Yeast Haspin Kinase Regulates Polarity Cues Necessary for Mitotic Spindle Positioning and Is Required to Tolerate Mitotic Arrest

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

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

Molecular Biology of the Cell ◽

10.1091/mbc.e14-08-1330 ◽

2015 ◽

Vol 26 (7) ◽

pp. 1286-1295 ◽

Cited By ~ 12

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.

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

Decision letter: Optogenetic dissection of mitotic spindle positioning in vivo

10.7554/elife.38198.053 ◽

2018 ◽

Keyword(s):

Mitotic Spindle ◽

Spindle Positioning

Download Full-text

Faculty Opinions recommendation of Anthrax toxin receptor 2a controls mitotic spindle positioning.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717977638.793470905 ◽

2013 ◽

Author(s):

Cecilia Moens

Keyword(s):

Mitotic Spindle ◽

Anthrax Toxin ◽

Spindle Positioning ◽

Toxin Receptor

Download Full-text

Spindle Checkpoint Insufficiency in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v110.11.873.873 ◽

2007 ◽

Vol 110 (11) ◽

pp. 873-873

Author(s):

Dominik Schnerch ◽

Julia Felthaus ◽

Monika Engelhardt ◽

Ralph M. Waesch

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Mitotic Spindle ◽

Myeloid Leukemia ◽

Mitotic Checkpoint ◽

Mitotic Arrest ◽

Cyclin B ◽

Resting Cells ◽

Chromosome Attachment ◽

Acute Myeloid

Abstract Chromosomal instability and aneuploidy are hallmarks of most human malignancies. Various mechanisms have been shown to give rise to numerical chromosome aberrations. Compromised function of the spindle assembly checkpoint (SAC) is generally regarded as one of the most powerful ways to drive genome instability. The SAC is a mitotic checkpoint mechanism ensuring the equal segregation of the mitotic chromosomes onto the developing daughter cells. Unfaithful mitotic surveillance by the SAC favors chromosomal misdistribution as error-prone chromosome attachment to the mitotic spindle does not induce a strong mitotic arrest by interference with anaphase promoting complex (APC)-dependent proteolysis. The APC is an important ubiquitin ligase that triggers the transition from mitosis into G1-phase by targeted proteolysis of mitotic regulators such as cyclin B and securin. The SAC prevents the proteolysis of those regulator proteins in the presence of mitotic aberrancies by inhibition of the APC. This leads to a delayed progression through mitosis and provides time to recover from defective chromosomal spindle attachment. SAC malfunction weakens the tight control on chromosome attachment and tension across the kinetochore favoring chromosomal misdistribution. We performed expression analyses of key proteins in SAC signaling in acute myeloid leukemia (AML). We found the SAC-components Bub1 and BubR1 to be down-regulated in most of the investigated AML cell lines. Functional assays revealed a defective mitotic arrest mechanism in comparison to SAC-competent cell lines after exposure to the microtubule disrupting agent nocodazole. This finding was accompanied by the observation of a decline in cyclin B and securin levels despite severe damage to the mitotic spindle induced by nocodazole. Expression of cyclin B and securin in the presence of spindle damage could be stabilized by proteasome inhibition. We established a shRNA-based model to evaluate the effects of BubR1- and/or Bub1-repression to levels found among AML cell lines to directly compare the Bub1/BubR1 knockdown phenotype with the investigated AML cell lines. Interestingly, BubR1 knockdown was sufficient to generate a phenotype resembling the behavior of our AML cell lines. Further experiments revealed a strong relation between premature degradation of cyclin B and the degree of BubR1 downregulation. Given the potent role of BubR1 in the generation of a mitotic arrest deficient phenotype, we addressed the BubR1 expression levels in a number of patients exhibiting karyotype abnormalities. Primary myeloid blast cells were stimulated with cytokines to force the largely resting cells into an actively dividing state. The maximum expression level of BubR1 in G2/M was used to define SAC-compentent and SAC-deficient populations. Strikingly, six out of eight (6/8) primary AML samples exhibited BubR1 expression patterns resembling the BubR1-knockdown model suggesting deficient mitotic surveillance in most of the primary AML samples. Since SAC deficiency is an important mechanism in creating numerical chromosomal aberrations and genetic instability, our findings underline a role for impaired SAC function in rise and progression of AML.

Download Full-text