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

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.

Download Full-text

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.

Download Full-text

Cdk1-dependent destabilization of long astral microtubules is required for spindle orientation

10.1101/2020.05.23.111989 ◽

2020 ◽

Cited By ~ 1

Author(s):

Divya Singh ◽

Nadine Schmidt ◽

Franziska Müller ◽

Tanja Bange ◽

Alexander W. Bird

Keyword(s):

Mitotic Spindle ◽

Cell Shape ◽

Microtubule Dynamics ◽

Spindle Orientation ◽

Cell Cortex ◽

Spindle Positioning ◽

Tissue Organization ◽

Microtubule Stability ◽

Astral Microtubule ◽

Early Mitosis

AbstractThe precise execution of mitotic spindle orientation in response to cell shape cues is important for tissue organization and development. The presence of astral microtubules extending from the centrosome towards the cell cortex is essential for this process, but little is understood about the contribution of astral microtubule dynamics to spindle positioning, or how astral microtubule dynamics are regulated spatiotemporally. The mitotic regulator Cdk1-CyclinB promotes destabilization of centrosomal microtubules and increased microtubule dynamics as cells transition from interphase to mitosis, but how Cdk1 activity specifically modulates astral microtubule stability, and whether it impacts spindle positioning, is unknown. Here we uncover a mechanism revealing that Cdk1 destabilizes astral microtubules to ensure spindle reorientation in response to cell shape. Phosphorylation of the EB1-dependent microtubule plus-end tracking protein GTSE1 by Cdk1 in early mitosis abolishes its interaction with EB1 and recruitment to microtubule plus-ends. Loss of Cdk1 activity, or mutation of phosphorylation sites in GTSE1, induces recruitment of GTSE1 to growing microtubule plus-ends in mitosis. This decreases the catastrophe frequency of astral microtubules, and causes an increase in the number of long astral microtubules reaching the cell cortex, which restrains the ability of cells to reorient spindles along the long cellular axis in early mitosis. Astral microtubules must thus not only be present, but also dynamic to allow the spindle to reorient in response to cell shape, a state achieved by selective destabilization of long astral microtubules via Cdk1.

Download Full-text

LGN regulates mitotic spindle orientation during epithelial morphogenesis

The Journal of Cell Biology ◽

10.1083/jcb.200910021 ◽

2010 ◽

Vol 189 (2) ◽

pp. 275-288 ◽

Cited By ~ 130

Author(s):

Zhen Zheng ◽

Huabin Zhu ◽

Qingwen Wan ◽

Jing Liu ◽

Zhuoni Xiao ◽

...

Keyword(s):

Mitotic Spindle ◽

Mdck Cells ◽

Apical Cell ◽

Cell Polarization ◽

Epithelial Morphogenesis ◽

Spindle Orientation ◽

Cell Cortex ◽

Dividing Cells ◽

Cortical Localization ◽

Lateral Cell

Coordinated cell polarization and mitotic spindle orientation are thought to be important for epithelial morphogenesis. Whether spindle orientation is indeed linked to epithelial morphogenesis and how it is controlled at the molecular level is still unknown. Here, we show that the NuMA- and Gα-binding protein LGN is required for directing spindle orientation during cystogenesis of MDCK cells. LGN localizes to the lateral cell cortex, and is excluded from the apical cell cortex of dividing cells. Depleting LGN, preventing its cortical localization, or disrupting its interaction with endogenous NuMA or Gα proteins all lead to spindle misorientation and abnormal cystogenesis. Moreover, artificial mistargeting of endogenous LGN to the apical membrane results in a near 90° rotation of the spindle axis and profound cystogenesis defects that are dependent on cell division. The normal apical exclusion of LGN during mitosis appears to be mediated by atypical PKC. Thus, cell polarization–mediated spatial restriction of spindle orientation determinants is critical for epithelial morphogenesis.

Download Full-text

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.

Download Full-text

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.

Download Full-text

CDK5RAP2 functions in centrosome to spindle pole attachment and DNA damage response

The Journal of Cell Biology ◽

10.1083/jcb.200912163 ◽

2010 ◽

Vol 189 (1) ◽

pp. 23-39 ◽

Cited By ~ 77

Author(s):

Alexis R. Barr ◽

John V. Kilmartin ◽

Fanni Gergely

Keyword(s):

Dna Damage ◽

Cell Fate ◽

Mitotic Spindle ◽

Brain Size ◽

Neurodevelopmental Disorder ◽

Spindle Pole ◽

Spindle Positioning ◽

Dna Damage Signaling ◽

Spindle Poles ◽

G2 Cell Cycle Arrest

The centrosomal protein, CDK5RAP2, is mutated in primary microcephaly, a neurodevelopmental disorder characterized by reduced brain size. The Drosophila melanogaster homologue of CDK5RAP2, centrosomin (Cnn), maintains the pericentriolar matrix (PCM) around centrioles during mitosis. In this study, we demonstrate a similar role for CDK5RAP2 in vertebrate cells. By disrupting two evolutionarily conserved domains of CDK5RAP2, CNN1 and CNN2, in the avian B cell line DT40, we find that both domains are essential for linking centrosomes to mitotic spindle poles. Although structurally intact, centrosomes lacking the CNN1 domain fail to recruit specific PCM components that mediate attachment to spindle poles. Furthermore, we show that the CNN1 domain enforces cohesion between parental centrioles during interphase and promotes efficient DNA damage–induced G2 cell cycle arrest. Because mitotic spindle positioning, asymmetric centrosome inheritance, and DNA damage signaling have all been implicated in cell fate determination during neurogenesis, our findings provide novel insight into how impaired CDK5RAP2 function could cause premature depletion of neural stem cells and thereby microcephaly.

Download Full-text

LET-99 determines spindle position and is asymmetrically enriched in response to PAR polarity cues inC. elegansembryos

Development ◽

10.1242/dev.129.19.4469 ◽

2002 ◽

Vol 129 (19) ◽

pp. 4469-4481 ◽

Cited By ~ 7

Author(s):

Meng-Fu Bryan Tsou ◽

Adam Hayashi ◽

Leah R. DeBella ◽

Garth McGrath ◽

Lesilee S. Rose

Keyword(s):

Asymmetric Cell Division ◽

Spindle Pole ◽

Cellular Polarity ◽

Wild Type ◽

Spindle Positioning ◽

C Elegans ◽

Spindle Poles ◽

Nuclear Rotation ◽

Spindle Position ◽

Asymmetric Regulation

Asymmetric cell division depends on coordinating the position of the mitotic spindle with the axis of cellular polarity. We provide evidence that LET-99 is a link between polarity cues and the downstream machinery that determines spindle positioning in C. elegans embryos. In let-99 one-cell embryos, the nuclear-centrosome complex exhibits a hyperactive oscillation that is dynein dependent, instead of the normal anteriorly directed migration and rotation of the nuclear-centrosome complex. Furthermore, at anaphase in let-99 embryos the spindle poles do not show the characteristic asymmetric movements typical of wild type animals. LET-99 is a DEP domain protein that is asymmetrically enriched in a band that encircles P lineage cells. The LET-99 localization pattern is dependent on PAR polarity cues and correlates with nuclear rotation and anaphase spindle pole movements in wild-type embryos, as well as with changes in these movements in par mutant embryos. In particular, LET-99 is uniformly localized in one-cell par-3 embryos at the time of nuclear rotation. Rotation fails in spherical par-3 embryos in which the eggshell has been removed, but rotation occurs normally in spherical wild-type embryos. The latter results indicate that nuclear rotation in intact par-3 embryos is dictated by the geometry of the oblong egg and are consistent with the model that the LET-99 band is important for rotation in wild-type embryos. Together, the data indicate that LET-99 acts downstream of PAR-3 and PAR-2 to determine spindle positioning, potentially through the asymmetric regulation of forces on the spindle.

Download Full-text

NuMA-microtubule interactions are critical for spindle orientation and the morphogenesis of diverse epidermal structures

eLife ◽

10.7554/elife.12504 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 57

Author(s):

Lindsey Seldin ◽

Andrew Muroyama ◽

Terry Lechler

Keyword(s):

Cell Fate ◽

Mitotic Spindle ◽

Cell Types ◽

Epidermal Differentiation ◽

Spindle Orientation ◽

Direct Function ◽

Spindle Positioning ◽

Adult Mice ◽

Neonatal Lethality ◽

The Matrix

Mitotic spindle orientation is used to generate cell fate diversity and drive proper tissue morphogenesis. A complex of NuMA and dynein/dynactin is required for robust spindle orientation in a number of cell types. Previous research proposed that cortical dynein/dynactin was sufficient to generate forces on astral microtubules (MTs) to orient the spindle, with NuMA acting as a passive tether. In this study, we demonstrate that dynein/dynactin is insufficient for spindle orientation establishment in keratinocytes and that NuMA’s MT-binding domain, which targets MT tips, is also required. Loss of NuMA-MT interactions in skin caused defects in spindle orientation and epidermal differentiation, leading to neonatal lethality. In addition, we show that NuMA-MT interactions are also required in adult mice for hair follicle morphogenesis and spindle orientation within the transit-amplifying cells of the matrix. Loss of spindle orientation in matrix cells results in defective differentiation of matrix-derived lineages. Our results reveal an additional and direct function of NuMA during mitotic spindle positioning, as well as a reiterative use of spindle orientation in the skin to build diverse structures.

Download Full-text

Mitochondria-driven assembly of a cortical anchor for mitochondria and dynein

The Journal of Cell Biology ◽

10.1083/jcb.201702022 ◽

2017 ◽

Vol 216 (10) ◽

pp. 3061-3071 ◽

Cited By ~ 22

Author(s):

Lauren M. Kraft ◽

Laura L. Lackner

Keyword(s):

Plasma Membrane ◽

Mitotic Spindle ◽

Cell Cortex ◽

Dual Function ◽

Mitochondrial Inheritance ◽

Cellular Functions ◽

Core Component ◽

Spindle Positioning ◽

The Core ◽

Contact Sites

Interorganelle contacts facilitate communication between organelles and impact fundamental cellular functions. In this study, we examine the assembly of the MECA (mitochondria–endoplasmic reticulum [ER]–cortex anchor), which tethers mitochondria to the ER and plasma membrane. We find that the assembly of Num1, the core component of MECA, requires mitochondria. Once assembled, Num1 clusters persistently anchor mitochondria to the cell cortex. Num1 clusters also function to anchor dynein to the plasma membrane, where dynein captures and walks along astral microtubules to help orient the mitotic spindle. We find that dynein is anchored by Num1 clusters that have been assembled by mitochondria. When mitochondrial inheritance is inhibited, Num1 clusters are not assembled in the bud, and defects in dynein-mediated spindle positioning are observed. The mitochondria-dependent assembly of a dual-function cortical anchor provides a mechanism to integrate the positioning and inheritance of the two essential organelles and expands the function of organelle contact sites.

Download Full-text

A centrosomal antigen localized on intermediate filaments and mitotic spindle poles

Journal of Cell Science ◽

10.1242/jcs.97.2.259 ◽

1990 ◽

Vol 97 (2) ◽

pp. 259-271

Author(s):

B. Buendia ◽

C. Antony ◽

F. Verde ◽

M. Bornens ◽

E. Karsenti

Keyword(s):

Monoclonal Antibody ◽

Intermediate Filaments ◽

Mitotic Spindle ◽

Mdck Cells ◽

Large Fraction ◽

Human Lymphocytes ◽

Spindle Poles ◽

Pericentriolar Material ◽

Xenopus Egg

A monoclonal antibody (CTR2611) raised against centrosomes isolated from human lymphocytes (KE37) stains the pericentriolar material and intermediate filaments in the same cells. In MDCK cells, where most of the microtubules do not originate from the pericentriolar region during interphase, the antigen is distributed along intermediate filaments. At the onset of mitosis, a large fraction of the CTR2611 antigen associates with the minus-end domain of the microtubules of the mitotic spindle but not with the pericentriolar region itself. Treatment of mitotic MDCK cells with taxol leads to the assembly of many microtubule asters in the cytoplasm at the expense of the mitotic spindle. The CTR2611 antigen is present in the center of each of these asters. Similar asters can also be produced in vitro by adding taxol to concentrated Xenopus egg mitotic cytoplasm. Again, the antigen is found close to the center of the asters. These results suggest that CTR2611 antigen is associated with a material involved in microtubule nucleation or microtubule minus-end stabilization. The monoclonal antibody recognizes a 74 × 10(3) Mr polypeptide and other polypeptides at 120 × 10(3) Mr and 170 × 10(3) Mr. The 74 × 10(3) Mr polypeptide is found in all species examined so far, suggesting that it contains a highly conserved epitope.

Download Full-text