The centrosome is generally maintained at the center of the cell. In animal cells, centrosome centration is powered by the pulling force of microtubules, which is dependent on cytoplasmic dynein. However, it is unclear how dynein brings the centrosome to the cell center, i.e., which structure inside the cell functions as a substrate to anchor dynein. Here, we provide evidence that a population of dynein, which is located on intracellular organelles and is responsible for organelle transport toward the centrosome, generates the force required for centrosome centration in Caenorhabditis elegans embryos. By using the database of full-genome RNAi in C. elegans, we identified dyrb-1, a dynein light chain subunit, as a potential subunit involved in dynein anchoring for centrosome centration. DYRB-1 is required for organelle movement toward the minus end of the microtubules. The temporal correlation between centrosome centration and the net movement of organelle transport was found to be significant. Centrosome centration was impaired when Rab7 and RILP, which mediate the association between organelles and dynein in mammalian cells, were knocked down. These results indicate that minus end-directed transport of intracellular organelles along the microtubules is required for centrosome centration in C. elegans embryos. On the basis of this finding, we propose a model in which the reaction forces of organelle transport generated along microtubules act as a driving force that pulls the centrosomes toward the cell center. This is the first model, to our knowledge, providing a mechanical basis for cytoplasmic pulling force for centrosome centration.endosome | lysosome | pronuclear migration | the centrosome-organelle mutual pulling model | yolk granule

t he centrosome is a major microtubule-organizing center in animal cells, and its intracellular positioning is critical for defining intracellular architecture. the centrosome positions itself at the cell center. centrosome centration depends on the microtubule cytoskeleton. to accomplish robust centration regardless of the cell size or cell shape, it has been assumed that the force mediated by the microtubules depends on microtubule length. however, a concrete mechanism to generate forces to pull the centrosome in a microtubule lengthdependent manner has been elusive. recently, we successfully demonstrated that centrosome-directed movement of intracellular organelles along microtubules drives centrosome centration in the Caenorhabditis elegans early embryo. based on this observation, we proposed the centrosome-organelle mutual pulling model in which the reaction forces of organelle transport generated along microtubules act as a driving force that pulls the centrosomes toward the cell center. this is the first experiment-based model that accounts for the microtubule length-dependent pulling force generated in the cytoplasm contributing to centrosome centration. intriguingly, this model is consistent with a recent estimation that the pulling force is proportional to the cubic length of microtubules. The Emergence of Order in Cell ArchitectureThe cell can be compared to a city. A population of macromolecules lives their lives inside the cell, and they utilize the a novel mechanism of microtubule length-dependent force to pull centrosomes toward the cell center

Mitochondrial heat shock protein 70 (mthsp70) functions as a mitochondrial import motor and is essential in mitochondrial biogenesis and energy generation in eukaryotic cells. HSP-6 (hsp70F) is a nematode orthologue of mthsp70. Knockdown of HSP-6 by RNA interference in young adult nematodes caused a reduction in the levels of ATP-2, HSP-60 and CLK-1, leading to abnormal mitochondrial morphology and lower ATP levels. As a result, RNA interference-treated worms had lower motility, defects in oogenesis, earlier accumulation of autofluorescent material, and a shorter life span. These are the major phenotypes observed during the aging of worms, suggesting that the reduction of HSP-6 causes early aging or progeria-like phenotypes. The amount of HSP-6 became dramatically reduced at the expected mean life span in not only wild-type but also in long and short life span mutant worms (wild-type, daf-2, and daf-16). Mitochondrial HSP-60 and ATP-2 were also reduced following the reduction of HSP-6 during aging. These results suggest that the reduction of HSP-6 causes defects in mitochondrial function at the final stage of aging, leading to mortality.Mitochondria are major organelles that carry out cellular oxidation and produce most of the cellular ATP by oxidative phosphorylation. Mitochondria also play essential roles in controlling cell viability and proliferation (1, 2). A large number of studies have shown the essential roles of mitochondria in development and differentiation (3, 4). Furthermore, mitochondria are thought to be deeply involved in the aging process, based on the free radical theory of aging, because mitochondria are a major source of reactive oxygen species (ROS) 3 (5, 6). Accumulating evidence supports this idea. For example, abnormal mitochondria accumulate during aging (7), and enforced breakdown of mtDNA or their repair mechanism causes premature aging in mice (8,9). In Caenorhabditis elegans, mutations in mev-1 (a subunit of the enzyme succinate dehydrogenase cytochrome b, a component of complex II of the mitochondrial electron transport chain) and gas-1 (a subunit of mitochondrial NADH-ubiquinone oxidoreductase, a component of complex I of the mitochondrial electron transport chain) increase ROS production and sensitivity to stress, resulting in a shorter life span (10 -13).A mammalian mitochondrial heat shock protein 70 (mthsp70, also known as mortalin or Grp75) has been shown to function as a mitochondrial protein import motor and is involved in mitochondrial biogenesis (14, 15). It has also been shown to be involved in the production of ROS (16), cell proliferation (17), and the regulation of life span (18) in mammalian cells. Increased expression of HSP-6 (hsp70F), the predicted C. elegans orthologue of mthsp70, by the introduction of an extra hsp-6 gene copies extended the life span of C. elegans (19). In contrast, deletion mutations of SSC1, the yeast orthologue of mthsp70, were lethal (20 -22), and knockdown of mthsp70 caused growth arrest in human cancer cells (17,23). Recently, it has been ...

We report that free tubulin subunits in the Caenorhabditis elegans embryo accumulate in the nascent spindle region, independent of spindle formation. We propose that this newly identified mechanism of accumulation of free tubulin and other molecules at the nascent spindle region contributes to efficient assembly of the mitotic spindle.

Cytoplasmic streaming refers to a collective movement of cytoplasm observed in many cell types. The mechanism of meiotic cytoplasmic streaming (MeiCS) in Caenorhabditis elegans zygotes is puzzling as the direction of the flow is not predefined by cell polarity and occasionally reverses. Here, we demonstrate that the endoplasmic reticulum (ER) network structure is required for the collective flow. Using a combination of RNAi, microscopy and image processing of C. elegans zygotes, we devise a theoretical model, which reproduces and predicts the emergence and reversal of the flow. We propose a positive-feedback mechanism, where a local flow generated along a microtubule is transmitted to neighbouring regions through the ER. This, in turn, aligns microtubules over a broader area to self-organize the collective flow. The proposed model could be applicable to various cytoplasmic streaming phenomena in the absence of predefined polarity. The increased mobility of cortical granules by MeiCS correlates with the efficient exocytosis of the granules to protect the zygotes from osmotic and mechanical stresses.

Remodeling of the embryo surface after fertilization is mediated by the exocytosis of cortical granules derived from the Golgi complex. This process is essential for oocyte-to-embryo transition in many species. However, how the fertilization signal reaches the cortical granules for their timely exocytosis is largely unknown. In Caenorhabditis elegans, the recruitment of separase, a downstream effector of the fertilization signal, to the cortical granules is essential for exocytosis because separase is required for membrane fusion. However, the molecule that recruits separase to the cortical granules remains unidentified. In this study, we found that Rab6, a Golgi-associated GTPase, is essential to recruit separase to the cortical granules in C. elegans embryos. Knockdown of the rab-6.1 gene, a Rab6 homologue in C. elegans, resulted in failure of the membrane fusion step of cortical granule exocytosis. Using a transgenic strain that expresses GFP-fused RAB-6.1, we found that RAB-6.1 temporarily co-localized with separase on the cortical granules for a few minutes and then was dispersed in the cytoplasm concomitantly with membrane fusion. We found that RAB-6.1 as well as cyclin-dependent kinase (CDK)-1 and anaphase promoting complex/cyclosome (APC/C) was required to recruit separase to the cortical granules. RAB-6.1 was not required for the chromosome segregation process, unlike CDK-1, APC/C, and SEP-1. The results indicate that RAB-6.1 is required specifically for the membrane fusion step of exocytosis and for the recruitment of separase to the granules. Thus, RAB-6.1 is an important molecule for the timely exocytosis of the cortical granules during oocyte-to-embryo transition.

Cell polarization is required to define body axes during development. The position of spatial cues for polarization is critical to direct the body axes. In Caenorhabditis elegans zygotes, the sperm-derived pronucleus/centrosome complex (SPCC) serves as the spatial cue to specify the anterior-posterior axis. Approximately 30 minutes after fertilization, the contractility of the cell cortex is relaxed near the SPCC, which is the earliest sign of polarization and called symmetry breaking (SB). It is unclear how the position of SPCC at SB is determined after fertilization. Here, we show that SPCC drifts dynamically through the cell-wide flow of the cytoplasm, called meiotic cytoplasmic streaming. This flow occasionally brings SPCC to the opposite side of the sperm entry site before SB. Our results demonstrate that cytoplasmic flow determines stochastically the position of the spatial cue of the body axis, even in an organism like C. elegans for which development is stereotyped. [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Repulsive guidance molecules (RGMs) are evolutionarily conserved proteins implicated in repulsive axon guidance. Here we report the function of the Caenorhabditis elegans ortholog DRAG-1 in axon branching. The axons of hermaphrodite-specific neurons (HSNs) extend dorsal branches at the region abutting the vulval muscles. The drag-1 mutants exhibited defects in HSN axon branching in addition to a small body size phenotype. DRAG-1 expression in the hypodermal cells was required for the branching of the axons. Although DRAG-1 is normally expressed in the ventral hypodermis excepting the vulval region, its ectopic expression in vulval precursor cells was sufficient to induce the branching. The C-terminal glycosylphosphatidylinositol anchor of DRAG-1 was important for its function, suggesting that DRAG-1 should be anchored to the cell surface. Genetic analyses suggested that the membrane receptor UNC-40 acts in the same pathway with DRAG-1 in HSN branching. We propose that DRAG-1 expressed in the ventral hypodermis signals via the UNC-40 receptor expressed in HSNs to elicit branching activity of HSN axons.