Functional Analysis of Cytoplasmic Dynein Heavy Chain inCaenorhabditis eleganswith Fast-acting Temperature-sensitive Mutations

Abstract

Cytoplasmic dynein, a minus-end-directed microtubule motor, has been implicated in many cellular and developmental processes. Identification of specific cellular processes that rely directly on dynein would be facilitated by a means to induce specific and rapid inhibition of its function. We have identified conditional variants of a Caenorhabditis elegans dynein heavy chain (DHC-1) that lose function within a minute of a modest temperature upshift. Mutant embryos generated at elevated temperature show defects in centrosome separation, pronuclear migration, rotation of the centrosome/nucleus complex, bipolar spindle assembly, anaphase chromosome segregation, and cytokinesis. Our analyses of mutant embryos generated at permissive temperature and then upshifted quickly just before events of interest indicate that DHC-1 is required specifically for rotation of the centrosome/nucleus complex, for chromosome congression to a well ordered metaphase plate, and for timely initiation of anaphase. Our results do not support the view that DHC-1 is required for anaphase B separation of spindle poles and chromosomes. A P-loop mutation identified in two independent dominant temperature-sensitive alleles of dhc-1, when engineered into the DHC1 gene of Saccharomyces cerevisiae, conferred a dominant temperature-sensitive dynein loss-of-function phenotype. This suggests that temperature-sensitive mutations can be created for time-resolved function analyses of dyneins and perhaps other P-loop proteins in a variety of model systems. INTRODUCTIONThe active transport of proteins, RNAs, organelles, and chromosomes to specific destinations within cells is essential for normal development. In animal cells, two major classes of force-producing motor proteins, kinesins and dyneins, use the energy generated by ATP hydrolysis to drive the active transport of cargoes along microtubules, which act as directional tracks. Dyneins and kinesins with C-terminal motor domains move toward microtubule minus-ends, whereas kinesins with N-terminal motor domains move toward plusends (reviewed in Sawin and Scholey, 1991; Higuchi and Endow, 2002;Vale, 2003). The diverse forms of dyneins and kinesins and substantial experimental work have established that, despite the simplicity of bidirectional microtubule tracks, force generation and transport along them are complex. Specific processes require particular motors or sets of motors, and those motors are subject to precise regulatory controls. Although the full complement of microtubule motors has been identified in several model organisms, their functions and hence the mechanisms of many active transport processes remain poorly defined.We focus here on questions about the functions of cytoplasmic dynein in mitosis and early patterning in C. elegans embryos. Cytoplasmic dynein is a multisubunit complex composed of two identical ϳ500-kDa heavy chains (DHCs) and several intermediate, light intermediate, and light chains (reviewed in Milisav, 1998;King, 2000;Vale, 2003). DHC is the force-producing component of...

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).…”

“…Surprisingly, we found that although the microtubule localization of DNC-1 appeared normal in oj35 embryos, the majority of the protein appeared to localize into posterior structures resembling P granules. In addition, the centrosome labeling appeared brighter than that in wild type, a phenotype reminiscent of defects in dynein heavy chain function (Schmidt et al 2005).…”

Mitotic Chromosome Biorientation in Fission Yeast Is Enhanced by Dynein and a Minus-end–directed, Kinesin-like Protein

“…Alternatively, congression can be brought about by attachment of one of the pole-proximal sister kinetochores to MTs that grow from the distant pole (Nicklas, 1997). In this case congression is presumably driven by minus end-directed motors pulling the distal kinetochore toward the pole it faces (Savoian et al, 2000;Sharp et al, 2000, Schmidt et al, 2005.…”

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in <em>C. elegans</em> Embryos

“…The position of the centrosomes is indicative of the stage of the cell cycle and is helpful in timing anoxia experiments. Chromosomes are marked by ruIs32 [unc-119 (+) pie-1::GFP::H2B], which encodes a GFP labeled histone protein and is driven by a germ-line specific promoter 5 . Thus, the movement and localization of chromosomes within blastomeres or oocytes can be followed.…”

“…Produce or obtain appropriate transgenic C. elegans strain of interest using transgenic methodologies or from Caenorhabditis Genetics Stock Center (CGC) or colleague. In this case we are using strain TH32 (pie-1::tbg-1::GFP; pie-1::GFP::H2B) 5 to visualize chromosomes and centrosomes as markers for cell division. 2.…”