Essential roles of KIF4 and its binding partner PRC1 in organized central spindle midzone formation

Cytokinesis in animal cells depends on spindle-derived spatial cues that culminate in Rho activation, and thereby actomyosin assembly, in a narrow equatorial band. Although the nature, origin, and variety of such cues have long been obscure, one component is certainly the Rho activator Ect2. Here we describe the behavior and function of Ect2 in echinoderm embryos, showing that Ect2 migrates from spindle midzone to astral microtubules in anaphase and that Ect2 shapes the pattern of Rho activation in incipient furrows. Our key finding is that Ect2 and its binding partner Cyk4 accumulate not only at normal furrows, but also at furrows that form in the absence of associated spindle, midzone, or chromosomes. In all these cases, the cell assembles essentially the same cytokinetic signaling ensemble—opposed astral microtubules decorated with Ect2 and Cyk4. We conclude that if multiple signals contribute to furrow induction in echinoderm embryos, they likely converge on the same signaling ensemble on an analogous cytoskeletal scaffold.

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Tektin 2 is required for central spindle microtubule organization and the completion of cytokinesis

The Journal of Cell Biology ◽

10.1083/jcb.200711160 ◽

2008 ◽

Vol 181 (4) ◽

pp. 595-603 ◽

Cited By ~ 16

Author(s):

Thomas M. Durcan ◽

Elizabeth S. Halpin ◽

Trisha Rao ◽

Nicholas S. Collins ◽

Emily K. Tribble ◽

...

Keyword(s):

Cell Formation ◽

Binucleate Cell ◽

Aurora B ◽

Microtubule Organization ◽

Spindle Microtubule ◽

Central Spindle ◽

Daughter Cells ◽

Spindle Poles ◽

Spindle Midzone ◽

Interfering Rna

During anaphase, the nonkinetochore microtubules in the spindle midzone become compacted into the central spindle, a structure which is required to both initiate and complete cytokinesis. We show that Tektin 2 (Tek2) associates with the spindle poles throughout mitosis, organizes the spindle midzone microtubules during anaphase, and assembles into the midbody matrix surrounding the compacted midzone microtubules during cytokinesis. Tek2 small interfering RNA (siRNA) disrupts central spindle organization and proper localization of MKLP1, PRC1, and Aurora B to the midzone and prevents the formation of a midbody matrix. Video microscopy revealed that loss of Tek2 results in binucleate cell formation by aberrant fusion of daughter cells after cytokinesis. Although a myosin II inhibitor, blebbistatin, prevents actin-myosin contractility, the microtubules of the central spindle are compacted. Strikingly, Tek2 siRNA abolishes this actin-myosin–independent midzone microtubule compaction. Thus, Tek2-dependent organization of the central spindle during anaphase is essential for proper midbody formation and the segregation of daughter cells after cytokinesis.

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Aurora B and Kif2A control microtubule length for assembly of a functional central spindle during anaphase

The Journal of Cell Biology ◽

10.1083/jcb.201302123 ◽

2013 ◽

Vol 202 (4) ◽

pp. 623-636 ◽

Cited By ~ 45

Author(s):

Ryota Uehara ◽

Yuki Tsukada ◽

Tomoko Kamasaki ◽

Ina Poser ◽

Kinya Yoda ◽

...

Keyword(s):

Experimental Data ◽

Steady State ◽

Cell Division ◽

Self Organization ◽

Aurora B ◽

Spindle Formation ◽

Central Spindle ◽

Model Simulations ◽

Spindle Midzone ◽

Organization Mechanism

The central spindle is built during anaphase by coupling antiparallel microtubules (MTs) at a central overlap zone, which provides a signaling scaffold for the regulation of cytokinesis. The mechanisms underlying central spindle morphogenesis are still poorly understood. In this paper, we show that the MT depolymerase Kif2A controls the length and alignment of central spindle MTs through depolymerization at their minus ends. The distribution of Kif2A was limited to the distal ends of the central spindle through Aurora B–dependent phosphorylation and exclusion from the spindle midzone. Overactivation or inhibition of Kif2A affected interchromosomal MT length and disorganized the central spindle, resulting in uncoordinated cell division. Experimental data and model simulations suggest that the steady-state length of the central spindle and its symmetric position between segregating chromosomes are predominantly determined by the Aurora B activity gradient. On the basis of these results, we propose a robust self-organization mechanism for central spindle formation.

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Relocation of Aurora B from centromeres to the central spindle at the metaphase to anaphase transition requires MKlp2

The Journal of Cell Biology ◽

10.1083/jcb.200403084 ◽

2004 ◽

Vol 166 (2) ◽

pp. 167-172 ◽

Cited By ~ 192

Author(s):

Ulrike Gruneberg ◽

Rüdiger Neef ◽

Reiko Honda ◽

Erich A. Nigg ◽

Francis A. Barr

Keyword(s):

Chromosome Segregation ◽

Aurora B ◽

Dual Control ◽

Aurora B Kinase ◽

Central Spindle ◽

Binding Partner ◽

Mitotic Kinases ◽

Spatial Regulation

Mitotic kinases of the Polo and Aurora families are key regulators of chromosome segregation and cytokinesis. Here, we have investigated the role of MKlp1 and MKlp2, two vertebrate mitotic kinesins essential for cytokinesis, in the spatial regulation of the Aurora B kinase. Previously, we have demonstrated that MKlp2 recruits Polo-like kinase 1 (Plk1) to the central spindle in anaphase. We now find that in MKlp2 but not MKlp1-depleted cells the Aurora B–INCENP complex remains at the centromeres and fails to relocate to the central spindle. MKlp2 exerts dual control over Aurora B localization, because it is a binding partner for Aurora B, and furthermore for the phosphatase Cdc14A. Cdc14A can dephosphorylate INCENP and may contribute to its relocation to the central spindle in anaphase. We propose that MKlp2 is involved in the localization of Plk1, Aurora B, and Cdc14A to the central spindle during anaphase, and that the integration of signaling by these proteins is necessary for proper cytokinesis.

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Kinesin-like Protein CHO1 Is Required for the Formation of Midbody Matrix and the Completion of Cytokinesis in Mammalian Cells

Molecular Biology of the Cell ◽

10.1091/mbc.01-10-0504 ◽

2002 ◽

Vol 13 (6) ◽

pp. 1832-1845 ◽

Cited By ~ 97

Author(s):

Jurgita Matuliene ◽

Ryoko Kuriyama

Keyword(s):

Binding Site ◽

Mammalian Cells ◽

Cytoplasmic Bridge ◽

Central Spindle ◽

Atp Binding Site ◽

Daughter Cells ◽

Dividing Cells ◽

Spindle Midzone ◽

Segregation Of Chromosomes

CHO1 is a mammalian kinesin-like motor protein of the MKLP1 subfamily. It associates with the spindle midzone during anaphase and concentrates to a midbody matrix during cytokinesis. CHO1 was originally implicated in karyokinesis, but the invertebrate homologues of CHO1 were shown to function in the midzone formation and cytokinesis. To analyze the role of the protein in mammalian cells, we mutated the ATP-binding site of CHO1 and expressed it in CHO cells. Mutant protein (CHO1F′) was able to interact with microtubules via ATP-independent microtubule-binding site(s) but failed to accumulate at the midline of the central spindle and affected the localization of endogenous CHO1. Although the segregation of chromosomes, the bundling of midzone microtubules, and the initiation of cytokinesis proceeded normally in CHO1F′-expressing cells, the completion of cytokinesis was inhibited. Daughter cells were frequently entering interphase while connected by a microtubule-containing cytoplasmic bridge from which the dense midbody matrix was missing. Depletion of endogenous CHO1 via RNA-mediated interference also affected the formation of midbody matrix in dividing cells, caused the disorganization of midzone microtubules, and resulted in abortive cytokinesis. Thus, CHO1 may not be required for karyokinesis, but it is essential for the proper midzone/midbody formation and cytokinesis in mammalian cells.

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Kif15 functions as an active mechanical ratchet

Molecular Biology of the Cell ◽

10.1091/mbc.e18-03-0151 ◽

2018 ◽

Vol 29 (14) ◽

pp. 1743-1752 ◽

Cited By ~ 6

Author(s):

Toni McHugh ◽

Hauke Drechsler ◽

Andrew D. McAinsh ◽

Nicolas J. Carter ◽

Robert A. Cross

Keyword(s):

Optical Trap ◽

Spindle Assembly ◽

Central Spindle ◽

Bipolar Spindle ◽

Binding Partner ◽

Loading Direction

Kif15 is a kinesin-12 that contributes critically to bipolar spindle assembly in humans. Here we use force-ramp experiments in an optical trap to probe the mechanics of single Kif15 molecules under hindering or assisting loads and in a variety of nucleotide states. While unloaded Kif15 is established to be highly processive, we find that under hindering loads, Kif15 takes <∼10 steps. As hindering load is increased, Kif15 forestep:backstep ratio decreases exponentially, with stall occurring at 6 pN. In contrast, under assisting loads, Kif15 detaches readily and rapidly, even from its AMPPNP state. Kif15 mechanics thus depend markedly on the loading direction. Kif15 interacts with a binding partner, Tpx2, and we show that Tpx2 locks Kif15 to microtubules under both hindering and assisting loads. Overall, our data predict that Kif15 in the central spindle will act as a mechanical ratchet, supporting spindle extension but resisting spindle compression.

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The role of centrosomes and astral microtubules during asymmetric division of Drosophila neuroblasts

Development ◽

10.1242/dev.128.7.1137 ◽

2001 ◽

Vol 128 (7) ◽

pp. 1137-1145 ◽

Cited By ~ 5

Author(s):

M.G. Giansanti ◽

M. Gatti ◽

S. Bonaccorsi

Keyword(s):

Cell Fate ◽

Cleavage Plane ◽

Mitotic Division ◽

Spindle Orientation ◽

Central Spindle ◽

Spindle Rotation ◽

Spindle Midzone ◽

Fate Determinants ◽

Cell Fate Determinants

Drosophila neuroblasts are stem cells that divide asymmetrically to produce another large neuroblast and a smaller ganglion mother cell (GMC). During neuroblast division, several cell fate determinants, such as Miranda, Prospero and Numb, are preferentially segregated into the GMC, ensuring its correct developmental fate. The accurate segregation of these determinants relies on proper orientation of the mitotic spindle within the dividing neuroblast, and on the correct positioning of the cleavage plane. In this study we have analyzed the role of centrosomes and astral microtubules in neuroblast spindle orientation and cytokinesis. We examined neuroblast division in asterless (asl) mutants, which, although devoid of functional centrosomes and astral microtubules, form well-focused anastral spindles that undergo anaphase and telophase. We show that asl neuroblasts assemble a normal cytokinetic ring around the central spindle midzone and undergo unequal cytokinesis. Thus, astral microtubules are not required for either signaling or positioning cytokinesis in Drosophila neuroblasts. Our results indicate that the cleavage plane is dictated by the positioning of the central spindle midzone within the cell, and suggest a model on how the central spindle attains an asymmetric position during neuroblast mitosis. We have also analyzed the localization of Miranda during mitotic division of asl neuroblasts. This protein accumulates in morphologically regular cortical crescents but these crescents are mislocalized with respect to the spindle orientation. This suggests that astral microtubules mediate proper spindle rotation during neuroblast division.

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Chromokinesin Kif4 promotes proper anaphase in mouse oocyte meiosis

Molecular Biology of the Cell ◽

10.1091/mbc.e18-10-0666 ◽

2019 ◽

Vol 30 (14) ◽

pp. 1691-1704 ◽

Cited By ~ 6

Author(s):

Carissa M. Heath ◽

Sarah M. Wignall

Keyword(s):

Family Member ◽

Chromosome Segregation ◽

Mouse Oocyte ◽

Aurora B ◽

Localization Pattern ◽

Binding Partner ◽

Mouse Oocytes ◽

Oocyte Meiosis ◽

Spindle Midzone ◽

Insight Into

Oocytes of many species lack centrioles and therefore form acentriolar spindles. Despite the necessity of oocyte meiosis for successful reproduction, how these spindles mediate accurate chromosome segregation is poorly understood. We have gained insight into this process through studies of the kinesin-4 family member Kif4 in mouse oocytes. We found that Kif4 localizes to chromosomes through metaphase and then largely redistributes to the spindle midzone during anaphase, transitioning from stretches along microtubules to distinct ring-like structures; these structures then appear to fuse together by telophase. Kif4’s binding partner PRC1 and MgcRacGAP, a component of the centralspindlin complex, have a similar localization pattern, demonstrating dynamic spindle midzone organization in oocytes. Kif4 knockdown results in defective midzone formation and longer spindles, revealing new anaphase roles for Kif4 in mouse oocytes. Moreover, inhibition of Aurora B/C kinases results in Kif4 mislocalization and causes anaphase defects. Taken together, our work reveals essential roles for Kif4 during the meiotic divisions, furthering our understanding of mechanisms promoting accurate chromosome segregation in acentriolar oocytes.

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Functional midbody assembly in the absence of a central spindle

The Journal of Cell Biology ◽

10.1083/jcb.202011085 ◽

2022 ◽

Vol 221 (3) ◽

Author(s):

Sophia M. Hirsch ◽

Frances Edwards ◽

Mimi Shirasu-Hiza ◽

Julien Dumont ◽

Julie C. Canman

Keyword(s):

Microtubule Assembly ◽

Primary Role ◽

Intercellular Bridge ◽

Contractile Ring ◽

Spindle Microtubule ◽

Central Spindle ◽

Family Protein ◽

Spindle Midzone ◽

Ring Constriction ◽

Spindle Microtubules

Contractile ring constriction during cytokinesis is thought to compact central spindle microtubules to form the midbody, an antiparallel microtubule bundle at the intercellular bridge. In Caenorhabditis elegans, central spindle microtubule assembly requires targeting of the CLASP family protein CLS-2 to the kinetochores in metaphase and spindle midzone in anaphase. CLS-2 targeting is mediated by the CENP-F–like HCP-1/2, but their roles in cytokinesis and midbody assembly are not known. We found that although HCP-1 and HCP-2 mostly function cooperatively, HCP-1 plays a more primary role in promoting CLS-2–dependent central spindle microtubule assembly. HCP-1/2 codisrupted embryos did not form central spindles but completed cytokinesis and formed functional midbodies capable of supporting abscission. These central spindle–independent midbodies appeared to form via contractile ring constriction–driven bundling of astral microtubules at the furrow tip. This work suggests that, in the absence of a central spindle, astral microtubules can support midbody assembly and that midbody assembly is more predictive of successful cytokinesis than central spindle assembly.

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Intrinsic Stacking Interactions of Natural and Artificial Nucleobases

10.26434/chemrxiv.11400405 ◽

2019 ◽

Author(s):

Drew P. Harding ◽

Laura J. Kingsley ◽

Glen Spraggon ◽

Steven Wheeler

Keyword(s):

Gas Phase ◽

Electrostatic Interactions ◽

Density Functional ◽

Molecular Descriptors ◽

Stacking Interactions ◽

Interaction Energies ◽

Functional Theory ◽

Binding Partner ◽

Heavy Atoms ◽

Wide Range

The intrinsic (gas-phase) stacking energies of natural and artificial nucleobases were explored using density functional theory (DFT) and correlated ab initio methods. Ranking the stacking strength of natural nucleobase dimers revealed a preference in binding partner similar to that seen from experiments, namely G > C > A > T > U. Decomposition of these interaction energies using symmetry-adapted perturbation theory (SAPT) showed that these dispersion dominated interactions are modulated by electrostatics. Artificial nucleobases showed a similar stacking preference for natural nucleobases and were also modulated by electrostatic interactions. A robust predictive multivariate model was developed that quantitively predicts the maximum stacking interaction between natural and a wide range of artificial nucleobases using molecular descriptors based on computed electrostatic potentials (ESPs) and the number of heavy atoms. This model should find utility in designing artificial nucleobase analogs that exhibit stacking interactions comparable to those of natural nucleobases. Further analysis of the descriptors in this model unveil the origin of superior stacking abilities of certain nucleobases, including cytosine and guanine.

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