Grégory Giannone

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.

Lamellipodial Actin Mechanically Links Myosin Activity with Adhesion-Site Formation

Cell motility proceeds by cycles of edge protrusion, adhesion, and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. We find that myosin II pulls the rear of the lamellipodial actin network, causing upward bending, edge retraction, and initiation of new adhesion sites. The network then separates from the edge and condenses over the myosin. Protrusion resumes as lamellipodial actin regenerates from the front and extends rearward until it reaches newly assembled myosin, initiating the next cycle. Upward bending, observed by evanescence and electron microscopy, results in ruffle formation when adhesion strength is low. Correlative fluorescence and electron microscopy shows that the regenerating lamellipodium forms a cohesive, separable layer of actin above the lamellum. Thus, actin polymerization periodically builds a mechanical link, the lamellipodium, connecting myosin motors with the initiation of adhesion sites, suggesting that the major functions driving motility are coordinated by a biomechanical process.

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 fibronectins 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 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 αvβ3 and deletion of β3 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.

The cancer glycocalyx mechanically primes integrin-mediated growth and survival

Malignancy is associated with altered expression of glycans and glycoproteins that contribute to the cellular glycocalyx. We constructed a glycoprotein expression signature, which revealed that metastatic tumours upregulate expression of bulky glycoproteins. A computational model predicted that these glycoproteins would influence transmembrane receptor spatial organization and function. We tested this prediction by investigating whether bulky glycoproteins in the glycocalyx promote a tumour phenotype in human cells by increasing integrin adhesion and signalling. Our data revealed that a bulky glycocalyx facilitates integrin clustering by funnelling active integrins into adhesions and altering integrin state by applying tension to matrix-bound integrins, independent of actomyosin contractility. Expression of large tumour-associated glycoproteins in non-transformed mammary cells promoted focal adhesion assembly and facilitated integrin-dependent growth factor signalling to support cell growth and survival. Clinical studies revealed that large glycoproteins are abundantly expressed on circulating tumour cells from patients with advanced disease. Thus, a bulky glycocalyx is a feature of tumour cells that could foster metastasis by mechanically enhancing cell-surface receptor function.

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.

Dynamic Superresolution Imaging of Endogenous Proteins on Living Cells at Ultra-High Density

Versatile superresolution imaging methods, able to give dynamic information of endogenous molecules at high density, are still lacking in biological science. Here, superresolved images and diffusion maps of membrane proteins are obtained on living cells. The method consists of recording thousands of single-molecule trajectories that appear sequentially on a cell surface upon continuously labeling molecules of interest. It allows studying any molecules that can be labeled with fluorescent ligands including endogenous membrane proteins on living cells. This approach, named universal PAINT (uPAINT), generalizes the previously developed point-accumulation-for-imaging-in-nanoscale-topography (PAINT) method for dynamic imaging of arbitrary membrane biomolecules. We show here that the unprecedented large statistics obtained by uPAINT on single cells reveal local diffusion properties of specific proteins, either in distinct membrane compartments of adherent cells or in neuronal synapses.

Super-Resolution Imaging Reveals That AMPA Receptors Inside Synapses Are Dynamically Organized in Nanodomains Regulated by PSD95

The spatiotemporal organization of neurotransmitter receptors in postsynaptic membranes is a fundamental determinant of synaptic transmission and information processing by the brain. Using four independent super-resolution light imaging methods and EM of genetically tagged and endogenous receptors, we show that, in rat hippocampal neurons, AMPARs are often highly concentrated inside synapses into a few clusters of ∼70 nm that contain ∼20 receptors. AMPARs are stabilized reversibly in these nanodomains and diffuse freely outside them. Nanodomains are dynamic in their shape and position within synapses and can form or disappear within minutes, although they are mostly stable for up to 1 h. AMPAR nanodomains are often, but not systematically, colocalized with clusters of the scaffold protein PSD95, which are generally of larger size than AMPAR nanoclusters. PSD95 expression level regulates AMPAR nanodomain size and compactness in parallel to miniature EPSC amplitude. Monte Carlo simulations further indicate the impact of AMPAR concentration in clusters on the efficacy of synaptic transmission. The observation that AMPARs are highly concentrated in nanodomains, instead of diffusively distributed in the PSD as generally thought, has important consequences on our understanding of excitatory neurotransmission. Furthermore, our results indicate that glutamatergic synaptic transmission is controlled by the nanometer-scale regulation of the size of these highly concentrated nanodomains.

Nanometer Analysis of Cell Spreading on Matrix-Coated Surfaces Reveals Two Distinct Cell States and STEPs

When mouse embryonic fibroblasts in suspension contact a matrix-coated surface, they rapidly adhere and spread. Using total internal reflection fluorescence microscopy of dye-loaded fibroblasts to quantify cell-substrate contact, we found that increasing the surface matrix density resulted in faster spreading initiation whereas lamellipodial dynamics during spreading were unaltered. After spreading initiation, most cells spread in an anisotropic manner through stochastic, transient extension periods (STEPs) with approximately 30 STEPs over 10 min to reach an area of 1300 micro m(2) +/- 300 micro m(2). A second mode of spreading, increased in serum-deprived cells, lacked STEPs and spread in a rapid, isotropic manner for 1-4 min. This isotropic mode was characterized by a high rate of area increase, 340 micro m(2)/min with 78% of the cell edge extending. Anisotropic cells spread slower via STEPs, 126 micro m(2)/min with 34% of the edge extending. During the initial 2-4 min of fast, isotropic spreading, centripetal flow of actin was low (0.8 micro m/min) whereas in anisotropic cells it was high from early times (4.7 micro m/min). After initial isotropic spreading, rearward actin movement increased and isotropic cells displayed STEPs similar to anisotropic cells. Thus, the two cell states display dramatically different spreading whereas long-term motility is based on STEPs.

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.

Calcium Rises Locally Trigger Focal Adhesion Disassembly and Enhance Residency of Focal Adhesion Kinase at Focal Adhesions

Focal adhesion kinase (FAK) activity and Ca2+ 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 Ca2+ sensor fused to FAK or to a FAK-related non-kinase domain, to measure simultaneously local Ca2+ variations at FA sites and FA dynamics. Discrete subcellular Ca2+ oscillators initiate both propagating and abortive Ca2+ waves in migrating U87 astrocytoma cells. Ca2+-dependent FA disassembly occurs when the Ca2+ wave reaches individual FAs, indicating that local but not global Ca2+ 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 Ca2+ elevation. FAK-related non-kinase domain-Ycam had a faster, Ca2+-insensitive recovery half-time (11 s), which is consistent with the effect of Ca2+ 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 Ca2+ and FAK autophosphorylation was correlated to intracellular Ca2+ levels, we propose that local Ca2+ 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.