Microtubule-severing activity of AAA+ ATPase Katanin is essential for female meiotic spindle assembly

The evolutionarily conserved microtubule (MT)-severing AAA-ATPase enzyme Katanin is emerging as a critical regulator of MT dynamics. In Caenorhabditis elegans, Katanin MT-severing activity is essential for meiotic spindle assembly but is toxic for the mitotic spindle. Here we analyzed Katanin dynamics in C. elegans and deciphered the role of Katanin phosphorylation in the regulation of its activity and stability. Katanin is abundant in oocytes, and its levels drop after meiosis, but unexpectedly, a significant fraction is present throughout embryogenesis, where it is dynamically recruited to the centrosomes and chromosomes during mitosis. We show that the minibrain kinase MBK-2, which is activated during meiosis, phosphorylates Katanin at multiple serines. We demonstrate unequivocally that Katanin phosphorylation at a single residue is necessary and sufficient to target Katanin for proteasomal degradation after meiosis, whereas phosphorylation at the other sites only inhibits Katanin ATPase activity stimulated by MTs. Our findings suggest that cycles of phosphorylation and dephosphorylation fine-tune Katanin level and activity to deliver the appropriate MT-severing activity during development.

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MEI-1/MEI-2 katanin-like microtubule severing activity is required for Caenorhabditis elegans meiosis

Genes & Development ◽

10.1101/gad.14.9.1072 ◽

2000 ◽

Vol 14 (9) ◽

pp. 1072-1084

Author(s):

Martin Srayko ◽

Dan W. Buster ◽

Omar A. Bazirgan ◽

Francis J. McNally ◽

Paul E. Mains

Keyword(s):

Caenorhabditis Elegans ◽

Hela Cells ◽

Mitotic Spindle ◽

Subcellular Location ◽

Meiotic Spindle ◽

Aaa Atpase ◽

C Elegans ◽

Microtubule Severing ◽

Acid Protein ◽

Spindle Function

The Caenorhabditis elegans meiotic spindle is morphologically distinct from the first mitotic spindle, yet both structures form in the same cytoplasm ∼20 minutes apart. Themei-1 and mei-2 genes of C. elegans are required for the establishment of the oocyte meiotic spindle but are not required for mitotic spindle function. mei-1 encodes an AAA ATPase family member with similarity to the p60 catalytic subunit of the heterodimeric sea urchin microtubule-severing protein, katanin. We report that mei-2 encodes a 280-amino acid protein containing a region similar to the p80-targeting subunit of katanin. MEI-1 and MEI-2 antibodies decorate the polar ends of meiotic spindle microtubules and meiotic chromatin. We find that the subcellular location of MEI-2 depends on wild-type mei-1 activity and vice versa. These experiments, combined with MEI-1 and MEI-2's similarity to p60 and p80 katanin, suggest that the C. elegans proteins function as a complex. In support of this idea, MEI-1 and MEI-2 physically associate in HeLa cells. Furthermore, co-expression of MEI-1 and MEI-2 in HeLa cells results in the disassembly of microtubules. These data lead us to conclude that MEI-1/MEI-2 microtubule-severing activity is required for meiotic spindle organization in C. elegans.

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Caenorhabditis elegansoocyte meiotic spindle pole assembly requires microtubule severing and the calponin homology domain protein ASPM-1

Molecular Biology of the Cell ◽

10.1091/mbc.e13-11-0687 ◽

2014 ◽

Vol 25 (8) ◽

pp. 1298-1311 ◽

Cited By ~ 33

Author(s):

Amy A. Connolly ◽

Valerie Osterberg ◽

Sara Christensen ◽

Meredith Price ◽

Chenggang Lu ◽

...

Keyword(s):

Family Member ◽

Spindle Assembly ◽

Spindle Pole ◽

Meiotic Spindle ◽

Calponin Homology Domain ◽

Calponin Homology ◽

Microtubule Severing ◽

Monopolar Spindle ◽

Meiotic Spindles ◽

Domain Protein

In many animals, including vertebrates, oocyte meiotic spindles are bipolar but assemble in the absence of centrosomes. Although meiotic spindle positioning in oocytes has been investigated extensively, much less is known about their assembly. In Caenorhabditis elegans, three genes previously shown to contribute to oocyte meiotic spindle assembly are the calponin homology domain protein encoded by aspm-1, the katanin family member mei-1, and the kinesin-12 family member klp-18. We isolated temperature-sensitive alleles of all three and investigated their requirements using live-cell imaging to reveal previously undocumented requirements for aspm-1 and mei-1. Our results indicate that bipolar but abnormal oocyte meiotic spindles assemble in aspm-1(-) embryos, whereas klp-18(-) and mei-1(-) mutants assemble monopolar and apolar spindles, respectively. Furthermore, two MEI-1 functions—ASPM-1 recruitment to the spindle and microtubule severing—both contribute to monopolar spindle assembly in klp-18(-) mutants. We conclude that microtubule severing and ASPM-1 both promote meiotic spindle pole assembly in C. elegans oocytes, whereas the kinesin 12 family member KLP-18 promotes spindle bipolarity.

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Faculty Opinions recommendation of Subunit Interactions and cooperativity in the microtubule-severing AAA ATPase spastin.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717147906.792452920 ◽

2012 ◽

Author(s):

Claire Walczak

Keyword(s):

Subunit Interactions ◽

Aaa Atpase ◽

Microtubule Severing

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Cellular Samurai: katanin and the severing of microtubules

Journal of Cell Science ◽

10.1242/jcs.113.16.2821 ◽

2000 ◽

Vol 113 (16) ◽

pp. 2821-2827 ◽

Cited By ~ 5

Author(s):

L. Quarmby

Keyword(s):

Epithelial Cells ◽

Essential Role ◽

Aaa Atpase ◽

C Elegans ◽

Microtubule Severing ◽

Biochemical Studies ◽

New Evidence

Recent biochemical studies of the AAA ATPase, katanin, provide a foundation for understanding how microtubules might be severed along their length. These in vitro studies are complemented by a series of recent reports of direct in vivo observation of microtubule breakage, which indicate that the in vitro phenomenon of catalysed microtubule severing is likely to be physiological. There is also new evidence that microtubule severing by katanin is important for the production of non-centrosomal microtubules in cells such as neurons and epithelial cells. Although it has been difficult to establish the role of katanin in mitosis, new genetic evidence indicates that a katanin-like protein, MEI-1, plays an essential role in meiosis in C. elegans. Finally, new proteins involved in the severing of axonemal microtubules have been discovered in the deflagellation system of Chlamydomonas.

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