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Dystonia-causing mutant torsinA inhibits cell adhesion and neurite extension through interference with cytoskeletal dynamics.

Early onset torsion dystonia is a movement disorder inherited as an autosomal dominant syndrome with reduced penetrance. Symptoms appear to result from altered neuronal circuitry within the brain with no evidence of neuronal loss. Most cases are caused by loss of a glutamic acid residue in the AAA+ chaperone protein, torsinA, encoded in the DYT1 gene. In this study, torsinA was found to move in conjunction with vimentin in three cell culture paradigms-recovery from microtubule depolymerization, expression of a dominant-negative form of kinesin light chain and respreading after trypsinization. Co-immune precipitation studies revealed association between vimentin and torsinA in a complex including other cytoskeletal elements, actin and tubulin, as well as two proteins previously shown to interact with torsinA-the motor protein, kinesin light chain 1, and the nuclear envelope protein, LAP1. Morphologic and functional differences related to vimentin were noted in primary fibroblasts from patients carrying this DYT1 mutation as compared with controls, including an increased perinuclear concentration of vimentin and a delayed rate of adhesion to the substratum. Overexpression of mutant torsinA inhibited neurite extension in human neuroblastoma cells, with torsinA and vimentin immunoreactivity enriched in the perinuclear region and in cytoplasmic inclusions. Collectively, these studies suggest that mutant torsinA interferes with cytoskeletal events involving vimentin, possibly by restricting movement of these particles/filaments, and hence may affect development of neuronal pathways in the brain.

Actins↗

Mutant huntingtin aggregates impair mitochondrial movement and trafficking in cortical neurons.

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine repeat in the huntingtin gene (Htt). Mitochondrial defects and protein aggregates are characteristic of affected neurons. Recent studies suggest that these aggregates impair cellular transport mechanisms by interacting with cytoskeletal components and molecular motors. Here, we investigated whether mutant Htt alters mitochondrial trafficking and morphology in primary cortical neurons. We demonstrate that full-length mutant Htt was more effective than N-terminal mutant Htt in blocking mitochondrial movement, an effect that correlated with its heightened expression in the cytosolic compartment. Aggregates impaired the passage of mitochondria along neuronal processes, causing mitochondria to accumulate adjacent to aggregates and become immobilized. Furthermore, mitochondrial trafficking was reduced specifically at sites of aggregates while remaining unaltered in regions lacking aggregates. We conclude that in cortical neurons, an early event in HD pathophysiology is the aberrant mobility and trafficking of mitochondria caused by cytosolic Htt aggregates.

Animals↗

MgcRacGAP regulates cortical activity through RhoA during cytokinesis.

Although Rho GTPases regulate multiple cellular events, their role in cell division is still obscure. Here we show that expression of a GTPase-activating protein (GAP)-deficient mutant (R386A) of the Rho regulator MgcRacGAP induces abnormal cortical activity during cytokinesis in U2OS cells. Multiple large blebs were observed in cells expressing MgcRacGAP R386A from the onset of anaphase to the late stage of cell division. When mitotic blebbing was excessive, cytokinesis was inhibited, and cells with micronuclei were generated. It has been reported that blebbing is caused by abnormal cortical activity. The MgcRacGAP R386A-induced abnormal cortical activity was inhibited by the dominant negative form of RhoA, but not Rac1 or Cdc42. Moreover, expression of constitutively active RhoA also induced drastic cortical activity during cytokinesis. Unlike apoptotic blebbing, MgcRacGAP R386A-induced blebbing was not inhibited by the ROCK inhibitor Y-27632, suggesting that MgcRacGAP regulates cortical activity during cytokinesis through a novel signaling pathway. We propose that MgcRacGAP plays a pivotal role in cytokinesis by regulating cortical movement through RhoA.

Cell Division↗

Viscoelastic response of fibroblasts to tension transmitted through adherens junctions.

Cytoplasmic deformation was monitored by observing the displacements of 200-nm green fluorescent beads microinjected into the cytoplasm of Swiss 3T3 fibroblasts. We noted a novel protrusion of nonruffling cell margins that was accompanied by axial flow of beads and cytoplasmic vesicles as far as 50 microm behind the protruding plasma membrane. Fluorescent analog cytochemistry and immunofluorescence localization of F-actin, alpha-actinin, N-cadherin, and beta-catenin showed that the protruding margins of deforming cells were mechanically coupled to neighboring cells by adherens junctions. Observations suggested that protrusion resulted from passive linear deformation in response to tensile stress exerted by centripetal contraction of the neighboring cell. The time dependence of cytoplasmic strain calculated from the displacements of beads and vesicles was fit quantitatively by a Kelvin-Voight model for a viscoelastic solid with a mean limiting strain of 0.58 and a mean strain rate of 4.3 x 10(-3) s(-1). In rare instances, the deforming cell and its neighbor spontaneously became uncoupled, and recoil of the protruding margin was observed. The time dependence of strain during recoil also fit a Kelvin-Voight model with similar parameters, suggesting that the kinetics of deformation primarily reflect the mechanical properties of the deformed cell rather than the contractile properties of its neighbor. The existence of mechanical coupling between adjacent fibroblasts through adherens junctions and the viscoelastic responses of cells to tension transmitted directly from cell to cell are factors that must be taken into account to fully understand the role of fibroblasts in such biological processes as wound closure and extracellular matrix remodeling during tissue development.

3T3 Cells↗

Contrasting patterns of mitochondrial redistribution in the early lineages of Caenorhabditis elegans and Acrobeloides sp. PS1146.

We compared the redistribution of mitochondria in the early embryos of Caenorhabditis elegans (C. elegans) and Acrobeloides sp. PS1146 (Acrobeloides)--two nematode species where the mechanisms for embryonic axis specification are different even though subsequent development is remarkably similar. During the first cell cycle of C. elegans, mitochondria move with the bulk cytoplasmic flows that are directed toward the sperm pronucleus and aggregate at the posterior cortex during the period known as "pseudocleavage." In contrast, in Acrobeloides embryos, where prominent cytoplasmic rearrangements are absent, mitochondria that are initially distributed loosely around the pronuclei and the cytoplasm are relocated around the mitotic spindle prior to cell division. Interestingly, this rearrangement is reiterated only in the germline and not the somatic lineage. In both species, the location of the mitochondria immediately prior to cell division correlates with the known location of the germline determinants, P granules, leading us to speculate that they may be associated.

Animals↗

The cell in the field of gravity and the centrifugal field.

It appears that the literature and logic that the earth's gravity has been one factor in the limitation of cell size, as well as being an important influence on the diversity of cell types and sizes throughout biological evolution. Analysis of the literature reveals an inverse relationship between the centrifugal force needed for intracellular stratification and cell size. The cells studied ranged in size from approximately 1 mm (amphibian eggs, Pelomyxa) to 0.01 mm (erythrocyte, lymphocyte), and g-forces ranged from about 100 g to 100 000 g respectively. Stratification within cell nuclei and organelles requires even greater forces, presumably because of their smaller size. Extrapolation from centrifugal forces to the force of gravity, and from the full stratification to the initial sedimentation of cell parts suggests a hypothesis for the evolutionary survival and existence of cells in the field of gravity. Average cell size results, in part, from the physical equilibrium between the destructive influence of the force of gravity and the protective role of diffusion and the cytoskeleton. At increased forces of gravity the cell size would thus be decreased, whereas at lower gravitational forces and weightlessness cell size would be expected to increase. Mechanisms of protection of giant cells against internal sedimentation are based on protoplasmic motion, thin and elongated shape of the cell body, increased cytoplasmic viscosity, and a reduced range of specific gravity of cell components, relative to the ground-plasm. The nucleolus, due to its higher density, is considered as a possible trigger of mitosis.

Animals↗

Some aspects of the morphology of insulin-secreting cells.

By using a spectrum of microscopical techniques such as thin sectioning, autoradiography, cytochemistry and freeze-etching, it is possible to unravel the main cytological events associated with synthesis, intracellular transport, storage and discharge of the hormone insulin by B-cell of the islet of Langerhans.

Animals↗

Subcellular distribution of calcium within root meristem cells.

The barley root (Hordeum vulgare) of 3-day old seedlings were treated with potassium oxalate to study the distribution of calcium among plant cell organelles. The best results were obtained by previously treating the root with 10(-3) M CaCl2 followed by an incubation for 20 min with potassium oxalate. The unstained sections for electronmicroscopy showed the deposits of calcium oxalate as a granular or a dark layer on the mitochondria, endoplasmic reticulum and the nucleare envelope. Within all the cells, dark bodies, like vacuoles, were noticed.

Calcium↗

Role of rab proteins in epithelial membrane traffic.

Small GTPase rab proteins play an important role in various aspects of membrane traffic, including cargo selection, vesicle budding, vesicle motility, tethering, docking, and fusion. Recent data suggest also that rabs, and their divalent effector proteins, organize organelle subdomains and as such may define functional organelle identity. Most rabs are ubiquitously expressed. However, some rabs are preferentially expressed in epithelial cells where they appear intimately associated with the epithelial-specific transcytotic pathway and/or tight junctions. This review discusses the role of rabs in epithelial membrane transport.

Animals↗

Molecular aspects of membrane trafficking in paramecium.

Results achieved in the molecular biology of Paramecium have shed new light on its elaborate membrane trafficking system. Paramecium disposes not only of the standard routes (endoplasmic reticulum --> Golgi --> lysosomes or secretory vesicles; endo- and phagosomes --> lysosomes/digesting vacuoles), but also of some unique features, e.g. and elaborate phagocytic route with the cytoproct and membrane recycling to the cytopharynx, as well as the osmoregulatory system with multiple membrane fusion sites. Exocytosis sites for trichocysts (dense-core secretory vesicles), parasomal sacs (coated pits), and terminal cisternae (early endosomes) display additional regularly arranged predetermined fusion/fission sites, which now can be discussed on a molecular basis. Considering the regular, repetitive arrangements of membrane components, availability of mutants for complementation studies, sensitivity to gene silencing, and so on, Paramecium continues to be a valuable model system for analyzing membrane interactions. This review intends to set a new baseline for ongoing work along these lines.

Animals↗