Cytoskeleton of mouse embryo fibroblasts. Electron microscopy of platinum replicas

We have studied the distribution of cytoskeletal elements in detergent-extracted mouse embryo fibroblasts using the platinum replica technique. It was shown that lamelloplasm can be subdivided into three zones: 1) the ruffle edge with dense microfilament meshwork; 2) the sparse zone adjacent to the ruffle edge and containing relatively few cytoskeletal elements; 3) the lamella proper occupied with a three-dimensional network of microfilaments, microtubules, intermediate filaments; this zone contained adhesion plaques corresponding to cell-substrate focal contacts and associated with the microfilament bundle ends. The cytoskeleton structure of the central (endoplasm) region of the cell was markedly different from that of the lamelloplasm. Its main feature was a dense microfilament sheath at the dorsal cell surface. Sites of microfilament bundle convergence can be visualized near the nucleus after partial removal of the sheath by more complete detergent extraction.     

Effect of Metalaxyl on the incidence and severity of Pearl millet downy mildew disease in Northern Nigeria

Pearl millet downy mildew (DM) incidence, severity and yield losses of two pearl millet varieties (local and improved) due to the disease were determined in the field. Significant differences in the disease incidence and severity were recorded in the plots sown with metalaxyl-treated seeds and those sown with non-treated seeds, indicating the efficacy of the fungicide on the fungus. Yield losses due to non-treatment of seeds with metalaxyl was 40.88 and 45.39% in a local variety and 43.00 and 18.60% in an improved variety in the 2000 and 2001 cropping seasons respectively. Significant differences between plots sown with metalaxyl-treated and those sown with non-treated seeds were obtained for other yield components such as 1000-grains weight, panicle length and weight.     

Analysis of the local organization and dynamics of cellular actin networks

A ctin filaments, with the aid of multiple accessory proteins, self-assemble into a variety of network patterns. We studied the organization and dynamics of the actin network in nonadhesive regions of cells bridging fibronectin-coated adhesive strips. The network was formed by actin nodes associated with and linked by myosin II and containing the formin disheveled-associated activator of morphogenesis 1 (DAAM1) and the cross-linker filamin A (FlnA). After Latrunculin A (LatA) addition, actin nodes appeared to be more prominent and demonstrated drift-diffusion motion. Superresolution microscopy revealed that, in untreated cells, DAAM1 formed patches with a similar spatial arrangement to the actin nodes. Node movement (diffusion coefficient and velocity) in LatA-treated cells was dependent on the level and activity of myosin IIA, DAAM1, and FlnA. Based on our results, we developed a computational model of the dynamic formin-filamin-actin asters that can self-organize into a contractile actomyosin network. We suggest that such networks are critical for connecting distant parts of the cell to maintain the mechanical coherence of the cytoplasm.     

Gene expression and the evolution of phenotypic diversity in social wasps

Background  

Organisms are capable of developing different phenotypes by altering the genes they express. This phenotypic plasticity provides a means for species to respond effectively to environmental conditions. One of the most dramatic examples of phenotypic plasticity occurs in the highly social hymenopteran insects (ants, social bees, and social wasps), where distinct castes and sexes all arise from the same genes. To elucidate how variation in patterns of gene expression affects phenotypic variation, we conducted a study to simultaneously address the influence of developmental stage, sex, and caste on patterns of gene expression in Vespula wasps. Furthermore, we compared the patterns found in this species to those found in other taxa in order to investigate how variation in gene expression leads to phenotypic evolution.     

A new dimension in retrograde flow: centripetal movement of engulfed particles

Centripetal motion of surface-adherent particles is a classic experimental system for studying surface dynamics on a eukaryotic cell. To investigate bead migration over the entire cell surface, we have developed an experimental assay using multinuclear giant fibroblasts, which provide expanded length scales and an unambiguous frame of reference. Beads coated by adhesion ligands concanavalin A or fibronectin are placed in specific locations on the cell using optical tweezers, and their subsequent motion is tracked over time. The adhesion, as well as velocity and directionality of their movement, expose distinct regions of the cytoplasm and membrane. Beads placed on the peripheral lamella initiate centripetal motion, whereas beads placed on the central part of the cell attach to a stationary cortex and do not move. Careful examination by complementary three-dimensional methods shows that the motion of a bead placed on the cell periphery takes place after engulfment into the cytoplasm, whereas stationary beads, placed near the cell center, are not engulfed. These results demonstrate that centripetal motion of adhering particles may occur inside as well as outside the cell. Inhibition of actomyosin activity is used to explore requirements for engulfment and aspects of the bead movement. Centripetal movement of adherent particles seems to depend on mechanisms distinct from those driving overall cell contractility.     

Magic touch: how does cell-cell adhesion trigger actin assembly?

The formation of cadherin-mediated cell-cell junctions is accompanied by a profound remodeling of the actin cytoskeleton. The Arp2/3 complex and its activator cortactin drive the assembly of branching actin-filament arrays, and formin-1 promotes the nucleation of non-branching actin filaments. Recruitment of these actin nucleators to nascent adhesions, formation of the links between them and the cytoplasmic cadherin domain, and triggering of their actin-polymerizing functions are vital steps in the development of cell-cell junctions. In this article, I discuss recent results showing that proteins involved in the regulation of actin polymerization have crucial roles in the development and maintenance of the cadherin junctions.     

Disruption of microtubules in living cells by tyrphostin AG-1714

Tyrphostin AG-1714 and several related molecules with the general structure of nitro-benzene malononitrile (BMN) disrupt microtubules in a large variety of cultured cells. This process can be inhibited by the stabilization of microtubules with taxol or by pretreatment of the cells with pervanadate, which inhibits tyrosine phosphatases and increases the overall levels of phosphotyrosine in cells. Unlike other microtubule-disrupting drugs such as nocodazole or colchicine, tyrphostin AG-1714 does not interfere with microtubule polymerization or stability in vitro, suggesting that the effect of this tyrphostin on microtubules is indirect. These results imply an involvement of protein tyrosine phosphorylation in the regulation of overall microtubule dynamics. Tyrphostins of AG-1714 type could thus be powerful tools for the identification of such microtubule regulatory pathways.     

YAP/TAZ as mechanosensors and mechanotransducers in regulating organ size and tumor growth

Organ size is controlled by the concerted action of biochemical and physical processes. Although mechanical forces are known to regulate cell and tissue behavior, as well as organogenesis, the precise molecular events that integrate mechanical and biochemical signals to control these processes are not fully known. The recently delineated Hippo-tumor suppressor network and its two nuclear effectors, YAP and TAZ, shed light on these mechanisms. YAP and TAZ are proto-oncogene proteins that respond to complex physical milieu represented by the rigidity of the extracellular matrix, cell geometry, cell density, cell polarity and the status of the actin cytoskeleton. Here, we review the current knowledge of how YAP and TAZ function as mechanosensors and mechanotransducers. We also suggest that by deciphering the mechanical and biochemical signals controlling YAP/TAZ function, we will gain insights into new strategies for cancer treatment and organ regeneration.     

Cortactin releases the brakes in actin- based motility by enhancing WASP-VCA detachment from Arp2/3 branches

Cortactin is involved in invadopodia and podosome formation [1], pathogens and endosome motility [2], and persistent lamellipodia protrusion [ [3] and [4] ]; its overexpression enhances cellular motility and metastatic activity [ [5] , [6] , [7] and [8] ]. Several mechanisms have been proposed to explain cortactin's role in Arp2/3-driven actin polymerization [ [9] and [10] ], yet its direct role in cell movement remains unclear. We use a biomimetic system to study the mechanism of cortactin-mediated regulation of actin-driven motility [11]. We tested the role of different cortactin variants that interact with Arp2/3 complex and actin filaments distinctively. We show that wild-type cortactin significantly enhances the bead velocity at low concentrations. Single filament experiments show that cortactin has no significant effect on actin polymerization and branch stability, whereas it strongly affects the branching rate driven by Wiskott-Aldrich syndrome protein (WASP)-VCA fragment and Arp2/3 complex. These results lead us to propose that cortactin plays a critical role in translating actin polymerization at a bead surface into motion, by releasing WASP-VCA from the new branching site. This enhanced release has two major effects: it increases the turnover rate of branching per WASP molecule, and it decreases the friction-like force caused by the binding of the moving surface with respect to the growing actin network.     

Physical model for self-organization of actin cytoskeleton and adhesion complexes at the cell front

Cell motion is driven by interplay between the actin cytoskeleton and the cell adhesions in the front part of the cell. The actin network segregates into lamellipodium and lamellum, whereas the adhesion complexes are characteristically distributed underneath the actin system. Here, we suggest a computational model for this characteristic organization of the actin-adhesion system. The model is based on the ability of the adhesion complexes to sense mechanical forces, the stick-slip character of the interaction between the adhesions and the moving actin network, and a hypothetical propensity of the actin network to disintegrate upon sufficiently strong stretching stresses. We identify numerically three possible types of system organization, all observed in living cells: two states in which the actin network exhibits segregation into lamellipodium and lamellum, whereas the cell edge either remains stationary or moves, and a state where the actin network does not undergo segregation. The model recovers the asynchronous fluctuations and outward bulging of the cell edge, and the dependence of the edge protrusion velocity on the rate of the nascent adhesion generation, the membrane tension, and the substrate rigidity.