Biomedical subjects

Olivier Pertz

Publications and source records attributed to Olivier Pertz.

10 recordsLinked to original sources

PubMed2006

Spatiotemporal dynamics of RhoA activity in migrating cells.

Rho family GTPases regulate the actin and adhesion dynamics that control cell migration. Current models postulate that Rac promotes membrane protrusion at the leading edge and that RhoA regulates contractility in the cell body. However, there is evidence that RhoA also regulates membrane protrusion. Here we use a fluorescent biosensor, based on a novel design preserving reversible membrane interactions, to visualize the spatiotemporal dynamics of RhoA activity during cell migration. In randomly migrating cells, RhoA activity is concentrated in a sharp band directly at the edge of protrusions. It is observed sporadically in retracting tails, and is low in the cell body. RhoA activity is also associated with peripheral ruffles and pinocytic vesicles, but not with dorsal ruffles induced by platelet-derived growth factor (PDGF). In contrast to randomly migrating cells, PDGF-induced membrane protrusions have low RhoA activity, potentially because PDGF strongly activates Rac, which has previously been shown to antagonize RhoA activity. Our data therefore show that different extracellular cues induce distinct patterns of RhoA signalling during membrane protrusion.

3T3 Cells↗

PubMed2006

Neutrophil polarization: spatiotemporal dynamics of RhoA activity support a self-organizing mechanism.

Chemoattractants like fMet-Leu-Phe (fMLP) induce neutrophils to polarize with phosphatidylinositol 3,4,5-trisphosphate (PIP3) and protrusive F-actin at the front and actomyosin contraction at the sides and back. RhoA and its downstream effector, myosin II, mediate the "backness" response, which locally inhibits the "frontness" response and constrains its location to one part of the cell. In living HL-60 cells, we used a fluorescent PIP3 probe or a single-chain FRET biosensor for RhoA-GTP to assess spatial distribution of frontness or backness responses, respectively, during the first 3 min after exposure to a uniform concentration of fMLP. Increased PIP3 signal or RhoA activity initially localized randomly about the cell's periphery but progressively redistributed to the front or to the back and sides, respectively. Cells rendered unable to mount the frontness response (by inhibiting actin polymerization or Gi, a trimeric G protein) responded to a micropipette source of attractant by localizing RhoA activity at the up-gradient edge. We infer that protrusive F-actin, induced by the frontness response, constrains the spatial distribution of backness by locally reducing activation of RhoA, thereby reducing its active form at the front. Mutual incompatibility of frontness and backness is responsible for self-organization of neutrophil polarity.

Actins↗

PubMed2006

Functional proteometrics for cell migration.

BACKGROUND: Advances in living cellular fluorescence biosensors and computerized microscopy enable a vision of fully automated high-resolution measurements of the detailed intracellular molecular dynamics directly linked to cellular behaviors. Given the heterogeneity of cell populations, a statistically relevant study of molecular-cellular dynamics is a key motivation for improved automation. METHODS: We explored automating computerized, microscope-based data extraction and analyses that monitor cell locomotion, rates of mitoses, and spatiotemporal activities of intracellular proteins via ratiometric fluorescent biosensors in mouse fibroblasts. Novel image processing methods included K-means clustering segmentation preprocessing followed by modified discrete, normalized cross-correlational alignment of two-color images; ratiometric processing for fluorescence resonance energy transfer (FRET) measurements; and intracellular spatial distribution measurements of RhoA GTPase activity. RESULTS: The interdivision time was 19.4 h (mean) +/- 6.0 h (SD) (n = 7) for the GFP-histone cells in the two-by-two field that was scanned for 72 h. After registration and ratioing of the cells with the RhoA biosensor, increases in both cell protrusion and retraction were coincident with to increases in RhoA activity. CONCLUSIONS: These advances lay the foundation for extracting and correlating measurements characterizing the functional relationships of spatial localization and protein activation with features of cell migration such as velocity, polarization, protrusion, retraction, and mitosis.

Animals↗

PubMed2004

Vinculin modulation of paxillin-FAK interactions regulates ERK to control survival and motility.

Cells lacking vinculin are highly metastatic and motile. The reasons for this finding have remained unclear. Both enhanced survival and motility are critical to metastasis. Here, we show that vinculin null (vin-/-) cells and cells expressing a vinculin Y822F mutant have increased survival due to up-regulated activity of extracellular signal-regulated kinase (ERK). This increase is shown to result from vinculin's modulation of paxillin-FAK interactions. A vinculin fragment (amino acids 811-1066) containing the paxillin binding site restored apoptosis and suppressed ERK activity in vin-/- cells. Both vinY822F and vin-/- cells exhibit increased interaction between paxillin and focal adhesion kinase (FAK) and increased paxillin and FAK phosphorylation. Transfection with paxillin Y31FY118F dominant-negative mutant in these cells inhibits ERK activation and restores apoptosis. The enhanced motility of vin-/- and vinY822F cells is also shown to be due to a similar mechanism. Thus, vinculin regulates survival and motility via ERK by controlling the accessibility of paxillin for FAK interaction.

Animals↗

PubMed2004

Designing biosensors for Rho family proteins--deciphering the dynamics of Rho family GTPase activation in living cells.

Rho family GTPases are molecular switches that couple changes in the extracellular environment to intracellular signal transduction pathways. Their ability to regulate behaviors such as cell motility suggests very tight kinetic and spatial control of their activity, which is missed in most biochemical assays. Fluorescent probes that non-invasively report the changing subcellular location of Rho GTPase activity in single living cells are now allowing us to examine spatio-temporal regulation of the activity of these proteins, and are providing new biological insights. Several strategies can be used to construct such probes, and there are advantages and disadvantages associated with the diverse probe designs.

Animals↗

PubMed2003

Homoassociation of VE-cadherin follows a mechanism common to "classical" cadherins.

Vascular endothelial cadherin (VE-cadherin/cadherin5) is specifically expressed in adherens junctions of endothelial cells and exerts important functions in cell-cell adhesion as well as signal transduction. To analyze the mechanism of VE-cadherin homoassociation, the ectodomains CAD1-5 were connected by linker sequences to the N terminus of the coiled-coil domain of cartilage matrix protein (CMP). The chimera VECADCMP were expressed in mammalian cells. The trimeric coiled-coil domain leads to high intrinsic domain concentrations and multivalency promoting self-association. Ca(2+)-dependent homophilic association of VECADCMP was detected in solid phase assays and cross-linking experiments. A striking analogy to homoassociation of type I ("classical") cadherins like E, N or P-cadherin was observed when interactions in VECADCMP and between these trimeric proteins were analyzed by electron microscopy. Ca(2+)-dependent ring-like and double ring-like arrangements suggest interactions between domains 1 and 2 of the ectodomains, which may be correlated with lateral and adhesive contacts in the adhesion process. Association to complexes composed of two VECADCMP molecules was also demonstrated by chemical cross-linking. No indication for an antiparallel association of VECAD ectodomains to hexameric complexes as proposed by Legrand et al. was found. Instead the data suggest that homoassociation of VE-cadherin follows the conserved mechanism of type I cadherins.

Animals↗

PubMed2002

Calcium-dependent homoassociation of E-cadherin by NMR spectroscopy: changes in mobility, conformation and mapping of contact regions.

Cadherins are calcium-dependent cell surface proteins that mediate homophilic cellular adhesion. The calcium-induced oligomerization of the N-terminal two domains of epithelial cadherin (ECAD12) was followed by NMR spectroscopy in solution over a large range of protein (10 microM-5 mM) and calcium (0-5 mM) concentrations. Several spectrally distinct states could be distinguished that correspond to a calcium-free monomeric form, a calcium-bound monomeric form, and to calcium-bound higher oligomeric forms. Chemical shift changes between these different states define calcium-binding residues as well as oligomerization contacts. Information about the relative orientation and mobility of the ECAD12 domains in the various states was obtained from weak alignment and 15N relaxation experiments. The data indicate that the calcium-free ECAD12 monomer adopts a flexible, kinked conformation that occludes the dimer interface observed in the ECAD12 crystal structure. In contrast, the calcium-bound monomer is already in a straight, non-flexible conformation where this interface is accessible. This mechanism provides a rational for the calcium-induced adhesiveness. Oligomerization induces chemical shift changes in an area of domain CAD1 that is centered at residue Trp-2. These shift changes extend to almost the entire surface of domain CAD1 at high (5 mM) protein concentrations. Smaller additional clusters of shift perturbations are observed around residue A80 in CAD1 and K160 in CAD2. According to weak alignment and relaxation data, the symmetry of a predominantly dimeric solution aggregate at 0.6 mM ECAD12 differs from the approximate C2-symmetry of the crystalline dimer.

Amino Acid Sequence↗

PubMed2002

Structure/function relationships in the minicollagen of Hydra nematocysts.

The minicollagens found in the inner layer of the Hydra nematocyst walls are the smallest collagens known with 12-16 Gly-X-Y repeats. Minicollagen-1, the best characterized member of this protein family so far, consists of a central collagen triple helix of 12 nm in length flanked at both ends by a polyproline stretch and a conserved cysteine-rich domain. The cysteine-rich tails are proposed to function in the assembly of soluble minicollagen trimers to high molecular structures by a switch of the disulfide linkage from intramolecular to intermolecular bonds. In this study, we investigate the trimeric nature of minicollagen-1 and its capacity to form disulfide-linked polymers in vitro. A fusion protein of minicollagen-1 with maltose-binding protein is secreted as a soluble trimer with only intrachain and no interchain disulfide bridges as confirmed by melting the collagen triple helix under reducing and non-reducing conditions. The conversion of minicollagen-1 trimers to monomers takes place between 40 and 55 degrees C with the melting point being approximately 45 degrees C. Oxidative reshuffling of the minicollagen-1 trimers leads to the formation of high molecular aggregates, which upon reduction show distinct polytrimeric states. Minicollagen trimers in isolated nematocyst capsules proved to be sensitive to SDS and were engaged in polymeric structures with additional cross-links that were resistant to reducing agent.

Animals↗

PubMed2002

Analysis of heterophilic and homophilic interactions of cadherins using the c-Jun/c-Fos dimerization domains.

Cadherin-mediated cell-cell adhesion is initiated by cis dimerization of cadherin ectodomains at the cell surface followed by an antiparallel trans interaction of dimers on opposing cells. To resolve open questions concerning the molecular details and specificity of cis and trans interactions, ectodomains of E- and P-cadherin were analyzed by chemical cross-linking and by electron microscopy. At the high intrinsic concentration created by artificial oligomerization the N-terminal cadherin (CAD)-domain of P-cadherin are forming ring-like cis dimers. At 2 mm Ca(2+)-associated rings involving two cis dimers indicate trans contacts in electron micrographs. cis and trans interactions were further analyzed by heterodimerization of the ectodomains of E-cadherin (ECAD) and P-cadherin (PCAD) through the leucine zipper domains of c-Jun and c-Fos. ECADJun/ECADFos dimers predominantly form ring-like cis dimers at 1 mm Ca(2+) and double-ringed trans contacts above 2 mm Ca(2+). The Ca(2+)-dependent tetrameric trans contacts of ECADJun/ECADFos dimers are also detectable after chemical cross-linking. Only cis contacts but no trans interactions are observed for heterodimers of ECADFos and the Trp-2 to Ala mutant ECADW2AJun arguing for a decisive role of Trp-2 in trans but not cis interaction. Neither cis nor trans interaction was found for heterodimers of ECADJun and PCADFos suggesting that specificity for homophilic interactions already exists at the level of cis dimerization.

Animals↗

PubMed2002

E-cadherin is a ligand for integrin alpha2beta1.

E-cadherin is a 120-kDa transmembrane glycoprotein expressed mainly on the surface of epithelial cells. The best characterised function of E-cadherin is homotypic, calcium-dependent cell-cell adhesion; however, the observation that E-cadherin is also capable of interacting with the alphaEbeta7 integrin to mediate leukocyte cell-cell adhesion [Nature 372 (1994) 190] suggests that it also participates in heterotypic interactions. To investigate the possibility that E-cadherin may interact with integrins expressed on non-leukocytic cells, cell adhesion and solid-phase receptor-ligand binding experiments were performed using a pentameric E-cadherin construct designed to detect low affinity, high avidity interactions. HT1080 human fibrosarcoma cells specifically adhered to pentameric E-cadherin, and this adhesion was inhibited by anti-functional monoclonal antibodies directed against the integrin alpha2 and beta1 subunits, but not by a series of antibodies recognising other subunits. This suggested that the E-cadherin receptor was alpha2beta1, a previously characterised collagen/laminin receptor. Pentameric E-cadherin, but not monomeric E-cadherin, specifically bound, in a divalent cation-dependent manner, to both purified alpha2beta1 and to a recombinant form of the A-domain of the alpha2 subunit, which has been shown to be a major ligand-binding site within this and other integrins. These findings demonstrate that E-cadherin can interact with alpha2beta1 and suggest that heterotypic interactions between E-cadherin and integrins may be more common than originally thought.

Cadherins↗