The doublecortin-family kinase ZYG-8DCLK1 regulates microtubule dynamics and motor-driven forces to promote the stability of C. elegans acentrosomal spindles.

Although centrosomes help organize spindles in most cell types, oocytes of most species lack these structures. During acentrosomal spindle assembly in C. elegans oocytes, 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. Simultaneously, microtubule dynamics must be precisely controlled to maintain spindle length and organization. How forces and dynamics are tuned to create a stable bipolar structure is poorly understood. 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. We found that ZYG-8 depletion from oocytes resulted in overelongated spindles with pole and midspindle defects. Importantly, experiments with monopolar spindles revealed that ZYG-8 depletion led to excess outward forces within the spindle and suggested a potential role for this protein in regulating the force-generating motor BMK-1/kinesin-5. Further, we found that ZYG-8 is also required for proper microtubule dynamics within the oocyte spindle and that kinase activity is required for its function during both meiosis and mitosis. Altogether, our findings reveal new roles for ZYG-8 in oocytes and provide insights into how acentrosomal spindles are stabilized to promote faithful meiosis.

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

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.

The Drosophila CG1674 gene encodes a synaptopodin 2-like related protein that localizes to the Z-disc and is required for normal flight muscle development and function.

BACKGROUND: To identify novel myofibrillar components of the Drosophila flight muscles, we carried out a proteomic analysis of chemically demembranated flight muscle myofibrils, and characterized the knockdown phenotype of a novel gene identified in the screen, CG1674. RESULTS: The CG1674 protein has some similarity to vertebrate synaptopodin 2-like, and when expressed as a FLAG-tagged fusion protein, it was localized during development to the Z-disc and cytoplasm. Knockdown of CG1674 expression affected the function of multiple muscle types, and defective flight in adults was accompanied by large actin-rich structures in the flight muscles that resembled overgrown Z-discs. Localization of CG1674 to the Z-disc depended predominantly upon presence of the Z-disc component alpha-actinin, but also depended upon other Z-disc components, including Mask, Zasp52, and Sals. We also observed re-localization of FLAG-CG1674 to the nucleus in Alpha-actinin and sals knockdown animals. CONCLUSIONS: These studies identify and characterize a previously unreported myofibrillar component of Drosophila muscle that is necessary for proper myofibril assembly during development.

The doublecortin-family kinase ZYG-8DCLK1 regulates motor activity to achieve proper force balance in C. elegans acentrosomal spindles.

Although centrosomes help organize spindles in most cell types, oocytes of most species lack these structures. During acentrosomal spindle assembly in C. elegans oocytes, 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.