scholarly journals Control of Mitotic Spindle Position by the Saccharomyces cerevisiae Formin Bni1p

1999 ◽  
Vol 144 (5) ◽  
pp. 947-961 ◽  
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.

scholarly journals Cell shape impacts on the positioning of the mitotic spindle with respect to the substratum

2015 ◽  
Vol 26 (7) ◽  
pp. 1286-1295 ◽  
Author(s):  
Francisco Lázaro-Diéguez ◽  
Iaroslav Ispolatov ◽  
Anne Müsch
Keyword(s):  
Mitotic Spindle ◽  
Cell Shape ◽  
Spindle Assembly Checkpoint ◽  
Mdck Cells ◽  
Spindle Pole ◽  
Spindle Orientation ◽  
Cell Cortex ◽  
Spindle Positioning ◽  
Spindle Poles ◽  
Spindle Position

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.

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

1997 ◽  
Vol 138 (5) ◽  
pp. 1023-1040 ◽  
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.

scholarly journals The Role of Actin in Spindle Orientation Changes during the Saccharomyces cerevisiae Cell Cycle

1999 ◽  
Vol 146 (5) ◽  
pp. 1019-1032 ◽  
Author(s):  
Chandra L. Theesfeld ◽  
Javier E. Irazoqui ◽  
Kerry Bloom ◽  
Daniel J. Lew
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Spindle Orientation ◽  
Cortical Actin ◽  
Yeast Saccharomyces Cerevisiae ◽  
Daughter Cells ◽  
M Phase ◽  
Actin Cables

In the budding yeast Saccharomyces cerevisiae, the mitotic spindle must align along the mother-bud axis to accurately partition the sister chromatids into daughter cells. Previous studies showed that spindle orientation required both astral microtubules and the actin cytoskeleton. We now report that maintenance of correct spindle orientation does not depend on F-actin during G2/M phase of the cell cycle. Depolymerization of F-actin using Latrunculin-A did not perturb spindle orientation after this stage. Even an early step in spindle orientation, the migration of the spindle pole body (SPB), became actin-independent if it was delayed until late in the cell cycle. Early in the cell cycle, both SPB migration and spindle orientation were very sensitive to perturbation of F-actin. Selective disruption of actin cables using a conditional tropomyosin double-mutant also led to de- fects in spindle orientation, even though cortical actin patches were still polarized. This suggests that actin cables are important for either guiding astral microtubules into the bud or anchoring them in the bud. In addition, F-actin was required early in the cell cycle for the development of the actin-independent spindle orientation capability later in the cell cycle. Finally, neither SPB migration nor the switch from actin-dependent to actin-independent spindle behavior required B-type cyclins.

scholarly journals Plk1 regulates spindle orientation by phosphorylating NuMA in human cells

2018 ◽  
Vol 1 (6) ◽  
pp. e201800223 ◽  
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.

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

2014 ◽  
Vol 25 (18) ◽  
pp. 2720-2734 ◽  
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.

scholarly journals Dynamic Behavior of Microtubules during Dynein-dependent Nuclear Migrations of Meiotic Prophase in Fission Yeast

2001 ◽  
Vol 12 (12) ◽  
pp. 3933-3946 ◽  
Author(s):  
Ayumu Yamamoto ◽  
Chihiro Tsutsumi ◽  
Hiroaki Kojima ◽  
Kazuhiro Oiwa ◽  
Yasushi Hiraoka
Keyword(s):  
Fission Yeast ◽  
Dynamic Behavior ◽  
Meiotic Prophase ◽  
Fluorescent Protein ◽  
Cortical Microtubule ◽  
Spindle Pole Body ◽  
Cytoplasmic Dynein ◽  
Spindle Pole ◽  
Microtubule Assembly ◽  
Cell Cortex

During meiotic prophase in fission yeast, the nucleus migrates back and forth between the two ends of the cell, led by the spindle pole body (SPB). This nuclear oscillation is dependent on astral microtubules radiating from the SPB and a microtubule motor, cytoplasmic dynein. Here we have examined the dynamic behavior of astral microtubules labeled with the green fluorescent protein during meiotic prophase with the use of optical sectioning microscopy. During nuclear migrations, the SPB mostly follows the microtubules that extend toward the cell cortex. SPB migrations start when these microtubules interact with the cortex and stop when they disappear, suggesting that these microtubules drive nuclear migrations. The microtubules that are followed by the SPB often slide along the cortex and are shortened by disassembly at their ends proximal to the cortex. In dynein-mutant cells, where nuclear oscillations are absent, the SPB never migrates by following microtubules, and microtubule assembly/disassembly dynamics is significantly altered. Based on these observations, together with the frequent accumulation of dynein at a cortical site where the directing microtubules interact, we propose a model in which dynein drives nuclear oscillation by mediating cortical microtubule interactions and regulating the dynamics of microtubule disassembly at the cortex.

scholarly journals The CLIP-170 Homologue Bik1p Promotes the Phosphorylation and Asymmetric Localization of Kar9p

2006 ◽  
Vol 17 (1) ◽  
pp. 178-191 ◽  
Author(s):  
Jeffrey K. Moore ◽  
Sonia D'Silva ◽  
Rita K. Miller
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Mitotic Spindle ◽  
Phosphorylation Site ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Basic Domain ◽  
Pole Body ◽  
Two Hybrid ◽  
Insight Into

Accurate positioning of the mitotic spindle in Saccharomyces cerevisiae is coordinated with the asymmetry of the two poles and requires the microtubule-to-actin linker Kar9p. The asymmetric localization of Kar9p to one spindle pole body (SPB) and microtubule (MT) plus ends requires Cdc28p. Here, we show that the CLIP-170 homologue Bik1p binds directly to Kar9p. In the absence of Bik1p, Kar9p localization is not restricted to the daughter-bound SPB, but it is instead found on both SPBs. Kar9p is hypophosphorylated in bik1Δ mutants, and Bik1p binds to both phosphorylated and unphosphorylated isoforms of Kar9p. Furthermore, the two-hybrid interaction between full-length KAR9 and the cyclin CLB5 requires BIK1. The binding site of Clb5p on Kar9p maps to a short region within the basic domain of Kar9p that contains a conserved phosphorylation site, serine 496. Consistent with this, Kar9p is found on both SPBs in clb5Δ mutants at a frequency comparable with that seen in kar9-S496A strains. Together, these data suggest that Bik1p promotes the phosphorylation of Kar9p on serine 496, which affects its asymmetric localization to one SPB and associated cytoplasmic MTs. These findings provide further insight into a mechanism for directing centrosomal inheritance.

Ady3p Links Spindle Pole Body Function to Spore Wall Synthesis in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1439-1450
Author(s):  
Mark E Nickas ◽  
Aaron M Neiman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Spindle Pole Body ◽  
Leading Edge ◽  
Spindle Pole ◽  
Spore Wall ◽  
Pole Body ◽  
Prospore Membrane ◽  
Sporulation Efficiency ◽  
Spore Walls

Abstract Spore formation in Saccharomyces cerevisiae requires the de novo synthesis of prospore membranes and spore walls. Ady3p has been identified as an interaction partner for Mpc70p/Spo21p, a meiosis-specific component of the outer plaque of the spindle pole body (SPB) that is required for prospore membrane formation, and for Don1p, which forms a ring-like structure at the leading edge of the prospore membrane during meiosis II. ADY3 expression has been shown to be induced in midsporulation. We report here that Ady3p interacts with additional components of the outer and central plaques of the SPB in the two-hybrid assay. Cells that lack ADY3 display a decrease in sporulation efficiency, and most ady3Δ/ady3Δ asci that do form contain fewer than four spores. The sporulation defect in ady3Δ/ady3Δ cells is due to a failure to synthesize spore wall polymers. Ady3p forms ring-like structures around meiosis II spindles that colocalize with those formed by Don1p, and Don1p rings are absent during meiosis II in ady3Δ/ady3Δ cells. In mpc70Δ/mpc70Δ cells, Ady3p remains associated with SPBs during meiosis II. Our results suggest that Ady3p mediates assembly of the Don1p-containing structure at the leading edge of the prospore membrane via interaction with components of the SPB and that this structure is involved in spore wall formation.

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