Search

In migrating cells, integrin-based focal adhesions (FAs) assemble in protruding lamellipodia in association with rapid filamentous actin (F-actin) assembly and retrograde flow. How dynamic F-actin is coupled to FA is not known. We analyzed the role of vinculin in integrating F-actin and FA dynamics by vinculin gene disruption in primary fibroblasts. Vinculin slowed F-actin flow in maturing FA to establish a lamellipodium-lamellum border and generate high extracellular matrix (ECM) traction forces. In addition, vinculin promoted nascent FA formation and turnover in lamellipodia and inhibited the frequency and rate of FA maturation. Characterization of a vinculin point mutant that specifically disrupts F-actin binding showed that vinculin-F-actin interaction is critical for these functions. However, FA growth rate correlated with F-actin flow speed independently of vinculin. Thus, vinculin functions as a molecular clutch, organizing leading edge F-actin, generating ECM traction, and promoting FA formation and turnover, but vinculin is dispensible for FA growth.

Migration and proliferation of cardiac fibroblasts (CFs) play an important role in the myocardial remodeling process. While many factors have been identified that regulate CF growth and migration, less is known about the signaling mechanisms involved in these processes. Here, we utilized Cre-LoxP technology to obtain focal adhesion kinase (FAK)deficient adult mouse CFs and studied how FAK functioned in modulating cell adhesion, proliferation, and migration of these cells. Treatment of [FAK.sup.flox/flox] CFs with Ad/Cre virus caused over 70% reduction of FAK protein levels within a cell population. FAK-deficient CFs showed no changes in focal adhesions, cell morphology, or protein expression levels of vinculin, talin, or paxillin; proline-rich tyrosine kinase 2 (Pyk2) expression and activity were increased. Knockdown of FAK protein in CFs increased PDGF-BB-induced proliferation, while it reduced PDGFBB-induced migration. Adhesion to fibronectin was not altered. To distinguish between the function of FAK and Pyk2, FAK function was inhibited via adenoviral-mediated overexpression of the natural FAK inhibitor FAK-related nonkinase (FRNK). Ad/FRNK had no effect on Pyk2 expression, inhibited the PDGF-BB-induced migration, but did not change the PDGF-BB-induced proliferation. FAK deficiency had only modest effects on increasing PDGF-BB activation of p38 and JNK MAPKs, with no alteration in the ERK response vs. control cells. These results demonstrate that FAK is required for the PDGF-BB-induced migratory response of adult mouse CFs and suggest that FAK could play an essential role in the wound-healing response that occurs in numerous cardiac pathologies. extracellular matrix; cytoskeleton; fibroblast

Aims: Desminopathies comprise hereditary myopathies and cardiomyopathies caused by mutations in the intermediate filament protein desmin that lead to severe and often lethal degeneration of striated muscle tissue. Animal and single cell studies hinted that this degeneration process is associated with massive ultrastructural defects correlating with increased susceptibility of the muscle to acute mechanical stress. The underlying mechanism of mechanical susceptibility, and how muscle degeneration develops over time, however, has remained elusive. Methods: Here, we investigated the effect of a desmin mutation on the formation, differentiation, and contractile function of in vitro‐engineered three‐dimensional micro‐tissues grown from muscle stem cells (satellite cells) isolated from heterozygous R349P desmin knock‐in mice. Results: Micro‐tissues grown from desmin‐mutated cells exhibited spontaneous unsynchronised contractions, higher contractile forces in response to electrical stimulation, and faster force recovery compared with tissues grown from wild‐type cells. Within 1 week of culture, the majority of R349P desmin‐mutated tissues disintegrated, whereas wild‐type tissues remained intact over at least three weeks. Moreover, under tetanic stimulation lasting less than 5 s, desmin‐mutated tissues partially or completely ruptured, whereas wild‐type tissues did not display signs of damage. Conclusions: Our results demonstrate that the progressive degeneration of desmin‐mutated micro‐tissues is closely linked to extracellular matrix fibre breakage associated with increased contractile forces and unevenly distributed tensile stress. This suggests that the age‐related degeneration of skeletal and cardiac muscle in patients suffering from desminopathies may be similarly exacerbated by mechanical damage from high‐intensity muscle contractions. We conclude that micro‐tissues may provide a valuable tool for studying the organization of myocytes and the pathogenic mechanisms of myopathies. [ABSTRACT FROM AUTHOR]

Two‐dimensional (2D) cancer models have been the standard for drug development over the past few years, but they frequently do not resemble in vivo properties adequately. 3D models are superior in many aspects and are, therefore, more similar to human pathophysiology. Over the past years, the emerging field of biofabrication has made significant advances, resulting in even more sophisticated 3D models. With this study, a hydrogel is created for biofabrication that is suitable for mimicking the tumor microenvironment in vitro and is further tested as a new vascularized melanoma model in vivo. The alginate/hyaluronic acid/gelatin bioink shows good shape‐fidelity, high cell survival rates, and enables successful cultivation of melanoma cells and adipose‐derived stem cells as well as cell differentiation in vitro. In vivo, in the arteriovenous loop model, it proves to be a unique method to study melanoma progression, tumor vascularization, and ultimately and reliably metastases in an isolated and controlled environment. These results show that this 3D model is very application‐oriented for molecular research and therapy development. [ABSTRACT FROM AUTHOR]

Integrins regulate cell behavior through the assembly of multiprotein complexes at the site of cell adhesion. Parvins are components of such a multiprotein complex. They consist of three members (α, β-, and γ-parvin), form a functional complex with integrin-linked kinase (ILK) and PINCH, and link integrins to the actin cytoskeleton. Whereas α- and β-parvins are widely expressed, γ-parvin has been reported to be expressed in hematopoietic organs. In the present study, we report the expression pattern of the parvins in hematopoietic cells and the phenotypic analysis of γ-parvin-deficient mice. Whereas α-parvin is not expressed in hematopoietic cells, β-parvin is only found in myeloid cells and γ-parvin is present in both cells of the myeloid and lymphoid lineages, where it binds ILK. Surprisingly, loss of γ-parvin expression had no effect on blood cell differentiation, proliferation, and survival and no consequence for the T-cell-dependent antibody response and lymphocyte and dendritic cell migration. These data indicate that despite the high expression of γ-parvin in hematopoietic cells it must play a more subtle role for blood cell homeostasis. [ABSTRACT FROM AUTHOR]

One of the most commonly assessed parameters in cellular analyses is the proliferative activity of a cell population. The fluorescence ubiquitin cell cycle indicator (FUCCI)-based system allows live and in vivo observation of cell cycle progression. Based on the mutually exclusive activity of two fluorescently labeled proteins cdt1 and geminin during the G0/1 and S/G2/M phases of the cell cycle, individual cells can be assigned to their respective cell cycle phase by fluorescence imaging of the nucleus. Here, we describe the generation of NIH/3T3 cells containing the FUCCI reporter system by lentiviral transduction and their use in 3D culture assays. The protocol can be adapted to other cell lines., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)