scholarly journals Microtubule end tethering of a processive kinesin-8 motor Kif18b is required for spindle positioning

2018 ◽  
Vol 217 (7) ◽  
pp. 2403-2416 ◽  
Author(s):  
Toni McHugh ◽  
Agata A. Gluszek ◽  
Julie P.I. Welburn
Keyword(s):  
Cell Division ◽  
Mitotic Spindle ◽  
Essential Role ◽  
Gene Knockout ◽  
Spindle Orientation ◽  
Binding Region ◽  
Spindle Positioning ◽  
Astral Microtubule ◽  
In Vitro Reconstitution

Mitotic spindle positioning specifies the plane of cell division during anaphase. Spindle orientation and positioning are therefore critical to ensure symmetric division in mitosis and asymmetric division during development. The control of astral microtubule length plays an essential role in positioning the spindle. In this study, using gene knockout, we show that the kinesin-8 Kif18b controls microtubule length to center the mitotic spindle at metaphase. Using in vitro reconstitution, we reveal that Kif18b is a highly processive plus end–directed motor that uses a C-terminal nonmotor microtubule-binding region to accumulate at growing microtubule plus ends. This region is regulated by phosphorylation to spatially control Kif18b accumulation at plus ends and is essential for Kif18b-dependent spindle positioning and regulation of microtubule length. Finally, we demonstrate that Kif18b shortens microtubules by increasing the catastrophe rate of dynamic microtubules. Overall, our work reveals that Kif18b uses its motile properties to reach microtubule ends, where it regulates astral microtubule length to ensure spindle centering.

scholarly journals Microtubule end tethering of a processive Kinesin-8 motor Kif18b is required for spindle positioning

2018 ◽  
Author(s):  
Toni McHugh ◽  
Agata Gluszek-Kustusz ◽  
Julie P.I. Welburn
Keyword(s):  
Cell Division ◽  
Mitotic Spindle ◽  
Essential Role ◽  
Gene Knockout ◽  
Integrated Approach ◽  
Spindle Orientation ◽  
Binding Region ◽  
Spindle Positioning ◽  
Symmetric Division ◽  
Astral Microtubule

AbstractMitotic spindle positioning specifies the plane of cell division during anaphase. Spindle orientation and positioning is therefore critical to ensure symmetric division in mitosis and asymmetric division during development. The control of astral microtubule length plays an essential role in positioning the spindle. Here we show using gene knockout that the Kinesin-8 Kif18b controls microtubule length to center the mitotic spindle at metaphase. Using an integrated approach, we reveal that Kif18b is a highly processive plus end-directed motor that uses a C-terminal non-motor microtubule-binding region to accumulate at growing microtubule plus ends. This region is regulated by phosphorylation to spatially control Kif18b accumulation at plus ends and is essential for Kif18b-dependent spindle positioning and regulation of microtubule length. Finally, we demonstrate that Kif18b shortens microtubules by increasing the catastrophe rate of dynamic microtubules. Overall, our work reveals that Kif18b utilizes its motile properties to reach microtubule ends where it regulates astral microtubule length to ensure spindle centering.

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

2020 ◽  
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.

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

eLife ◽  
2016 ◽  
Vol 5 ◽  
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.

scholarly journals The mitotic spindle protein CKAP2 potently increases formation and stability of microtubules

eLife ◽  
2022 ◽  
Vol 11 ◽  
Author(s):  
Thomas S McAlear ◽  
Susanne Bechstedt
Keyword(s):  
Cell Division ◽  
Growth Factor ◽  
Mitotic Spindle ◽  
Apparent Rate Constant ◽  
Microtubule Dynamics ◽  
Proliferation Marker ◽  
Microtubule Growth ◽  
Large Rearrangements ◽  
And Function

Cells increase microtubule dynamics to make large rearrangements to their microtubule cytoskeleton during cell division. Changes in microtubule dynamics are essential for the formation and function of the mitotic spindle, and misregulation can lead to aneuploidy and cancer. Using in vitro reconstitution assays we show that the mitotic spindle protein Cytoskeleton-Associated Protein 2 (CKAP2) has a strong effect on nucleation of microtubules by lowering the critical tubulin concentration 100-fold. CKAP2 increases the apparent rate constant ka of microtubule growth by 50-fold and increases microtubule growth rates. In addition, CKAP2 strongly suppresses catastrophes. Our results identify CKAP2 as the most potent microtubule growth factor to date. These finding help explain CKAP2's role as an important spindle protein, proliferation marker, and oncogene.

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 Physical Description of Mitotic Spindle Orientation During Cell Division

2009 ◽  
Vol 96 (3) ◽  
pp. 195a-196a
Author(s):  
Andrea Jiménez-Dalmaroni ◽  
Manuel Théry ◽  
Victor Racine ◽  
Michel Bornens ◽  
Frank Jülicher
Keyword(s):  
Cell Division ◽  
Mitotic Spindle ◽  
Spindle Orientation ◽  
Physical Description

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 HMMR acts in the PLK1-dependent spindle positioning pathway and supports neural development

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Marisa Connell ◽  
Helen Chen ◽  
Jihong Jiang ◽  
Chia-Wei Kuan ◽  
Abbas Fotovati ◽  
...  
Keyword(s):  
Cell Division ◽  
Neural Development ◽  
Cell Types ◽  
Spindle Orientation ◽  
Spindle Positioning ◽  
Over Expression ◽  
Oriented Cell Division ◽  
Neonatal Lethality ◽  
Cortical Localization ◽  
Dependent Pathway

Oriented cell division is one mechanism progenitor cells use during development and to maintain tissue homeostasis. Common to most cell types is the asymmetric establishment and regulation of cortical NuMA-dynein complexes that position the mitotic spindle. Here, we discover that HMMR acts at centrosomes in a PLK1-dependent pathway that locates active Ran and modulates the cortical localization of NuMA-dynein complexes to correct mispositioned spindles. This pathway was discovered through the creation and analysis of Hmmr-knockout mice, which suffer neonatal lethality with defective neural development and pleiotropic phenotypes in multiple tissues. HMMR over-expression in immortalized cancer cells induces phenotypes consistent with an increase in active Ran including defects in spindle orientation. These data identify an essential role for HMMR in the PLK1-dependent regulatory pathway that orients progenitor cell division and supports neural development.

scholarly journals Robust control of mitotic spindle orientation in the developing epidermis

2010 ◽  
Vol 191 (5) ◽  
pp. 915-922 ◽  
Author(s):  
Nicholas D. Poulson ◽  
Terry Lechler
Keyword(s):  
Cell Division ◽  
Robust Control ◽  
Progenitor Cells ◽  
Mitotic Spindle ◽  
Asymmetric Cell Division ◽  
Transcriptional Regulator ◽  
Spindle Orientation ◽  
Control Points ◽  
External Cues ◽  
Increase Surface Area

Progenitor cells must balance self-amplification and production of differentiated progeny during development and homeostasis. In the epidermis, progenitors divide symmetrically to increase surface area and asymmetrically to promote stratification. In this study, we show that individual epidermal cells can undergo both types of division, and therefore, the balance is provided by the sum of individual cells’ choices. In addition, we define two control points for determining a cell’s mode of division. First is the expression of the mouse Inscuteable gene, which is sufficient to drive asymmetric cell division (ACD). However, there is robust control of division orientation as excessive ACDs are prevented by a change in the localization of NuMA, an effector of spindle orientation. Finally, we show that p63, a transcriptional regulator of stratification, does not control either of these processes. These data have uncovered two important regulatory points controlling ACD in the epidermis and allow a framework for analysis of how external cues control this important choice.

scholarly journals Cell contacts orient some cell division axes in the Caenorhabditis elegans embryo.

1995 ◽  
Vol 129 (4) ◽  
pp. 1071-1080 ◽  
Author(s):  
B Goldstein
Keyword(s):  
Cell Division ◽  
Caenorhabditis Elegans ◽  
Mitotic Spindle ◽  
Spindle Orientation ◽  
Cell Contact ◽  
Specific Cell ◽  
Cell Contacts ◽  
Established Cell ◽  
A Site ◽  
Cell Cell

Cells of the early Caenorhabditis elegans embryo divide in an invariant pattern. Here I show that the division axes of some early cells (EMS and E) are controlled by specific cell-cell contacts (EMS-P2 or E-P3 contact). Altering the orientation of contact between these cells alters the axis along which the mitotic spindle is established, and hence the orientation of cell division. Contact-dependent mitotic spindle orientation appears to work by establishing a site of the type described by Hyman and White (1987. J. Cell Biol. 105:2123-2135) in the cortex of the responding cell: one centrosome moves toward the site of cell-cell contact during centrosome rotation in both intact embryos and reoriented cell pairs. The effect is especially apparent when two donor cells are placed on one side of the responding cell: both centrosomes are "captured," pulling the nucleus to one side of the cell. No centrosome rotation occurs in the absence of cell-cell contact, nor in nocodazole-treated cell pairs. The results suggest that some of the cortical sites described by Hyman and White are established cell autonomously (in P1, P2, and P3), and some are established by cell-cell contact (in EMS and E). Additional evidence presented here suggests that in the EMS cell, contact-dependent spindle orientation ensures a cleavage plane that will partition developmental information, received by induction, to one of EMS's daughter cells.

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