Acentrosomal spindle assembly and maintenance in <i>Caenorhabditis elegans</i> oocytes requires a kinesin-12 nonmotor microtubule interaction domain

Wolff, Ian D.; Hollis, Jeremy; Wignall, Sarah M.

doi:10.1091/mbc.e22-05-0153

Cited by 4 publications

(4 citation statements)

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“…Our hypothesis that outward forces are activated upon ZYG-8 depletion raised the possibility that this protein may normally suppress a factor that generates these forces. Since KLP-18/kinesin-12 provides the major outward sorting force in C. elegans oocytes (Wolff et al, 2016; Wolff et al, 2022b), we first sought to explore the possibility that ZYG-8 regulates KLP-18. To this end, we co-depleted KLP-18 and ZYG-8, to determine if there was evidence for excess outward force under these conditions.…”

Section: Resultsmentioning

confidence: 99%

“…However, mechanisms must also exist to limit these outward forces, so that the spindle can remain stable after it forms ( Figure 8A ). Previous studies revealed that KLP-18/kinesin-12 and BMK-1/kinesin-5 produce outward forces, while dynein produces a counter-balancing inward force Wolff et al, 2016; Cavin-Meza et al, 2022a; Wolff et al, 2022b). We have now identified ZYG-8 as another factor that is important for this force balance.…”

Section: Discussionmentioning

confidence: 99%

“…KLP-18/kinesin-12, a plus-end directed microtubule motor, sorts microtubule minus ends outwards to establish bipolarity; depletion of KLP-18 results in a monopolar spindle in which microtubules minus ends coalesce into a single pole at the center with plus ends radiating outwards (Wignall and Villeneuve, 2009; Wolff et al, 2016b). KLP-18 is also required to maintain spindle bipolarity; if KLP-18 is inactivated after the bipolar spindle has already formed, the two poles converge together to create a monopolar spindle (Wolff et al, 2022b).…”

Section: Introductionmentioning

confidence: 99%

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The doublecortin-family kinase ZYG-8^DCLK1regulates motor activity to achieve proper force balance inC. elegansacentrosomal spindles

Czajkowski,

Divekar,

Wignall

2023

Preprint

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Although centrosomes help organize spindles in most cell types, oocytes of most species lack these structures. During acentrosomal spindle assembly inC. elegansoocytes, microtubule minus ends are sorted outwards away from the chromosomes where they form poles, but then these outward forces must be balanced to form a stable bipolar structure. How proper force balance is achieved in these spindles is not known. Here, we have gained insight into this question through studies of ZYG-8, a conserved doublecortin-family kinase; the mammalian homolog of this microtubule-associated protein is upregulated in many cancers and has been implicated in cell division, but the mechanisms by which it functions are poorly understood. Interestingly, we found that ZYG-8 depletion from oocytes resulted in spindles that were over-elongated, suggesting that there was excess outward force following ZYG-8 removal. Experiments with monopolar spindles confirmed this hypothesis and revealed a role for ZYG-8 in regulating the force-generating motor BMK-1/kinesin-5. Importantly, further investigation revealed that kinase activity is required for the function of ZYG-8 in both meiosis and mitosis. Altogether, our results support a model in which ZYG-8 regulates motor-driven forces within the oocyte spindle, thus identifying a new function for a doublecortin-family protein in cell division.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The doublecortin-family kinase ZYG-8^DCLK1regulates motor activity to achieve proper force balance inC. elegansacentrosomal spindles

Czajkowski,

Divekar,

Wignall

2023

Preprint

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“…A number of factors required for acentrosomal spindle assembly and pole formation in C. elegans have been identified: MEI-1/2 katanin promote the generation of short microtubules [4], KLP-15/16 bundle these microtubules [5,6], KLP-18 and MESP-1 sort microtubules to establish bipolarity [3], ASPM-1 binds to microtubule minus ends and functions to focus the spindle poles in conjunction with dynein [7,8], and KLP-7 MCAK acts to limit the number of spindle poles and inhibit excess microtubule nucleation [2,9,10]. Rapid inactivation or depletion of MEI-1, KLP-18, or dynein causes defects in spindle structure [8,11,12], demonstrating that these factors are required for spindle maintenance. However, whether any other pole associated-proteins stabilize the spindle to maintain bipolarity is not known.…”

Section: Introductionmentioning

confidence: 99%

ZYG-9ch-TOG promotes the stability of acentrosomal poles via regulation of spindle microtubules in C. elegans oocyte meiosis

et al. 2022

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During mitosis, centrosomes serve as microtubule organizing centers that guide the formation of a bipolar spindle. However, oocytes of many species lack centrosomes; how meiotic spindles establish and maintain these acentrosomal poles remains poorly understood. Here, we show that the microtubule polymerase ZYG-9ch-TOG is required to maintain acentrosomal pole integrity in C. elegans oocyte meiosis. We exploited the auxin inducible degradation system to remove ZYG-9 from pre-formed spindles within minutes; this caused the poles to split apart and an unstable multipolar structure to form. Depletion of TAC-1, a protein known to interact with ZYG-9 in mitosis, caused loss of proper ZYG-9 localization and similar spindle phenotypes, further demonstrating that ZYG-9 is required for pole integrity. However, depletion of ZYG-9 or TAC-1 surprisingly did not affect the assembly or stability of monopolar spindles, suggesting that these proteins are not required for acentrosomal pole structure per se. Moreover, fluorescence recovery after photobleaching (FRAP) revealed that ZYG-9 turns over rapidly at acentrosomal poles, displaying similar turnover dynamics to tubulin itself, suggesting that ZYG-9 does not play a static structural role at poles. Together, these data support a global role for ZYG-9 in regulating the stability of bipolar spindles and demonstrate that the maintenance of acentrosomal poles requires factors beyond those acting to organize the pole structure itself.

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Newfound features of meiotic chromosome organization that promote efficient congression and segregation in Caenorhabditis elegans oocytes

Horton

Divekar

Wignall

2022

MBoC

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Although end-on microtubule-kinetochore attachments typically drive chromosome alignment, C. elegans oocytes do not form these connections. Instead, microtubule bundles run laterally alongside chromosomes and a ring-shaped protein complex facilitates congression (the “ring complex”/RC). Here, we report new aspects of RC and chromosome structure that are required for congression and segregation. First, we found that in addition to encircling the outside of each homologous chromosome pair (bivalent), the RC also forms internal subloops that wrap around the domains where cohesion is lost during the first meiotic division; cohesin removal could therefore disengage these subloops in anaphase, enabling RC removal from chromosomes. Additionally, we discovered new features of chromosome organization that facilitate congression. Analysis of a mutant that forms bivalents with a fragile, unresolved homolog interface revealed that these bivalents are usually able to biorient on the spindle, with lateral microtubule bundles running alongside them and constraining the chromosome arms such that the two homologs are pointed to opposite spindle poles. This biorientation facilitates congression, as mono-oriented bivalents exhibited reduced polar ejection forces that resulted in congression defects. Thus, despite not forming end-on attachments, chromosome biorientation promotes congression in C. elegans oocytes. Our work therefore reveals novel features of chromosome organization in oocytes and highlights the importance of proper chromosome structure for faithful segregation during the meiotic divisions. [Media: see text] [Media: see text] [Media: see text]

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Acentrosomal spindle assembly and maintenance in Caenorhabditis elegans oocytes requires a kinesin-12 nonmotor microtubule interaction domain

Cited by 4 publications

References 82 publications

The doublecortin-family kinase ZYG-8^DCLK1regulates motor activity to achieve proper force balance inC. elegansacentrosomal spindles

The doublecortin-family kinase ZYG-8^DCLK1regulates motor activity to achieve proper force balance inC. elegansacentrosomal spindles

ZYG-9ch-TOG promotes the stability of acentrosomal poles via regulation of spindle microtubules in C. elegans oocyte meiosis

Newfound features of meiotic chromosome organization that promote efficient congression and segregation in Caenorhabditis elegans oocytes

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Acentrosomal spindle assembly and maintenance in Caenorhabditis elegans oocytes requires a kinesin-12 nonmotor microtubule interaction domain

Cited by 4 publications

References 82 publications

The doublecortin-family kinase ZYG-8DCLK1regulates motor activity to achieve proper force balance inC. elegansacentrosomal spindles

The doublecortin-family kinase ZYG-8DCLK1regulates motor activity to achieve proper force balance inC. elegansacentrosomal spindles

ZYG-9ch-TOG promotes the stability of acentrosomal poles via regulation of spindle microtubules in C. elegans oocyte meiosis

Newfound features of meiotic chromosome organization that promote efficient congression and segregation in Caenorhabditis elegans oocytes

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The doublecortin-family kinase ZYG-8^DCLK1regulates motor activity to achieve proper force balance inC. elegansacentrosomal spindles

The doublecortin-family kinase ZYG-8^DCLK1regulates motor activity to achieve proper force balance inC. elegansacentrosomal spindles