A Unique Set of Centrosome Proteins Requires Pericentrin for Spindle-Pole Localization and Spindle Orientation

All known mechanisms of mitotic spindle orientation rely on astral microtubules. We report that even in the absence of astral microtubules, metaphase spindles in MDCK and HeLa cells are not randomly positioned along their x-z dimension, but preferentially adopt shallow β angles between spindle pole axis and substratum. The nonrandom spindle positioning is due to constraints imposed by the cell cortex in flat cells that drive spindles that are longer and/or wider than the cell's height into a tilted, quasidiagonal x-z position. In rounder cells, which are taller, fewer cortical constraints make the x-z spindle position more random. Reestablishment of astral microtubule–mediated forces align the spindle poles with cortical cues parallel to the substratum in all cells. However, in flat cells, they frequently cause spindle deformations. Similar deformations are apparent when confined spindles rotate from tilted to parallel positions while MDCK cells progress from prometaphase to metaphase. The spindle disruptions cause the engagement of the spindle assembly checkpoint. We propose that cell rounding serves to maintain spindle integrity during its positioning.

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The yeast centrosome translates the positional information of the anaphase spindle into a cell cycle signal

The Journal of Cell Biology ◽

10.1083/jcb.200705197 ◽

2007 ◽

Vol 179 (3) ◽

pp. 423-436 ◽

Cited By ~ 77

Author(s):

Hiromi Maekawa ◽

Claire Priest ◽

Johannes Lechner ◽

Gislene Pereira ◽

Elmar Schiebel

Keyword(s):

Spindle Pole Body ◽

Coiled Coil ◽

Positional Information ◽

Spindle Pole ◽

Mitotic Exit ◽

Spindle Orientation ◽

Additional Function ◽

A Cell ◽

Cytoplasmic Microtubules ◽

Guanosine Triphosphatase

The spindle orientation checkpoint (SPOC) of budding yeast delays mitotic exit when cytoplasmic microtubules (MTs) are defective, causing the spindle to become misaligned. Delay is achieved by maintaining the activity of the Bfa1–Bub2 guanosine triphosphatase–activating protein complex, an inhibitor of mitotic exit. In this study, we show that the spindle pole body (SPB) component Spc72, a transforming acidic coiled coil–like molecule that interacts with the γ-tubulin complex, recruits Kin4 kinase to both SPBs when cytoplasmic MTs are defective. This allows Kin4 to phosphorylate the SPB-associated Bfa1, rendering it resistant to inactivation by Cdc5 polo kinase. Consistently, forced targeting of Kin4 to both SPBs delays mitotic exit even when the anaphase spindle is correctly aligned. Moreover, we present evidence that Spc72 has an additional function in SPOC regulation that is independent of the recruitment of Kin4. Thus, Spc72 provides a missing link between cytoplasmic MT function and components of the SPOC.

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Control of Mitotic Spindle Position by the Saccharomyces cerevisiae Formin Bni1p

The Journal of Cell Biology ◽

10.1083/jcb.144.5.947 ◽

1999 ◽

Vol 144 (5) ◽

pp. 947-961 ◽

Cited By ~ 113

Author(s):

Laifong Lee ◽

Saskia K. Klee ◽

Marie Evangelista ◽

Charles Boone ◽

David Pellman

Keyword(s):

Saccharomyces Cerevisiae ◽

Mitotic Spindle ◽

Cortical Microtubule ◽

Spindle Pole Body ◽

Spindle Pole ◽

Spindle Orientation ◽

Cell Cortex ◽

Cortical Actin ◽

Bud Site ◽

Spindle Position

Alignment of the mitotic spindle with the axis of cell division is an essential process in Saccharomyces cerevisiae that is mediated by interactions between cytoplasmic microtubules and the cell cortex. We found that a cortical protein, the yeast formin Bni1p, was required for spindle orientation. Two striking abnormalities were observed in bni1Δ cells. First, the initial movement of the spindle pole body (SPB) toward the emerging bud was defective. This phenotype is similar to that previously observed in cells lacking the kinesin Kip3p and, in fact, BNI1 and KIP3 were found to be in the same genetic pathway. Second, abnormal pulling interactions between microtubules and the cortex appeared to cause preanaphase spindles in bni1Δ cells to transit back and forth between the mother and the bud. We therefore propose that Bni1p may localize or alter the function of cortical microtubule-binding sites in the bud. Additionally, we present evidence that other bipolar bud site determinants together with cortical actin are also required for spindle orientation.

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Role of astral microtubules and actin in spindle orientation and migration in the budding yeast, Saccharomyces cerevisiae.

The Journal of Cell Biology ◽

10.1083/jcb.119.3.583 ◽

1992 ◽

Vol 119 (3) ◽

pp. 583-593 ◽

Cited By ~ 182

Author(s):

R E Palmer ◽

D S Sullivan ◽

T Huffaker ◽

D Koshland

Keyword(s):

Saccharomyces Cerevisiae ◽

Chromosome Segregation ◽

Mother Cell ◽

Fold Increase ◽

Spindle Pole ◽

Spindle Orientation ◽

Temperature Sensitive ◽

Chromosome Movement ◽

Yeast Saccharomyces Cerevisiae ◽

Cytoskeletal Elements

In the yeast Saccharomyces cerevisiae, before the onset of anaphase, the spindle apparatus is always positioned with one spindle pole at, or through, the neck between the mother cell and the growing bud. This spindle orientation enables proper chromosome segregation to occur during anaphase, allowing one replicated genome to be segregated into the bud and the other to remain in the mother cell. In this study, we synchronized a population of cells before the onset of anaphase such that > 90% of the cells in the population had spindles with the correct orientation, and then disrupted specific cytoskeletal elements using temperature-sensitive mutations. Disruption of either the astral microtubules or actin function resulted in improper spindle orientation in approximately 40-50% of the cells. When cells with disrupted astral microtubules or actin function entered into anaphase, there was a 100-200-fold increase in the frequency of binucleated cell bodies. Thus, the maintenance of proper spindle orientation by these cytoskeletal elements was essential for proper chromosome segregation. These data are consistent with the model that proper spindle orientation is maintained by directly or indirectly tethering the astral microtubules to the actin cytoskeleton. After nuclear migration, but before anaphase, bulk chromosome movement occurs within the nucleus apparently because the chromosomes are attached to a mobile spindle. The frequency and magnitude of bulk chromosome movement is greatly diminished by disruption of the astral microtubules but not by disruption of the nonkinetochore spindle microtubules. These results suggest that astral microtubules are not only important for spindle orientation before anaphase, but they also mediate force on the spindle, generating spindle displacement and in turn chromosome movement. Potential roles for this force in spindle assembly and orientation are discussed.

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Plk1 regulates spindle orientation by phosphorylating NuMA in human cells

Life Science Alliance ◽

10.26508/lsa.201800223 ◽

2018 ◽

Vol 1 (6) ◽

pp. e201800223 ◽

Cited By ~ 14

Author(s):

Shrividya Sana ◽

Riya Keshri ◽

Ashwathi Rajeevan ◽

Sukriti Kapoor ◽

Sachin Kotak

Keyword(s):

Cell Division ◽

Mitotic Spindle ◽

Molecular Mechanisms ◽

Spindle Pole ◽

Spindle Orientation ◽

Cell Cortex ◽

Division Plane ◽

Evolutionarily Conserved ◽

Cortical Localization ◽

Proper Orientation

Proper orientation of the mitotic spindle defines the correct division plane and is essential for accurate cell division and development. In metazoans, an evolutionarily conserved complex comprising of NuMA/LGN/Gαi regulates proper orientation of the mitotic spindle by orchestrating cortical dynein levels during metaphase. However, the molecular mechanisms that modulate the spatiotemporal dynamics of this complex during mitosis remain elusive. Here, we report that acute inactivation of Polo-like kinase 1 (Plk1) during metaphase enriches cortical levels of dynein/NuMA/LGN and thus influences spindle orientation. We establish that this impact of Plk1 on cortical levels of dynein/NuMA/LGN is through NuMA, but not via dynein/LGN. Moreover, we reveal that Plk1 inhibition alters the dynamic behavior of NuMA at the cell cortex. We further show that Plk1 directly interacts and phosphorylates NuMA. Notably, NuMA-phosphorylation by Plk1 impacts its cortical localization, and this is needed for precise spindle orientation during metaphase. Overall, our finding connects spindle-pole pool of Plk1 with cortical NuMA and answers a long-standing puzzle about how spindle-pole Plk1 gradient dictates proper spindle orientation for error-free mitosis.

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Influence of the centrosome in cytokinesis of brown algae: polyspermic zygotes of Scytosiphon lomentaria (Scytosiphonales,Phaeophyceae)

Journal of Cell Science ◽

10.1242/jcs.115.12.2541 ◽

2002 ◽

Vol 115 (12) ◽

pp. 2541-2548

Author(s):

Chikako Nagasato ◽

Taizo Motomura

Keyword(s):

Brown Algae ◽

Spindle Pole ◽

Spindle Orientation ◽

Daughter Cells ◽

Male Gametes ◽

Spindle Poles ◽

Scytosiphon Lomentaria ◽

Polar Spindle ◽

Spindle Position ◽

The Relationship

We examined the relationship between the spindle orientation and the determination site of cytokinesis in brown algal cells using polyspermic zygotes of Scytosiphon lomentaria. When two male gametes fuse with one female gamete, the zygote has two pairs of centrioles derived from male gametes and three chloroplasts from two male and one female gametes. Just before mitosis, two pairs of centrioles duplicate and migrate towards the future mitotic poles. Spindle MTs develop and three or four spindle poles are formed. In a tri-polar spindle, one pair of centrioles shifts away from the spindle, otherwise, two pairs of centrioles exist adjoining at one spindle pole. Chromosomes arrange at several equators of the spindle. As a result of these multipolar mitoses, three or four daughter nuclei developed. Subsequently, these daughter nuclei form a line along the long axis of the cell. Cell partition always takes place between daughter nuclei, perpendicular to the long axis of the cell. Three or four daughter cells are produced by cytokinesis. Some of the daughter cells after cytokinesis do not have a nucleus, but all of them always contain the centrosome and chloroplast. Therefore, the number of daughter cells always coincides with the number of centrosomes or microtubule organizing centers (MTOCs). These results show that the cytokinetic plane in the brown algae is determined by the position of centrosomes after mitosis and is not dependent on the spindle position.

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A lysine deacetylase Hos3 is targeted to the bud neck and involved in the spindle position checkpoint

Molecular Biology of the Cell ◽

10.1091/mbc.e13-10-0619 ◽

2014 ◽

Vol 25 (18) ◽

pp. 2720-2734 ◽

Cited By ~ 7

Author(s):

Mengqiao Wang ◽

Ruth N. Collins

Keyword(s):

Spindle Pole Body ◽

Neck Region ◽

Spindle Pole ◽

Mitotic Exit ◽

Spindle Orientation ◽

Lysine Deacetylase ◽

Bud Neck ◽

Morphogenesis Checkpoint ◽

Spindle Position

An increasing number of cellular activities can be regulated by reversible lysine acetylation. Targeting the enzymes responsible for such posttranslational modifications is instrumental in defining their substrates and functions in vivo. Here we show that a Saccharomyces cerevisiae lysine deacetylase, Hos3, is asymmetrically targeted to the daughter side of the bud neck and to the daughter spindle pole body (SPB). The morphogenesis checkpoint member Hsl7 recruits Hos3 to the neck region. Cells with a defect in spindle orientation trigger Hos3 to load onto both SPBs. When associated symmetrically with both SPBs, Hos3 functions as a spindle position checkpoint (SPOC) component to inhibit mitotic exit. Neck localization of Hos3 is essential for its symmetric association with SPBs in cells with misaligned spindles. Our data suggest that Hos3 facilitates cross-talk between the morphogenesis checkpoint and the SPOC as a component of the intricate monitoring of spindle orientation after mitotic entry and before commitment to mitotic exit.

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Kinesin-related KIP3 of Saccharomyces cerevisiae Is Required for a Distinct Step in Nuclear Migration

The Journal of Cell Biology ◽

10.1083/jcb.138.5.1023 ◽

1997 ◽

Vol 138 (5) ◽

pp. 1023-1040 ◽

Cited By ~ 135

Author(s):

Todd M. DeZwaan ◽

Eric Ellingson ◽

David Pellman ◽

David M. Roof

Keyword(s):

Saccharomyces Cerevisiae ◽

Mitotic Spindle ◽

Nuclear Migration ◽

Spindle Pole ◽

Live Cells ◽

Spindle Orientation ◽

Chain Gene ◽

Null Mutant ◽

Bud Neck ◽

Bud Site

Spindle orientation and nuclear migration are crucial events in cell growth and differentiation of many eukaryotes. Here we show that KIP3, the sixth and final kinesin-related gene in Saccharomyces cerevisiae, is required for migration of the nucleus to the bud site in preparation for mitosis. The position of the nucleus in the cell and the orientation of the mitotic spindle was examined by microscopy of fixed cells and by time-lapse microscopy of individual live cells. Mutations in KIP3 and in the dynein heavy chain gene defined two distinct phases of nuclear migration: a KIP3-dependent movement of the nucleus toward the incipient bud site and a dynein-dependent translocation of the nucleus through the bud neck during anaphase. Loss of KIP3 function disrupts the unidirectional movement of the nucleus toward the bud and mitotic spindle orientation, causing large oscillations in nuclear position. The oscillatory motions sometimes brought the nucleus in close proximity to the bud neck, possibly accounting for the viability of a kip3 null mutant. The kip3 null mutant exhibits normal translocation of the nucleus through the neck and normal spindle pole separation kinetics during anaphase. Simultaneous loss of KIP3 and kinesin-related KAR3 function, or of KIP3 and dynein function, is lethal but does not block any additional detectable movement. This suggests that the lethality is due to the combination of sequential and possibly overlapping defects. Epitope-tagged Kip3p localizes to astral and central spindle microtubules and is also present throughout the cytoplasm and nucleus.

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