scholarly journals The spindle assembly function of Caenorhabditis elegans katanin does not require microtubule-severing activity

2011 ◽  
Vol 22 (9) ◽  
pp. 1550-1560 ◽  
Author(s):  
Karen Perry McNally ◽  
Francis J. McNally
Keyword(s):  
Caenorhabditis Elegans ◽  
Spindle Pole ◽  
Catalytic Subunit ◽  
Meiotic Spindle ◽  
Regulatory Subunit ◽  
Loss Of Function ◽  
Microtubule Binding ◽  
Microtubule Severing ◽  
Terminal Domain ◽  
Meiotic Spindles

Katanin is a heterodimeric microtubule-severing protein that is conserved among eukaryotes. Loss-of-function mutations in the Caenorhabditis elegans katanin catalytic subunit, MEI-1, cause specific defects in female meiotic spindles. To determine the relationship between katanin’s microtubule-severing activity and its role in meiotic spindle formation, we analyzed the MEI-1(A338S) mutant. Unlike wild-type MEI-1, which mediated disassembly of microtubule arrays in Xenopus fibroblasts, MEI-1(A338S) had no effect on fibroblast microtubules, indicating a lack of microtubule-severing activity. In C. elegans, MEI-1(A338S) mediated assembly of extremely long bipolar meiotic spindles. In contrast, a nonsense mutation in MEI-1 caused assembly of meiotic spindles without any poles as assayed by localization of the spindle-pole protein, ASPM-1. These results indicated that katanin protein, but not katanin’s microtubule-severing activity, is required for assembly of acentriolar meiotic spindle poles. To understand the nonsevering activities of katanin, we characterized the N-terminal domain of the katanin catalytic subunit. The N-terminal domain was necessary and sufficient for binding to the katanin regulatory subunit. The katanin regulatory subunit in turn caused a dramatic change in the microtubule-binding properties of the N-terminal domain of the catalytic subunit. This unique bipartite microtubule-binding structure may mediate the spindle-pole assembly activity of katanin during female meiosis.

scholarly journals Katanin maintains meiotic metaphase chromosome alignment and spindle structure in vivo and has multiple effects on microtubules in vitro

2014 ◽  
Vol 25 (7) ◽  
pp. 1037-1049 ◽  
Author(s):  
Karen McNally ◽  
Evan Berg ◽  
Daniel B. Cortes ◽  
Veronica Hernandez ◽  
Paul E. Mains ◽  
...  
Keyword(s):  
Point Mutations ◽  
Metaphase Plate ◽  
Meiotic Spindle ◽  
Loss Of Function ◽  
Microtubule Organization ◽  
Bipolar Structure ◽  
Microtubule Severing ◽  
Meiotic Spindles

Assembly of Caenorhabditis elegans female meiotic spindles requires both MEI-1 and MEI-2 subunits of the microtubule-severing ATPase katanin. Strong loss-of-function mutants assemble apolar intersecting microtubule arrays, whereas weaker mutants assemble bipolar meiotic spindles that are longer than wild type. To determine whether katanin is also required for spindle maintenance, we monitored metaphase I spindles after a fast-acting mei-1(ts) mutant was shifted to a nonpermissive temperature. Within 4 min of temperature shift, bivalents moved off the metaphase plate, and microtubule bundles within the spindle lengthened and developed a high degree of curvature. Spindles eventually lost bipolar structure. Immunofluorescence of embryos fixed at increasing temperature indicated that MEI-1 was lost from spindle microtubules before loss of ASPM-1, indicating that MEI-1 and ASPM-1 act independently at spindle poles. We quantified the microtubule-severing activity of purified MEI-1/MEI-2 complexes corresponding to six different point mutations and found a linear relationship between microtubule disassembly rate and meiotic spindle length. Previous work showed that katanin is required for severing at points where two microtubules intersect in vivo. We show that purified MEI-1/MEI-2 complexes preferentially sever at intersections between two microtubules and directly bundle microtubules in vitro. These activities could promote parallel/antiparallel microtubule organization in meiotic spindles.

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

2014 ◽  
Vol 25 (8) ◽  
pp. 1298-1311 ◽  
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.

scholarly journals Genetic and Molecular Characterization of the Caenorhabditis elegans Gene, mel-26, a Postmeiotic Negative Regulator of MEI-1, a Meiotic-Specific Spindle Component

Genetics ◽  
1998 ◽  
Vol 150 (1) ◽  
pp. 119-128
Author(s):  
M Rhys Dow ◽  
Paul E Mains
Keyword(s):  
Caenorhabditis Elegans ◽  
Protein Interactions ◽  
Negative Regulator ◽  
Meiotic Spindle ◽  
Loss Of Function ◽  
Protein Protein Interactions ◽  
Gain Of Function ◽  
Recessive Mutations ◽  
Female Germline

Abstract We have previously described the gene mei-1, which encodes an essential component of the Caenorhabditis elegans meiotic spindle. When ectopically expressed after the completion of meiosis, mei-1 protein disrupts the function of the mitotic cleavage spindles. In this article, we describe the cloning and the further genetic characterization of mel-26, a postmeiotic negative regulator of mei-1. mel-26 was originally identified by a gain-of-function mutation. We have reverted this mutation to a loss-of-function allele, which has recessive phenotypes identical to the dominant defects of its gain-of-function parent. Both the dominant and recessive mutations of mel-26 result in mei-1 protein ectopically localized in mitotic spindles and centrosomes, leading to small and misoriented cleavage spindles. The loss-of-function mutation was used to clone mel-26 by transformation rescue. As suggested by genetic results indicating that mel-26 is required only maternally, mel-26 mRNA was expressed predominantly in the female germline. The gene encodes a protein that includes the BTB motif, which is thought to play a role in protein-protein interactions.

scholarly journals The Caenorhabditis elegans Microtubule-severing Complex MEI-1/MEI-2 Katanin Interacts Differently with Two Superficially Redundant β-Tubulin Isotypes

2004 ◽  
Vol 15 (1) ◽  
pp. 142-150 ◽  
Author(s):  
Chenggang Lu ◽  
Martin Srayko ◽  
Paul E. Mains
Keyword(s):  
Caenorhabditis Elegans ◽  
Genetic Background ◽  
Morphological Changes ◽  
Meiotic Spindle ◽  
Tubulin Isotypes ◽  
Microtubule Severing ◽  
Tubulin Isotype ◽  
Embryonic Viability ◽  
Β Tubulin

The microtubule-severing protein complex katanin is required for a variety of important microtubule-base morphological changes in both animals and plants. Caenorhabditis elegans katanin is encoded by the mei-1 and mei-2 genes and is required for oocyte meiotic spindle formation and must be inactivated before the first mitotic cleavage. We identified a mutation, sb26, in the tbb-2 β-tubulin gene that partially inhibits MEI-1/MEI-2 activity: sb26 rescues lethality caused by ectopic MEI-1/MEI-2 expression during mitosis, and sb26 increases meiotic defects in a genetic background where MEI-1/MEI-2 activity is lower than normal. sb26 does not interfere with MEI-1/MEI-2 microtubule localization, suggesting that this mutation likely interferes with severing. Tubulin deletion alleles and RNA-mediated interference revealed that TBB-2 and the other germline enriched β-tubulin isotype, TBB-1, are redundant for embryonic viability. However, limiting MEI-1/MEI-2 activity in these experiments revealed that MEI-1/MEI-2 preferentially interacts with TBB-2–containing microtubules. Our results demonstrate that these two superficially redundant β-tubulin isotypes have functionally distinct roles in vivo.

scholarly journals Cytoplasmic Dynein Light Intermediate Chain Is Required for Discrete Aspects of Mitosis in Caenorhabditis elegans

2001 ◽  
Vol 12 (10) ◽  
pp. 2921-2933 ◽  
Author(s):  
John H. Yoder ◽  
Min Han
Keyword(s):  
Caenorhabditis Elegans ◽  
Nucleotide Binding ◽  
Cytoplasmic Dynein ◽  
Meiotic Spindle ◽  
Phenotypic Characterization ◽  
Loss Of Function ◽  
Dynein Motor ◽  
Centrosome Separation ◽  
Transporter Family ◽  
Intermediate Chain

We describe phenotypic characterization of dli-1, the Caenorhabditis elegans homolog of cytoplasmic dynein light intermediate chain (LIC), a subunit of the cytoplasmic dynein motor complex. Animals homozygous for loss-of-function mutations indli-1 exhibit stochastic failed divisions in late larval cell lineages, resulting in zygotic sterility. dli-1 is required for dynein function during mitosis. Depletion of thedli-1 gene product through RNA-mediated gene interference (RNAi) reveals an early embryonic requirement. One-celldli-1(RNAi) embryos exhibit failed cell division attempts, resulting from a variety of mitotic defects. Specifically, pronuclear migration, centrosome separation, and centrosome association with the male pronuclear envelope are defective indli-1(RNAi) embryos. Meiotic spindle formation, however, is not affected in these embryos. DLI-1, like its vertebrate homologs, contains a putative nucleotide-binding domain similar to those found in the ATP-binding cassette transporter family of ATPases as well as other nucleotide-binding and -hydrolyzing proteins. Amino acid substitutions in a conserved lysine residue, known to be required for nucleotide binding, confers complete rescue in a dli-1mutant background, indicating this is not an essential domain for DLI-1 function.

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

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.

Two domains of p80 katanin regulate microtubule severing and spindle pole targeting by p60 katanin

2000 ◽  
Vol 113 (9) ◽  
pp. 1623-1633 ◽  
Author(s):  
K.P. McNally ◽  
O.A. Bazirgan ◽  
F.J. McNally
Keyword(s):  
Mitotic Spindle ◽  
Binding Proteins ◽  
Spindle Pole ◽  
Negative Regulator ◽  
Microtubule Binding ◽  
Microtubule Severing ◽  
Spindle Poles ◽  
Wd40 Domain

The assembly and function of the mitotic spindle requires the activity of a number of microtubule-binding proteins. Some microtubule-binding proteins bind microtubules in vitro but do not co-localize with microtubules in interphase cells. Instead these proteins associate with specific subregions of the mitotic spindle. Katanin, a heterodimeric microtubule-severing ATPase, is found localized at mitotic spindle poles. In this paper we demonstrate that human p60 katanin and the C-terminal domain of human p80 katanin both bind microtubules in vitro. Association of these two proteins results in an increased microtubule affinity and increased microtubule-severing activity in vitro. Association of these subunits in transfected HeLa cells increases microtubule disassembly activity and targeting to spindle poles. The N-terminal WD40 domain of p80 katanin acts as a negative regulator of microtubule disassembly activity and is also required for spindle pole localization, possibly through interactions with another spindle-pole protein. These results support a model in which katanin is targeted to spindle poles through a combination of direct microtubule binding by the p60 subunit and through interactions between the WD40 domain and an unknown protein. We propose that both domains of p80 are essential in precisely regulating katanin's activity in vivo.

scholarly journals Dynactin-dependent cortical dynein and spherical spindle shape correlate temporally with meiotic spindle rotation in Caenorhabditis elegans

2015 ◽  
Vol 26 (17) ◽  
pp. 3030-3046 ◽  
Author(s):  
Marina E. Crowder ◽  
Jonathan R. Flynn ◽  
Karen P. McNally ◽  
Daniel B. Cortes ◽  
Kari L. Price ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Spherical Shape ◽  
Nematode Species ◽  
Meiotic Spindle ◽  
Present Evidence ◽  
Basic Domain ◽  
C Elegans ◽  
Polar Bodies ◽  
Spindle Rotation ◽  
Meiotic Spindles

Oocyte meiotic spindles orient with one pole juxtaposed to the cortex to facilitate extrusion of chromosomes into polar bodies. In Caenorhabditis elegans, these acentriolar spindles initially orient parallel to the cortex and then rotate to the perpendicular orientation. To understand the mechanism of spindle rotation, we characterized events that correlated temporally with rotation, including shortening of the spindle in the pole-to pole axis, which resulted in a nearly spherical spindle at rotation. By analyzing large spindles of polyploid C. elegans and a related nematode species, we found that spindle rotation initiated at a defined spherical shape rather than at a defined spindle length. In addition, dynein accumulated on the cortex just before rotation, and microtubules grew from the spindle with plus ends outward during rotation. Dynactin depletion prevented accumulation of dynein on the cortex and prevented spindle rotation independently of effects on spindle shape. These results support a cortical pulling model in which spindle shape might facilitate rotation because a sphere can rotate without deforming the adjacent elastic cytoplasm. We also present evidence that activation of spindle rotation is promoted by dephosphorylation of the basic domain of p150 dynactin.

scholarly journals Localization of the mei-1 gene product of Caenorhaditis elegans, a meiotic-specific spindle component.

1994 ◽  
Vol 126 (1) ◽  
pp. 199-209 ◽  
Author(s):  
S Clark-Maguire ◽  
P E Mains
Keyword(s):  
Regulatory Network ◽  
Meiotic Spindle ◽  
Loss Of Function ◽  
Wild Type ◽  
Female Meiosis ◽  
Gain Of Function ◽  
Spindle Poles ◽  
Meiotic Spindles ◽  
Female Germline ◽  
Meiosis I

Genetic evidence suggests that the product of the mei-1 gene of Caenorhabditis elegans is specifically required for meiosis in the female germline. Loss-of-function mei-1 mutations block meiotic spindle formation while a gain-of-function allele instead results in spindle defects during the early mitotic cleavages. In this report, we use immunocytochemistry to examine the localization of the mei-1 product in wild-type and mutant embryos. During metaphase of meiosis I in wild-type embryos, mei-1 protein was found throughout the spindle but was more concentrated toward the poles. At telophase I, mei-1 product colocalized with the chromatin at the spindle poles. The pattern was repeated during meiosis II but no mei-1 product was visible during the subsequent mitotic cleavages. The mei-1 gain-of-function allele resulted in ectopic mei-1 staining in the centers of the microtubule-organizing centers during interphase and in the spindles during the early cleavages. This aberrant localization is probably responsible for the poorly formed and misoriented cleavage spindles characteristic of the mutation. We also examined the localization of mei-1(+) product in the presence of mutations of genes that genetically interact with mei-1 alleles. mei-2 is apparently required to localize mei-1 product to the spindle during meiosis while mel-26 acts as a postmeiotic inhibitor. We conclude that mei-1 encodes a novel spindle component, one that is specialized for the acentriolar meiotic spindles unique to female meiosis. The genes mei-2 and mel-26 are part of a regulatory network that confines mei-1 activity to meiosis.

scholarly journals Caenorhabditis elegans Aurora A kinase is required for the formation of spindle microtubules in female meiosis

2015 ◽  
Vol 26 (23) ◽  
pp. 4187-4196 ◽  
Author(s):  
Eisuke Sumiyoshi ◽  
Yuma Fukata ◽  
Satoshi Namai ◽  
Asako Sugimoto
Keyword(s):  
Caenorhabditis Elegans ◽  
Kinase Activity ◽  
Active Form ◽  
Meiotic Spindle ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Female Meiosis ◽  
Germinal Vesicle Breakdown ◽  
Meiotic Spindles ◽  
Spindle Microtubules

In many animals, female meiotic spindles are assembled in the absence of centrosomes, the major microtubule (MT)-organizing centers. How MTs are formed and organized into meiotic spindles is poorly understood. Here we report that, in Caenorhabditis elegans, Aurora A kinase/AIR-1 is required for the formation of spindle microtubules during female meiosis. When AIR-1 was depleted or its kinase activity was inhibited in C. elegans oocytes, although MTs were formed around chromosomes at germinal vesicle breakdown (GVBD), they were decreased during meiotic prometaphase and failed to form a bipolar spindle, and chromosomes were not separated into two masses. Whereas AIR-1 protein was detected on and around meiotic spindles, its kinase-active form was concentrated on chromosomes at prometaphase and on interchromosomal MTs during late anaphase and telophase. We also found that AIR-1 is involved in the assembly of short, dynamic MTs in the meiotic cytoplasm, and these short MTs were actively incorporated into meiotic spindles. Collectively our results suggest that, after GVBD, the kinase activity of AIR-1 is continuously required for the assembly and/or stabilization of female meiotic spindle MTs.

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