Paul E. Mains

Genetic analysis of the proximal portion of the mouse t complex: Evidence for a second inversion within t haplotypes

Genomic sequences derived from the mouse t complex by a microdissection cloning technique have been used as tools to obtain high resolution genetic maps of the wild-type and t haplotype forms of the most proximal portion of chromosome 17. Genetic mapping was performed through a recombinant inbred strain analysis and an analysis of partial t haplotypes. The accumulated data demonstrate the existence of a large inversion of genetic material, encompassing the loci of T and qk, within the proximal portion of t haplotypes. This newly described proximal inversion and the previously described distal inversion provide an explanation for the suppression of recombination observed along the length of t haplotype DNA in heterozygous mice.

The Caenorhabditis elegans nonmuscle myosin genes nmy-1 and nmy-2 function as redundant components of the let-502/Rho-binding kinase and mel-11/myosin phosphatase pathway during embryonic morphogenesis

Rho-binding kinase and the myosin phosphatase targeting subunit regulate nonmuscle contractile events in higher eukaryotes. Genetic evidence indicates that the C. elegans homologs regulate embryonic morphogenesis by controlling the actin-mediated epidermal cell shape changes that transform the spherical embryo into a long, thin worm. LET-502/Rho-binding kinase triggers elongation while MEL-11/myosin phosphatase targeting subunit inhibits this contractile event. We describe mutations in the nonmuscle myosin heavy chain gene nmy-1 that were isolated as suppressors of the mel-11 hypercontraction phenotype. However, a nmy-1 null allele displays elongation defects less severe than mutations in let-502 or in the single nonmuscle myosin light chain gene mlc-4. This results because nmy-1 is partially redundant with another nonmuscle myosin heavy chain, nmy-2, which was previously known only for its role in anterior/posterior polarity and cytokinesis in the early embryo. At the onset of elongation, NMY-1 forms filamentous-like structures similar to actin, and LET-502 is interspersed with these structures, where it may trigger contraction. MEL-11, which inhibits elongation, is initially cytoplasmic. In response to LET-502 activity, MEL-11 becomes sequestered away from the contractile apparatus, to the plasma membrane, when elongation commences. Upon completion of morphogenesis, MEL-11 again appears in the cytoplasm where it may halt actin/myosin contraction.

Kinesin-1 mediates translocation of the meiotic spindle to the oocyte cortex through KCA-1, a novel cargo adapter

In animals, female meiotic spindles are attached to the egg cortex in a perpendicular orientation at anaphase to allow the selective disposal of three haploid chromosome sets into polar bodies. We have identified a complex of interacting Caenorhabditis elegans proteins that are involved in the earliest step in asymmetric positioning of anastral meiotic spindles, translocation to the cortex. This complex is composed of the kinesin-1 heavy chain orthologue, UNC-116, the kinesin light chain orthologues, KLC-1 and -2, and a novel cargo adaptor, KCA-1. Depletion of any of these subunits by RNA interference resulted in meiosis I metaphase spindles that remained stationary at a position several micrometers from the cell cortex during the time when wild-type spindles translocated to the cortex. After this prolonged stationary period, unc-116(RNAi) spindles moved to the cortex through a partially redundant mechanism that is dependent on the anaphase-promoting complex. This study thus reveals two sequential mechanisms for translocating anastral spindles to the oocyte cortex.

The mbk-2 kinase is required for inactivation of MEI-1/katanin in the one-cell Caenorhabditis elegans embryo

The Caenorhabditis elegans early embryo is widely used to study the regulation of microtubule‐related processes. In a screen for mutants affecting the first cell division, we isolated a temperature‐sensitive mutation affecting pronuclear migration and spindle positioning, phenotypes typically linked to microtubule or centrosome defects. In the mutant, microtubules are shorter and chromosome segregation is impaired, while centrosome organization appears normal. The mutation corresponds to a strong loss of function in mbk‐2, a conserved serine/threonine kinase. The microtubule‐related defects are due to the postmeiotic persistence of MEI‐1, a homologue of the microtubule‐severing protein katanin. In addition, P‐granule distribution is abnormal in mbk‐2 mutants, consistent with genetic evidence that mbk‐2 has other functions and with the requirement of mbk‐2 activity at the one‐cell stage. We propose that mbk‐2 potentiates the degradation of MEI‐1 and other proteins, possibly via direct phosphorylation.

UNC-89 (obscurin) binds to MEL-26, a BTB-domain protein, and affects the function of MEI-1 (katanin) in striated muscle of Caenorhabditis elegans

UNC-89 (obscurin) interacts with MEL-26, a BTB-domain protein/adaptor for cullin-3. MEL-26 colocalizes with UNC-89 at M-lines. Mutations in MEL-26, CUL-3 (cullin-3), and MEI-1 (katanin) result in a muscle phenotype similar to that of unc-89 mutants. The level of MEI-1 is reduced in unc-89 mutants, suggesting that normally UNC-89 inhibits CUL-3/MEL-26 in muscle.

The BTB Protein MEL-26 Promotes Cytokinesis in C. elegans by a CUL-3-Independent Mechanism

BACKGROUND The initiation of a cleavage furrow is essential to separate cells during cytokinesis, but little is known about the mechanisms controlling this actin-driven process. Previous studies in C. elegans embryos revealed that inactivation of the CUL-3-based E3 ligase activator rfl-1 results in an aberrant microtubule network, ectopic furrowing during pronuclear migration, and defects during cytokinesis. RESULTS Here, we show that MEL-26, a substrate-specific adaptor of the CUL-3-based E3 ligase, is required for efficient cell separation and cleavage furrow ingression during the C. elegans early mitotic divisions. Loss of MEL-26 function leads to delayed onset and slow ingression of cytokinesis furrows that frequently regress. Conversely, increased levels of MEL-26 in cul-3(RNAi) and rfl-1 mutant embryos cause a hypercontractile cortex, with several simultaneously ingressing furrows during pronuclear migration. MEL-26 accumulates at cleavage furrows and binds the actin-interacting protein POD-1. Importantly, POD-1 is not a substrate of the MEL-26/CUL-3 ligase but is required to localize MEL-26 to the cortex. CONCLUSIONS Our results suggest that MEL-26 not only acts as a substrate-specific adaptor within the MEL-26/CUL-3 complex, but also promotes cytokinesis by a CUL-3- and microtubule-independent mechanism.

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

Oocyte meiotic spindles are bipolar but assemble without centrosomes. Three Caenorhabditis elegans genes that contribute are that for the calponin homology domain protein, aspm-1; the katanin mei-1; and the kinesin-12 family member klp-18. The results indicate that both microtubule severing and ASPM-1 promote pole assembly, whereas KLP-18 promotes bipolarity.

The Caenorhabditis elegans ing-3 Gene Regulates Ionizing Radiation-Induced Germ-Cell Apoptosis in a p53-Associated Pathway

The inhibitor of growth (ING) family of type II tumor suppressors are encoded by five genes in mammals and by three genes in Caenorhabditis elegans. All ING proteins contain a highly conserved plant homeodomain (PHD) zinc finger. ING proteins are activated by stresses, including ionizing radiation, leading to the activation of p53. ING proteins in mammals and yeast have recently been shown to read the histone code in a methylation-sensitive manner to regulate gene expression. Here we identify and characterize ing-3, the C. elegans gene with the highest sequence identity to the human ING3 gene. ING-3 colocalizes with chromatin in embryos, the germline, and somatic cells. The ing-3 gene is part of an operon but is also transcribed from its own promoter. Both ing-3(RNAi) and ing-3 mutant strains demonstrate that the gene likely functions in concert with the C. elegans p53 homolog, cep-1, to induce germ-cell apoptosis in response to ionizing radiation. Somatically, the ing-3 mutant has a weak kinker uncoordinated (kinker Unc) phenotype, indicating a possible neuronal function.

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

Caenorhabditis elegans bivalents are positioned between dense bundles of microtubules within female meiotic spindles. Rapid inactivation of katanin after meiotic spindle assembly causes loss of organized microtubule bundles and displacement of bivalents from the metaphase plate. Purified katanin can preferentially sever at intersections between microtubules.

The Role of Protein Phosphatase 4 in Regulating Microtubule Severing in the Caenorhabditis elegans Embryo

MEI-1, the catalytic subunit of the Caenorhabditis elegans “katanin” microtubule-severing complex, is required for meiotic spindle formation. However, MEI-1 must be inactivated after the completion of meiosis to allow formation of the first mitotic spindle. Recent work demonstrated that post-meiotic MEI-1 undergoes ubiquitin-dependent degradation mediated by two independent pathways. Here we describe another level of MEI-1 regulation involving the protein phosphatase 4 (PP4) complex. The PP4 R1 regulatory subunit protein phosphatase four regulatory subunit 1 (ppfr-1) was identified in an RNA interference (RNAi) screen for suppressors of a mei-1(gf) allele that is refractory to post-meiotic degradation. RNAi to the PP4 catalytic subunit PPH-4.1 or to the α4 regulatory PPFR-4 also suppressed lethality of ectopic MEI-1. These results suggest that PP4(+) activates MEI-1, and therefore loss of PP4 decreases ectopic MEI-1(gf) activity. PPH-4.1 and MEI-1 co-immunoprecipitate with one another, indicating that the PP4 complex likely regulates MEI-1 activity directly rather than through an intermediate. The ppfr-1 mutant has subtle meiotic defects indicating that PPFR-1 also regulates MEI-1 during meiosis. MBK-2 is the only kinase known to phosphorylate MEI-1 and triggers post-meiotic MEI-1 degradation. However, genetic interactions between PP4 and mbk-2 were not consistent with an antagonistic relationship between the phosphatase and kinase. Additionally, reducing PP4 in mei-1(gf) did not change the level or localization of post-meiotic MEI-1. Thus, by making use of a genetic background where MEI-1 is ectopically expressed, we have uncovered a third mechanism of MEI-1 regulation, one based on phosphorylation but independent of degradation. The redundant regulatory pathways likely contribute in different ways to the rapid and precise post-meiotic inactivation of MEI-1 microtubule-severing activity.