Microtubule cytoskeleton and morphogenesis in the amoebae of the myxomycete Physarum polycephalum

Summary— The amoebae of the myxomycete Physarum polycephalum are of interest in order to analyze the morphogenesis of the microtubule and microfilament cytoskeleton during cell cycle and flagellation. The amoebal interphase microtubule cytoskeleton consists of 2 distinct levels of organization, which correspond to different physiological roles. The first level is composed of the 2 kinetosomes or centrioles and their associated structures. The anterior and posterior kinetosomes forming the anterior and posterior flagella are morphologically distinguishable. Each centriole plays a role in the morphogenesis of its associated satellites and specific microtubule arrays. The 2 distinct centrioles correspond to the 2 successive maturation stages of the pro-centrioles which are built during prophase. The second level of organization consists of a prominent microtubule organizing center (mtoc 1) to which the anterior centriole is attached at least during interphase. This mtoc plays a role in the formation of the mitotic pole. These observations based on ultrastructural and physiological analyses of the amoebal cystoskeleton are now being extended to the biochemical level. The complex formed by the 2 centrioles and the mtoc 1 has been purified without modifying the microtubule-nucleating activity of the mtoc 1. Several microtubule-associated proteins have been characterized by their ability to bind taxol-stabilized microtubules. Their functions (e.g., microtubule assembly, protection of microtubules against dilution or cold treatment, phosphorylating and ATPase activities) are under investigation. These biochemical approaches could allow in vitro analysis of the morphogenesis of the amoebal microtubule cytoskeleton.     

COLLAGEN SYNTHESIS AND CELL GROWTH IN CHICK EMBRYO FIBROBLASTS: INFLUENCE OF COLCHICINE,CYTOCHALASIN B AND CONCANAVALIN A

Culturing of chick embryo fibroblasts in the presence of colchicine or cytochalasin B with and without concanavalin A (Con A) demonstrated that colchicine induces greater neosynthesis of endocellular type I collagen, whereas cytochalasin B boosts secretion. The effects are modified by the addition of Con A, which increases α₂more than a1 chain production.³H-thymidine incorporation is unaffected by cytochalasin B, but stimulated by colchicine. Con A neutralizes the stimulatory action of colchicine. It would therefore seem that Con A exerts transmembrane control of effects induced by colchicine and cytochalasin B by binding to cell surface receptors and so triggering rearrangement of the cytoskeleton.     

The Saccharomyces cerevisiae phosphotyrosyl phosphatase activator proteins are required for a subset of the functions disrupted by protein phosphatase 2A mutations

In Saccharomyces cerevisiae, PTPA is encoded by two genes, YPA1 and YPA2. In order to examine the biological role of PTPA as potential regulator of protein phosphatase 2A (PP2A), we compared the phenotypes of the ypaDelta mutants with these of PP2A-deficient strains. While deletion of both YPA genes is lethal, deletion of YPA1 alone results in a phenotype resembling that of PP2A-deficient strains in specific aspects such as aberrant bud morphology, abnormal actin distribution, and similar growth defects under various growth conditions. These phenotypes were even more pronounced when YPA1 was deleted in a pph21Delta genetic background. Moreover, ypaDelta mutants are hypersensitive to nocodazole and show inappropriate mitotic spindle formation as previously described for mutants in the catalytic subunit of PP2A, suggesting that Ypa, like PP2A, has a function in mitotic spindle formation. These results are consistent with an in vivo role of Ypa as a regulator of PP2A. However, unlike a PP2A-deficient strain, ypaDelta mutants do not show a G2 arrest. Therefore, Ypa does not seem to play a role in the regulation of PP2A at this stage of the cell cycle. These results imply that Ypa regulates a specific subset of PP2A functions, possibly by controlling the subunit composition of PP2A.     

Direct observations of the mechanical behaviors of the cytoskeleton in living fibroblasts

Cytoskeletal proteins tagged with green fluorescent protein were used to directly visualize the mechanical role of the cytoskeleton in determining cell shape. Rat embryo (REF 52) fibroblasts were deformed using glass needles either uncoated for purely physical manipulations, or coated with laminin to induce attachment to the cell surface. Cells responded to uncoated probes in accordance with a three-layer model in which a highly elastic nucleus is surrounded by cytoplasmic microtubules that behave as a jelly-like viscoelastic fluid. The third, outermost cortical layer is an elastic shell under sustained tension. Adhesive, laminin-coated needles caused focal recruitment of actin filaments to the contacted surface region and increased the cortical layer stiffness. This direct visualization of actin recruitment confirms a widely postulated model for mechanical connections between extracellular matrix proteins and the actin cytoskeleton. Cells tethered to laminin-treated needles strongly resisted elongation by actively contracting. Whether using uncoated probes to apply simple deformations or laminin-coated probes to induce surface-to-cytoskeleton interaction we observed that experimentally applied forces produced exclusively local responses by both the actin and microtubule cytoskeleton. This local accomodation and dissipation of force is inconsistent with the proposal that cellular tensegrity determines cell shape.     

A role for p21-activated kinase in endothelial cell migration

The serine/threonine p21-activated kinase (PAK) is an effector for Rac and Cdc42, but its role in regulating cytoskeletal organization has been controversial. To address this issue, we investigated the role of PAK in migration of microvascular endothelial cells. We found that a dominant negative (DN) mutant of PAK significantly inhibited cell migration and increased stress fibers and focal adhesions. The DN effect mapped to the most NH(2)-terminal proline-rich SH3-binding sequence. Observation of a green fluorescent protein-tagged alpha-actinin construct in living cells revealed that the DN construct had no effect on membrane ruffling, but dramatically inhibited stress fiber and focal contact motility and turnover. Constitutively active PAK inhibited migration equally well and also increased stress fibers and focal adhesions, but had a somewhat weaker effect on their dynamics. In contrast to their similar effects on motility, DN PAK decreased cell contractility, whereas active PAK increased contractility. Active PAK also increased myosin light chain (MLC) phosphorylation, as indicated by staining with an antibody to phosphorylated MLC, whereas DN PAK had little effect, despite the increase in actin stress fibers. These results demonstrate that although PAK is not required for extension of lamellipodia, it has substantial effects on cell adhesion and contraction. These data suggest a model in which PAK plays a role coordinating the formation of new adhesions at the leading edge with contraction and detachment at the trailing edge.     

Identification of a suppressor of the Dictyostelium profilin-minus phenotype as a CD36/LIMP-II homologue

Profilin is an ubiquitous G-actin binding protein in eukaryotic cells. Lack of both profilin isoforms in Dictyostelium discoideum resulted in impaired cytokinesis and an arrest in development. A restriction enzyme-mediated integration approach was applied to profilin-minus cells to identify suppressor mutants for the developmental phenotype. A mutant with wild-type-like development and restored cytokinesis was isolated. The gene affected was found to code for an integral membrane glycoprotein of a predicted size of 88 kD containing two transmembrane domains, one at the NH2 terminus and the other at the COOH terminus. It is homologous to mammalian CD36/LIMP-II and represents the first member of this family in D. discoideum, therefore the name DdLIMP is proposed. Targeted disruption of the lmpA gene in the profilin-minus background also rescued the mutant phenotype. Immunofluorescence revealed a localization in vesicles and ringlike structures on the cell surface. Partially purified DdLIMP bound specifically to PIP2 in sedimentation and gel filtration assays. A direct interaction between DdLIMP and profilin could not be detected, and it is unclear how far upstream in a regulatory cascade DdLIMP might be positioned. However, the PIP2 binding of DdLIMP points towards a function via the phosphatidylinositol pathway, a major regulator of profilin.     

Expression of pattern in plants: combining molecular and calculus-based biophysical paradigms

Pattern formation in plant meristems occurs across a broad scale. At the topographical level (large scale), tissue folding in the meristem is responsible for the initiation of new organs in specific phyllotactic patterns and also determines organ shape. At the cellular level (small scale), oriented cell division and microtubule-based cellulose reinforcement control cell pattern and growth direction. I argue here that structural specification at each scale is highly efficient if the pertinent gene activity is manifested in two complementary biophysical categories. At large scale, one category is the tendency of the formative tissue to fold with a certain spatial periodicity determined by its material properties (e.g., bending stiffness from cellulose content). This latent tendency is formalized in a differential equation for physical buckling. The second category at this scale comprises boundary conditions that specify how the latent tendency is manifested as topography: whether tissue humps occur as whorls or Fibonacci spirals. This versatile combinatorial format accounts for the relative stability of alternative organ patterning as well as alternative organ shaping (e.g., stamens vs. carpels). It also accounts for the structural shifts seen in normal development and after mutation or chemical/physical intervention. At small scale, the latent differential activity is the tendency for groups of dividing cells to co-align their cytoskeletons. The curvature of the surface opposes this tendency. The least curved part of a new primordium is its quasicylindrical midportion. There, by aligning microtubules and cellulose coherently around the organ, a new growth direction is set. Thus large-scale buckling produces curvature variation, which, in turn, affects the localization and orientation of the cytoskeleton. This scheme for the coherent production of diverse geometrical features, involving calculus at two structural levels, is supported by complex organogenetic responses to simple physical intervention. Also, many morphological alternatives, wild type vs. mutant, reflect single changes in parameters in this differential-integral format.     

p47(phox) PX domain of NADPH oxidase targets cell membrane via moesin-mediated association with the actin cytoskeleton

Activation of phagocytic NADPH oxidase requires association of its cytosolic subunits with the membrane-bound flavocytochrome. Extensive phosphorylation of the p47(phox) subunit of NADPH oxidase marks the initiation of this activation process. The p47(phox) subunit then translocates to the plasma membrane, bringing the p67(phox) subunit to cytochrome b558 to form the active NADPH oxidase complex. However, the detailed mechanism for targeting the p47(phox) subunit to the cell membrane during activation still remains unclear. Here, we show that the p47(phox) PX domain is responsible for translocating the p47(phox) subunit to the plasma membrane for subsequent activation of NADPH oxidase. We also demonstrate that translocation of the p47(phox) PX domain to the plasma membrane is not due to interactions with phospholipids but rather to association with the actin cytoskeleton. This association is mediated by direct interaction between the p47(phox) PX domain and moesin.