine (SD 2TLH) in addition to 3 mM or 10 mM aminotriazole (Wong et al 2007). …”

Section: Interaction Analysismentioning

confidence: 99%

The Mps1 Kinase Modulates the Recruitment and Activity of Cnn1CENP-T at Saccharomyces cerevisiae Kinetochores

et al. 2015

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Kinetochores are conserved protein complexes that bind the replicated chromosomes to the mitotic spindle and then direct their segregation. To better comprehend Saccharomyces cerevisiae kinetochore function, we dissected the phospho-regulated dynamic interaction between conserved kinetochore protein Cnn1 CENP-T , the centromere region, and the Ndc80 complex through the cell cycle. Cnn1 localizes to kinetochores at basal levels from G1 through metaphase but accumulates abruptly at anaphase onset. How Cnn1 is recruited and which activities regulate its dynamic localization are unclear. We show that Cnn1 harbors two kinetochore-localization activities: a C-terminal histone-fold domain (HFD) that associates with the centromere region and a N-terminal Spc24/Spc25 interaction sequence that mediates linkage to the microtubule-binding Ndc80 complex. We demonstrate that the established Ndc80 binding site in the N terminus of Cnn1, Cnn1 , should be extended with flanking residues, Cnn1 , to allow near maximal binding affinity to Ndc80. Cnn1 localization was proposed to depend on Mps1 kinase activity at Cnn1-S74, based on in vitro experiments demonstrating the Cnn1-Ndc80 complex interaction. We demonstrate that from G1 through metaphase, Cnn1 localizes via both its HFD and N-terminal Spc24/Spc25 interaction sequence, and deletion or mutation of either region results in anomalous Cnn1 kinetochore levels. At anaphase onset (when Mps1 activity decreases) Cnn1 becomes enriched mainly via the N-terminal Spc24/Spc25 interaction sequence. In sum, we provide the first in vivo evidence of Cnn1 preanaphase linkages with the kinetochore and enrichment of the linkages during anaphase. KEYWORDS Cnn1; Mps1; kinetochore; centromere; CENP-T K INETOCHORES are large protein structures that assemble hierarchically on the centromeres (CEN) of replicated chromosomes (sister chromatids). They biorient each sister chromatid pair to the microtubules (MTs) of the mitotic spindle and orchestrate chromatid segregation into the daughter cells (Cheeseman 2014;Malvezzi and Westermann 2014). At the core of each kinetochore lies a protein network named KMN (KLN1/Spc105, MIS12/Mtw1, and NDC80/Ndc80 complexes) that bridges the CEN and MTs (Cheeseman et al. 2006;Westermann et al. 2007). The Ndc80 complex attaches kinetochores to the MTs via its outer Ndc80/Nuf2 dimer (Wei et al. 2007;Ciferri et al. 2008;Alushin et al. 2010), while its CEN-proximal Spc24/Spc25 dimer interacts with a putatively CEN-associated protein, known as Cnn1 in budding yeast (CENP-T in metazoans). The N-terminal domain of Cnn1 and CENP-T hook onto the interface of the Spc24/Spc25 dimer (Malvezzi et al. 2013;Nishino et al. 2013). In its C terminus, Cnn1 harbors a histonefold domain (HFD) (Schleiffer et al. 2012), which may associate with CEN DNA, as does CENP-T (Hori et al. 2008;Nishino et al. 2012).Cnn1 levels at kinetochores are low from G1 through metaphase but increase two-to threefold at anaphase entry and drop back to base level at anaphase exit. Cnn1 interacts with the...

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Section: Interaction Analysismentioning

confidence: 99%

The Mps1 Kinase Modulates the Recruitment and Activity of Cnn1CENP-T at Saccharomyces cerevisiae Kinetochores

et al. 2015

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“…The Dam1 complex locates along microtubules, but not at kinetochores during their microtubule lattice association Tanaka et al 2007). Subsequently this complex is loaded onto kinetochores and plays an important role, perhaps in association with the Ndc80 complex, in tethering them at microtubule plus ends Shang et al 2003;Wong et al 2007) as well as in pulling them poleward as microtubules depolymerize (Fig. 1, step 2, end-on pulling; Fig.…”

Section: Interface Of Kinetochore Microtubule Interaction: the Ndc80 mentioning

confidence: 99%

Bi-orienting chromosomes: acrobatics on the mitotic spindle

Tanaka

2008

Chromosoma

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To maintain their genetic integrity, eukaryotic cells must segregate their chromosomes properly to opposite poles during mitosis. This process mainly depends on the forces generated by microtubules that attach to kinetochores. During prometaphase, kinetochores initially interact with a single microtubule that extends from a spindle pole and then move towards a spindle pole. Subsequently, microtubules that extend from the other spindle pole also interact with kinetochores and, eventually, each sister kinetochore attaches to microtubules that extend from opposite poles (sister kinetochore bi-orientation). If sister kinetochores interact with microtubules in wrong orientation, this must be corrected before the onset of anaphase. Here, I discuss the processes leading to biorientation and the mechanisms ensuring this pivotal state that is required for proper chromosome segregation.

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“…For several years it has been known that She1 localizes to the mitotic spindle, bud neck, spindle pole bodies, and astral MTs (Wong et al 2007(Wong et al , 2009(Wong et al , 2010. Previous work described She1's role in inhibiting dynein activity until it is required for spindle positioning in anaphase ) and showed that She1 phosphorylation by Ipl1 is required to promote spindle disassembly .…”

Section: Discussionmentioning

confidence: 99%

Regulation of Mitotic Spindle Disassembly by an Environmental Stress-Sensing Pathway in Budding Yeast

Pigula¹,

Drubin²,

Barnes

2014

Genetics

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Timely spindle disassembly is essential for coordination of mitotic exit with cytokinesis. In the budding yeast Saccharomyces cerevisiae, the microtubule-associated protein She1 functions in one of at least three parallel pathways that promote spindle disassembly. She1 phosphorylation by the Aurora kinase Ipl1 facilitates a role for She1 in late anaphase, when She1 contributes to microtubule depolymerization and shrinkage of spindle halves. By examining the genetic interactions of known spindle disassembly genes, we identified three genes in the environmental stress-sensing HOG (high-osmolarity glycerol response) pathway, SHO1, PBS2, and HOG1, and found they are necessary for proper localization of She1 to the anaphase spindle and for proper spindle disassembly. HOG pathway mutants exhibited spindle disassembly defects, as well as mislocalization of anillin-related proteins Boi1 and Boi2 from the bud neck. Moreover, Boi2, but not Boi1, plays a role in spindle disassembly that places Boi2 in a pathway with Sho1, Pbs2, and Hog1. Together, our data identify a process by which cells monitor events at the spindle and bud neck and describe a novel role for the HOG pathway in mitotic signaling. MICROTUBULE dynamics play important roles in every stage of mitosis. In early M phase, interpolar microtubules help drive the separation of the spindle pole bodies (SPBs) to establish a bipolar spindle. As these interpolar microtubules (ipMTs) lengthen, the SPBs separate due to sliding forces generated by opposing microtubules originating from the opposite poles. In addition to ipMTs, kinetochore microtubules (kMTs) are attached to the kinetochore of each chromatid. A third class of microtubules, astral microtubules (aMTs), attach the SPBs to the cell cortex and help position the spindle along the mother-bud axis in yeast or perpendicular to the division plane in mammalian cells. Together, these three classes of MTs form the mitotic spindle. Over the course of mitosis, the dynamics of microtubules are modulated by several classes of proteins including +TIP proteins such as EB1/Bim1, which stabilize microtubule plus ends and promote growth, crosslinking proteins such as Ase1, which stabilize lateral interactions between ipMTs of the midzone, motor proteins such as Cin8 and Kip1, which generate forces necessary to slide the ipMTs by each other, and other motor proteins such as Kar3 and dynein (reviewed in Westermann et al. 2007;Khmelinskii and Schiebel 2008) that are also responsible for generating forces on MTs.She1 is an important regulator of microtubule dynamics in budding yeast and it appears to have several functions in the cell Bergman et al. 2012;Markus et al. 2012). Beginning in G1, She1 localizes to the SPB and restricts the activity of dynein by inhibiting its interaction with dynactin until anaphase, when dynein is required for spindle positioning prior to elongation into the bud ). In M phase, She1 also localizes to the kinetochore and along spindle microtubules and to the bud neck . Upon completion of anaph...

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A Protein Interaction Map of the Mitotic Spindle

Cited by 90 publications

References 55 publications

The Mps1 Kinase Modulates the Recruitment and Activity of Cnn1CENP-T at Saccharomyces cerevisiae Kinetochores

The Mps1 Kinase Modulates the Recruitment and Activity of Cnn1CENP-T at Saccharomyces cerevisiae Kinetochores

Bi-orienting chromosomes: acrobatics on the mitotic spindle

Regulation of Mitotic Spindle Disassembly by an Environmental Stress-Sensing Pathway in Budding Yeast

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