scholarly journals KLP-7 acts through the Ndc80 complex to limit pole number in C. elegans oocyte meiotic spindle assembly

2015 ◽  
Vol 210 (6) ◽  
pp. 917-932 ◽  
Author(s):  
Amy A. Connolly ◽  
Kenji Sugioka ◽  
Chien-Hui Chuang ◽  
Joshua B. Lowry ◽  
Bruce Bowerman
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Meiotic Spindle ◽  
Meiotic Cell ◽  
Bipolar Structure ◽  
C Elegans ◽  
Spindle Poles ◽  
Ndc80 Complex ◽  
Bipolar Spindles

During oocyte meiotic cell division in many animals, bipolar spindles assemble in the absence of centrosomes, but the mechanisms that restrict pole assembly to a bipolar state are unknown. We show that KLP-7, the single mitotic centromere–associated kinesin (MCAK)/kinesin-13 in Caenorhabditis elegans, is required for bipolar oocyte meiotic spindle assembly. In klp-7(−) mutants, extra microtubules accumulated, extra functional spindle poles assembled, and chromosomes frequently segregated as three distinct masses during meiosis I anaphase. Moreover, reducing KLP-7 function in monopolar klp-18(−) mutants often restored spindle bipolarity and chromosome segregation. MCAKs act at kinetochores to correct improper kinetochore–microtubule (k–MT) attachments, and depletion of the Ndc-80 kinetochore complex, which binds microtubules to mediate kinetochore attachment, restored bipolarity in klp-7(−) mutant oocytes. We propose a model in which KLP-7/MCAK regulates k–MT attachment and spindle tension to promote the coalescence of early spindle pole foci that produces a bipolar structure during the acentrosomal process of oocyte meiotic spindle assembly.

scholarly journals Multiple motors cooperate to establish and maintain acentrosomal spindle bipolarity in C. elegans oocyte meiosis

2021 ◽  
Author(s):  
Gabriel Cavin-Meza ◽  
Michelle M. Kwan ◽  
Sarah M. Wignall
Keyword(s):  
Chromosome Segregation ◽  
Meiotic Spindle ◽  
Complete Dissolution ◽  
C Elegans ◽  
Spindle Poles ◽  
Oocyte Meiosis ◽  
Monopolar Spindle ◽  
Spindle Stability ◽  
Bipolar Spindles ◽  
Spindle Structure

ABSTRACTWhile centrosomes organize spindle poles during mitosis, oocyte meiosis can occur in their absence. Spindles in human oocytes frequently fail to maintain bipolarity and consequently undergo chromosome segregation errors, making it important to understand mechanisms that promote acentrosomal spindle stability. To this end, we have optimized the auxin-inducible degron system in C. elegans to remove factors from pre-formed oocyte spindles within minutes and assess effects on spindle structure. This approach revealed that dynein is required to maintain the integrity of acentrosomal poles; removal of dynein from bipolar spindles caused pole splaying, and when coupled with a monopolar spindle induced by depletion of kinesin-12 motor KLP-18, dynein depletion led to a complete dissolution of the monopole. Surprisingly, we went on to discover that following monopole disruption, individual chromosomes were able to reorganize local microtubules and re-establish a miniature bipolar spindle that mediated chromosome segregation. This revealed the existence of redundant microtubule sorting forces that are undetectable when KLP-18 and dynein are active. We found that the kinesin-5 family motor BMK-1 provides this force, uncovering the first evidence that kinesin-5 contributes to C. elegans meiotic spindle organization. Altogether, our studies have revealed how multiple motors are working synchronously to establish and maintain bipolarity in the absence of centrosomes.

scholarly journals Kinetochore-mediated outward force promotes spindle pole separation in fission yeast

2019 ◽  
Vol 30 (22) ◽  
pp. 2802-2813 ◽  
Author(s):  
Yutaka Shirasugi ◽  
Masamitsu Sato
Keyword(s):  
Fission Yeast ◽  
Motor Proteins ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Double Knockout ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Bipolar Spindles ◽  
Meiosis I

Bipolar spindles are organized by motor proteins that generate microtubule-­dependent forces to separate the two spindle poles. The fission yeast Cut7 (kinesin-5) is a plus-end-directed motor that generates the outward force to separate the two spindle poles, whereas the minus-end-directed motor Pkl1 (kinesin-14) generates the inward force. Balanced forces by these antagonizing kinesins are essential for bipolar spindle organization in mitosis. Here, we demonstrate that chromosomes generate another outward force that contributes to the bipolar spindle assembly. First, it was noted that the cut7 pkl1 double knockout failed to separate spindle poles in meiosis I, although the mutant is known to succeed it in mitosis. It was assumed that this might be because meiotic kinetochores of bivalent chromosomes joined by cross-overs generate weaker tensions in meiosis I than the strong tensions in mitosis generated by tightly tethered sister kinetochores. In line with this idea, when meiotic mono-oriented kinetochores were artificially converted to a mitotic bioriented layout, the cut7 pkl1 mutant successfully separated spindle poles in meiosis I. Therefore, we propose that spindle pole separation is promoted by outward forces transmitted from kinetochores to spindle poles through microtubules.

scholarly journals CDK-1 inhibits meiotic spindle shortening and dynein-dependent spindle rotation in C. elegans

2011 ◽  
Vol 193 (7) ◽  
pp. 1229-1244 ◽  
Author(s):  
Marina L. Ellefson ◽  
Francis J. McNally
Keyword(s):  
Polar Body ◽  
Spindle Pole ◽  
Cyclin B ◽  
Meiotic Spindle ◽  
Anaphase Promoting Complex ◽  
C Elegans ◽  
Perpendicular Orientation ◽  
Spindle Poles ◽  
Spindle Rotation ◽  
Chromosome Separation

In animals, the female meiotic spindle is positioned at the egg cortex in a perpendicular orientation to facilitate the disposal of half of the chromosomes into a polar body. In Caenorhabditis elegans, the metaphase spindle lies parallel to the cortex, dynein is dispersed on the spindle, and the dynein activators ASPM-1 and LIN-5 are concentrated at spindle poles. Anaphase-promoting complex (APC) activation results in dynein accumulation at spindle poles and dynein-dependent rotation of one spindle pole to the cortex, resulting in perpendicular orientation. To test whether the APC initiates spindle rotation through cyclin B–CDK-1 inactivation, separase activation, or degradation of an unknown dynein inhibitor, CDK-1 was inhibited with purvalanol A in metaphase-I–arrested, APC-depleted embryos. CDK-1 inhibition resulted in the accumulation of dynein at spindle poles and dynein-dependent spindle rotation without chromosome separation. These results suggest that CDK-1 blocks rotation by inhibiting dynein association with microtubules and with LIN-5–ASPM-1 at meiotic spindle poles and that the APC promotes spindle rotation by inhibiting CDK-1.

scholarly journals Katanin controls mitotic and meiotic spindle length

2006 ◽  
Vol 175 (6) ◽  
pp. 881-891 ◽  
Author(s):  
Karen McNally ◽  
Anjon Audhya ◽  
Karen Oegema ◽  
Francis J. McNally
Keyword(s):  
Chromosome Segregation ◽  
Eukaryotic Cell ◽  
Meiotic Spindle ◽  
Mitotic Spindles ◽  
Wild Type ◽  
Female Meiosis ◽  
C Elegans ◽  
Microtubule Severing ◽  
Spindle Poles ◽  
Type C

Accurate control of spindle length is a conserved feature of eukaryotic cell division. Lengthening of mitotic spindles contributes to chromosome segregation and cytokinesis during mitosis in animals and fungi. In contrast, spindle shortening may contribute to conservation of egg cytoplasm during female meiosis. Katanin is a microtubule-severing enzyme that is concentrated at mitotic and meiotic spindle poles in animals. We show that inhibition of katanin slows the rate of spindle shortening in nocodazole-treated mammalian fibroblasts and in untreated Caenorhabditis elegans meiotic embryos. Wild-type C. elegans meiotic spindle shortening proceeds through an early katanin-independent phase marked by increasing microtubule density and a second, katanin-dependent phase that occurs after microtubule density stops increasing. In addition, double-mutant analysis indicated that γ-tubulin–dependent nucleation and microtubule severing may provide redundant mechanisms for increasing microtubule number during the early stages of meiotic spindle assembly.

scholarly journals Differentiation of Cytoplasmic and Meiotic Spindle Assembly MCAK Functions by Aurora B-dependent Phosphorylation

2004 ◽  
Vol 15 (6) ◽  
pp. 2895-2906 ◽  
Author(s):  
Ryoma Ohi ◽  
Tanuj Sapra ◽  
Jonathan Howard ◽  
Timothy J. Mitchison
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly ◽  
Microtubule Dynamics ◽  
Meiotic Spindle ◽  
Aurora B ◽  
Dependent Manner ◽  
Dynamic Nature ◽  
Spindle Microtubule ◽  
Bipolar Spindles

The KinI kinesin MCAK is a microtubule depolymerase important for governing spindle microtubule dynamics during chromosome segregation. The dynamic nature of spindle assembly and chromosome-microtubule interactions suggest that mechanisms must exist that modulate the activity of MCAK, both spatially and temporally. In Xenopus extracts, MCAK associates with and is stimulated by the inner centromere protein ICIS. The inner centromere kinase Aurora B also interacts with ICIS and MCAK raising the possibility that Aurora B may regulate MCAK activity as well. Herein, we demonstrate that recombinant Aurora B-INCENP inhibits Xenopus MCAK activity in vitro in a phosphorylation-dependent manner. Substituting endogenous MCAK in Xenopus extracts with the alanine mutant XMCAK-4A, which is resistant to inhibition by Aurora B-INCENP, led to assembly of mono-astral and monopolar structures instead of bipolar spindles. The size of these structures and extent of tubulin polymerization in XMCAK-4A extracts indicate that XM-CAK-4A is not defective for microtubule dynamics regulation throughout the cytoplasm. We further demonstrate that the ability of XMCAK-4A to localize to inner centromeres is abolished. Our results show that MCAK regulation of cytoplasmic and spindle-associated microtubules can be differentiated by Aurora B-dependent phosphorylation, and they further demonstrate that this regulation is required for bipolar meiotic spindle assembly.

scholarly journals CENP-32 is required to maintain centrosomal dominance in bipolar spindle assembly

2015 ◽  
Vol 26 (7) ◽  
pp. 1225-1237 ◽  
Author(s):  
Shinya Ohta ◽  
Laura Wood ◽  
Iyo Toramoto ◽  
Ken-Ichi Yagyu ◽  
Tatsuo Fukagawa ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Additional Data ◽  
Weak Interactions ◽  
Metaphase Plate ◽  
Spindle Pole ◽  
Animal Cells ◽  
Central Spindle ◽  
Spindle Poles ◽  
Spindle Microtubules ◽  
Bipolar Spindles

Centrosomes nucleate spindle formation, direct spindle pole positioning, and are important for proper chromosome segregation during mitosis in most animal cells. We previously reported that centromere protein 32 (CENP-32) is required for centrosome association with spindle poles during metaphase. In this study, we show that CENP-32 depletion seems to release centrosomes from bipolar spindles whose assembly they had previously initiated. Remarkably, the resulting anastral spindles function normally, aligning the chromosomes to a metaphase plate and entering anaphase without detectable interference from the free centrosomes, which appear to behave as free asters in these cells. The free asters, which contain reduced but significant levels of CDK5RAP2, show weak interactions with spindle microtubules but do not seem to make productive attachments to kinetochores. Thus CENP-32 appears to be required for centrosomes to integrate into a fully functional spindle that not only nucleates astral microtubules, but also is able to nucleate and bind to kinetochore and central spindle microtubules. Additional data suggest that NuMA tethers microtubules at the anastral spindle poles and that augmin is required for centrosome detachment after CENP-32 depletion, possibly due to an imbalance of forces within the spindle.

scholarly journals Microtubule Assembly and Pole Coalescence: Early Steps in C. elegans Oocyte Meiosis I Spindle Assembly

2020 ◽  
Author(s):  
Chien-Hui Chuang ◽  
Aleesa J. Schlientz ◽  
Jie Yang ◽  
Bruce Bowerman
Keyword(s):  
Molecular Mechanisms ◽  
Spindle Assembly ◽  
Nuclear Lamina ◽  
Spindle Pole ◽  
Microtubule Assembly ◽  
Bipolar Structure ◽  
C Elegans ◽  
Nuclear Envelope Breakdown ◽  
Oocyte Meiosis ◽  
Meiosis I

ABSTRACTHow oocytes assemble bipolar meiotic spindles in the absence of centrosomes as microtubule organizing centers remains poorly understood. We have used live cell imaging in C. elegans to investigate requirements for the nuclear lamina and for conserved regulators of microtubule dynamics during oocyte meiosis I spindle assembly, assessing these requirements with respect to recently identified spindle assembly steps. We show that the nuclear lamina is required for microtubule bundles to form a cage-like structure that appears shortly after oocyte nuclear envelope breakdown and surrounds the oocyte chromosomes, although bipolar spindles still assembled in its absence. Although two conserved regulators of microtubule nucleation, RAN-1 and γ-tubulin, are not required for bipolar spindle assembly, both contribute to normal levels of spindle-associated microtubules and spindle assembly dynamics. Finally, the XMAP215 ortholog ZYG-9 and the nearly identical minus-end directed kinesins KLP-15/16 are required for proper assembly of the early cage-like structure of microtubule bundles, and for early spindle pole foci to coalesce into a bipolar structure. Our results provide a framework for assigning molecular mechanisms to recently described steps in C. elegans oocyte meiosis I spindle assembly.

scholarly journals Microtubule assembly and pole coalescence: early steps in Caenorhabditiselegans oocyte meiosis I spindle assembly

Biology Open ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. bio052308
Author(s):  
Chien-Hui Chuang ◽  
Aleesa J. Schlientz ◽  
Jie Yang ◽  
Bruce Bowerman
Keyword(s):  
Molecular Mechanisms ◽  
Spindle Assembly ◽  
Nuclear Lamina ◽  
Spindle Pole ◽  
Microtubule Assembly ◽  
Bipolar Structure ◽  
C Elegans ◽  
Nuclear Envelope Breakdown ◽  
Oocyte Meiosis ◽  
Meiosis I

ABSTRACTHow oocytes assemble bipolar meiotic spindles in the absence of centrosomes as microtubule organizing centers remains poorly understood. We have used live cell imaging in Caenorhabditis elegans to investigate requirements for the nuclear lamina and for conserved regulators of microtubule dynamics during oocyte meiosis I spindle assembly, assessing these requirements with respect to recently identified spindle assembly steps. We show that the nuclear lamina is required for microtubule bundles to form a peripheral cage-like structure that appears shortly after oocyte nuclear envelope breakdown and surrounds the oocyte chromosomes, although bipolar spindles still assembled in its absence. Although two conserved regulators of microtubule nucleation, RAN-1 and γ-tubulin, are not required for bipolar spindle assembly, both contribute to normal levels of spindle-associated microtubules and spindle assembly dynamics. Finally, the XMAP215 ortholog ZYG-9 and the nearly identical minus-end directed kinesins KLP-15/16 are required for proper assembly of the early cage-like structure of microtubule bundles, and for early spindle pole foci to coalesce into a bipolar structure. Our results provide a framework for assigning molecular mechanisms to recently described steps in C. elegans oocyte meiosis I spindle assembly.

scholarly journals TRIM37 prevents formation of condensate-organized ectopic spindle poles to ensure mitotic fidelity

2021 ◽  
Vol 220 (7) ◽  
Author(s):  
Franz Meitinger ◽  
Dong Kong ◽  
Midori Ohta ◽  
Arshad Desai ◽  
Karen Oegema ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Ubiquitin Ligase ◽  
Spindle Pole ◽  
Mitotic Entry ◽  
Centriole Duplication ◽  
Mulibrey Nanism ◽  
Spindle Poles ◽  
Pathological Mechanism ◽  
Pericentriolar Material ◽  
Centrosomal Proteins

Centrosomes are composed of a centriolar core surrounded by pericentriolar material that nucleates microtubules. The ubiquitin ligase TRIM37 localizes to centrosomes, but its centrosomal roles are not yet defined. We show that TRIM37 does not control centriole duplication, structure, or the ability of centrioles to form cilia but instead prevents assembly of an ectopic centrobin-scaffolded structured condensate that forms by budding off of centrosomes. In ∼25% of TRIM37-deficient cells, the condensate organizes an ectopic spindle pole, recruiting other centrosomal proteins and acquiring microtubule nucleation capacity during mitotic entry. Ectopic spindle pole–associated transient multipolarity and multipolar segregation in TRIM37-deficient cells are suppressed by removing centrobin, which interacts with and is ubiquitinated by TRIM37. Thus, TRIM37 ensures accurate chromosome segregation by preventing the formation of centrobin-scaffolded condensates that organize ectopic spindle poles. Mutations in TRIM37 cause the disorder mulibrey nanism, and patient-derived cells harbor centrobin condensate-organized ectopic poles, leading us to propose that chromosome missegregation is a pathological mechanism in this disorder.

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

mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
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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