Cytoplasmic Dynein Light Intermediate Chain Is Required for Discrete Aspects of Mitosis inCaenorhabditis elegans

Abstract

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 in dli-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 the dli-1 gene product through RNA-mediated gene interference (RNAi) reveals an early embryonic requirement. One-cell dli-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 in dli-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-1 mutant background, indicating this is not an essential domain for DLI-1 function. INTRODUCTIONCytoplasmic dynein is a large multisubunit complex composed of two motor proteins, the heavy chains, and several associated subunits that have been named light, light intermediate, and intermediate chains. These subunits have no sequence homology; instead, the names reflect their relative molecular weights. Cytoplasmic dynein is the major microtubule minus-end-directed motor protein and has been implicated in a variety of cellular processes, during both interphase and mitosis. Membranous vesicle transport, endoplasmic reticulum-to-Golgi transport, and axonal retrograde transport are all dynein-dependent processes (Lacey and Haimo, 1992;Dillman et al., 1996;Presley et al., 1997).Dynein's mitotic roles are numerous. Function blocking antibody experiments against heavy chain in vertebrate cells have shown the motor protein is required for proper spindle formation and centrosome separation (Vaisberg et al., 1993). Similar results were observed in Drosophila heavy chain mutants with additional observations suggesting dynein is required to maintain centrosome association with the nuclear envelope (Robinson et al., 1999). In both Aspergillus nidulans and Neurospora crassa, dynein heavy chain mutations reveal a role for the complex in nuclear migration (Plamann et al., 1994;Xiang et al., 1995). Other recent work suggests dynein may mediate microtubule binding at the kinetochore (Wordeman and Mitchison, 1995).The one-cell Caenorhabditis elegans embryo is also an excellent model system for investigating the roles of dynein, its subunits, and the proteins with which it interacts. When cytoplasmic dynein heavy chain (dhc-1) was eliminated through RNA-mediated gene interference (RNAi), all of the above-mentioned phenotypes were o...

SPD-3 Is Required for Spindle Alignment in Caenorhabditis elegans Embryos and Localizes to Mitochondria

“…DISCUSSION SPD-3 and dynein: We have shown that oj35 embryos have defects in meiosis, maternal pronuclear migration, and mitotic spindle alignment leading to embryonic lethality. Interestingly, these defects are similar to those seen in weak loss-of-function of the dynein motor protein or components of its activating protein complex, dynactin (Skop and White 1998;Gonczy et al 1999;Yoder and Han 2001;Schmidt et al 2005). In addition, we showed that SPD-3 is required for proper localization or expression of the dynactin components DNC-2 (p50 dynamitin) and DNC-1 (p150glued).…”

“…NM-068635. 1 processes including meiosis, pronuclear migration, centrosome separation, and chromosome segregation (Skop and White 1998;Gonczy et al 1999;Yoder and Han 2001). Generally, it is believed that spindle alignment occurs by the capture of astral microtubule plus ends at the cell cortex by localized or locally activated dynein/ dynactin complexes, followed by shortening of the captured microtubules (White and Strome 1996;Tsou et al 2002).…”

Dlic1 deficiency impairs ciliogenesis of photoreceptors by destabilizing dynein

“…The function of the DLIC protein was first implicated in the control of the mitotic spindle and the assembly of centrioles by the fact that DLIC1, not DLIC2, specifically interacted with pericentrin (PCNT) in Cos-7 cells [7]. Mutations in the gene encoding DLIC or depleting its gene product by RNAi were shown to result in a variety of mitotic defects from yeast to mammals [8][9][10][11]. DLIC1 also participates in intracellular vesicle transport via forming a complex with small GTPases Rab4 and Rab11, respectively [12,13].…”

Mutations inCaenorhabditis elegansCytoplasmic Dynein Components Reveal Specificity of Neuronal Retrograde Cargo

“…1(ku266), did not complement js351 and had identical misaccumulation phenotypes (Yoder and Han, 2001) (data not shown). Sequencing the dli-1 gene in js351 identified a stop codon (CAA to TAA) at amino acid Gln 218 .…”

“…pJHY10, a StuI-XhoI fragment of dli-1 containing 3.5 kb upstream, 1.6 kb genomic open reading frame, and 450 bp of the 3Ј untranslated region, was used for rescue (Yoder and Han, 2001). pJHY10 at 15 ng/l and pRF4 at 150 ng/l were injected into dli-1(js351) /dpy-20(e1282) unc-31(e169) unc-26(e205); jsIs37.…”