Gerald Burgstaller

Plectin-controlled keratin cytoarchitecture affects MAP kinases involved in cellular stress response and migration.

Plectin is a major intermediate filament (IF)–based cytolinker protein that stabilizes cells and tissues mechanically, regulates actin filament dynamics, and serves as a scaffolding platform for signaling molecules. In this study, we show that plectin deficiency is a cause of aberrant keratin cytoskeleton organization caused by a lack of orthogonal IF cross-linking. Keratin networks in plectin-deficient cells were more susceptible to osmotic shock–induced retraction from peripheral areas, and their okadaic acid–induced disruption (paralleled by stress-activated MAP kinase p38 activation) proceeded faster. Basal activities of the MAP kinase Erk1/2 and of the membrane-associated upstream protein kinases c-Src and PKCδ were significantly elevated, and increased migration rates, as assessed by in vitro wound-closure assays and time-lapse microscopy, were observed. Forced expression of RACK1, which is the plectin-binding receptor protein for activated PKCδ, in wild-type keratinocytes elevated their migration potential close to that of plectin-null cells. These data establish a link between cytolinker-controlled cytoarchitecture/scaffolding functions of keratin IFs and specific MAP kinase cascades mediating distinct cellular responses.

Actin cytoskeleton remodelling via local inhibition of contractility at discrete microdomains

Activation of conventional protein kinase C by phorbol ester triggers the Src-dependent remodelling of the actin cytoskeleton and the formation of podosomes in vascular smooth muscle cells. Rearrangement of actin cytoskeleton in response to phorbol-12,13-dibutyrate is characterised by the simultaneous disassembly of peripheral actin stress fibres and focal adhesions, focal de novo actin polymerisation and actomyosin contraction in the cell center, indicating a spatially and temporally segregated, differential modulation of actin-cytoskeleton stability and turnover. Taking advantage of the prominent actin cytoskeleton in A7r5 cells we show here, that the molecular basis for the local inhibition of contractility is the specific recruitment of p190RhoGAP to specialised microdomains at the focal adhesion/stress fibre interface, which are constitutively enriched in cortactin. The microdomains contain structurally altered actin filaments inaccessible to phalloidin. However, the filaments remain decorated with high molecular weight tropomyosins. Clustering of cortactin during podosome formation causes the rapid, local dispersion of myosin and tropomyosin, and interferes with the F-actin binding of h1calponin, consistent with a RhoGAP-mediated reduction of contractility. Phorbol ester-induced podosome formation is efficiently blocked by expression of constitutively active Dia1, which leads to the dispersion of cortactin. The results provide direct evidence for the spatially restricted inhibition of contractility via the recruitment and accumulation of cortactin and p190RhoGAP.

Keeping the Vimentin Network under Control: Cell–Matrix Adhesion–associated Plectin 1f Affects Cell Shape and Polarity of Fibroblasts

Mature focal adhesions and fibrillar adhesions act as anchorage sites for vimentin filaments, with plectin isoform 1f being the crucial linker protein. Plectin serves as a nucleation and assembly center for the de novo formation of vimentin networks. Anchored vimentin creates a resilient cage-like core structure that affects cell shape.

Mechanosensing through focal adhesion-anchored intermediate filaments

Integrin‐based mechanotransduction involves a complex focal adhesion (FA)‐associated machinery that is able to detect and respond to forces exerted either through components of the extracellular matrix or the intracellular contractile actomyosin network. Here, we show a hitherto unrecognized regulatory role of vimentin intermediate filaments (IFs) in this process. By studying fibroblasts in which vimentin IFs were decoupled from FAs, either because of vimentin deficiency (V0) or loss of vimentin network anchorage due to deficiency in the cytolinker protein plectin (P0), we demonstrate attenuated activation of the major mechanosensor molecule FAK and its downstream targets Src, ERK1/2, and p38, as well as an up‐regulation of the compensatory feedback loop acting on RhoA and myosin light chain. In line with these findings, we show strongly reduced FA turnover rates in P0 fibroblasts combined with impaired directional migration, formation of protrusions, and up‐regulation of “stretched” high‐affinity integrin complexes. By exploiting tension‐independent conditions, we were able to mechanistically link these defects to diminished cytoskeletal tension in both P0 and V0 cells. Our data provide important new insights into molecular mechanisms underlying cytoskeleton‐regulated mechanosensing, a feature that is fundamental for controlled cell movement and tumor progression.—Gregor, M., Osmanagic‐Myers, S., Burgstaller, G., Wolfram, M., Fischer, I., Walko, G., Resch, G. P., Jörgl, A., Herrmann, H., Wiche, G. Mechanosensing through focal adhesion‐anchored intermediate filaments. FASEB J. 28, 715–729 (2014). www.fasebj.org

The role of calponin in the gene profile of metastatic cells: inhibition of metastatic cell motility by multiple calponin repeats

Metastasis of diseased cells is the basic event leading to death in individuals with cancer. Establishment of metastasis requires that tumour cells migrate from the site of the primary tumour into the circulation system, escape from the vasculature and form secondary tumours at novel sites. These processes depend to a large degree on cytoskeletal remodeling. We show here that multiple copies of the short actin‐binding module CLIK23 from human or Caenorhabditis elegans calponin proteins effectively inhibit cell motility on two dimensional matrices and suppress soft agar colony formation of metastatic melanoma and adenocarcinoma cells of murine and human origin. Ectopic expression of CLIK23 modules for 30 days results in the formation of multinucleated cells. The repeat displays true modular behaviour, resulting in increased cytoskeletal effects in direct correlation with the increase in number of modules. Our results demonstrate that the role of calponin in the signature profile of metastasising cells is that of a mechanical stabiliser of the actin cytoskeleton, which interferes with actin turnover by binding at a unique interface along the actin filament.

Purification of Smooth Muscle Actin

Publisher Summary This article describes the method used for purifying polymerization-competent actin from mammalian or avian visceral smooth muscle. Actin purified from mammalian or avian skeletal muscle is a readily available probe to study the potential interaction of a putative actin-binding protein or recombinant fragment in vitro . A primary source of contamination is the co-purification of tropomyosin. Moreover, because the total amount of actin present in smooth muscle is reduced compared to that in skeletal muscle, the yield is lower. The most convenient source of actin for relatively large yields is the powder remaining after the extensive drying of muscle homogenates in acetone. Resuspend the pellet in about 50 ml G-actin buffer and dialyze against 2 l of G-actin buffer overnight with two to three changes of buffer. Centrifuge the dialyzed actin solution to remove polymerized or aggregated actin at 32,000xg for 60 min.