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Regulation of the MEI-1/MEI-2 Microtubule-Severing Katanin Complex in Early Caenorhabditis elegans Development.

ABSTRACT: After fertilization, rapid changes of the Caenorhabditis elegans cytoskeleton occur in the transition from meiosis to mitosis, requiring precise regulation. The MEI-1/MEI-2 katanin microtubule-severing complex is essential for meiotic spindle formation but must be quickly inactivated to allow for proper formation of the mitotic spindle. MEI-1/MEI-2 inactivation is dependent on multiple redundant pathways. The primary pathway employs the MEL-26 substrate adaptor for the CUL-3/cullin-based E3 ubiquitin ligase, which targets MEI-1 for proteosomal degradation. Here, we used quantitative antibody staining to measure MEI-1 levels to determine how other genes implicated in MEI-1 regulation act relative to CUL-3/MEL-26 The anaphase-promoting complex/cyclosome, APC/C, the DYRK (Dual-specificity tyrosine-regulated kinase), MBK-2, and the CUL-2-based E3 ubiquitin ligase act together to degrade MEI-1, in parallel to MEL-26/CUL-3 CUL-2 is known to keep MEL-26 low during meiosis, so CUL-2 apparently changes its target from MEL-26 in meiosis to MEI-1 in mitosis. RFL-1, an activator of cullin E3 ubiquitin ligases, activates CUL-2 but not CUL-3 for MEI-1 elimination. HECD-1 (HECT/Homologous to the E6AP carboxyl terminus domain) E3 ligase acts as a MEI-1 activator in meiosis but functions as an inhibitor during mitosis, without affecting levels of MEI-1 or MEI-2 Our results highlight the multiple layers of MEI-1 regulation that are required during the switch from the meiotic to mitotic modes of cell division.

SUBMITTER: Beard SM

PROVIDER: S-EPMC5068946 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description:The Caenorhabditis elegans meiotic spindle is morphologically distinct from the first mitotic spindle, yet both structures form in the same cytoplasm approximately 20 minutes apart. The mei-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.

| S-EPMC316576 | biostudies-literature

STRIPAK regulation of katanin microtubule severing in the Caenorhabditis elegans embryo.

Project description:Microtubule severing plays important role in cell structure and cell division. The microtubule severing protein katanin, composed of the MEI-1/MEI-2 subunits in Caenorhabditis elegans, is required for oocyte meiotic spindle formation; however, it must be inactivated for mitosis to proceed as continued katanin expression is lethal. Katanin activity is regulated by 2 ubiquitin-based protein degradation pathways. Another ubiquitin ligase, HECD-1, the homolog of human HECTD1/HECT domain E3 ubiquitin protein ligase 1, regulates katanin activity without affecting katanin levels. In other organisms, HECD-1 is a component of the striatin-interacting kinase phosphatase complex, which affects cell proliferation and a variety of signaling pathways. Here we conducted a systematic screen of how mutations in striatin-interacting kinase phosphatase components affect katanin function in C. elegans. Striatin-interacting kinase phosphatase core components (FARL-11, CASH-1, LET-92, and GCK-1) were katanin inhibitors in mitosis and activators in meiosis, much like HECD-1. By contrast, variable components (SLMP-1, OTUB-2) functioned as activators of katanin activity in mitosis, indicating they may function to alter striatin-interacting kinase phosphatase core function. The core component CCM-3 acted as an inhibitor at both divisions, while other components (MOB-4, C49H3.6) showed weak interactions with katanin mutants. Additional experiments indicate that katanin may be involved with the centralspindlin complex and a tubulin chaperone. HECD-1 shows ubiquitous expression in the cytoplasm throughout meiosis and early development. The differing functions of the different subunits could contribute to the diverse functions of the striatin-interacting kinase phosphatase complex in C. elegans and other organisms.

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Microtubule severing by the katanin complex is activated by PPFR-1-dependent MEI-1 dephosphorylation.

Project description:Katanin is an evolutionarily conserved microtubule (MT)-severing complex implicated in multiple aspects of MT dynamics. In Caenorhabditis elegans, the katanin homologue MEI-1 is required for meiosis, but must be inactivated before mitosis. Here we show that PPFR-1, a regulatory subunit of a trimeric protein phosphatase 4 complex, enhanced katanin MT-severing activity during C. elegans meiosis. Loss of ppfr-1, similarly to the inactivation of MT severing, caused a specific defect in meiosis II spindle disassembly. We show that a fraction of PPFR-1 was degraded after meiosis, contributing to katanin inactivation. PPFR-1 interacted with MEL-26, the substrate recognition subunit of the CUL-3 RING E3 ligase (CRL3(MEL-26)), which also targeted MEI-1 for post-meiotic degradation. Reversible protein phosphorylation of MEI-1 may ensure temporal activation of the katanin complex during meiosis, whereas CRL3(MEL-26)-mediated degradation of both MEI-1 and its activator PPFR-1 ensure efficient katanin inactivation in the transition to mitosis.

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Regulation of microtubule severing by katanin subunits during neuronal development.

Project description:Katanin, the microtubule-severing protein, consists of a subunit termed P60 that breaks the lattice of the microtubule and another subunit termed P80, the functions of which are not well understood. Data presented here show that the ratio of P60 to P80 varies markedly in different tissues, at different phases of development, and regionally within the neuron. P80 is more concentrated in the cell body and less variable during development, whereas P60 often shows concentrations in the distal tips of processes as well as dramatic spikes in expression at certain developmental stages. Overexpression of P60 at various stages in the differentiation of cultured hippocampal neurons results in substantial loss of microtubule mass and a diminution in total process length. In comparison, overexpression of P80, which is thought to augment the severing of microtubules by P60, results in a milder loss of microtubule mass and diminution in process length. At the developmental stage corresponding to axogenesis, overexpression of P60 decreases the total number of processes extended by the neuron, whereas overexpression of P80 produces the opposite result, suggesting that the effects on neuronal morphology are dependent on the degree of microtubule severing and loss of polymer. The microtubules that occupy the axon are notably more resistant to depolymerization in response to excess P60 or P80 than microtubules elsewhere in the neuron, suggesting that regional differences in the susceptibility of microtubules to severing proteins may be a critical factor in the generation and maintenance of neuronal polarity.

| S-EPMC1201504 | biostudies-literature

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

Project description: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.

| S-EPMC3084677 | biostudies-literature

Phosphorylation of the microtubule-severing AAA+ enzyme Katanin regulates C. elegans embryo development.

Project description: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.

| S-EPMC7265321 | biostudies-literature

The Mammalian Family of Katanin Microtubule-Severing Enzymes.

Project description:The katanin family of microtubule-severing enzymes is critical for cytoskeletal rearrangements that affect key cellular processes like division, migration, signaling, and homeostasis. In humans, aberrant expression, or dysfunction of the katanins, is linked to developmental, proliferative, and neurodegenerative disorders. Here, we review current knowledge on the mammalian family of katanins, including an overview of evolutionary conservation, functional domain organization, and the mechanisms that regulate katanin activity. We assess the function of katanins in dividing and non-dividing cells and how their dysregulation promotes impaired ciliary signaling and defects in developmental programs (corticogenesis, gametogenesis, and neurodevelopment) and contributes to neurodegeneration and cancer. We conclude with perspectives on future katanin research that will advance our understanding of this exciting and dynamic class of disease-associated enzymes.

| S-EPMC8369831 | biostudies-literature

Microtubule-binding domains in Katanin p80 subunit are essential for severing activity in C. elegans.

Project description:Microtubule-severing enzymes (MSEs), such as Katanin, Spastin, and Fidgetin play essential roles in cell division and neurogenesis. They damage the microtubule (MT) lattice, which can either destroy or amplify the MT cytoskeleton, depending on the cellular context. However, little is known about how they interact with their substrates. We have identified the microtubule-binding domains (MTBD) required for Katanin function in C. elegans. Katanin is a heterohexamer of dimers containing a catalytic subunit p60 and a regulatory subunit p80, both of which are essential for female meiotic spindle assembly. Here, we report that p80-like(MEI-2) dictates Katanin binding to MTs via two MTBDs composed of basic patches. Substituting these patches reduces Katanin binding to MTs, compromising its function in female meiotic-spindle assembly. Structural alignments of p80-like(MEI-2) with p80s from different species revealed that the MTBDs are evolutionarily conserved, even if the specific amino acids involved vary. Our findings highlight the critical importance of the regulatory subunit (p80) in providing MT binding to the Katanin complex.

| S-EPMC10853069 | biostudies-literature

Proteomic Analysis of the Mammalian Katanin Family of Microtubule-severing Enzymes Defines Katanin p80 subunit B-like 1 (KATNBL1) as a Regulator of Mammalian Katanin Microtubule-severing.

Project description:The Katanin family of microtubule-severing enzymes is critical for remodeling microtubule-based structures that influence cell division, motility, morphogenesis and signaling. Katanin is composed of a catalytic p60 subunit (A subunit, KATNA1) and a regulatory p80 subunit (B subunit, KATNB1). The mammalian genome also encodes two additional A-like subunits (KATNAL1 and KATNAL2) and one additional B-like subunit (KATNBL1) that have remained poorly characterized. To better understand the factors and mechanisms controlling mammalian microtubule-severing, we have taken a mass proteomic approach to define the protein interaction module for each mammalian Katanin subunit and to generate the mammalian Katanin family interaction network (Katan-ome). Further, we have analyzed the function of the KATNBL1 subunit and determined that it associates with KATNA1 and KATNAL1, it localizes to the spindle poles only during mitosis and it regulates Katanin A subunit microtubule-severing activity in vitro Interestingly, during interphase, KATNBL1 is sequestered in the nucleus through an N-terminal nuclear localization signal. Finally KATNB1 was able to compete the interaction of KATNBL1 with KATNA1 and KATNAL1. These data indicate that KATNBL1 functions as a regulator of Katanin A subunit microtubule-severing activity during mitosis and that it likely coordinates with KATNB1 to perform this function.

| S-EPMC4858946 | biostudies-literature

An anchoring complex recruits katanin for microtubule severing at the plant cortical nucleation sites.

Project description:Microtubules are severed by katanin at distinct cellular locations to facilitate reorientation or amplification of dynamic microtubule arrays, but katanin targeting mechanisms are poorly understood. Here we show that a centrosomal microtubule-anchoring complex is used to recruit katanin in acentrosomal plant cells. The conserved protein complex of Msd1 (also known as SSX2IP) and Wdr8 is localized at microtubule nucleation sites along the microtubule lattice in interphase Arabidopsis cells. Katanin is recruited to these sites for efficient release of newly formed daughter microtubules. Our cell biological and genetic studies demonstrate that Msd1-Wdr8 acts as a specific katanin recruitment factor to cortical nucleation sites (but not to microtubule crossover sites) and stabilizes the association of daughter microtubule minus ends to their nucleation sites until they become severed by katanin. Molecular coupling of sequential anchoring and severing events by the evolutionarily conserved complex renders microtubule release under tight control of katanin activity.

| S-EPMC8211667 | biostudies-literature

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