Collagen-Based Biomimetic Systems to Study the Biophysical Tumour Microenvironment

The extracellular matrix (ECM) is a pericellular network of proteins and other molecules that provides mechanical support to organs and tissues. ECM biophysical properties such as topography, elasticity and porosity strongly influence cell proliferation, differentiation and migration. The cell’s perception of the biophysical microenvironment (mechanosensing) leads to altered gene expression or contractility status (mechanotransduction). Mechanosensing and mechanotransduction have profound implications in both tissue homeostasis and cancer. Many solid tumours are surrounded by a dense and aberrant ECM that disturbs normal cell functions and makes certain areas of the tumour inaccessible to therapeutic drugs. Understanding the cell-ECM interplay may therefore lead to novel and more effective therapies. Controllable and reproducible cell culturing systems mimicking the ECM enable detailed investigation of mechanosensing and mechanotransduction pathways. Here, we discuss ECM biomimetic systems. Mainly focusing on collagen, we compare and contrast structural and molecular complexity as well as biophysical properties of simple 2D substrates, 3D fibrillar collagen gels, cell-derived matrices and complex decellularized organs. Finally, we emphasize how the integration of advanced methodologies and computational methods with collagen-based biomimetics will improve the design of novel therapies aimed at targeting the biophysical and mechanical features of the tumour ECM to increase therapy efficacy

Nanoclustering as a dominant feature of plasma membrane organization

Early studies have revealed that some mammalian plasma membrane proteins exist in small nanoclusters. The advent of super-resolution microscopy has corroborated and extended this picture, and led to the suggestion that many, if not most, membrane proteins are clustered at the plasma membrane at nanoscale lengths. In this Commentary, we present selected examples of glycosylphosphatidyl-anchored proteins, Ras family members and several immune receptors that provide evidence for nanoclustering. We advocate the view that nanoclustering is an important part of the hierarchical organization of proteins in the plasma membrane. According to this emerging picture, nanoclusters can be organized on the mesoscale to form microdomains that are capable of supporting cell adhesion, pathogen binding and immune cell-cell recognition amongst other functions. Yet, a number of outstanding issues concerning nanoclusters remain open, including the details of their molecular composition, biogenesis, size, stability, function and regulation. Notions about these details are put forth and suggestions are made about nanocluster function and why this general feature of protein nanoclustering appears to be so prevalent.Postprint (published version

Using Magnetic Probes to Study Receptor Clustering in Live Cells

During pathogen recognition T-Cell Receptors form microclusters which are believed to be the central signalling units. These structures could hold the secret behind the exceptional sensitivity of T-Cells in distinguishing single triggering ‘agonist’ peptides against a background of thousands. We have developed a biophysical approach based on magnetic tweezers that allows us to study the players involved in these receptor clusters and their dynamics. We use antibody functionalized magnetic beads to target CD3, a subunit of the TCR Complex to induce TCR clustering. Using magnetic tweezers, we move the beads along the cell membrane and simultaneously measure trafficking of co-receptors and proteins involved in the complex using confocal fluorescence microscopy and fluorescence recovery after photobleaching (FRAP). We study co-receptor CD6, which is considered a co-stimulator for cell activation during cluster formation. Our findings suggest that while CD6 is not physically associated with TCR complex, it gets recruited into the TCR clusters. There it is partially immobilized and moves along as clusters are displaced. The diffusion coefficient of CD6 is higher in bead-stimulated cells, whereas CD6 outside clusters diffuse faster than those within clusters. We are also downscaling this method to induce formation of receptor nanoclusters, in order to explore the effects of physical receptor oligomerization on the activity of TCR and Epidermal Growth Factor Receptors

MT1-MMP directs force-producing proteolytic contacts that drive tumor cell invasion

International audienceUnraveling the mechanisms that govern the formation and function of invadopodia is essential towards the prevention of cancer spread. Here, we characterize the ultrastructural organization, dynamics and mechanical properties of collagenotytic invadopodia forming at the interface between breast cancer cells and a physiologic fibrillary type I collagen matrix. Our study highlights an uncovered role for MT1-MMP in directing invadopodia assembly independent of its proteolytic activity. Electron microscopy analysis reveals a polymerized Arp2/3 actin network at the concave side of the curved invadopodia in association with the collagen fibers. Actin polymerization is shown to produce pushing forces that repel the confining matrix fibers, and requires MT1-MMP matrix-degradative activity to widen the matrix pores and generate the invasive pathway. A theoretical model is proposed whereby pushing forces result from actin assembly and frictional forces in the actin meshwork due to the curved geometry of the matrix fibers that counterbalance resisting forces by the collagen fibers

Changes in membrane sphingolipid composition modulate dynamics and adhesion of integrin nanoclusters

Sphingolipids are essential constituents of the plasma membrane (PM) and play an important role in signal transduction by modulating clustering and dynamics of membrane receptors. Changes in lipid composition are therefore likely to influence receptor organisation and function, but how this precisely occurs is difficult to address given the intricacy of the PM lipid-network. Here, we combined biochemical assays and single molecule dynamic approaches to demonstrate that the local lipid environment regulates adhesion of integrin receptors by impacting on their lateral mobility. Induction of sphingomyelinase (SMase) activity reduced sphingomyelin (SM) levels by conversion to ceramide (Cer), resulting in impaired integrin adhesion and reduced integrin mobility. Dual-colour imaging of cortical actin in combination with single molecule tracking of integrins showed that this reduced mobility results from increased coupling to the actin cytoskeleton brought about by Cer formation. As such, our data emphasizes a critical role for the PM local lipid composition in regulating the lateral mobility of integrins and their ability to dynamically increase receptor density for efficient ligand binding in the process of cell adhesion

Response of serum proteome in patients undergoing infrarenal aortic aneurysm repair.

BACKGROUND: Postoperative organ dysfunction in conventional surgery for abdominal aortic aneurysm (AAA) is associated with a complex inflammatory reaction, with activation of coagulation and fibrinolysis. A prospective,observational study was performed to define the complex plasma proteomic changes after AAA repair and to identify factor(s) that may affect myocardial function in uncomplicated procedures. METHODS: Ten patients undergoing infrarenal AAA repair were investigated. Eight subjects subjected to major abdominal surgery served as controls. Hemodynamic changes were continuously monitored by using the pressure recording analytical method technique. The time course of plasma proteins was investigated after induction of anesthesia and at different times after surgery (6 h, 12 h, 24 h, 36 h) by using two-dimensional difference gel electrophoresis, matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and Western blot. The effects of plasma on the functional properties of isolated rat ventricular myocytes were also investigated. RESULTS: In AAA patients alone, 18 spots were found to change more than two-fold in expression level, spot identification revealing an increased thrombin generation 6 h after surgery. At the same time cardiac cycle efficiency significantly reduced versus baseline (-0.5 +/- 0.9 vs. 0.18 +/- 0.3 in AAA patients, P < 0.01; 0.4 +/- 0.1 vs. 0.2 +/- 0.3 in control surgery, not significant; P < 0.01 group x time interaction at ANOVA). Plasma obtained 6 h after AAA surgery dose-dependently inhibited contractile function of control rat myocytes (percent shortening fell by 51% with 10% of AAA plasma and was abolished with 20% of AAA plasma, P < 0.001 for both). The inhibitory response was abolished by thrombin antagonism. CONCLUSIONS: These findings show for the first time the possible role of thrombin generation within the complex activation of inflammatory response in causing hemodynamic instability in the early postoperative period after AAA surger

Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

LFA-1 is a leukocyte specific β2 integrin that plays a major role in regulating adhesion and migration of different immune cells. Recent data suggest that LFA-1 on mature dendritic cells (mDCs) may function as a chemokine-inducible anchor during homing of DCs through the afferent lymphatics into the lymph nodes, by transiently switching its molecular conformational state. However, the role of LFA-1 mobility in this process is not yet known, despite that the importance of lateral organization and dynamics for LFA-1-mediated adhesion regulation is broadly recognized. Using single particle tracking approaches we here show that LFA-1 exhibits higher mobility on resting mDCs compared to monocytes. Lymphoid chemokine CCL21 stimulation of the LFA-1 high affinity state on mDCs, led to a significant reduction of mobility and an increase on the fraction of stationary receptors, consistent with re-activation of the receptor. Addition of soluble monomeric ICAM-1 in the presence of CCL21 did not alter the diffusion profile of LFA-1 while soluble ICAM-1 nano-aggregates in the presence of CCL21 further reduced LFA-1 mobility and readily bound to the receptor. Overall, our results emphasize the importance of LFA-1 lateral mobility across the membrane on the regulation of integrin activation and its function as adhesion receptor. Importantly, our data show that chemokines alone are not sufficient to trigger the high affinity state of the integrin based on the strict definition that affinity refers to the adhesion capacity of a single receptor to its ligand in solution. Instead our data indicate that nanoclustering of the receptor, induced by multi-ligand binding, is required to maintain stable cell adhesion once LFA-1 high affinity state is transiently triggered by inside-out signals.Peer ReviewedPostprint (published version

Actomyosin-dependent dynamic spatial patterns of cytoskeletal components drive mesoscale podosome organization

Podosomes are cytoskeletal structures crucial for cell protrusion and matrix remodelling in osteoclasts, activated endothelial cells, macrophages and dendritic cells. In these cells, hundreds of podosomes are spatially organized in diversely shaped clusters. Although we and others established individual podosomes as micron-sized mechanosensing protrusive units, the exact scope and spatiotemporal organization of podosome clustering remain elusive. By integrating a newly developed extension of Spatiotemporal Image Correlation Spectroscopy with novel image analysis, we demonstrate that F-actin, vinculin and talin exhibit directional and correlated flow patterns throughout podosome clusters. Pattern formation and magnitude depend on the cluster actomyosin machinery. Indeed, nanoscopy reveals myosin IIA-decorated actin filaments interconnecting multiple proximal podosomes. Extending well-beyond podosome nearest neighbours, the actomyosin-dependent dynamic spatial patterns reveal a previously unappreciated mesoscale connectivity throughout the podosome clusters. This directional transport and continuous redistribution of podosome components provides a mechanistic explanation of how podosome clusters function as coordinated mechanosensory area

Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells

The C-type lectin dendritic cell (DC)–specific intercellular adhesion molecule grabbing non-integrin (DC-SIGN; CD209) facilitates binding and internalization of several viruses, including HIV-1, on DCs, but the underlying mechanism for being such an efficient phagocytic pathogen-recognition receptor is poorly understood. By high resolution electron microscopy, we demonstrate a direct relation between DC-SIGN function as viral receptor and its microlocalization on the plasma membrane. During development of human monocyte-derived DCs, DC-SIGN becomes organized in well-defined microdomains, with an average diameter of 200 nm. Biochemical experiments and confocal microscopy indicate that DC-SIGN microdomains reside within lipid rafts. Finally, we show that the organization of DC-SIGN in microdomains on the plasma membrane is important for binding and internalization of virus particles, suggesting that these multimolecular assemblies of DC-SIGN act as a docking site for pathogens like HIV-1 to invade the host