Biomedical subjects

Grégory Giannone

Publications and source records attributed to Grégory Giannone.

9 recordsLinked to original sources

PubMed2006

Lateral membrane waves constitute a universal dynamic pattern of motile cells.

We have monitored active movements of the cell circumference on specifically coated substrates for a variety of cells including mouse embryonic fibroblasts and T cells, as well as wing disk cells from fruit flies. Despite having different functions and being from multiple phyla, these cell types share a common spatiotemporal pattern in their normal membrane velocity; we show that protrusion and retraction events are organized in lateral waves along the cell membrane. These wave patterns indicate both spatial and temporal long-range periodic correlations of the actomyosin gel.

Actomyosin↗

PubMed2006

Substrate rigidity and force define form through tyrosine phosphatase and kinase pathways.

Cell forces define cell morphology, alterations in which are caused by tyrosine kinase and phosphatase mutations, which implies a causal linkage. Recent studies have shown that phosphotyrosine signaling is involved in force sensing for cells on flat surfaces. Early force-dependent activation of Src family kinases by phosphatases or cytoskeleton stretch leads to the activation of downstream signaling. In addition, force generation by cells depends on a feedback mechanism between matrix rigidity or force generation and myosin contractility. Components of the force-sensing pathway are linked to the integrin-cytoskeleton complex at sites of force application and serve as scaffolds for signaling processes. Thus, early events in force detection are mechanically induced cytoskeletal changes that result in biochemical signals to mechanoresponsive pathways that then regulate cell form.

Adherens Junctions↗

PubMed2004

Dynamic phase transitions in cell spreading.

We monitored isotropic spreading of mouse embryonic fibroblasts on fibronectin-coated substrates. Cell adhesion area versus time was measured via total internal reflection fluorescence microscopy. Spreading proceeds in well-defined phases. We found a power-law area growth with distinct exponents in three sequential phases, which we denote as basal, continuous, and contractile spreading. High resolution differential interference contrast microscopy was used to characterize local membrane dynamics at the spreading front. Fourier power spectra of membrane velocity reveal the sudden development of periodic membrane retractions at the transition from continuous to contractile spreading. We propose that the classification of cell spreading into phases with distinct functional characteristics and protein activity serves as a paradigm for a general program of a phase classification of cellular phenotype.

Animals↗

PubMed2004

Calcium rises locally trigger focal adhesion disassembly and enhance residency of focal adhesion kinase at focal adhesions.

Focal adhesion kinase (FAK) activity and Ca(2+) signaling led to a turnover of focal adhesions (FAs) required for cell spreading and migration. We used yellow Cameleon-2 (Ycam), a fluorescent protein-based Ca(2+) sensor fused to FAK or to a FAK-related non-kinase domain, to measure simultaneously local Ca(2+) variations at FA sites and FA dynamics. Discrete subcellular Ca(2+) oscillators initiate both propagating and abortive Ca(2+) waves in migrating U87 astrocytoma cells. Ca(2+)-dependent FA disassembly occurs when the Ca(2+) wave reaches individual FAs, indicating that local but not global Ca(2+) increases trigger FA disassembly. An unexpectedly rapid flux of FAK between cytosolic and FA compartments was revealed by fluorescence recovery after photobleaching studies. The FAK-Ycam recovery half-time (17 s) at FAs was slowed (to 29 s) by Ca(2+) elevation. FAK-related non-kinase domain-Ycam had a faster, Ca(2+)-insensitive recovery half-time (11 s), which is consistent with the effect of Ca(2+) on FAK-Ycam dynamics not being due to a general modification of the dynamics of FA components. Because FAK association at FAs was prolonged by Ca(2+) and FAK autophosphorylation was correlated to intracellular Ca(2+) levels, we propose that local Ca(2+) elevations increase the residency of FAK at FAs, possibly by means of tyrosine phosphorylation of FAK, thereby leading to increased activation of its effectors involved in FA disassembly.

Blotting, Western↗

PubMed2004

Periodic lamellipodial contractions correlate with rearward actin waves.

Cellular lamellipodia bind to the matrix and probe its rigidity through forces generated by rearward F-actin transport. Cells respond to matrix rigidity by moving toward more rigid matrices using an unknown mechanism. In spreading and migrating cells we find local periodic contractions of lamellipodia that depend on matrix rigidity, fibronectin binding and myosin light chain kinase (MLCK). These contractions leave periodic rows of matrix bound beta3-integrin and paxillin while generating waves of rearward moving actin bound alpha-actinin and MLCK. The period between contractions corresponds to the time for F-actin to move across the lamellipodia. Shortening lamellipodial width by activating cofilin decreased this period proportionally. Increasing lamellipodial width by Rac signaling activation increased this period. We propose that an actin bound, contraction-activated signaling complex is transported locally from the tip to the base of the lamellipodium, activating the next contraction/extension cycle.

Actin Depolymerizing Factors↗

PubMed2003

Talin1 is critical for force-dependent reinforcement of initial integrin-cytoskeleton bonds but not tyrosine kinase activation.

Cells rapidly transduce forces exerted on extracellular matrix contacts into tyrosine kinase activation and recruitment of cytoskeletal proteins to reinforce integrin-cytoskeleton connections and initiate adhesion site formation. The relationship between these two processes has not been defined, particularly at the submicrometer level. Using talin1-deficient cells, it appears that talin1 is critical for building early mechanical linkages. Deletion of talin1 blocked laser tweezers, force-dependent reinforcement of submicrometer fibronectin-coated beads and early formation of adhesion sites in response to force, even though Src family kinases, focal adhesion kinase, and spreading were activated normally. Recruitment of vinculin and paxillin to sites of force application also required talin1. FilaminA had a secondary role in strengthening fibronectin-integrin-cytoskeleton connections and no role in stretch-dependent adhesion site assembly. Thus, force-dependent activation of tyrosine kinases is independent of early force-dependent structural changes that require talin1 as part of a critical scaffold.

Cell Adhesion↗

PubMed2003

Two-piconewton slip bond between fibronectin and the cytoskeleton depends on talin.

Mechanical forces on matrix-integrin-cytoskeleton linkages are crucial for cell viability, morphology and organ function. The production of force depends on the molecular connections from extracellular-matrix-integrin complexes to the cytoskeleton. The minimal matrix complex causing integrin-cytoskeleton connections is a trimer of fibronectin's integrin-binding domain FNIII7-10 (ref. 4). Here we report a specific, molecular slip bond that was broken repeatedly by a force of 2 pN at the cellular loading rate of 60 nm x s(-1); this occurred with single trimer beads but not with monomer. Talin1, which binds to both integrins and actin filaments in vitro, is required for the 2-pN slip bond and rapid cytoskeleton binding. Further, inhibition of fibronectin binding to alpha(v)beta3 and deletion of beta3 markedly decreases the 2-pN force peak. We suggest that talin1 initially forms a molecular slip bond between closely packed fibronectin-integrin complexes and the actin cytoskeleton, which can apply a low level of force to fibronectin until many bonds form or a signal is received to activate a force response.

3T3 Cells↗

PubMed2003

Delphinidin, an active compound of red wine, inhibits endothelial cell apoptosis via nitric oxide pathway and regulation of calcium homeostasis.

1. Epidemiological studies have suggested that moderate consumption of natural dietary polyphenolic compounds might reduce the risk of cardiovascular disease and also protect against cancer. The present study investigates the effects of delphinidin, an anthocyanin present in red wine, on bovine aortic endothelial cells apoptosis. 2. Based on flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling analysis and detection of mitochondrial cytochrome c release, we show that delphinidin (10(-2) g l(-1)) alone had no effect either on necrosis or on apoptosis, but it significantly reduced apoptosis elicited by actinomycin D (1 micro g ml(-1), 24 h) and 7beta-hydroxycholesterol (10 micro g ml(-1), 18 h). 3. The protective effect of delphinidin was abolished by inhibitors of nitric oxide-synthase (NOS) (L-NA, 100 micro M and SMT, 100 micro M), guanylyl cyclase (ODQ, 100 micro M) and MAP kinase (PD98059, 30 micro M). 4. Western blot analysis and protein detection by confocal microscopy demonstrate that the antiapoptotic effect of delphinidin was associated with an increased endothelial NOS expression mediated by a MAP kinase pathway. 5. Finally, delphinidin alone had no effect on cytosolic-free calcium ([Ca(2+)](i)), but normalized the changes in [Ca(2+)](i) produced by actinomycin D towards the control values, suggesting that the antiapoptotic effect of delphinidin is associated with the maintenance of [Ca(2+)](i) in the physiological range. 6. All of the observed effects of delphinidin may preserve endothelium integrity, the alteration of which lead to pathologies including cardiovascular diseases, such as atherosclerosis, and is often associated with cancers. In conclusion, the protective effect of delphinidin against endothelial cell apoptosis contributes to understand the potential benefits of a consumption rich in polyphenols.

Animals↗

PubMed2002

FAK-mediated inhibition of vascular smooth muscle cell migration by the tetraspanin CD9.

Migration of vascular smooth muscle cells (SMC) towards the intima is a key event in vascular proliferative diseases. We investigated a potential role for the tetraspanin CD9 in this process in a wound migration assay. Aortic SMC from CD9 knock-out mice had higher migration rates and the presumably stimulatory anti-CD9 antibody ALMA-1 inhibited migration of human SMC. The signaling pathways responsible for this inhibitory effect were investigated. In migrating CD9-/- SMC, stress fiber formation was decreased and focal adhesions were smaller and more diffusely distributed, consistent with an inhibition of integrin clustering. In migrating mouse SMC expressing CD9, focal adhesion kinase (FAK) tyrosine phosphorylation was doubled. No differences in intracellular calcium signaling were observed between CD9+/+ and CD9-/- SMC during migration. We suggest that CD9 in hibits SMC migration by a stimulation of both stress fiber formation and integrin clustering, leading to a stimulation of FAK phosphorylation.

Actins↗