Emmanuelle Planus

Médecine/sciences (M/S), revue internationale dans le domaine de la recherche biologique, médicale et en santé

Integrin endocytosis is essential for many fundamental cellular processes. Whether and how the internalization impacts cellular mechanics remains elusive. Whereas previous studies reported the contribution of the integrin activator, talin, in force development, the involvement of inhibitors is less documented. We identified ICAP-1 as an integrin inhibitor involved in mechanotransduction by co-working with NME2 to control clathrin-mediated endocytosis of integrins at the edge of focal adhesions (FA). Loss of ICAP-1 enables β3-integrin-mediated force generation independently of β1 integrin. β3-integrin-mediated forces were associated with a decrease in β3 integrin dynamics stemming from their reduced diffusion within adhesion sites and slow turnover of FA. The decrease in β3 integrin dynamics correlated with a defect in integrin endocytosis. ICAP-1 acts as an adaptor for clathrin-dependent endocytosis of integrins. ICAP-1 controls integrin endocytosis by interacting with NME2, a key regulator of dynamin-dependent clathrin-coated pits fission. Control of clathrin-mediated integrin endocytosis by an inhibitor is an unprecedented mechanism to tune forces at FA.

Bordetella pertussis, the causative agent of whopping cough, produces an adenylate cyclase toxin (CyaA) that plays a key role in the host colonization by targeting innate immune cells which express CD11b/CD18, the cellular receptor of CyaA. CyaA is also able to invade non-phagocytic cells, via a unique entry pathway consisting in a direct translocation of its catalytic domain across the cytoplasmic membrane of the cells. Within the cells, CyaA is activated by calmodulin to produce high levels of cyclic adenosine monophosphate (cAMP) and alter cellular physiology. In this study, we explored the effects of CyaA toxin on the cellular and molecular structure remodeling of A549 alveolar epithelial cells. Using classical imaging techniques, biochemical and functional tests, as well as advanced cell mechanics method, we quantify the structural and functional consequences of the massive increase of intracellular cyclic AMP induced by the toxin: cell shape rounding associated to adhesion weakening process, actin structure remodeling for the cortical and dense components, increase in cytoskeleton stiffness, and inhibition of migration and repair. We also show that, at low concentrations (0.5 nM), CyaA could significantly impair the migration and wound healing capacities of the intoxicated alveolar epithelial cells. As such concentrations might be reached locally during B. pertussis infection, our results suggest that the CyaA, beyond its major role in disabling innate immune cells, might also contribute to the local alteration of the epithelial barrier of the respiratory tract, a hallmark of pertussis.

Bordetella pertussis , the causative agent of whopping cough, produces an adenylate cyclase toxin (CyaA) that plays a key role in the host colonization by targeting innate immune cells which express CD11b/CD18, the cellular receptor of CyaA. CyaA is also able to invade non-phagocytic cells, via a unique entry pathway consisting in a direct translocation of its catalytic domain across the cytoplasmic membrane of the cells. Within the cells, CyaA is activated by calmodulin to produce high levels of cyclic adenosine monophosphate (cAMP) and alter cellular physiology. In this study, we explored the effects of CyaA toxin on the cellular and molecular structure remodeling of A549 alveolar epithelial cells. Using classical imaging techniques, biochemical and functional tests, as well as advanced cell mechanics method, we quantify the structural and functional consequences of the massive increase of intracellular cyclic AMP induced by the toxin: cell shape rounding associated to adhesion weakening process, actin structure remodeling for the cortical and dense components, increase in cytoskeleton stiffness, and inhibition of migration and repair. We also show that, at the low concentrations that may be found in vivo during B. pertussis infection, CyaA impairs the migration and wound healing capacities of the intoxicated alveolar epithelial cells. Our results suggest that the CyaA, beyond its major role in disabling innate immune cells, might also contribute to the local alteration of the epithelial barrier of the respiratory tract, that is an hallmark of pertussis .

Endothelial integrity relies on a mechanical crosstalk between intercellular and cell-matrix interactions. This cross-talk is compromised in hemorrhagic vascular lesions of patients carrying loss-of-function mutations in CCM genes. RhoA/ROCK-dependent cytoskeletal remodeling is central to the disease as it causes unbalanced cell adhesion towards increased cell-extracellular matrix adhesions and destabilized cell-cell junctions. Our study reveals that CCM proteins directly orchestrate ROCK1 and ROCK2 complementary roles on the mechanics of the endothelium. CCM proteins act as a scaffold promoting ROCK2 interactions with VE-cadherin and limiting ROCK1 kinase activity. Loss of CCM1 produces excessive ROCK1-dependent actin stress fibers and destabilizes intercellular junctions. Silencing of ROCK1 but not ROCK2 restores the adhesive and mechanical homeostasis of CCM1/2-depleted endothelial monolayers and rescues cardiovascular defects of ccm1 mutant zebrafish embryos. Conversely, knocking down of Rock2 but not Rock1 in WT zebrafish embryos generates defects reminiscent of the ccm1 mutant phenotypes. Our study uncovers the role of the CCM complex in controlling ROCK1 and ROCK2 to preserve endothelial integrity and drive heart morphogenesis. Moreover, it identifies solely the ROCK1 isoform as therapeutic target for the CCM disease.

During total liquid ventilation, lung cells are exposed to perfluorocarbon (PFC) whose chemophysical properties highly differ from standard aqueous cell feeding medium (DMEM). We herein perform a systematic study of structural and mechanical properties of A549 alveolar epithelial cells in order to characterize their response to PFC exposure, using DMEM as control condition. Changes in F-actin structure, focal adhesion density and glycocalyx distribution are evaluated by confocal fluorescent microscopy. Changes in cell mechanics and adhesion are measured by multiscale magnetic twisting cytometry (MTC). Two different microrheological models (single Voigt and power law) are used to analyze the cell mechanics characterized by cytoskeleton (CSK) stiffness and characteristic relaxation times. Cell–matrix adhesion is analyzed using a stochastic multibond deadhesion model taking into account the non-reversible character of the cell response, allowing us to quantify the adhesion weakness and the number of associated bonds. The roles of F-actin structure and glycocalyx layer are evaluated by depolymerizing F-actin and degrading glycocalyx, respectively. Results show that PFC exposure consistently induces F-actin remodeling, CSK softening and adhesion weakening. These results demonstrate that PFC triggers an alveolar epithelial cell response herein evidenced by a decay in intracellular CSK tension, an adhesion weakening and a glycocalyx layer redistribution. These PFC-induced cell adjustments are consistent with the hypothesis that cells respond to a decrease in adhesion energy at cell surface. This adhesion energy can be even further reduced in the presence of surfactant adsorbed at the cell surface.

Background Information: The adenylate cyclase (CyaA) toxin is a major virulent factor of Bordetella pertussis, the causative agent of whooping cough. CyaA toxin is able to invade eukaryotic cells where it produces high levels of cyclic adenosine monophosphate (cAMP) affecting cellular physiology. Whether CyaA toxin can modulate cell matrix adhesion and mechanics of infected cells remains largely unknown.Results: In this study, we use a recently proposed multiple bond force spectroscopy (MFS) with an atomic force microscope to assess the early phase of cell adhesion (maximal detachment and local rupture forces) and cell rigidity (Young's modulus) in alveolar epithelial cells (A549) for toxin exposure 95%) at CyaA concentration of 0.5 nM, but a significant effect (≈81%) at 10 nM. MFS performed on A549 for three different concentrations (0.5, 5 and 10 nM) demonstrates that CyaA toxin significantly affects both cell adhesion (detachment forces are decreased) and cell mechanics (Young's modulus is increased). CyaA toxin (at 0.5 nM) assessed at three indentation/retraction speeds (2, 5 and 10 μm/s) significantly affects global detachment forces, local rupture events and Young modulus compared with control conditions, while an enzymatically inactive variant CyaAE5 has no effect. These results reveal the loading rate dependence of the multiple bonds newly formed between the cell and integrin‐specific coated probe as well as the individual bond kinetics which are only slightly affected by the patho‐physiological dose of CyaA toxin. Finally, theory of multiple bond force rupture enables us to deduce the bond number N which is reduced by a factor of 2 upon CyaA exposure (N ≈ 6 versus N ≈ 12 in control conditions).Conclusions: MFS measurements demonstrate that adhesion and mechanical properties of A549 are deeply affected by exposure to the CyaA toxin but not to an enzymatically inactive variant. This indicates that the alteration of cell mechanics triggered by CyaA is a consequence of the increase in intracellular cAMP in these target cells.Significance: These results suggest that mechanical and adhesion properties of the cells appear as pertinent markers of cytotoxicity of CyaA toxin.

Background Information Integrin‐mediated adhesion is a key process by which cells physically connect with their environment, and express sensitivity and adaptation through mechanotransduction. A critical step of cell adhesion is the formation of the first bonds which individually generate weak contacts (∼tens pN) but can sustain thousand times higher forces (∼tens nN) when associated. Results We propose an experimental validation by multiple bond force spectroscopy (MFS) of a stochastic model predicting adhesion reinforcement permitted by non‐cooperative, multiple bonds on which force is homogeneously distributed (called parallel bond configuration). To do so, spherical probes (diameter: 6.6 μm), specifically coated by RGD‐peptide to bind integrins, are used to statically indent and homogenously stretch the multiple bonds created for short contact times (2 s) between the bead and the surface of epithelial cells (A549). Using different separation speeds (v = 2, 5, 10 μm/s) and measuring cellular Young's modulus as well as the local stiffness preceding local rupture events, we obtain cell‐by‐cell the effective loading rates both at the global cell level and at the local level of individual constitutive bonds. Local rupture forces are in the range: , whereas global rupture (detachment) forces reach . Global and local rupture forces both exhibit linear dependencies with the effective loading rate, the slopes of these two linear relationships providing an estimate of the number of independent integrin bonds constituting the tested multiple bond structure (∼12). Conclusions The MFS method enables to validate the reinforcement of integrin‐mediated adhesion induced by the multiple bond configuration in which force is homogeneously distributed amongst parallel bonds. Local rupture events observed in the course of a spectroscopy manoeuver (MFS) lead to rupture force values considered in the literature as single‐integrin bonds. Significance Adhesion reinforcement permitted by the parallel multiple bond association is particularly challenging to verify for two reasons: first, it is difficult to control precisely the direction of forces experimentally, and second, because both global and local bond rupture forces depend on the effective loading rate applied to the bond. Here, we propose an integrin‐specific MFS method capable of detecting bond number and characterising bond configuration and its impact on adhesion strength.

There was an error published in J. Cell Sci. 130 , [626-636][1]. The name of the author Alexander Kyumurkov was incorrect in the original author list. The correct spelling is given above. The authors apologise to the readers for any confusion that this error might have caused. [1]: /lookup/

Cell migration is a complex process requiring density and rigidity sensing of the microenvironment to adapt cell migratory speed through focal adhesion and actin cytoskeleton regulation. ICAP-1 (also known as ITGB1BP1), a β1 integrin partner, is essential for ensuring integrin activation cycle and focal adhesion formation. We show that ICAP-1 is monoubiquitylated by Smurf1, preventing ICAP-1 binding to β1 integrin. The non-ubiquitylatable form of ICAP-1 modifies β1 integrin focal adhesion organization and interferes with fibronectin density sensing. ICAP-1 is also required for adapting cell migration in response to substrate stiffness in a β1-integrin-independent manner. ICAP-1 monoubiquitylation regulates rigidity sensing by increasing MRCKα (also known as CDC42BPA)-dependent cell contractility through myosin phosphorylation independently of substrate rigidity. We provide evidence that ICAP-1 monoubiquitylation helps in switching from ROCK2-mediated to MRCKα-mediated cell contractility. ICAP-1 monoubiquitylation serves as a molecular switch to coordinate extracellular matrix density and rigidity sensing thus acting as a crucial modulator of cell migration and mechanosensing.