Ingo Thievessen

SILAC Mouse for Quantitative Proteomics Uncovers Kindlin-3 as an Essential Factor for Red Blood Cell Function

Stable isotope labeling by amino acids in cell culture (SILAC) has become a versatile tool for quantitative, mass spectrometry (MS)-based proteomics. Here, we completely label mice with a diet containing either the natural or the (13)C(6)-substituted version of lysine. Mice were labeled over four generations with the heavy diet, and development, growth, and behavior were not affected. MS analysis of incorporation levels allowed for the determination of incorporation rates of proteins from blood cells and organs. The F2 generation was completely labeled in all organs tested. SILAC analysis from various organs lacking expression of beta1 integrin, beta-Parvin, or the integrin tail-binding protein Kindlin-3 confirmed their absence and disclosed a structural defect of the red blood cell membrane skeleton in Kindlin-3-deficient erythrocytes. The SILAC-mouse approach is a versatile tool by which to quantitatively compare proteomes from knockout mice and thereby determine protein functions under complex in vivo conditions.

Vinculin–actin interaction couples actin retrograde flow to focal adhesions, but is dispensable for focal adhesion growth

Vinculin functions as a molecular clutch that organizes leading edge F-actin, generates traction, and promotes focal adhesion formation and turnover but not adhesion growth.

Integrin-linked kinase stabilizes myotendinous junctions and protects muscle from stress-induced damage.

Skeletal muscle expresses high levels of integrin-linked kinase (ILK), predominantly at myotendinous junctions (MTJs) and costameres. ILK binds the cytoplasmic domain of β1 integrin and mediates phosphorylation of protein kinase B (PKB)/Akt, which in turn plays a central role during skeletal muscle regeneration. We show that mice with a skeletal muscle–restricted deletion of ILK develop a mild progressive muscular dystrophy mainly restricted to the MTJs with detachment of basement membranes and accumulation of extracellular matrix. Endurance exercise training enhances the defects at MTJs, leads to disturbed subsarcolemmal myofiber architecture, and abrogates phosphorylation of Ser473 as well as phosphorylation of Thr308 of PKB/Akt. The reduction in PKB/Akt activation is accompanied by an impaired insulin-like growth factor 1 receptor (IGF-1R) activation. Coimmunoprecipitation experiments reveal that the β1 integrin subunit is associated with the IGF-1R in muscle cells. Our data identify the β1 integrin–ILK complex as an important component of IGF-1R/insulin receptor substrate signaling to PKB/Akt during mechanical stress in skeletal muscle.

Three-dimensional force microscopy of cells in biopolymer networks.

We describe a technique for the quantitative measurement of cell-generated forces in highly nonlinear three-dimensional biopolymer networks that mimic the physiological situation of living cells. We computed forces of MDA-MB-231 breast carcinoma cells from the measured network deformations around the cells using a finite-element approach based on a constitutive equation that captures the complex mechanical properties of diverse biopolymers such as collagen gels, fibrin gels and Matrigel. Our measurements show that breast carcinoma cells cultured in collagen gels generated nearly constant forces regardless of the collagen concentration and matrix stiffness. Furthermore, time-lapse force measurements showed that these cells migrated in a gliding motion with alternating phases of high and low contractility, elongation, migratory speed and persistence.

Vinculin, cell mechanics and tumour cell invasion

The focal adhesion protein, vinculin, is important for transmitting mechanical forces and orchestrating mechanical signalling events. Deregulation of vinculin results in altered cell adhesion, contractility, motility and growth, all of which are important processes in cancer metastasis. This review summarises recent reports on the role of vinculin in cellular force generation and signalling, and discusses implications for a role of vinculin in promoting cancer cell migration in 3D environments.

Hedgehog signaling in normal urothelial cells and in urothelial carcinoma cell lines.

Constitutive activation of hedgehog signaling, often caused by PTCH1 inactivation and leading to inappropriate activation of GLI target genes, is crucial for the development of several human tumors including basal cell carcinoma of the skin and medulloblastoma. The PTCH1 gene at 9q22 is also considered as a candidate tumor suppressor in transitional cell carcinoma (TCC), of which >50% show LOH in this region. However, only rare mutations have been found in PTCH1. We have therefore investigated GLI‐dependent promoter activity and expression of hedgehog pathway components in TCC cell lines and proliferating normal urothelial cells. Normal urothelial cells cultured in serum‐free medium, but not TCC lines exhibited low, but significant promoter activity under standard growth conditions. Accordingly, GLI1‐3 and PTCH1 mRNAs were expressed at moderate levels, and sonic hedgehog (SHH) mRNA expression was low to undetectable. In co‐transfection experiments GLI1 increased promoter activity significantly in one TCC line and further in normal urothelial cells, but less strongly in other TCC lines. Expression patterns of GLI factor mRNAs did not correlate with inducibility. No significant effects of SHH or cyclopamine on proliferation were observed, ruling out autocrine effects. However, SHH induced GLI‐dependent promoter activity in normal urothelial cells. Taken together, our data suggest that the hedgehog pathway is weakly active in normal adult urothelial cells and of limited importance in TCC.

Vinculin is required for cell polarization, migration, and extracellular matrix remodeling in 3D collagen

Vinculin is filamentous (F)‐actin‐binding protein enriched in integrin‐based adhesions to the extracellular matrix (ECM). Whereas studies in 2‐dimensional (2D) tissue culture models have suggested that vinculin negatively regulates cell migration by promoting cytoskeleton‐ECM coupling to strengthen and stabilize adhesions, its role in regulating cell migration in more physiologic, 3‐dimensional (3D) environments is unclear. To address the role of vinculin in 3D cell migration, we analyzed the morphodynamics, migration, and ECM remodeling of primary murine embryonic fibroblasts (MEFs) with cre/loxP‐mediated vinculin gene disruption in 3D collagen I cultures. We found that vinculin promoted 3D cell migration by increasing directional persistence. Vinculin was necessary for persistent cell protrusion, cell elongation, and stable cell orientation in 3D collagen, but was dispensable for lamellipodia formation, suggesting that vinculin‐mediated cell adhesion to the ECM is needed to convert actin‐based cell protrusion into persistent cell shape change and migration. Consistent with this finding, vinculin was necessary for efficient traction force generation in 3D collagen without affecting myosin II activity and promoted 3D collagen fiber alignment and macroscopical gel contraction. Our results suggest that vinculin promotes directionally persistent cell migration and tension‐dependent ECM remodeling in complex 3D environments by increasing cell‐ECM adhesion and traction force generation.—Thievessen, I., Fakhri, N., Steinwachs, J., Kraus, V., McIsaac, R. S., Gao, L., Chen, B.‐C., Baird, M. A., Davidson, M. W., Betzig, E., Oldenbourg, R., Waterman, C., M., Fabry, B. Vinculin is required for cell polarization, migration, and extracellular matrix remodeling in 3D collagen. FASEB J. 29, 4555‐4567 (2015). www.fasebj.org

Cardiac fibroblasts require focal adhesion kinase for normal proliferation and migration

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(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 PDGF-BB-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.

γ-Parvin is dispensable for hematopoiesis, leukocyte trafficking, and T-cell-dependent antibody response

ABSTRACT 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.