Association of adenovirus with the microtubule organizing center

Adenoviruses (Ad) must deliver their genomes to the nucleus of the target cell to initiate an infection. Following entry into the cell and escape from the endosome, Ad traffics along the microtubule cytoskeleton toward the nucleus. In the final step in Ad trafficking, Ad must leave the microtubule and establish an association with the nuclear envelope. We hypothesized that in cells lacking a nucleus, the capsid moves to and associates with the microtubule organizing center (MTOC). To test this hypothesis, we established an experimental system to examine Ad trafficking in enucleated cells compared to Ad trafficking in intact, mock-enucleated cells. Enucleation of a monolayer of A549 human lung epithelial cells was accomplished by depolymerization of the actin cytoskeleton followed by centrifugation. Upon infection of enucleated cells with Cy3-labeled Ad, the majority of Ad capsid trafficked to a discrete, centrally located site which colocalized with pericentrin, a component of the MTOC. MTOC-associated Ad had escaped from endosomes and thus had direct access to MTOC components. Ad localization at this site was sensitive to the microtubule-depolymerizing agent nocodazole, but not to the microfilament-depolymerizing agent cytochalasin B, indicating that intact microtubules were required to maintain the localization with the MTOC. Ad localization to the MTOC in the enucleated cells was stable, as demonstrated by continuing Ad localization with pericentrin for more than 5 h after infection, a strong preference for Ad arrival at rather than Ad departure from the MTOC, and minimal redistribution of Ad between MTOCs within a single cell. In summary, the data demonstrate that the Ad capsid establishes a stable interaction with the MTOC when a nucleus is not present, suggesting that dissociation of Ad from microtubules likely requires nuclear factors.     

Activin-treated ectoderm has complete organizing center activity in Cynops embryos

The differentiation and organizer activity of newt ectoderm treated with activin A was studied in explantation and transplantation experiments. In the explantation experiments, ectoderm dissected from late morulae–early gastrulae stage embryos treated with a high concentration of activin A (100 ng/mL) formed only yolk-rich endodermal cells. Mesodermal tissues, such as notochord and muscle, were seldom found in these explants. When they were transplanted into the blastocoele of other early gastrulae, they formed part of the endoderm of the host embryo and induced a secondary axis with only posterior characters (including axial mesoderm and neural tissues). In contrast, whole secondary axes were induced when activin-treated ectoderm was transplanted into the ventral marginal zone (VMZ) of early blastulae. The transplanted pieces invaginated by themselves and differentiated into foregut structures including pharynx, stomach, and liver. These phenomena were also observed in experiments in which presumptive foregut was used instead of activin-treated ectoderm. These findings show that activin-treated ectoderm can act as the complete organizing center in Cynops .     

Protein complexes at the microtubule organizing center regulate bipolar spindle assembly

Bipolar spindle assembly is essential to genomic stability in dividing cells. Centrosomes or spindle pole bodies duplicated earlier at G1/S remain adjacent until triggered at mitotic onset to become bipolar. Pole reorientation is stabilized by microtubule interdigitation but mechanistic details for bipolarity remain incomplete. To investigate the contribution of spindle pole microtubule organizing center (MTOC) proteins in bipolarity, we applied genetic, structural and molecular biochemical analysis along with timelapse microscopy. Spindle formation was followed by an in vivo growth assay with the conditional allele cut7-22ts, encoding fission yeast mitotic Kinesin-5, essential for bipolarity. By analysis of double and triple mutant strains of MTOC alleles and cut7-22ts we found that stabilized microtubules or increased bundling can rescue cut7-22ts associated bipolarity defects. These changes to microtubule dynamics and organization occurred through two surface domains on γ-tubulin, a helix 11 domain and an adjacent site for binding MTOC protein Alp4. We demonstrate that Kinesin-14 Pkl1, known to oppose bipolarity, can bind to γ-tubulin at helix 11 and that mutation of either of two conserved residues in helix 11 can impair Kinesin-14 binding. Altering the Alp4/γ-tubulin interaction, conserved residues in helix 11 or deletion of pkl1 each are sufficient to rescue bipolarity in our cut7-22ts strain. Our findings provide novel insights into regulation of the bipolar mechanism through the MTOC complex.     

Yeast ubiquitin-like genes are involved in duplication of the microtubule organizing center

KAR1 is required for duplication of the Saccharomyces cerevisiae microtubule organizing center, the spindle pole body (SPB) (Rose, M.D., and G.R. Fink, 1987. Cell. 48:1047-1060). Suppressors of a kar1 allele defective for SPB duplication were isolated in two genes, CDC31 and DSK2 (Vallen, E.A., W.H., M. Winey, and M.D. Rose. 1994. Genetics. 137:407-422). To elucidate the role of DSK2 in SPB duplication, we cloned the gene and found it encodes a novel ubiquitin-like protein containing an NH2 terminus 36% identical to ubiquitin. The only other known yeast ubiquitin-like protein is encoded by the nucleotide excision repair gene RAD23 (Watkins, J.F.,P. Sung, L. Prakash, and S. Prakash. 1993. Mol. Cell. Bio. 13:7757-7765). Unlike ubiquitin, the NH2- terminal domain of Dsk2p is not cleaved from the protein, indicating that Dsk2p is not conjugated to other proteins. Although the DSK2-1 mutation alters a conserved residue in the Dsk2p ubiquitin-like domain, we detect no differences in Dsk2p or Cdc31p stability. Therefore, DSK2 does not act by interfering with ubiquitin-dependent protein degradation of these proteins. Although DSK2 is not essential, a strain deleted for both DSK2 and RAD23 is temperature sensitive for growth due to a block in SPB duplication. In addition, overexpression of DSK2 is toxic, and the DSK2-1 allele causes a block in SPB duplication. Therefore, DSK2 dosage is critical for SPB duplication. We determined that CDC31 gene function is downstream of DSK2 and KAR1. Dsk2p is a nuclear-enriched protein, and we propose that Dsk2p assists in Cdc31 assembly into the new SPB.     

Actin filament array during side branch initiation in protonema cells of the mossFunaria hygrometrica: an actin organizing center at the plasma membrane

Summary With an improved method to visualize the actin filament system it is possible to detect a small, peculiar accumulation of actin filaments under the prospective area of side branch formation inFunaria protonema cells. It consists of a ring-like configuration of actin filaments from which filaments radiate, preferentially along the plasma membrane. During the transition to tip growth the arrangement becomes loosened and eventually disappears whereas the filaments are concentrated in inner regions of the cytoplasm with a maximum in the apical dome.     

Stathmin regulates microtubule dynamics and microtubule organizing center polarization in activated T cells

Polarization of T cells involves reorientation of the microtubule organizing center (MTOC). Because activated ERK is localized at the immunological synapse, we investigated its role by showing that ERK activation is important for MTOC polarization. Suspecting that ERK phosphorylates a regulator of microtubules, we next focused on stathmin, a known ERK substrate. Our work indicates that during T cell activation, ERK is recruited to the synapse, allowing it to phosphorylate stathmin molecules near the immunological synapse. Supporting an important role of stathmin phosphorylation in T cell activation, we showed that T cell activation results in increased microtubule growth rate dependent on the presence of stathmin. The significance of this finding was demonstrated by results showing that CTLs from stathmin(-/-) mice displayed defective MTOC polarization and defective target cell cytolysis. These data implicate stathmin as a regulator of the microtubule network during T cell activation.     

Cassava, Manihot esculenta Crantz genetic resources: VI. Anatomy of a diversity center

Central Brasil is one of the four centers of diversity of Manihot species. Of 26 wild Manihot species reported to occur in this region, 20 were collected from two limited areas, each less than 100 km in diameter. These two areas are Goiás Velho and Corumbá de Goiás. Considering the Harlan concept of geographic patterns of variation of cultivated crops, it is assumed that the two areas are microcenters of cassava diversity. Topographic, soil and ethnological data of these species were collected and compared.     

Cytoskeleton: centrosom-in absentia.

Recent results challenge long-held assumptions that centrosomes are essential organizers of mitotic spindles, but suggest that they couple spindle behavior with developmental and cellular events, perhaps by nucleating astral microtubules which mediate interactions with other cytoskeletal components.     

Paxillin localizes to the lymphocyte microtubule organizing center and associates with the microtubule cytoskeleton

Paxillin is a focal adhesion-associated protein that functions as a multi-domain adapter protein, binding several structural and signaling molecules. alpha-Tubulin was identified as an interacting protein in a two-hybrid screen using the paxillin C-terminal LIM domain as a bait. In vitro binding assays with glutathione S-transferase-paxillin demonstrated an interaction of alpha-tubulin with the C terminus of paxillin. Another member of the tubulin family, gamma-tubulin, bound to both the N and the C terminus of paxillin. The interaction between paxillin and both alpha- and gamma-tubulin in vivo was confirmed by co-immunoprecipitation from human T lymphoblasts. Immunofluorescence studies revealed that, in adherent T cells, paxillin localized to sites of cell-matrix interaction as well as to a large perinuclear region. Confocal microscopy revealed that this region corresponds to the lymphocyte microtubule organizing center, where paxillin colocalizes with alpha- and gamma-tubulin. The localization of paxillin to this area was observed in cells in suspension as well as during adhesion to integrin ligands. These data constitute the first characterization of the interaction of paxillin with the microtubule cytoskeleton, and suggest that paxillin, in addition to its well established role at focal adhesions, could also be associated with the lymphocyte microtubule network.     

Pollen Tube Cytoskeleton: Structure andFunction

   apd: 3 July 2001     

Cytoskeleton of mollicutes

Mollicutes are a class of bacteria that lack a peptidoglycan layer but have various cell shapes. They perform chromosome segregation and binary fission in a well-organized manner. Especially, species with polarized cell morphology duplicate their membrane protrusion at a position adjacent to the original one and move the new protrusion laterally to the opposite end pole before cell division. The featured various cell shapes of Mollicutes are supported by cytoskeletal structures composed of proteins. Recent progress in the study of cytoskeletons of walled bacteria and genome sequencing has revealed that the cytoskeletons of Mollicutes are not common with those of other bacteria. Mollicutes have special cytoskeletal proteins and structures that are sometimes not shared even by other mollicute species.     

Macroecology: the organizing forces

The fundamental problem in ecology is the relationship between organisms and the physical world. This question is approached through the study of ecosystems as wholes. Pristine, autonomous Arctic lakes provide an invaluable starting point for such investigations. Studies on these lakes, initiated in 1958, indicate that the dominant fish populations assume a recognizable and repeatable structure, which if not disturbed by external forces, is maintained indefinitely. The observable characteristics are high biomass, large individual size, great age, uniformity of individuals despite great variation in age, and relatively few juveniles. This configuration expresses a state of least specific energy dissipation or least specific entropy production and may be regarded as a standing wave in the energy flow. Similar characteristics were found in the dominant species in a wide range of ecosystems worldwide. A state of least dissipation develops when two energy transport processes interfere with each other in the vicinity of thermodynamic equilibrium. Thus it is postulated that living organisms originated through the agonistic interaction between two energy transport processes within materials, initially occurring in an environment close to thermodynamic equilibrium. These two processes, fundamental to energy transfer, are recognized as the principle of least action and its diametrical opposite, the principle of most action. Each force dominates system behavior in a different time frame: most action in the short-term (ecological time) and least action over the long-term (evolutionary time). Interaction between the countervailing forces provides an understanding of a wide range of emergent ecological generalities such as succession, r- and K-selection, the stability and diversity of ecosystems, and the directionality of evolutionary change.