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Complementary roles for dynein and kinesins in the Xenopus egg cortical rotation.

Aligned vegetal subcortical microtubules in fertilized Xenopus eggs mediate the "cortical rotation", a translocation of the vegetal cortex and of dorsalizing factors toward the egg equator. Kinesin-related protein (KRP) function is essential for the cortical rotation, and dynein has been implicated indirectly; however, the role of neither microtubule motor protein family is understood. We examined the consequence of inhibiting dynein--dynactin-based transport by microinjection of excess dynamitin beneath the vegetal egg surface. Dynamitin introduced before the cortical rotation prevented formation of the subcortical array, blocking microtubule incorporation from deeper regions. In contrast, dynamitin injected after the microtubule array was fully established did not block cortical translocation, unlike inhibitory-KRP antibodies. During an early phase of cortical rotation, when microtubules showed a distinctive wavy organization, dynamitin disrupted microtubule alignment and perturbed cortical movement. These findings indicate that dynein is required for formation and early maintenance of the vegetal microtubule array, while KRPs are largely responsible for displacing the cortex once the microtubule tracks are established. Consistent with this model for the cortical rotation, photobleach analysis revealed both microtubules that translocated with the vegetal cytoplasm relative to the cortex, and ones that moved with the cortex relative to the cytoplasm.

Animals↗

Nucleoporins NPP-1, NPP-3, NPP-4, NPP-11 and NPP-13 are required for proper spindle orientation in C. elegans.

Nucleoporins are components of the nuclear pore, which is required for nucleo-cytoplasmic transport. We report a role for a subclass of nucleoporins in orienting the mitotic spindle in C. elegans embryos. RNAi-mediated depletion of any of five putative nucleoporins npp-1, npp-3, npp-4, npp-11, and npp-13 leads to indistinguishable spindle orientation defects. Transgenic worms expressing NPP-1::GFP or NPP-11::GFP show GFP localization at the nuclear envelope, consistent with their predicted function. NPP-1 interacts with the other nucleoporins in yeast two-hybrid assays, suggesting that the proteins affect spindle orientation by a common process. The failed orientation phenotype of npp-1(RNAi) is at least partially epistatic to the ectopic spindle rotation in the AB blastomere of par-3 mutant embryos. This suggests that NPP-1 contributes to the mechanics of spindle orientation. However, NPP-1 is also required for PAR-6 asymmetry at the two-cell stage, indicating that nucleoporins may be required to define cortical domains in the germ line blastomere P1. Nuclear envelope structure is abnormal in npp-1(RNAi) embryos, but the envelope maintains its integrity, and most nuclear proteins we assayed accumulate normally. These findings raise the possibility that these nucleoporins may have direct roles in orienting the mitotic spindle and the maintenance of cell polarity.

Adenosine Triphosphatases↗

High-resolution map and imprinting analysis of the Gtl2-Dnchc1 domain on mouse chromosome 12.

The imprinted Dlk1-Dio3 region on mouse chromosome 12 contains six imprinted genes and a number of maternally expressed snoRNAs and miRNAs. Here we present a high-resolution sequence analysis of the 1.1-Mb segment telomeric to Gtl2 in mouse and a homology comparison to the human. Ppp2r5c and Dnchc1 at the telomeric end of the analyzed sequence are biallelically expressed, suggesting that the imprinted domain does not extend beyond the paternally expressed Dio3 gene. RT-PCR experiments support the predicted presence of a maternally expressed intergenic transcript(s) encompassing Gtl2, Rian, and Mirg. These maternally expressed genes, and also the intergenic transcript(s), show pronounced expression in the adult mouse brain, whereas the paternally transcribed Dio3 and the nonimprinted Ppp2r5c and Dnchc1 are expressed in different tissues. Hence, tissue-specific coregulation of maternally expressed genes might be an important feature of this domain.

Animals↗

Rhodopsin's carboxy-terminal cytoplasmic tail acts as a membrane receptor for cytoplasmic dynein by binding to the dynein light chain Tctex-1.

The interaction of cytoplasmic dynein with its cargoes is thought to be indirectly mediated by dynactin, a complex that binds to the dynein intermediate chain. However, the roles of other dynein subunits in cargo binding have been unknown. Here we demonstrate that dynein translocates rhodopsin-bearing vesicles along microtubules. This interaction occurs directly between the C-terminal cytoplasmic tail of rhodopsin and Tctex-1, a dynein light chain. C-terminal rhodopsin mutations responsible for retinitis pigmentosa inhibit this interaction. Our results point to an alternative docking mechanism for cytoplasmic dynein, provide novel insights into the role of motor proteins in the polarized transport of post-Golgi vesicles, and shed light on the molecular basis of retinitis pigmentosa.

Amino Acid Sequence↗

The genomic structure of DCTN1, a candidate gene for limb-girdle muscular dystrophy (LGMD2B).

Dynactin is a required activator for the molecular motor cytoplasmic dynein, and is likely to be essential for normal neuronal development. Previously we mapped the human gene encoding the p150Glued subunit of dynactin to 2p13, in the vicinity of the locus linked to limb-girdle muscular dystrophy (LGMB2B). We now report the genomic organization of DCTN1. We have identified 32 exons in the gene which spans approximately 25 kb. Alternative splicing of several of the exons generates functionally distinct isoforms of the p150Glued polypeptide.

Alternative Splicing↗

The role of cytoplasmic dynein in the human brain developmental disease lissencephaly.

Lissencephaly is a brain developmental disorder characterized by disorganization of the cortical regions resulting from defects in neuronal migration. Recent evidence has implicated the human LIS-1 gene in Miller-Dieker lissencephaly and isolated lissencephaly sequence. LIS-1 is homologous to the fungal genes NudF and PAC1, which are involved in cytoplasmic dynein mediated nuclear transport, but it is also almost identical to a subunit of PAF acetylhydrolase, an enzyme which inactivates the lipid mediator platelet activating factor. Recent evidence from our laboratory has revealed that cytoplasmic dynein coimmunoprecipitates with LIS-1 in bovine brain cytosol, supporting a role in the dynein pathway in vertebrates. Overexpression of LIS-1 interferes with cell division, with noteworthy effects on chromosome attachment to the mitotic spindle and on the interaction of astral microtubules with the cell cortex. Other aspects of dynein function, such as the organization of the Golgi apparatus, are not affected. Together, these results suggest a role for LIS-1 in cytoplasmic dynein functions involving microtubule plus-ends. Furthermore, they suggest that mutations in LIS-1 may produce a lissencephalic phenotype either by interfering with the movement of neuronal nuclei within extending processes, or by interference with the division cycle of neuronal progenitor cells in the ventricular and subventricular zones of the developing nervous system.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

Mitotic motors in Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae provides a unique opportunity for study of the microtubule-based motor proteins that participate in mitotic spindle function. The genome of Saccharomyces encodes a relatively small and genetically tractable set of microtubule-based motor proteins. The single cytoplasmic dynein and five of the six kinesin-related proteins encoded have been implicated in mitotic spindle function. Each motor protein is unique in amino acid sequence. On account of functional overlap, no single motor is uniquely required for cell viability, however. The ability to create and analyze multiple mutants has allowed experimental dissection of the roles performed by each mitotic motor. Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle (kinesin-related Cin8p, Kip1p, Kip3p and Kar3p). Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell (dynein and kinesin-related Kip2p, Kip3p and Kar3p). The six motors apparently contribute three fundamental activities to spindle function: motility, microtubule cross-linking and regulation of microtubule dynamics.

Anaphase↗

Two approaches to isolate cytoplasmic dynein ATPase from Neurospora crassa.

Cytoplasmic dynein is a force-producing enzyme that, in association with dynactin, conducts minus-end directed transport of various organelles along microtubules. Biochemical analyses of cytoplasmic dynein and dynactin have been conducted primarily in vertebrate systems, whereas genetic analyses have been explored mainly in yeast and the filamentous fungi. To provide a complementary biochemical approach for the study of fungal dynein, we isolated/partially purified cytoplasmic dynein ATPase from the filamentous fungus Neurospora crassa. N. crassa dynein was partially purified by slightly modifying the existing procedures, described for mammalian cytoplasmic dynein that uses dynein-microtubule binding, followed by release with ATP and sucrose gradient fractionation. A novel approach was also used to isolate dynein-specific ATPase by gel filtration (Sepharose CL-4B). The K(m), ATP obtained by isolating dynein ATPase using gel filtration was similar to that obtained by using conventional method, suggests that contaminant proteins do not interfere with the dynein ATPase activity. Like vertebrate dynein, N. crassa dynein is a general NTPase with highest activity toward ATP, and only the ATPase activity is stimulated by microtubules. The K(m), ATP for N. crassa cytoplasmic dynein is 10- to 15-fold higher than that of the vertebrate enzyme.

Adenosine Triphosphate↗

The BMP homolog Gbb provides a retrograde signal that regulates synaptic growth at the Drosophila neuromuscular junction.

We show that the BMP ortholog Gbb can signal by a retrograde mechanism to regulate synapse growth of the Drosophila neuromuscular junction (NMJ). gbb mutants have a reduced NMJ synapse size, decreased neurotransmitter release, and aberrant presynaptic ultrastructure. These defects are similar to those we observe in mutants of BMP receptors and Smad transcription factors. However, whereas these BMP receptors and signaling components are required in the presynaptic motoneuron, Gbb expression is required in large part in postsynaptic muscles; gbb expression in muscle rescues key aspects of the gbb mutant phenotype. Consistent with this notion, we find that blocking retrograde axonal transport by overexpression of dominant-negative p150/Glued in neurons inhibits BMP signaling in motoneurons. These experiments reveal that a muscle-derived BMP retrograde signal participates in coordinating neuromuscular synapse development and growth.

Adenine Nucleotides↗

Dynein at the cortex.

Cytoplasmic dynein is a minus end directed microtubule motor protein with numerous functions during interphase and mitosis. Recent evidence has identified several roles mediated by a fraction of cytoplasmic dynein associated with the cell cortex. So far, these include nuclear migration, mitotic spindle orientation, and cytoskeletal reorientation during wound healing, but others are likely. The possibility that a cortically bound form of dynein might represent its most ancient evolutionary state is discussed.

Animals↗

Dynactin.

Explore the source record for details and available documents.

Dynactin Complex↗

Chromosome movement: dynein-out at the kinetochore.

Cell biologists have long speculated that a minus end-directed motor localized at kinetochores contributes to the poleward movement of chromosomes during mitosis. Two recent studies provide direct evidence that cytoplasmic dynein can perform this function.

Animals↗

Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization.

In migrating adherent cells such as fibroblasts and endothelial cells, the microtubule-organizing center (MTOC) reorients toward the leading edge [1-3]. MTOC reorientation repositions the Golgi toward the front of the cell [1] and contributes to directional migration [4]. The mechanism of MTOC reorientation and its relation to the formation of stabilized microtubules (MTs) in the leading edge, which occurs concomitantly with MTOC reorientation [3], is unknown. We show that serum and the serum lipid, lysophosphatidic acid (LPA), increased Cdc42 GTP levels and triggered MTOC reorientation in serum-starved wounded monolayers of 3T3 fibroblasts. Cdc42, but not Rho or Rac, was both sufficient and necessary for LPA-stimulated MTOC reorientation. MTOC reorientation was independent of Cdc42-induced changes in actin and was not blocked by cytochalasin D. Inhibition of dynein or dynactin blocked LPA- and Cdc42-stimulated MTOC reorientation. LPA also stimulates a Rho/mDia pathway that selectively stabilizes MTs in the leading edge [5, 6]; however, activators and inhibitors of MTOC reorientation and MT stabilization showed that each response was regulated independently. These results establish an LPA/Cdc42 signaling pathway that regulates MTOC reorientation in a dynein-dependent manner. MTOC reorientation and MT stabilization both act to polarize the MT array in migrating cells, yet these processes act independently and are regulated by separate Rho family GTPase-signaling pathways.

3T3 Cells↗

Nuclear envelope: torn apart at mitosis.

The findings of two recent studies suggest a novel mechanism for nuclear envelope breakdown in which cytoplasmic dynein anchored on the outside of the nucleus generates tension, thus triggering tearing of the nuclear envelope.

Animals↗