scholarly journals An Asp–CaM complex is required for centrosome–pole cohesion and centrosome inheritance in neural stem cells

2015 ◽  
Vol 211 (5) ◽  
pp. 987-998 ◽  
Author(s):  
Todd Schoborg ◽  
Allison L. Zajac ◽  
Carey J. Fagerstrom ◽  
Rodrigo X. Guillen ◽  
Nasser M. Rusan
Keyword(s):  
Stem Cells ◽  
Cell Polarity ◽  
Mitotic Spindle ◽  
Function Analysis ◽  
Tissue Homeostasis ◽  
Cell Cortex ◽  
Cross Link ◽  
Spindle Poles ◽  
Spindle Microtubules ◽  
Spindle Function

The interaction between centrosomes and mitotic spindle poles is important for efficient spindle formation, orientation, and cell polarity. However, our understanding of the dynamics of this relationship and implications for tissue homeostasis remains poorly understood. Here we report that Drosophila melanogaster calmodulin (CaM) regulates the ability of the microcephaly-associated protein, abnormal spindle (Asp), to cross-link spindle microtubules. Both proteins colocalize on spindles and move toward spindle poles, suggesting that they form a complex. Our binding and structure–function analysis support this hypothesis. Disruption of the Asp–CaM interaction alone leads to unfocused spindle poles and centrosome detachment. This behavior leads to randomly inherited centrosomes after neuroblast division. We further show that spindle polarity is maintained in neuroblasts despite centrosome detachment, with the poles remaining stably associated with the cell cortex. Finally, we provide evidence that CaM is required for Asp’s spindle function; however, it is completely dispensable for Asp’s role in microcephaly suppression.

scholarly journals Optogenetic EB1 inactivation shortens metaphase spindles by disrupting cortical force-producing interactions with astral microtubules

2021 ◽  
Author(s):  
Alessandro Dema ◽  
Jeffrey van Haren ◽  
Torsten Wittmann
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Live Cells ◽  
Cell Cortex ◽  
Reversible Inactivation ◽  
Molecular Systems ◽  
Spindle Poles ◽  
Spindle Dynamics ◽  
Spindle Function ◽  
Transient Relaxation

Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to spindle function: Kinetochore microtubules attach and transmit forces to chromosomes, antiparallel interpolar microtubules support spindle structure, and astral microtubules connect spindle poles to the cell cortex [1,2]. In mammalian cells, End Binding (EB) proteins associate with all growing microtubule plus ends throughout the cell cycle and serve as adaptors for a diverse group of +TIPs that control microtubule dynamics and interactions with other intracellular structures [3]. Because binding of many +TIPs to EB1 and thus microtubule-end association is switched off by mitotic phosphorylation [4-6] the mitotic function of EBs remains poorly understood. To analyze how EB1 and associated +TIPs on different spindle microtubule populations contribute to mitotic spindle dynamics, we use a light sensitive EB1 variant, π-EB1, that allows local, acute and reversible inactivation of +TIP association with growing microtubule ends in live cells [7]. We find that acute π-EB1 photoinactivation results in rapid and reversible metaphase spindle shortening and transient relaxation of tension across the central spindle. However, in contrast to interphase, π-EB1 photoinactivation does not inhibit microtubule growth in metaphase, but instead increases astral microtubule length and number. Yet, in the absence of EB1 activity astral microtubules fail to engage the cortical dynein/dynactin machinery and spindle poles move away from regions of π-EB1 photoinactivation. In conclusion, our optogenetic approach reveals mitotic EB1 functions that remain hidden in genetic experiments likely due to compensatory molecular systems regulating vertebrate spindle dynamics.

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 The kinesin-8 Kip3 scales anaphase spindle length by suppression of midzone microtubule polymerization

2014 ◽  
Vol 204 (6) ◽  
pp. 965-975 ◽  
Author(s):  
Rania S. Rizk ◽  
Katherine A. DiScipio ◽  
Kathleen G. Proudfoot ◽  
Mohan L. Gupta
Keyword(s):  
Mitotic Spindle ◽  
Molecular Mechanisms ◽  
Spindle Microtubule ◽  
Microtubule Polymerization ◽  
Sister Chromatids ◽  
Final Length ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
Spindle Function ◽  
Yeast Mutants

Mitotic spindle function is critical for cell division and genomic stability. During anaphase, the elongating spindle physically segregates the sister chromatids. However, the molecular mechanisms that determine the extent of anaphase spindle elongation remain largely unclear. In a screen of yeast mutants with altered spindle length, we identified the kinesin-8 Kip3 as essential to scale spindle length with cell size. Kip3 is a multifunctional motor protein with microtubule depolymerase, plus-end motility, and antiparallel sliding activities. Here we demonstrate that the depolymerase activity is indispensable to control spindle length, whereas the motility and sliding activities are not sufficient. Furthermore, the microtubule-destabilizing activity is required to counteract Stu2/XMAP215-mediated microtubule polymerization so that spindle elongation terminates once spindles reach the appropriate final length. Our data support a model where Kip3 directly suppresses spindle microtubule polymerization, limiting midzone length. As a result, sliding forces within the midzone cannot buckle spindle microtubules, which allows the cell boundary to define the extent of spindle elongation.

scholarly journals Chromosomal attachments set length and microtubule number in the Saccharomyces cerevisiae mitotic spindle

2014 ◽  
Vol 25 (25) ◽  
pp. 4034-4048 ◽  
Author(s):  
Natalie J. Nannas ◽  
Eileen T. O’Toole ◽  
Mark Winey ◽  
Andrew W. Murray
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Simple Model ◽  
Mitotic Spindle ◽  
Cell Types ◽  
Yeast Saccharomyces Cerevisiae ◽  
Spindle Poles ◽  
Length Regulation ◽  
Spindle Microtubules ◽  
Different Cell Types

The length of the mitotic spindle varies among different cell types. A simple model for spindle length regulation requires balancing two forces: pulling, due to micro­tubules that attach to the chromosomes at their kinetochores, and pushing, due to interactions between microtubules that emanate from opposite spindle poles. In the budding yeast Saccharomyces cerevisiae, we show that spindle length scales with kinetochore number, increasing when kinetochores are inactivated and shortening on addition of synthetic or natural kinetochores, showing that kinetochore–microtubule interactions generate an inward force to balance forces that elongate the spindle. Electron microscopy shows that manipulating kinetochore number alters the number of spindle microtubules: adding extra kinetochores increases the number of spindle microtubules, suggesting kinetochore-based regulation of microtubule number.

scholarly journals Complexities of Microtubule Population Dynamics within the Mitotic Spindle

2019 ◽  
Author(s):  
Aaron R. Tipton ◽  
Gary J. Gorbsky
Keyword(s):  
Mitotic Spindle ◽  
Cell Cortex ◽  
Spindle Microtubule ◽  
Sister Chromatids ◽  
Two Component ◽  
Chromosome Attachment ◽  
Complex Landscape ◽  
Spindle Microtubules ◽  
Insight Into ◽  
Slow Turnover

AbstractThe mitotic spindle functions to move chromosomes to alignment at metaphase, then segregate sister chromatids during anaphase. Analysis of spindle microtubule kinetics utilizing fluorescence dissipation after photoactivation described two main populations, a slow and a fast turnover population, historically taken to reflect kinetochore versus non-kinetochore microtubules respectively. This two component demarcation seems likely oversimplified. Microtubule turnover may vary among different spindle microtubules, regulated by spatial distribution and interactions with other microtubules and with organelles such as kinetochores, chromosome arms, and the cell cortex. How turnover among various spindle microtubules is differentially regulated and its significance remains unclear. We tested the concept of kinetochore versus non-kinetochore microtubules by disrupting kinetochores through depletion of the Ndc80 complex. In the absence of functional kinetochores, microtubule dynamics still exhibited slow and fast turnover populations, though proportions and timings of turnover were altered. Importantly, the data obtained following Hec1/Ndc80 depletion suggests other sub-populations, in addition to kinetochore microtubules, contribute to the slow turnover population. Further manipulation of spindle microtubules revealed a complex landscape. Dissection of the dynamics of microtubule populations will provide a greater understanding of mitotic spindle kinetics and insight into roles in facilitating chromosome attachment, movement, and segregation during mitosis.

scholarly journals SLK-dependent activation of ERMs controls LGN–NuMA localization and spindle orientation

2014 ◽  
Vol 205 (6) ◽  
pp. 791-799 ◽  
Author(s):  
Mickael Machicoane ◽  
Cristina A. de Frutos ◽  
Jenny Fink ◽  
Murielle Rocancourt ◽  
Yannis Lombardi ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Molecular Level ◽  
Spindle Orientation ◽  
Cell Cortex ◽  
Mitotic Apparatus ◽  
Mitotic Entry ◽  
Spindle Microtubules ◽  
Force Generator

Mitotic spindle orientation relies on a complex dialog between the spindle microtubules and the cell cortex, in which F-actin has been recently implicated. Here, we report that the membrane–actin linkers ezrin/radixin/moesin (ERMs) are strongly and directly activated by the Ste20-like kinase at mitotic entry in mammalian cells. Using microfabricated adhesive substrates to control the axis of cell division, we found that the activation of ERMs plays a key role in guiding the orientation of the mitotic spindle. Accordingly, impairing ERM activation in apical progenitors of the mouse embryonic neocortex severely disturbed spindle orientation in vivo. At the molecular level, ERM activation promotes the polarized association at the mitotic cortex of leucine-glycine-asparagine repeat protein (LGN) and nuclear mitotic apparatus (NuMA) protein, two essential factors for spindle orientation. We propose that activated ERMs, together with Gαi, are critical for the correct localization of LGN–NuMA force generator complexes and hence for proper spindle orientation.

scholarly journals Cytokinetic astralogy

2009 ◽  
Vol 187 (6) ◽  
pp. 757-759 ◽  
Author(s):  
Julie C. Canman
Keyword(s):  
Mitotic Spindle ◽  
Direct Contact ◽  
Cell Cortex ◽  
Animal Cells ◽  
Division Plane ◽  
Cell Biol ◽  
Spindle Microtubules

Division plane specification in animal cells has long been presumed to involve direct contact between microtubules of the anaphase mitotic spindle and the cell cortex. In this issue, von Dassow et al. (von Dassow et al. 2009. J. Cell. Biol. doi:10.1083/jcb.200907090) challenge this assumption by showing that spindle microtubules can effectively position the division plane at a distance from the cell cortex.

scholarly journals Mechanical and genetic separation of aster- and midzone-positioned cytokinesis

2008 ◽  
Vol 36 (3) ◽  
pp. 381-383 ◽  
Author(s):  
Henrik Bringmann
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Mitotic Spindle ◽  
Heterotrimeric G Proteins ◽  
Contractile Ring ◽  
Spindle Poles ◽  
Spindle Midzone ◽  
Spindle Microtubules ◽  
Two Populations ◽  
Provided Examples

The mitotic spindle positions the cytokinesis furrow. The cytokinesis furrow then forms and ingresses at the site of the mitotic spindle, between the spindle poles. Two populations of spindle microtubules are implicated in cytokinesis furrow positioning: radial microtubule arrays called asters and bundled non-kinetochore microtubules called the spindle midzone. Here I will discuss our recent results that provided examples of how aster-positioned and midzone-positioned cytokinesis can be mechanically and genetically separated. These experiments illustrate how asters and midzone contribute to cytokinesis. ASS (asymmetric spindle severing) is a mechanical way to spatially separate the aster and midzone signals. In Caenorhabditis elegans embryos, asters and midzone provide two consecutive signals that position the cytokinesis furrow. The first signal is positioned midway between the microtubule asters; the second signal is positioned over the spindle midzone. Aster and midzone contribution can also be genetically separated. Mutants in spd-1 have no detectable midzone and are defective in midzone-positioned but not aster-positioned cytokinesis. Disruption of the function of LET-99 and the heterotrimeric G-proteins GOA-1/GPA-16 and their regulator GPR-1/2 causes defects in aster-positioned cytokinesis but not in midzone-positioned cytokinesis. In order to understand aster-positioned cytokinesis we have to understand how microtubule asters spatially control the activity of LET-99, GPR-1/2 and GOA-1/GPA-16 and how the activity of these G-protein pathway components control the assembly of a contractile ring.

scholarly journals Microtubule pivoting enables mitotic spindle assembly in S. cerevisiae

2021 ◽  
Vol 220 (3) ◽  
Author(s):  
Kimberly K. Fong ◽  
Trisha N. Davis ◽  
Charles L. Asbury
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Force Generation ◽  
Initial Contact ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Mitotic Spindle Assembly ◽  
Spindle Microtubules ◽  
Pole Component

To assemble a bipolar spindle, microtubules emanating from two poles must bundle into an antiparallel midzone, where plus end–directed motors generate outward pushing forces to drive pole separation. Midzone cross-linkers and motors display only modest preferences for antiparallel filaments, and duplicated poles are initially tethered together, an arrangement that instead favors parallel interactions. Pivoting of microtubules around spindle poles might help overcome this geometric bias, but the intrinsic pivoting flexibility of the microtubule–pole interface has not been directly measured, nor has its importance during early spindle assembly been tested. By measuring the pivoting of microtubules around isolated yeast spindle poles, we show that pivoting flexibility can be modified by mutating a microtubule-anchoring pole component, Spc110. By engineering mutants with different flexibilities, we establish the importance of pivoting in vivo for timely pole separation. Our results suggest that passive thermal pivoting can bring microtubules from side-by-side poles into initial contact, but active minus end–directed force generation will be needed to achieve antiparallel alignment.

scholarly journals Fission yeast pkl1 is a kinesin-related protein involved in mitotic spindle function.

1996 ◽  
Vol 7 (10) ◽  
pp. 1639-1655 ◽  
Author(s):  
A L Pidoux ◽  
M LeDizet ◽  
W Z Cande
Keyword(s):  
Fission Yeast ◽  
Mitotic Spindle ◽  
Related Protein ◽  
Microtubule Organization ◽  
Spindle Poles ◽  
Similar Phenotype ◽  
Increased Sensitivity ◽  
Spindle Function ◽  
Related Proteins ◽  
Very High

We have used anti-peptide antibodies raised against highly conserved regions of the kinesin motor domain to identify kinesin-related proteins in the fission yeast Schizosaccharomyces pombe. Here we report the identification of a new kinesin-related protein, which we have named pkl1. Sequence homology and domain organization place pkl1 in the Kar3/ncd subfamily of kinesin-related proteins. Bacterially expressed pkl1 fusion proteins display microtubule-stimulated ATPase activity, nucleotide-sensitive binding, and bundling of microtubules. Immunofluorescence studies with affinity-purified antibodies indicate that the pkl1 protein localizes to the nucleus and the mitotic spindle. Pkl1 null mutants are viable but have increased sensitivity to microtubule-disrupting drugs. Disruption of pkl1+ suppresses mutations in another kinesin-related protein, cut7, which is known to act in the spindle. Overexpression of pkl1 to very high levels causes a similar phenotype to that seen in cut7 mutants: V-shaped and star-shaped microtubule structures are observed, which we interpret to be spindles with unseparated spindle poles. These observations suggest that pkl1 and cut7 provide opposing forces in the spindle. We propose that pkl1 functions as a microtubule-dependent motor that is involved in microtubule organization in the mitotic spindle.

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