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Biomedical subjects

Mario Gimona

Publications and source records attributed to Mario Gimona.

At least 19 recordsLinked to original sources
PubMed2006

Adhesions that mediate invasion.

Infiltration of new tissue areas requires that a mammalian cell overcomes the physical and biochemical barrier of the surrounding extracellular matrix. Cell migration during embryonic development, and growth, invasion and dispersal of metastatic tumor cells depend to a large extent on the controlled degradation of extracellular matrix components. Localized degradation of the surrounding matrix is seen at defined adhesive (podosomes) and/or protrusive (invadopodia) locations in a variety of normal cells and aggressive carcinoma cells, suggesting that these membrane-associated cellular devices have a central role in mediating polarized migration in cells that cross-tissue boundaries. Here, we will discuss the recent advances and developments in this field, and provide our provisional outlook into the future understanding of the principles of focal extracellular matrix degradation by podosomes and invadopodia.

Animals↗
PubMed2006

Protein linguistics - a grammar for modular protein assembly?

The correspondence between biology and linguistics at the level of sequence and lexical inventories, and of structure and syntax, has fuelled attempts to describe genome structure by the rules of formal linguistics. But how can we define protein linguistic rules? And how could compositional semantics improve our understanding of protein organization and functional plasticity?

Linguistics↗
PubMed2005

The actin cytoskeleton, RAS-cAMP signaling and mitochondrial ROS in yeast apoptosis.

The release of reactive oxygen species (ROS) by mitochondria instigates the pathways of programmed cell death in eukaryotic cells. Gourlay and Ayscough present intriguing experimental evidence that mutations in the genes encoding the regulatory proteins End3p and Sla1p, which influence actin dynamics in budding yeast, lead to a loss of mitochondrial membrane potential, resulting in ROS production and apoptosis. This effect can be suppressed by downregulation of the RAS-cAMP signaling pathway, thus establishing the existence of a new and complex regulatory network.

Actins↗
PubMed2005

Matrix-degrading podosomes in smooth muscle cells.

Activation of protein kinase C by phorbol esters triggers the remodelling of the actin cytoskeleton and the formation of podosomes in smooth muscle cells (SMCs). Regional control of actin dynamics at specialised microdomains results in a local reduction in contractile forces. The molecular basis for this local inhibition of contractility includes the clustering of cortactin during podosome formation (which precedes the rapid, local dispersion of myosin, tropomyosin and h1 calponin), and the specific recruitment of 110-kDa actin filament-associated protein (AFAP-110) and 190-kDa Rho-specific GTPase-activating protein (p190RhoGAP) to the microdomains. Podosome formation also correlates with cell polarisation, the induction of cell motility, and local degradation of the extracellular matrix. These findings may provide explanations for the complex mechanisms underlying SMC invasion in the course of the development of atherosclerotic lesions and restenosis, and support the concept that matrix degradation and the concomitant engagement of the molecular machinery initiating actin-based cell motility drive tissue invasion in smooth muscle.

Actin Cytoskeleton↗
PubMed2005

Podosome-mediated matrix resorption and cell motility in vascular smooth muscle cells.

The migration of vascular smooth muscle cells (VSMCs) is a principal factor for the development and progression of vascular diseases. In addition, phenotypic alteration from the contractile (differentiated) to the synthetic (dedifferentiated) state and a proteolytic process in the form of extra cellular matrix degradation are necessary for SMC invasion. The actual mechanism leading to the focal degradation of basement membrane matrix components and, hence, SMC migration within the tissue itself is, however, unclear. In response to phorbol ester [phorbol-12,13-dibutyrate (PDBu)], VSMCs in culture form podosomes, dynamic organelles critical for cell adhesion and substrate degradation that are typically found in invasive cells and cells that cross tissue boundaries. Here, we show that PDBu-stimulated VSMCs resorb the extracellular matrix at the sites of podosomes. Podosome formation correlates with an increased polarization of VSMCs on fibronectin- or collagen-coated flexible substrates in addition to a concomitant induction of cell motility. VSMCs embedded in reconstituted basement membrane support adopt the typical spindle-shaped morphology of differentiated SMCs in vivo and, after PDBu treatment, form peripheral lamellipodia and podosomes around their matrix-contacting surface. Our findings demonstrate that podosome formation is the potential mechanism underlying the ability of VSMCs to traverse the surrounding basement membrane and escape the barrier of the tunica media in vascular diseases.

Animals↗
PubMed2005

CLAMP, a novel microtubule-associated protein with EB-type calponin homology.

Microtubules (MTs) are polymers of alpha and beta tubulin dimers that mediate many cellular functions, including the establishment and maintenance of cell shape. The dynamic properties of MTs may be influenced by tubulin isotype, posttranslational modifications of tubulin, and interaction with microtubule-associated proteins (MAPs). End-binding (EB) family proteins affect MT dynamics by stabilizing MTs, and are the only MAPs reported that bind MTs via a calponin-homology (CH) domain (J Biol Chem 278 (2003) 49721-49731; J Cell Biol 149 (2000) 761-766). Here, we describe a novel 27 kDa protein identified from an inner ear organ of Corti library. Structural homology modeling demonstrates a CH domain in this protein similar to EB proteins. Northern and Western blottings confirmed expression of this gene in other tissues, including brain, lung, and testis. In the organ of Corti, this protein localized throughout distinctively large and well-ordered MT bundles that support the elongated body of mechanically stiff pillar cells of the auditory sensory epithelium. When ectopically expressed in Cos-7 cells, this protein localized along cytoplasmic MTs, promoted MT bundling, and efficiently stabilized MTs against depolymerization in response to high concentration of nocodazole and cold temperature. We propose that this protein, designated CLAMP, is a novel MAP and represents a new member of the CH domain protein family.

Amino Acid Sequence↗
PubMed2004

CRP2 is an autonomous actin-binding protein.

Cysteine-rich proteins (CRPs) have been shown to be involved in cell differentiation, transcriptional regulation and the organisation of the actin cytoskeleton. Thus far, the latter function has been inferred solely from the in vitro interaction of CRP1, CRP2, and CRP3 with alpha-actinin and zyxin. We show here that purified, recombinant CRP2 binds directly to F-actin in vitro in co-sedimentation assays. Using a green fluorescent protein (GFP)-tagged construct of CRP2 we analysed its localisation and dynamics in A7r5 rat smooth muscle cells. CRP2 was associated with the actin cytoskeleton and decorated actin stress fibres in a continuous fashion, unlike the periodic labelling pattern observed for alpha-actinin and zyxin, which also accumulate in focal adhesions. Using live video fluorescence microscopy we observed the behaviour of GFP-CRP2 during the dynamic rearrangement of the actin cytoskeleton in phorbol 12,13-dibutyrate-treated A7r5 cells. In contrast to the actin-binding proteins SM22alpha and alpha-actinin, GFP-CRP2 did not translocate into the podosomes induced by this treatment, but remained preferentially bound to the stress fibres, suggesting an actin filament-stabilising role for CRP2. When fused to the mitochondrial targeting sequence from the Listeria protein ActA, GFP-CRP2 was almost completely localised to mitochondria, but no significant recruitment of either alpha-actinin or zyxin could be observed. Taken together, our results demonstrate that CRP2 can bind to F-actin directly and that the association with the actin cytoskeleton is independent of alpha-actinin or zyxin localisation in the cell, thus questioning the role of CRP2 as a regulator of alpha-actinin function in vivo.

Actinin↗
PubMed2004

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.

Actins↗
PubMed2004

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 CLIK(23) 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 CLIK(23) 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.

Adenocarcinoma↗
PubMed2004

Tyrosine phosphorylation of calponins. Inhibition of the interaction with F-actin.

The phosphorylation-dephosphorylation of serine and threonine residues of calponin is known to modulate in vitro its interaction with F-actin and is thought to regulate several biological processes in cells, involving either of the calponin isoforms. Here, we identify, for the first time, tyrosine-phosphorylated calponin h3 within COS 7 cells, before and after their transfection with the pSV vector containing cDNA encoding the cytoplasmic, Src-related, tyrosine kinase, Fyn. We then describe the specific tyrosine phosphorylation in vitro of calponin h1 and calponin h3 by this kinase. 32P-labeling of tyrosine residues was monitored by combined autoradiography, immunoblotting with a specific phosphotyrosine monoclonal antibody and dephosphorylation with the phosphotyrosine-specific protein phosphatase, YOP. PhosphorImager analyses showed the incorporation of maximally 1.4 and 2.0 mol of 32P per mol of calponin h3 and calponin h1, respectively. As a result, 75% and 68%, respectively, of binding to F-actin was lost by the phosphorylated calponins. Furthermore, F-actin, added at a two- or 10-fold molar excess, did not protect, but rather increased, the extent of 32P-labeling in both calponins. Structural analysis of the tryptic phosphopeptides from each 32P-labeled calponin revealed a single, major 32P-peptide in calponin h3, with Tyr261 as the phosphorylation site. Tyr261 was also phosphorylated in calponin h1, together with Tyr182. Collectively, the data point to the potential involvement, at least in living nonmuscle cells, of tyrosine protein kinases and the conserved Tyr261, located in the third repeat motif of the calponin molecule, in a new level of regulation of the actin-calponin interaction.

Actins↗
PubMed2003

Podosome formation in cultured A7r5 vascular smooth muscle cells requires Arp2/3-dependent de-novo actin polymerization at discrete microdomains.

Phorbol ester triggers the conversion of focal adhesions into podosomes in A7r5 smooth muscle cells. Here we followed the dynamics of podosome formation using dual fluorescence live video and confocal microscopy, as well as interference reflection and evanescent wave microscopy. We show that podosomes form at the outer region of stress fiber bundles, at specialized sites where they are embedded in adhesion plaques at the basal surface of the plasma membrane, and that cortactin resides constitutively at these microdomains. We further demonstrate that the formation of podosomes requires Arp2/3-dependent actin polymerization at the stress fiber-focal adhesion interface. Concentration of Arp2/3 coincides with podosome formation and precedes the engagement of SM22 and alpha-actinin, while the focal adhesion components zyxin and vinculin redistribute only at later stages of podosome development. We thus suggest that the genesis of podosomes includes two steps, one requiring the early de novo polymerization of actin filaments, and a second, late phase characterized by the recruitment of focal adhesion components. Moreover, we provide evidence for the existence of an as yet unidentified region in close proximity to the focal adhesion-stress fiber interface, which marks the site of actin cytoskeleton remodeling and is a novel site of Arp2/3-dependent F-actin polymerization.

Actin Cytoskeleton↗
PubMed2003

Protein kinase C delta induces Src kinase activity via activation of the protein tyrosine phosphatase PTP alpha.

Previously we have shown that protein kinase C (PKC)-mediated reorganization of the actin cytoskeleton in smooth muscle cells is transmitted by the non-receptor tyrosine kinase, Src. Several authors have described how 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation of cells results in an increase of Src activity, but the mechanism of the PKC-mediated Src activation is unknown. Using PKC isozymes purified from Spodoptera frugiperda insect cells, we show here that PKC is not able to activate Src directly. Our data reveal that the PKC-dependent Src activation occurs via the activation of the protein tyrosine phosphatase (PTP) PTP alpha. PTP alpha becomes activated in vivo after TPA stimulation. Further, we show that PKC delta phosphorylates and activates only PTP alpha in vitro but not any other of the TPA-responsive PKC isozymes that are expressed in A7r5 rat aortic smooth muscle cells. To further substantiate our data, we show that cells lacking PKC delta have a markedly reduced PTP alpha and Src activity after 12-O-tetradecanoylphorbol-13-acetate stimulation. These data support a model in which the main mechanism of 12-O-tetradecanoylphorbol-13-acetate-induced Src activation is the direct phosphorylation and activation of PTP alpha by PKC delta, which in turn dephosphorylates and activates Src.

Actins↗
PubMed2003

Calponin repeats regulate actin filament stability and formation of podosomes in smooth muscle cells.

Phorbol ester induces actin cytoskeleton rearrangements in cultured vascular smooth muscle cells. Calponin and SM22 alpha are major components of differentiated smooth muscle and potential regulators of actin cytoskeleton interactions. Here we show that actin fibers decorated with h1 CaP remain stable, whereas SM22 alpha-decorated actin bundles undergo rapid reorganization into podosomes within 30 min of PDBu exposure. Ectopic expression of GFP alpha-actinin had no effect on the stability of the actin cytoskeleton and alpha-actinin was transported rapidly into PDBu-induced podosomes. Our results demonstrate the involvement of CaP and SM22 alpha in coordinating the balance between stabilization and dynamics of the actin cytoskeleton in mammalian smooth muscle. We provide evidence for the existence of two functionally distinct actin filament populations and introduce a molecular mechanism for the stabilization of the actin cytoskeleton by the unique actin-binding interface formed by calponin family-specific CLIK23 repeats.

Actin Cytoskeleton↗
PubMed2002

Conventional protein kinase C mediates phorbol-dibutyrate-induced cytoskeletal remodeling in a7r5 smooth muscle cells.

Phorbol dibutyrate (PDBu) induced the formation of podosome-like structures together with partial disassembly of actin stress fibers in A7r5 smooth muscle cells. These podosomes contained alpha-actinin, F-actin, and vinculin and exhibit a tubular, column-like structure arising perpendicularly from the bottom of PDBu-treated cells. The conventional protein kinase C (PKC) antagonist, GO6976, inhibited PDBu-induced cytoskeletal remodeling at 0.1 microM, whereas the novel PKC antagonist, rottlerin, was ineffective at 10 microM. PDBu induced the translocation of the conventional PKC-alpha but not the novel PKC-delta to the sites of podosome formation in A7r5 cells. Although partial disassembly of actin stress fibers was observed in both Y-27632- and PDBu-treated cells, focal adhesions were much reduced in number and size only in Y-27632-treated cells. Furthermore, PDBu restored focal adhesions in Y-27632-treated cells. Live video fluorescence microscopy of alpha-actinin GFP revealed a lag phase of about 20 min prior to the rapid formation and dynamic reorganization of podosomes during PDBu treatment. These findings suggest that conventional PKCs mediate PDBu-induced formation of dynamic podosome-like structures in A7r5 cells, and Rho-kinase is unlikely to be the underlying mechanism. The podosome columns could represent molecular scaffolds where PKC-alpha phosphorylates regulatory proteins necessary for Ca(2+) sensitization in smooth muscle cells.

Acetophenones↗
PubMed2002

The molecular basis for the autoregulation of calponin by isoform-specific C-terminal tail sequences.

The three genetic isoforms of calponin (CaP), h1, h2 and acidic, are distinguished mostly by their individual C-terminal tail sequences. Deletion of these sequences beyond the last homologous residue Cys273 increases actin filament association for all three isoforms, indicating a negative regulatory role for the unique tail regions. We have tested this hypothesis by constructing a series of deletion and substitution mutants for all three CaP isoforms. Here we demonstrate that the C-terminal sequences regulate actin association by altering the function of the second actin-binding site, ABS2, in CaP comprised of the three 29-residue calponin repeats. Removal of the inhibitory tail resulted in an increased binding and bundling activity, and caused a prominent re-localization of h2 CaP from the peripheral actin network to the central actin stress fibers in transfected A7r5 smooth muscle cells. Domain-swap experiments demonstrated that the tail sequence of h2 CaP can downregulate cytoskeletal association efficiently in all three CaP isoforms, whereas the tail of the smooth-muscle-specific h1 CaP variant had little effect. Site-directed mutagenesis further revealed that the negatively charged residues within the tail region are essential for this regulatory function. Finally we demonstrate that the tail sequences regulate the second actin-binding site (ABS2) and not the strong actin-binding ABS1 region in CaP.

Actins↗
PubMed2002

Protein kinase C induces actin reorganization via a Src- and Rho-dependent pathway.

We have investigated the mechanism of PKC-induced actin reorganization in A7r5 vascular smooth muscle cells. PKC activation by 12-O-tetradecanoylphorbol-13-acetate induces the disassembly of actin stress fibers concomitant with the appearance of membrane ruffles. PKC also induces rapid tyrosine phosphorylation in these cells. As we could show, utilizing the Src-specific inhibitor PP2 and a kinase-deficient c-Src mutant, actin reorganization is dependent on PKC-induced Src activation. Subsequently, the activity of the small G-protein RhoA is decreased, whereas Rac and Cdc42 activities remain unchanged. Disassembly of actin stress fibers could also be observed using the Rho kinase-specific inhibitor Y-27632, indicating that the decrease in RhoA activity on its own is responsible for actin reorganization. In addition, we show that tyrosine phosphorylation of p190RhoGAP is increased upon 12-O-tetradecanoylphorbol-13-acetate stimulation, directly linking Src activation to a decrease in RhoA activity. Our data provide substantial evidence for a model elucidating the molecular mechanisms of PKC-induced actin rearrangements.

Actins↗
PubMed2002

Functional plasticity of CH domains.

With the refinement of algorithms for the identification of distinct motifs from sequence databases, especially those using secondary structure predictions, new protein modules have been determined in recent years. Calponin homology (CH) domains were identified in a variety of proteins ranging from actin cross-linking to signaling and have been proposed to function either as autonomous actin binding motifs or serve a regulatory function. Despite the overall structural conservation of the unique CH domain fold, the individual modules display a quite striking functional variability. Analysis of the actopaxin/parvin protein family suggests the existence of novel (type 4 and type 5) CH domain families which require special attention, as they appear to be a good example for how CH domains may function as scaffolds for other functional motifs of different properties.

Actinin↗