Roles of the small GTPases RhoA and Rac1 in cell behavior

The Rho-family GTPases are proving to have a variety of biological functions apart from their well known effects on the cytoskeleton. Recent work indicates their involvement in signaling between the adhesion receptors integrin and syndecan, effects on the recruitment of beta-catenin to the nucleus, and potential roles in the nucleus as well as the cytoplasm

The actin crosslinking protein palladin modulates force generation and mechanosensitivity of tumor associated fibroblasts

Cells organize actin filaments into higher-order structures by regulating the composition, distribution and concentration of actin crosslinkers. Palladin is an actin crosslinker found in the lamellar actin network and stress fibers, which are critical for mechanosensing of the environment. Palladin also serves as a molecular scaffold for α-actinin, another key actin crosslinker. By virtue of its close interactions with actomyosin structures in the cell, palladin may play an important role in cell mechanics. However, the role of palladin in cellular force generation and mechanosensing has not been studied. Here, we investigate the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. Traction force microscopy revealed that tumor-associated fibroblasts generate larger forces on substrates of increased stiffness. Contrary to expectations, knocking down palladin increased the forces generated by cells and inhibited their ability to sense substrate stiffness for very stiff gels. This was accompanied by significant differences in actin organization, adhesion dynamics and altered myosin organization in palladin knock-down cells. Our results suggest that actin crosslinkers such as palladin and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis

Modulation of Corneal Fibroblast Contractility within Fibrillar Collagen Matrices

PURPOSE. To investigate the migratory and contractile behavior of isolated human corneal fibroblasts in fibrillar collagen matrices. METHODS. A telomerase-infected, extended-lifespan human corneal fibroblast cell line (HTK) was transfected by using a vector for enhanced green fluorescent protein (GFP)-α-actinin. Cells were plated at low density on top of or within 100-μm-thick fibrillar collagen lattices. After 18 hours to 7 days, time-lapse imaging was performed. At each 1- to 3-minute interval, GFP and Nomarski differential interference contrast (DIC) images were acquired in rapid succession. Serum-containing (S+) medium was used initially for perfusion. After 2 hours, perfusion was switched to either serum-free (S-) or S+ medium containing the Rho-kinase inhibitor Y-27632 for 1 to 2 hours. Finally, perfusion was changed back to S+ medium for 1 hour. RESULTS. Two to 4 days after plating, many cells underwent spontaneous contraction and/or relaxation in S+ medium. A decrease in the distance between consecutive α-actinin-dense bodies along stress fibers was measured during contraction, and focal adhesion and matrix displacements correlated significantly. Removal of serum or inhibition of Rho-kinase induced cell body elongation and relaxation of matrix stress, as confirmed using finite element modeling. Rapid formation and extension of pseudopodia and filopodia were also observed, and transient tractional forces were generated by these extending processes. CONCLUSIONS. Cultured human corneal fibroblasts can undergo rapid changes in the subcellular pattern of force generation that are mediated, in part, by Rho-kinase. Sarcomeric shortening of stress fibers in contracting corneal fibroblasts is also demonstrated for the first time

Identification of palladin isoforms and characterization of an isoform-specific interaction between Lasp-1 and palladin

Palladin is a recently described phosphoprotein with an important role in cytoskeletal organization. The major palladin isoform (90-92 kDa) binds to three actin-associated proteins (ezrin, VASP and [alpha]-actinin), suggesting that palladin functions as a cytoskeletal scaffold. Here, we describe the organization of the palladin gene, which encodes multiple isoforms, including one (140 kDa) with a similar localization pattern to 90 kDa palladin. Overexpression of the 90 kDa or 140 kDa isoforms in COS-7 cells results in rearrangements of the actin cytoskeleton into super-robust bundles and star-like arrays, respectively. Sequence analysis of 140 kDa palladin revealed a conserved binding site for SH3 domains, suggesting that it binds directly to the SH3-domain protein Lasp-1. Binding of 140 kDa palladin, but not 90 kDa palladin, to Lasp-1 was confirmed by yeast two-hybrid and GST-pull-down assays. Isoform-specific siRNA experiments suggested that 140 kDa palladin plays a role in recruiting Lasp-1 to stress fibers. These results add Lasp-1, an actin-binding protein with a crucial role in cell motility, to the growing list of palladin's binding partners, and suggest that 140 kDa palladin has a specialized function in organizing the actin arrays that participate in cell migration and/or cellular contractility

Morphology and Viscoelasticity of Actin Networks Formed with the Mutually Interacting Crosslinkers: Palladin and Alpha-actinin

Actin filaments and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. Even though cells have multiple actin binding proteins (ABPs) that exist simultaneously to maintain the structural and mechanical integrity of the cellular cytoskeleton, how these proteins work together to determine the properties of actin networks is not clearly understood. The ABP, palladin, is essential for the maintenance of cell morphology and the regulation of cell movement. Palladin coexists with -actinin in stress fibers and focal adhesions and binds to both actin and -actinin. To obtain insight into how mutually interacting actin crosslinking proteins modulate the properties of actin networks, we characterized the micro-structure and mechanics of actin networks crosslinked with palladin and -actinin. We first showed that palladin crosslinks actin filaments into bundled networks which are viscoelastic in nature. Our studies also showed that composite networks of -actinin/palladin/actin behave very similar to pure palladin or pure -actinin networks. However, we found evidence that palladin and -actinin synergistically modify network viscoelasticity. To our knowledge, this is the first quantitative characterization of the physical properties of actin networks crosslinked with two mutually interacting crosslinkers

An interaction between alpha-actinin and the beta 1 integrin subunit in vitro

A number of cytoskeletal-associated proteins that are concentrated in focal contacts, namely alpha-actinin, vinculin, talin, and integrin, have been shown to interact in vitro such that they suggest a potential link between actin filaments and the membrane. Because some of these interactions are of low affinity, we suspect the additional linkages also exist. Therefore, we have used a synthetic peptide corresponding to the cytoplasmic domain of beta 1 integrin and affinity chromatography to identify additional integrin-binding proteins. Here we report our finding of an interaction between the cytoplasmic domain of beta 1 integrin and the actin-binding protein alpha-actinin. Beta 1- integrin cytoplasmic domain peptide columns bound several proteins from Triton extracts of chicken embryo fibroblasts. One protein at approximately 100 kD was identified by immunoblot analysis as alpha- actinin. Solid phase binding assays indicated that alpha-actinin bound specifically and directly to the beta 1 peptide with relatively high affinity. Using purified heterodimeric chicken smooth muscle integrin (a beta 1 integrin) or the platelet integrin glycoprotein IIb/IIIa complex (a beta 3 integrin), binding of alpha-actinin was also observed in similar solid phase assays, albeit with a lower affinity than was seen using the beta 1 peptide. alpha-Actinin also bound specifically to phospholipid vesicles into which glycoprotein IIb/IIIa had been incorporated. These results lead us to suggest that this integrin-alpha- actinin linkage may contribute to the attachment of actin filaments to the membrane in certain locations

The role of palladin in actin organization and cell motility

Palladin is a widely expressed protein found in stress fibers, focal adhesions, growth cones, Z-discs, and other actin-based subcellular structures. It belongs to a small gene family that includes the Z-disc proteins myopalladin and myotilin, all of which share similar Ig-like domains. Recent advances have shown that palladin shares with myotilin the ability to bind directly to F-actin, and to crosslink actin filaments into bundles, in vitro. Studies in a variety of cultured cells suggest that the actin-organizing activity of palladin plays a central role in promoting cell motility. Correlative evidence also supports this hypothesis, as palladin levels are typically upregulated in cells that are actively migrating: in developing vertebrate embryos, in cells along a wound edge, and in metastatic cancer cells. Recently, a mutation in the human palladin gene was implicated in an unusually penetrant form of inherited pancreatic cancer, which has stimulated new ideas about the role of palladin in invasive cancer

Structural Characterization of the Interactions between Palladin and α-Actinin

The interaction between α-actinin and palladin, two actin-crosslinking proteins, is essential for proper bidirectional targeting of these proteins. As a first step toward understanding the role of this complex in organizing cytoskeletal actin, we have characterized binding interactions between the EF hand domain of α-actinin (Act-EF34) and peptides derived from palladin, and generated a NMR-derived structural model for the Act-EF34/palladin peptide complex. The critical binding site residues are similar to an actinin binding motif previously suggested for the complex between Act-EF34 and titin Z-repeats. The structure-based model of the Act-EF34/palladin peptide complex expands our understanding of binding specificity between the scaffold protein α-actinin and various ligands, which appears to require an α-helical motif containing four hydrophobic residues, common to many α–actinin ligands. We also provide evidence that the Family-X mutation in palladin, associated with a highly penetrant form of pancreatic cancer, does not interfere with α-actinin binding

Palladin Compensates for the Arp2/3 Complex and Supports Actin Structures during Listeria Infections

Palladin is an important component of motile actin-rich structures and nucleates branched actin filament arrays in vitro. Here we examine the role of palladin during Listeria monocytogenes infections in order to tease out novel functions of palladin. We show that palladin is co-opted by L. monocytogenes during its cellular entry and intracellular motility. Depletion of palladin resulted in shorter and misshapen comet tails, and when actin- or VASP-binding mutants of palladin were overexpressed in cells, comet tails disintegrated or became thinner. Comet tail thinning resulted in parallel actin bundles within the structures. To determine whether palladin could compensate for the Arp2/3 complex, we overexpressed palladin in cells treated with the Arp2/3 inhibitor CK-666. In treated cells, bacterial motility could be initiated and maintained when levels of palladin were increased. To confirm these findings, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells, L. monocytogenes failed to generate comet tails. When palladin was overexpressed in this Arp2/3 functionally null cell line, the ability of L. monocytogenes to generate comet tails was restored. Using purified protein components, we demonstrate that L. monocytogenes actin clouds and comet tails can be generated (in a cell-free system) by palladin in the absence of the Arp2/3 complex. Collectively, our results demonstrate that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility