LMX1B is essential for the maintenance of differentiated podocytes in adult kidneys.

Mutations of the LMX1B gene cause nail-patella syndrome, a rare autosomal-dominant disorder affecting the development of the limbs, eyes, brain, and kidneys. The characterization of conventional Lmx1b knockout mice has shown that LMX1B regulates the development of podocyte foot processes and slit diaphragms, but studies using podocyte-specific Lmx1b knockout mice have yielded conflicting results regarding the importance of LMX1B for maintaining podocyte structures. In order to address this question, we generated inducible podocyte-specific Lmx1b knockout mice. One week of Lmx1b inactivation in adult mice resulted in proteinuria with only minimal foot process effacement. Notably, expression levels of slit diaphragm and basement membrane proteins remained stable at this time point, and basement membrane charge properties also did not change, suggesting that alternative mechanisms mediate the development of proteinuria in these mice. Cell biological and biophysical experiments with primary podocytes isolated after 1 week of Lmx1b inactivation indicated dysregulation of actin cytoskeleton organization, and time-resolved DNA microarray analysis identified the genes encoding actin cytoskeleton-associated proteins, including Abra and Arl4c, as putative LMX1B targets. Chromatin immunoprecipitation experiments in conditionally immortalized human podocytes and gel shift assays showed that LMX1B recognizes AT-rich binding sites (FLAT elements) in the promoter regions of ABRA and ARL4C, and knockdown experiments in zebrafish support a model in which LMX1B and ABRA act in a common pathway during pronephros development. Our report establishes the importance of LMX1B in fully differentiated podocytes and argues that LMX1B is essential for the maintenance of an appropriately structured actin cytoskeleton in podocytes.

CD24 controls Src/STAT3 activity in human tumors.

CD24 is a glycosyl-phosphatidylinositol-anchored membrane protein that is frequently over-expressed in a variety of human carcinomas and is correlated with poor prognosis. In cancer cell lines, changes of CD24 expression can alter several cellular properties in vitro and tumor growth in vivo. However, little is known about how CD24 mediates these effects. Here we have analyzed the functional consequences of CD24 knock-down or over-expression in human cancer cell lines. Depletion of CD24 reduced cell proliferation and adhesion, enhanced apoptosis, and regulated the expression of various genes some of which were identified as STAT3 target genes. Loss of CD24 reduced STAT3 and FAK phosphorylation. Diminished STAT3 activity was confirmed by specific reporter assays. We found that reduced STAT3 activity after CD24 knock-down was accompanied by altered Src phosphorylation. Silencing of Src, similar to CD24, targeted the expression of prototype STAT3-regulated genes. Likewise, the over-expression of CD24 augmented Src-Y416 phosphorylation, the recruitment of Src into lipid rafts and the expression of STAT3-dependent target genes. An antibody to CD24 was effective in reducing tumor growth of A549 lung cancer and BxPC3 pancreatic cancer xenografts in mice. Antibody treatment affected the level of Src-phosphorylation in the tumor and altered the expression of STAT3 target genes. Our results provide evidence that CD24 regulates STAT3 and FAK activity and suggest an important role of Src in this process. Finally, the targeting of CD24 by antibodies could represent a novel route for tumor therapy.

Vinculin facilitates cell invasion into three-dimensional collagen matrices.

The cytoskeletal protein vinculin contributes to the mechanical link of the contractile actomyosin cytoskeleton to the extracellular matrix (ECM) through integrin receptors. In addition, vinculin modulates the dynamics of cell adhesions and is associated with decreased cell motility on two-dimensional ECM substrates. The effect of vinculin on cell invasion through dense three-dimensional ECM gels is unknown. Here, we report how vinculin expression affects cell invasion into three-dimensional collagen matrices. Cell motility was investigated in vinculin knockout and vinculin expressing wild-type mouse embryonic fibroblasts. Vinculin knockout cells were 2-fold more motile on two-dimensional collagen-coated substrates compared with wild-type cells, but 3-fold less invasive in 2.4 mg/ml three-dimensional collagen matrices. Vinculin knockout cells were softer and remodeled their cytoskeleton more dynamically, which is consistent with their enhanced two-dimensional motility but does not explain their reduced three-dimensional invasiveness. Importantly, vinculin-expressing cells adhered more strongly to collagen and generated 3-fold higher traction forces compared with vinculin knockout cells. Moreover, vinculin-expressing cells were able to migrate into dense (5.8 mg/ml) three-dimensional collagen matrices that were impenetrable for vinculin knockout cells. These findings suggest that vinculin facilitates three-dimensional matrix invasion through up-regulation or enhanced transmission of traction forces that are needed to overcome the steric hindrance of ECMs.

Inefficient clearance of dying cells in patients with SLE: anti-dsDNA autoantibodies, MFG-E8, HMGB-1 and other players.

Systemic lupus erythematosus (SLE) is a complex disease resulting from inflammatory responses of the immune system against several autoantigens. Inflammation is conditioned by the continuous presence of autoantibodies and leaked autoantigens, e.g. from not properly cleared dying and dead cells. Various soluble molecules and biophysical properties of the surface of apoptotic cells play significant roles in the appropriate recognition and further processing of dying and dead cells. We exemplarily discuss how Milk fat globule epidermal growth factor 8 (MFG-E8), biophysical membrane alterations, High mobility group box 1 (HMGB1), C-reactive protein (CRP), and anti-nuclear autoantibodies may contribute to the etiopathogenesis of the disease. Up to date knowledge about these key elements may provide new insights that lead to the development of new treatment strategies of the disease.

The role of the tissue microenvironment in the regulation of cancer cell motility and invasion.

During malignant neoplastic progression the cells undergo genetic and epigenetic cancer-specific alterations that finally lead to a loss of tissue homeostasis and restructuring of the microenvironment. The invasion of cancer cells through connective tissue is a crucial prerequisite for metastasis formation. Although cell invasion is foremost a mechanical process, cancer research has focused largely on gene regulation and signaling that underlie uncontrolled cell growth. More recently, the genes and signals involved in the invasion and transendothelial migration of cancer cells, such as the role of adhesion molecules and matrix degrading enzymes, have become the focus of research. In this review we discuss how the structural and biomechanical properties of extracellular matrix and surrounding cells such as endothelial cells influence cancer cell motility and invasion. We conclude that the microenvironment is a critical determinant of the migration strategy and the efficiency of cancer cell invasion.

Anchorage of vinculin to lipid membranes influences cell mechanical properties.

The focal adhesion protein vinculin (1066 residues) can be separated into a 95-kDa head and a 30-kDa tail domain. Vinculin's lipid binding sites localized on the tail, helix 3 (residues 944-978) and the unstructured C-terminal arm (residues 1052-1066, the so-called lipid anchor), influence focal adhesion turnover and are important for cell migration and adhesion. Using magnetic tweezers, we characterized the cell mechanical behavior in mouse embryonic fibroblast (MEF)-vin(-/-) cells transfected with EGFP-linked-vinculin deficient of the lipid anchor (vinDeltaC, residues 1-1051). MEF-vinDeltaC cells incubated with fibronectin-coated paramagnetic beads were less stiff, and more beads detached during these experiments compared to MEF-rescue cells. Cells expressing vinDeltaC formed fewer focal contacts as determined by confocal microscopy. Two-dimensional traction measurements showed that MEF-vinDeltaC cells generate less force compared to rescue cells. Attenuated traction forces were also found in cells that expressed vinculin with point mutations (R1060 and K1061 to Q) of the lipid anchor that impaired lipid binding. However, traction generation was not diminished in cells that expressed vinculin with impaired lipid binding caused by point mutations on helix 3. Mutating the src-phosphorylation site (Y1065 to F) resulted in reduced traction generation. These observations show that both the lipid binding and the src-phosphorylation of vinculin's C-terminus are important for cell mechanical behavior.

Up-regulation of Rho/ROCK signaling in sarcoma cells drives invasion and increased generation of protrusive forces.

Tumor cell invasion is the most critical step of metastasis. Determination of the mode of invasion within the particular tumor is critical for effective cancer treatment. Protease-independent amoeboid mode of invasion has been described in carcinoma cells and more recently in sarcoma cells on treatment with protease inhibitors. To analyze invasive behavior, we compared highly metastatic sarcoma cells with parental nonmetastatic cells. The metastatic cells exhibited a functional up-regulation of Rho/ROCK signaling and, similarly to carcinoma cells, an amoeboid mode of invasion. Using confocal and traction force microscopy, we showed that an up-regulation of Rho/ROCK signaling leads to increased cytoskeletal dynamics, myosin light chain localization, and increased tractions at the leading edge of the cells and that all of these contributed to increased cell invasiveness in a three-dimensional collagen matrix. We conclude that cells of mesenchymal origin can use the amoeboid nonmesenchymal mode of invasion as their primary invading mechanism and show the dependence of ROCK-mediated amoeboid mode of invasion on the increased capacity of cells to generate force.

CD24 induces localization of beta1 integrin to lipid raft domains.

The expression of the glycosyl phosphatidylinositol (GPI)-anchored protein CD24 correlates with poor prognosis in a variety of carcinomas. However, little is known about the cellular mechanisms of the CD24-mediated effects. In this study, we present evidence that CD24 affects the lateral localization of beta1 integrin. Using stably CD24-transfected A125 and MDA-MB-435S carcinoma cells we show that CD24 augments beta1-dependent cell motility and stimulates transmigration and invasion across a monolayer of endothelial cells. Furthermore, as demonstrated by sucrose density gradient centrifugation and Western Blot analysis, CD24 recruits beta1 integrin into lipid raft domains. We suggest that CD24 acts as a gate-keeper for lipid rafts, thereby regulating the activity of integrins and other proteins.

Stress fluctuations and motion of cytoskeletal-bound markers.

Cytoskeletal (CSK) dynamics such as remodeling and reorganization can be studied by tracking the spontaneous motion of CSK-bound particles. Particle motion is thought to be driven by local, ATP-dependent intracellular force fluctuations due to polymerization processes and motor proteins, and to be impeded by a viscoelastic, metastable cytoskeletal network. The mechanisms that link particle motion to force fluctuations and the CSK dynamics remain unclear. We report simultaneous measurements of the spontaneous motion of CSK-bound particles and of cellular force fluctuations. Cellular force fluctuations were measured by tracking fluorescent markers embedded in an elastic polyacrylamide hydrogel substrate that served as an extracellular matrix (ECM). The motion of CSK-bound particles and markers embedded in the ECM showed both persistence and superdiffusive behavior. Moreover, the movements of CSK-bound beads were temporally and spatially correlated with force fluctuations in the ECM. The findings suggest that the spontaneous motion of CSK-bound beads is driven not by random, local stress fluctuations within a viscoelastic continuum or cage, but rather by stress fluctuations within a tensed and constantly remodeling CSK network that transmits stresses over considerable distances to the ECM.

Focal adhesion kinase activity is required for actomyosin contractility-based invasion of cells into dense 3D matrices.

The focal adhesion kinase (FAK) regulates the dynamics of integrin-based cell adhesions important for motility. FAK's activity regulation is involved in stress-sensing and focal-adhesion turnover. The effect of FAK on 3D migration and cellular mechanics is unclear. We analyzed FAK knock-out mouse embryonic fibroblasts and cells expressing a kinase-dead FAK mutant, R454-FAK, in comparison to FAK wild-type cells. FAK knock-out and FAKR454/R454 cells invade dense 3D matrices less efficiently. These results are supported by FAK knock-down in wild-type fibroblasts and MDA-MB-231 human breast cancer cells showing reduced invasiveness. Pharmacological interventions indicate that in 3D matrices, cells deficient in FAK or kinase-activity behave similarly to wild-type cells treated with inhibitors of Src-activity or actomyosin-contractility. Using magnetic tweezers experiments, FAKR454/R454 cells are shown to be softer and exhibit impaired adhesion to fibronectin and collagen, which is consistent with their reduced 3D invasiveness. In line with this, FAKR454/R454 cells cannot contract the matrix in contrast to FAK wild-type cells. Finally, our findings demonstrate that active FAK facilitates 3D matrix invasion through increased cellular stiffness and transmission of actomyosin-dependent contractile force in dense 3D extracellular matrices.

The phenotype of cancer cell invasion controlled by fibril diameter and pore size of 3D collagen networks.

The behavior of cancer cells is strongly influenced by the properties of extracellular microenvironments, including topology, mechanics and composition. As topological and mechanical properties of the extracellular matrix are hard to access and control for in-depth studies of underlying mechanisms in vivo, defined biomimetic in vitro models are needed. Herein we show, how pore size and fibril diameter of collagen I networks distinctively regulate cancer cell morphology and invasion. Three-dimensional collagen I matrices with a tight control of pore size, fibril diameter and stiffness were reconstituted by adjustment of concentration and pH value during matrix reconstitution. At first, a detailed analysis of topology and mechanics of matrices using confocal laser scanning microscopy, image analysis tools and force spectroscopy indicate pore size and not fibril diameter as the major determinant of matrix elasticity. Secondly, by using two different breast cancer cell lines (MDA-MB-231 and MCF-7), we demonstrate collagen fibril diameter--and not pore size--to primarily regulate cell morphology, cluster formation and invasion. Invasiveness increased and clustering decreased with increasing fibril diameter for both, the highly invasive MDA-MB-231 cells with mesenchymal migratory phenotype and the MCF-7 cells with amoeboid migratory phenotype. As this behavior was independent of overall pore size, matrix elasticity is shown to be not the major determinant of the cell characteristics. Our work emphasizes the complex relationship between structural-mechanical properties of the extracellular matrix and invasive behavior of cancer cells. It suggests a correlation of migratory and invasive phenotype of cancer cells in dependence on topological and mechanical features of the length scale of single fibrils and not on coarse-grained network properties.

Physical break-down of the classical view on cancer cell invasion and metastasis.

Eight classical hallmarks of cancer have been proposed and are well-defined by using biochemical or molecular genetic methods, but are not yet precisely defined by cellular biophysical processes. To define the malignant transformation of neoplasms and finally reveal the functional pathway, which enables cancer cells to promote cancer progression, these classical hallmarks of cancer require the inclusion of specific biomechanical properties of cancer cells and their microenvironment such as the extracellular matrix and embedded cells such as fibroblasts, macrophages or endothelial cells. Nonetheless a main novel ninth hallmark of cancer is still elusive in classical tumor biological reviews, which is the aspect of physics in cancer disease by the natural selection of an aggressive (highly invasive) subtype of cancer cells. The physical aspects can be analyzed by using state-of-the-art biophysical methods. Thus, this review will present current cancer research in a different light and will focus on novel physical methods to investigate the aggressiveness of cancer cells from a biophysicist's point of view. This may lead to novel insights into cancer disease and will overcome classical views on cancer. In addition, this review will discuss how physics of cancer can help to reveal whether cancer cells will invade connective tissue and metastasize. In particular, this review will point out how physics can improve, break-down or support classical approaches to examine tumor growth even across primary tumor boundaries, the invasion of single or collective cancer cells, transendothelial migration of cancer cells and metastasis in targeted organs. Finally, this review will show how physical measurements can be integrated into classical tumor biological analysis approaches. The insights into physical interactions between cancer cells, the primary tumor and the microenvironment may help to solve some "old" questions in cancer disease progression and may finally lead to novel approaches for development and improvement of cancer diagnostics and therapies.

Phagocytized beads reduce the α5ß1 integrin facilitated invasiveness of cancer cells by regulating cellular stiffness.

Cell invasion through the extracellular matrix (ECM) of connective tissue is an important biomechanical process, which plays a prominent role in tumor progression. The malignancy of tumors depends mainly on the capacity of cancer cells to migrate and metastasize. A prerequisite for metastasis is the invasion of cancer cells through connective tissue to targeted organs. Cellular stiffness and cytoskeletal remodeling dynamics have been proposed to affect the invasiveness of cancer cells. Here, this study investigated whether highly invasive cancer cells are capable of invading into dense 3D-ECMs with an average pore-size of 1.3 or 3.0 µm when phagocytized beads (2.7 and 4.5 µm diameter) increased their cellular stiffness and reduced their cytoskeletal remodeling dynamics compared to weakly invasive cancer cells. The phagocytized beads decreased the invasiveness of the α5ß1(high) cancer cells into 3D-ECMs, whereas the invasiveness of the α5ß1(low) cancer cells was not affected. The effect of phagocytized beads on the highly invasive α5ß1(high) cells was abolished by specific knock-down of the α5 integrin subunit or addition of an anti-α5 integrin blocking antibody. Furthermore, the reduction of contractile forces using MLCK and ROCK inhibitors abolished the effect of phagocytized beads on the invasiveness of α5ß1(high) cells. In addition, the cellular stiffness of α5ß1(high) cells was increased after bead phagocytosis, whereas the bead phagocytosis did not alter the stiffness of α5ß1(low) cells. Taken together, the α5ß1 integrin dependent invasiveness was reduced after bead phagocytosis by altered biomechanical properties, suggesting that the α5ß1(high) cells need an appropriate intermediate cellular stiffness to overcome the steric hindrance of 3D-ECMs, whereas the α5ß1(low) cells were not affected by phagocytized beads.

Endothelial cell's biomechanical properties are regulated by invasive cancer cells.

Most cancer-related deaths are caused by the ability of cancer cells to metastasize. This process includes the dissemination of cancer cells from the primary tumor side and their migration to targeted organ sites. During the migration of cancer cells through the connective tissue microenvironment, which consists of endothelial cells and extracellular matrix components, biomechanical properties are crucial for the efficiency and speed of cancer cell invasion and subsequently, metastases formation. Biomechanics can enable cancer cells to migrate through tissue, transmigrate through basement membranes as well as endothelial monolayers and form metastases in targeted organs. The current focus of cancer research still lies on the investigation of cancer cell's biochemical and molecular capabilities such as molecular genetics and gene signaling, but these approaches ignore the mechanical nature of the invasion process of cancer cells. Moreover, even the role of the endothelium during the transmigration and invasion of cells is not clear, it has been seen as a passive barrier, but this could not explain all novel findings. This review discusses how cancer cells alter the structural, biochemical and mechanical properties of the endothelium to regulate their own invasiveness through extracellular matrices and hence, through the tissue microenvironment. Finally, this review sheds light on the mechanical properties of cancer cells and the interacting endothelium and points out the importance of the mechanical properties as a critical determinant for the efficiency of cancer cell invasion and the overall progression of cancer. In conclusion, the regulation of the endothelial cell's biomechanical properties by cancer cells is a critical determinant of cancer cell invasiveness and may affect the future development of new cancer treatments.

The biomechanical properties of 3d extracellular matrices and embedded cells regulate the invasiveness of cancer cells.

The malignancy of tumors depends on the biomechanical properties of cancer cells and their microenvironment, which enable cancer cells to migrate through the connective tissue, transmigrate through basement membranes and endothelial monolayers and form metastases in targeted organs. The current focus of cancer research is still based on biological capabilities such as molecular genetics and gene signaling, but these approaches ignore the mechanical nature of the invasion process of cancer cells. This review will focus on how structural, biochemical and mechanical properties of extracellular matrices (ECMs), and adjacent cells regulate the invasiveness of cancer cells. In addition, it presents how cancer cells create their own microenvironment by restructuring of the ECM and by interaction with stromal cells, which then further contribute to the progression of cancer disease. Finally, this review will point out that mechanical properties are a critical determinant for the efficiency of cancer cell invasion and the progression of cancer which might affect the future development of new cancer treatments.

Neoplastic progression of the human breast cancer cell line G3S1 is associated with elevation of cytoskeletal dynamics and upregulation of MT1-MMP.

The newly established breast cancer cell line G3S1, derived from EM-G3 breast cancer progenitors, was analyzed for functional changes related to neoplastic progression manifested by elevated invasiveness and enhanced capability to degrade gelatin. Degradation of gelatin and invasiveness of G3S1 cells was found to be dependent on the activity of matrix proteinases and actin cytoskeletal dynamics. Therefore, the expression and activity of these proteases was compared in G3S1 and EM-G3 cells. Despite enhanced capability of G3S1 cells to degrade gelatin, these cells exhibited lower levels of secreted extracellular matrix degrading proteases than parental EM-G3 cells. However, the expression of membrane-bound MT1-MMP was strongly elevated in G3S1 cells. While the degradation of gelatin was associated with invadopodia-like structures in both EM-G3 and G3S1 cells, the cytoskeletal remodeling dynamics was greatly elevated in G3S1 cells, suggesting that upregulation of MT1-MMP, together with elevation of cytoskeletal remodeling dynamics can effectively cause elevated invasiveness and enhanced matrix degrading capability in G3S1 cells.

The vinculin-DeltaIn20/21 mouse: characteristics of a constitutive, actin-binding deficient splice variant of vinculin.

BACKGROUND: The cytoskeletal adaptor protein vinculin plays a fundamental role in cell contact regulation and affects central aspects of cell motility, which are essential to both embryonal development and tissue homeostasis. Functional regulation of this evolutionarily conserved and ubiquitously expressed protein is dominated by a high-affinity, autoinhibitory head-to-tail interaction that spatially restricts ligand interactions to cell adhesion sites and, furthermore, limits the residency time of vinculin at these sites. To date, no mutants of the vinculin protein have been characterized in animal models. METHODOLOGY/PRINCIPAL FINDINGS: Here, we investigate vinculin-DeltaEx20, a splice variant of the protein lacking the 68 amino acids encoded by exon 20 of the vinculin gene VCL. Vinculin-DeltaEx20 was found to be expressed alongside with wild type protein in a knock-in mouse model with a deletion of introns 20 and 21 (VCL-DeltaIn20/21 allele) and shows defective head-to-tail interaction. Homozygous VCL-DeltaIn20/21 embryos die around embryonal day E12.5 showing cranial neural tube defects and exencephaly. In mouse embryonic fibroblasts and upon ectopic expression, vinculin-DeltaEx20 reveals characteristics of constitutive head binding activity. Interestingly, the impact of vinculin-DeltaEx20 on cell contact induction and stabilization, a hallmark of the vinculin head domain, is only moderate, thus allowing invasion and motility of cells in three-dimensional collagen matrices. Lacking both F-actin interaction sites of the tail, the vinculin-DeltaEx20 variant unveils vinculin's dynamic binding to cell adhesions independent of a cytoskeletal association, and thus differs from head-to-tail binding deficient mutants such as vinculin-T12, in which activated F-actin binding locks the protein variant to cell contact sites. CONCLUSIONS/SIGNIFICANCE: Vinculin-DeltaEx20 is an active variant supporting adhesion site stabilization without an enhanced mechanical coupling. Its presence in a transgenic animal reveals the potential of splice variants in the vinculin gene to alter vinculin function in vivo. Correct control of vinculin is necessary for embryonic development.