TPX2 regulates the localization and activity of Eg5 in the mammalian mitotic spindle

Mitotic spindle assembly requires the regulated activity of numerous spindle-associated proteins. In mammalian cells, the Kinesin-5 motor Eg5 interacts with the spindle assembly factor TPX2, but how this interaction contributes to spindle formation and function is not established. Using bacterial artificial chromosome technology, we generated cells expressing TPX2 lacking the Eg5 interaction domain. Spindles in these cells were highly disorganized with multiple spindle poles. The TPX2–Eg5 interaction was required for kinetochore fiber formation and contributed to Eg5 localization to spindle microtubules but not spindle poles. Microinjection of the Eg5-binding domain of TPX2 resulted in spindle elongation, indicating that the interaction of Eg5 with TPX2 reduces motor activity. Consistent with this possibility, we found that TPX2 reduced the velocity of Eg5-dependent microtubule gliding, inhibited microtubule sliding, and resulted in the accumulation of motor on microtubules. These results establish a novel function of TPX2 in regulating the location and activity of the mitotic motor Eg5.

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Poleward Transport of TPX2 in the Mammalian Mitotic Spindle Requires Dynein, Eg5, and Microtubule Flux

Molecular Biology of the Cell ◽

10.1091/mbc.e09-07-0601 ◽

2010 ◽

Vol 21 (6) ◽

pp. 979-988 ◽

Cited By ~ 54

Author(s):

Nan Ma ◽

U. S. Tulu ◽

Nick P. Ferenz ◽

Carey Fagerstrom ◽

Andrew Wilde ◽

...

Keyword(s):

Mitotic Spindle ◽

Spindle Assembly ◽

Fiber Formation ◽

Aurora A ◽

Mitotic Progression ◽

Mitotic Kinase ◽

Spindle Poles ◽

C Terminus ◽

Assembly Factor ◽

Microtubule Formation

TPX2 is a Ran-regulated spindle assembly factor that is required for kinetochore fiber formation and activation of the mitotic kinase Aurora A. TPX2 is enriched near spindle poles and is required near kinetochores, suggesting that it undergoes dynamic relocalization throughout mitosis. Using photoactivation, we measured the movement of PA-GFP-TPX2 in the mitotic spindle. TPX2 moves poleward in the half-spindle and is static in the interzone and near spindle poles. Poleward transport of TPX2 is sensitive to inhibition of dynein or Eg5 and to suppression of microtubule flux with nocodazole or antibodies to Kif2a. Poleward transport requires the C terminus of TPX2, a domain that interacts with Eg5. Overexpression of TPX2 lacking this domain induced excessive microtubule formation near kinetochores, defects in spindle assembly and blocked mitotic progression. Our data support a model in which poleward transport of TPX2 down-regulates its microtubule nucleating activity near kinetochores and links microtubules generated at kinetochores to dynein for incorporation into the spindle.

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NuMA regulates mitotic spindle assembly, structural dynamics and function via phase separation

Nature Communications ◽

10.1038/s41467-021-27528-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Mengjie Sun ◽

Mingkang Jia ◽

He Ren ◽

Biying Yang ◽

Wangfei Chi ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Structural Dynamics ◽

Mitotic Spindle ◽

Spindle Assembly ◽

Spindle Poles ◽

Mitotic Spindle Assembly ◽

Dynamics And Function ◽

And Function ◽

Liquid Liquid Phase Separation

AbstractA functional mitotic spindle is essential for accurate chromosome congression and segregation during cell proliferation; however, the underlying mechanisms of its assembly remain unclear. Here we show that NuMA regulates this assembly process via phase separation regulated by Aurora A. NuMA undergoes liquid-liquid phase separation during mitotic entry and KifC1 facilitates NuMA condensates concentrating on spindle poles. Phase separation of NuMA is mediated by its C-terminus, whereas its dynein-dynactin binding motif also facilitates this process. Phase-separated NuMA droplets concentrate tubulins, bind microtubules, and enrich crucial regulators, including Kif2A, at the spindle poles, which then depolymerizes spindle microtubules and promotes poleward spindle microtubule flux for spindle assembly and structural dynamics. In this work, we show that NuMA orchestrates mitotic spindle assembly, structural dynamics and function via liquid-liquid phase separation regulated by Aurora A phosphorylation.

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A centriolar FGR1 oncogene partner-like protein required for paraflagellar rod assembly, but not axoneme assembly in African trypanosomes

Open Biology ◽

10.1098/rsob.170218 ◽

2018 ◽

Vol 8 (7) ◽

pp. 170218 ◽

Cited By ~ 3

Author(s):

Jane Harmer ◽

Katie Towers ◽

Max Addison ◽

Sue Vaughan ◽

Michael L. Ginger ◽

...

Keyword(s):

Basal Body ◽

Mitotic Spindle ◽

Cell Biology ◽

Mammalian Cells ◽

Molecular Level ◽

African Trypanosomes ◽

Spindle Poles ◽

Microtubule Organizing Centres ◽

Polar Spindle ◽

And Function

Proteins of the FGR1 oncogene partner (or FOP) family are found at microtubule organizing centres (MTOCs) including, in flagellate eukaryotes, the centriole or flagellar basal body from which the axoneme extends. We report conservation of FOP family proteins, Tb FOPL and Tb OFD1, in the evolutionarily divergent sleeping sickness parasite Trypanosoma brucei , showing (in contrast with mammalian cells, where FOP is essential for flagellum assembly) depletion of a trypanosome FOP homologue, Tb FOPL, affects neither axoneme nor flagellum elongation. Instead, Tb FOPL depletion causes catastrophic failure in assembly of a lineage-specific, extra-axonemal structure, the paraflagellar rod (PFR). That depletion of centriolar Tb FOPL causes failure in PFR assembly is surprising because PFR nucleation commences approximately 2 µm distal from the basal body. When over-expressed with a C-terminal myc-epitope, Tb FOPL was also observed at mitotic spindle poles. Little is known about bi-polar spindle assembly during closed trypanosome mitosis, but indication of a possible additional MTOC function for Tb FOPL parallels MTOC localization of FOP-like protein TONNEAU1 in acentriolar plants. More generally, our functional analysis of Tb FOPL emphasizes significant differences in evolutionary cell biology trajectories of FOP-family proteins. We discuss how at the molecular level FOP homologues may contribute to flagellum assembly and function in diverse flagellates.

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Kinesin-5 Is Dispensable for Bipolar Spindle Formation and Elongation in Candida albicans, but Simultaneous Loss of Kinesin-14 Activity Is Lethal

mSphere ◽

10.1128/msphere.00610-19 ◽

2019 ◽

Vol 4 (6) ◽

Cited By ~ 1

Author(s):

Irsa Shoukat ◽

Corey Frazer ◽

John S. Allingham

Keyword(s):

Candida Albicans ◽

Mitotic Spindle ◽

Fungal Pathogens ◽

Spindle Assembly ◽

Spindle Pole ◽

Content Type ◽

Bipolar Spindle ◽

Spindle Elongation ◽

Simultaneous Loss ◽

Bipolar Spindles

ABSTRACT Mitotic spindles assume a bipolar architecture through the concerted actions of microtubules, motors, and cross-linking proteins. In most eukaryotes, kinesin-5 motors are essential to this process, and cells will fail to form a bipolar spindle without kinesin-5 activity. Remarkably, inactivation of kinesin-14 motors can rescue this kinesin-5 deficiency by reestablishing the balance of antagonistic forces needed to drive spindle pole separation and spindle assembly. We show that the yeast form of the opportunistic fungus Candida albicans assembles bipolar spindles in the absence of its sole kinesin-5, CaKip1, even though this motor exhibits stereotypical cell-cycle-dependent localization patterns within the mitotic spindle. However, cells lacking CaKip1 function have shorter metaphase spindles and longer and more numerous astral microtubules. They also show defective hyphal development. Interestingly, a small population of CaKip1-deficient spindles break apart and reform two bipolar spindles in a single nucleus. These spindles then separate, dividing the nucleus, and then elongate simultaneously in the mother and bud or across the bud neck, resulting in multinucleate cells. These data suggest that kinesin-5-independent mechanisms drive assembly and elongation of the mitotic spindle in C. albicans and that CaKip1 is important for bipolar spindle integrity. We also found that simultaneous loss of kinesin-5 and kinesin-14 (CaKar3Cik1) activity is lethal. This implies a divergence from the antagonistic force paradigm that has been ascribed to these motors, which could be linked to the high mitotic error rate that C. albicans experiences and often exploits as a generator of diversity. IMPORTANCE Candida albicans is one of the most prevalent fungal pathogens of humans and can infect a broad range of niches within its host. This organism frequently acquires resistance to antifungal agents through rapid generation of genetic diversity, with aneuploidy serving as a particularly important adaptive mechanism. This paper describes an investigation of the sole kinesin-5 in C. albicans, which is a major regulator of chromosome segregation. Contrary to other eukaryotes studied thus far, C. albicans does not require kinesin-5 function for bipolar spindle assembly or spindle elongation. Rather, this motor protein associates with the spindle throughout mitosis to maintain spindle integrity. Furthermore, kinesin-5 loss is synthetically lethal with loss of kinesin-14—canonically an opposing force producer to kinesin-5 in spindle assembly and anaphase. These results suggest a significant evolutionary rewiring of microtubule motor functions in the C. albicans mitotic spindle, which may have implications in the genetic instability of this pathogen.

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Mechanisms of Mitotic Spindle Assembly and Function

International Review of Cytology - A Survey of Cell Biology ◽

10.1016/s0074-7696(07)65003-7 ◽

2008 ◽

pp. 111-158 ◽

Cited By ~ 243

Author(s):

Claire E. Walczak ◽

Rebecca Heald

Keyword(s):

Mitotic Spindle ◽

Spindle Assembly ◽

Mitotic Spindle Assembly ◽

And Function

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ULTRASTRUCTURAL ANALYSIS OF MITOTIC SPINDLE ELONGATION IN MAMMALIAN CELLS IN VITRO

The Journal of Cell Biology ◽

10.1083/jcb.50.2.416 ◽

1971 ◽

Vol 50 (2) ◽

pp. 416-431 ◽

Cited By ~ 114

Author(s):

B. R. Brinkley ◽

Joiner Cartwright

Keyword(s):

Mitotic Spindle ◽

Mammalian Cells ◽

Chinese Hamster ◽

Spindle Axis ◽

Hamster Strain ◽

Early Anaphase ◽

Spindle Microtubules ◽

Spindle Elongation ◽

Elongation Process

The mitotic spindle of many mammalian cells undergoes an abrupt elongation at anaphase. In both cultured rat kangaroo (strain PtK1) and Chinese hamster (strain Don-C) fibroblasts, the distance from pole to pole at metaphase doubles during anaphase and telophase. In order to determine the organization and distribution of spindle microtubules during the elongation process, cells were fixed and flat embedded in Epon 812. Selected cells were photographed with the phase-contrast microscope and then serially sectioned perpendicular to the major spindle axis. Microtubule profiles were counted in selected sections, and the number was plotted with respect to position along the spindle axis. Interpretation of the distribution profiles indicated that not all interpolar microtubules extended from pole to pole. It is estimated that 55–70% of the interpolar microtubules are overlapped at the cell equator while 30–45% extend across the equator into both half spindles. This arrangement appeared to persist from early anaphase (before elongation) until telophase after the elongation process. Although sliding or shearing of microtubules may occur in the spindle, such appears not to be the mechanism by which the spindle elongates in anaphase. Instead, our data support the hypothesis that spindle elongation occurs by growth of prepositioned microtubules which "push" the poles apart.

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The G-protein regulatory (GPR) motif-containing Leu–Gly–Asn-enriched protein (LGN) and Giα3 influence cortical positioning of the mitotic spindle poles at metaphase in symmetrically dividing mammalian cells

European Journal of Cell Biology ◽

10.1016/j.ejcb.2006.08.002 ◽

2006 ◽

Vol 85 (12) ◽

pp. 1233-1240 ◽

Cited By ~ 28

Author(s):

Joe B. Blumer ◽

Ryoko Kuriyama ◽

Thomas W. Gettys ◽

Stephen M. Lanier

Keyword(s):

G Protein ◽

Mitotic Spindle ◽

Mammalian Cells ◽

Spindle Poles

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The Nek6 and Nek7 Protein Kinases Are Required for Robust Mitotic Spindle Formation and Cytokinesis

Molecular and Cellular Biology ◽

10.1128/mcb.01867-08 ◽

2009 ◽

Vol 29 (14) ◽

pp. 3975-3990 ◽

Cited By ~ 98

Author(s):

Laura O'Regan ◽

Andrew M. Fry

Keyword(s):

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Mitotic Spindles ◽

Mitotic Progression ◽

Serine Threonine Kinase ◽

Spindle Formation ◽

Spindle Poles ◽

And Function ◽

Interfering Rna ◽

Reduced Activity

ABSTRACT Nek6 and Nek7 are members of the NIMA-related serine/threonine kinase family. Previous work showed that they contribute to mitotic progression downstream of another NIMA-related kinase, Nek9, although the roles of these different kinases remain to be defined. Here, we carried out a comprehensive analysis of the regulation and function of Nek6 and Nek7 in human cells. By generating specific antibodies, we show that both Nek6 and Nek7 are activated in mitosis and that interfering with their activity by either depletion or expression of reduced-activity mutants leads to mitotic arrest and apoptosis. Interestingly, while completely inactive mutants and small interfering RNA-mediated depletion delay cells at metaphase with fragile mitotic spindles, hypomorphic mutants or RNA interference treatment combined with a spindle assembly checkpoint inhibitor delays cells at cytokinesis. Importantly, depletion of either Nek6 or Nek7 leads to defective mitotic progression, indicating that although highly similar, they are not redundant. Indeed, while both kinases localize to spindle poles, only Nek6 obviously localizes to spindle microtubules in metaphase and anaphase and to the midbody during cytokinesis. Together, these data lead us to propose that Nek6 and Nek7 play independent roles not only in robust mitotic spindle formation but also potentially in cytokinesis.

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Rapid microtubule-independent dynamics of Cdc20 at kinetochores and centrosomes in mammalian cells

The Journal of Cell Biology ◽

10.1083/jcb.200201135 ◽

2002 ◽

Vol 158 (5) ◽

pp. 841-847 ◽

Cited By ~ 90

Author(s):

Marko J. Kallio ◽

Victoria A. Beardmore ◽

Jasminder Weinstein ◽

Gary J. Gorbsky

Keyword(s):

Mitotic Spindle ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Anaphase Promoting Complex ◽

Late Prophase ◽

Checkpoint Signaling ◽

Spindle Poles ◽

Anaphase Onset ◽

Late Telophase ◽

Green Fluorescent

Cdc20 is a substrate adaptor and activator of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase whose activity is required for anaphase onset and exit from mitosis. A green fluorescent protein derivative, Cdc20–GFP, bound to centrosomes throughout the cell cycle and to kinetochores from late prophase to late telophase. We mapped distinct domains of Cdc20 that are required for association with kinetochores and centrosomes. FRAP measurements revealed extremely rapid dynamics at the kinetochores (t1/2 = 5.1 s) and spindle poles (t1/2 = 4.7 s). This rapid turnover is independent of microtubules. Rapid transit of Cdc20 through kinetochores may ensure that spindle checkpoint signaling at unattached/relaxed kinetochores can continuously inhibit APC/CCdc20 targeting of anaphase inhibitors (securins) throughout the cell until all the chromosomes are properly attached to the mitotic spindle.

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