Cellular Motility Driven by Assembly and Disassembly of Actin Filaments

Adhesions between fibroblastic cells and extracellular matrix have been studied extensively in vitro, but little is known about their in vivo counterparts. Here, we characterized the composition and function of adhesions in three-dimensional (3D) matrices derived from tissues or cell culture. "3D-matrix adhesions" differ from focal and fibrillar adhesions characterized on 2D substrates in their content of alpha5beta1 and alphavbeta3 integrins, paxillin, other cytoskeletal components, and tyrosine phosphorylation of focal adhesion kinase (FAK). Relative to 2D substrates, 3D-matrix interactions also display enhanced cell biological activities and narrowed integrin usage. These distinctive in vivo 3D-matrix adhesions differ in structure, localization, and function from classically described in vitro adhesions, and as such they may be more biologically relevant to living organisms.

http://science.sciencemag.org/content/sci/294/5547/1708.full.pdf

Taking Cell-Matrix Adhesions to the Third Dimension

Src family protein tyrosine kinases are activated following engagement of many different classes of cellular receptors and participate in signaling pathways that control a diverse spectrum of receptor-induced biological activities. While several of these kinases have evolved to play distinct roles in specific receptor pathways, there is considerable redundancy in the functions of these kinases, both with respect to the receptor pathways that activate these kinases and the downstream effectors that mediate their biological activities. This chapter reviews the evidence implicating Src family kinases in specific receptor pathways and describes the mechanisms leading to their activation, the targets that interact with these kinases, and the biological events that they regulate.

Cellular functions regulated by src family kinases

Focal adhesions are sites of tight adhesion to the underlying extracellular matrix developed by cells in culture. They provided a structural link between the actin cytoskeleton and the extracellular matrix and are regions of signal transduction that relate to growth control. The assembly of focal adhesions is regulated by the GTP-binding protein Rho. Rho stimulates contractility which, in cells that are tightly adherent to the substrate, generates isometric tension. In turn, this leads to the bundling of actin filaments and the aggregation of integrins (extracellular matrix receptors) in the plane of the membrane. The aggregation of integrins activates the focal adhesion kinase and leads to the assembly of a multicomponent signaling complex.

/pdf/focal-adhesions-contractility-and-signaling-ij3wyc7tuo.pdf

Focal adhesions, contractility, and signaling

Activated rhoA, a ras-related GTP-binding protein, stimulates the appearance of stress fibers, focal adhesions, and tyrosine phosphorylation in quiescent cells (Ridley, A.J., and A. Hall, 1992. Cell. 70:389-399). The pathway by which rho triggers these events has not been elucidated. Many of the agents that activate rho (e.g., vasopressin, endothelin, lysophosphatidic acid) stimulate the contractility of smooth muscle and other cells. We have investigated whether rho's induction of stress fibers, focal adhesions, and tyrosine phosphorylation is the result of its stimulation of contractility. We demonstrate that stimulation of fibroblasts with lysophosphatidic acid, which activates rho, induces myosin light chain phosphorylation. This precedes the formation of stress fibers and focal adhesions and is accompanied by increased contractility. Inhibition of contractility by several different mechanisms leads to inhibition of rho-induced stress fibers, focal adhesions, and tyrosine phosphorylation. In addition, when contractility is inhibited, integrins disperse from focal adhesions as stress fibers and focal adhesions disassemble. Conversely, upon stimulation of contractility, diffusely distributed integrins are aggregated into focal adhesions. These results suggest that activated rho stimulates contractility, driving the formation of stress fibers and focal adhesions and elevating tyrosine phosphorylation. A model is proposed to account for how contractility could promote these events.

/pdf/rho-stimulated-contractility-drives-the-formation-of-stress-6jnhqys6rn.pdf

Rho-stimulated contractility drives the formation of stress fibers and focal adhesions.

This article outlines the present knowledge of the architecture, molecular composition, and dynamics of focal contacts of adhesive animal cells. These structures, developed at the plasma membrane at sites where cells touch their substratum, are essential for cellular attachment in tissue formation during embryogenesis and wound healing. In tissue culture, they are particularly prominent and thus amenable to detailed investigation. Focal contacts consist of a cytoplasmic face, comprising cytoskeletal elements, a transmembrane connecting region, and a extracellular face composed of proteins of the extracellular matrix. The molecular anatomy of the numerous proteins involved, the basis for classifying them as structural or regulatory components, and their in vitro interactions are described. Based on this information, current models on the dynamics of their assembly and of possible regulatory mechanisms involving a variety of signal transduction pathways are discussed.

The molecular architecture of focal adhesions

Profilins as regulators of actin dynamics

http://www.jbc.org/content/274/53/37908.full.pdf

A role for polyproline motifs in the spinal muscular atrophy protein SMN. Profilins bind to and colocalize with smn in nuclear gems.

Profilins are small proteins involved in actin dynamics. In accordance with this function, they are found in all eukaryotes and are structurally highly conserved. However, their precise role in regulating actin-related functions is just beginning to emerge. This article recapitulates the wealth of information on structure, expression and functions accumulated on profilins from many different organisms in the 30 years after their discovery as actin-binding proteins. Emphasis is given to their interaction with a plethora of many different ligands in the cytoplasm as well as in the nucleus, which is considered the basis for their various activities and the significance of the tissue-specific expression of profilin isoforms.

The profile of profilins

Gephyrin is an essential component of the postsynaptic cortical protein network of inhibitory synapses. Gephyrin-based scaffolds participate in the assembly as well as the dynamics of receptor clusters by connecting the cytoplasmic domains of glycine and GABA A receptor polypeptides to two cytoskeletal systems, microtubules and microfilaments. Although there is evidence for a physical linkage between gephyrin and microtubules, the interaction between gephyrin and microfilaments is not well understood so far. Here, we show that neuronal gephyrin interacts directly with key regulators of microfilament dynamics, profilin I and neuronal profilin IIa, and with microfilament adaptors of the mammalian enabled (Mena)/vasodilator stimulated phosphoprotein (VASP) family, including neuronal Mena. Profilin and Mena/VASP coprecipitate with gephyrin from tissue and cells, and complex formation requires the E-domain of gephyrin, not the proline-rich central domain. Consequently, gephyrin is not a ligand for the proline-binding motif of profilins, as suspected previously. Instead, it competes with G-actin and phospholipids for the same binding site on profilin. Gephyrin, profilin, and Mena/VASP colocalize at synapses of rat spinal cord and cultivated neurons and in gephyrin clusters expressed in transfected cells. Thus, Mena/VASP and profilin can contribute to the postulated linkage between receptors, gephyrin scaffolds, and the microfilament system and may regulate the microfilament-dependent receptor packing density and dynamics at inhibitory synapses.

https://www.jneurosci.org/content/jneuro/23/23/8330.full.pdf

Martin Rothkegel

Papers

The molecular architecture of focal adhesions

Profilins as regulators of actin dynamics

A role for polyproline motifs in the spinal muscular atrophy protein SMN. Profilins bind to and colocalize with smn in nuclear gems.

The profile of profilins

Complex formation between the postsynaptic scaffolding protein gephyrin, profilin, and mena: A possible link to the microfilament system