Biology subjects

Helfer, E.

Publications and source records attributed to Helfer, E..

3 recordsLinked to original sources

bioRxiv2023

Physical mechanisms of red blood cell splenic filtration

The splenic interendothelial slits fulfill the essential function of continuously filtering red blood cells (RBCs) from the bloodstream to eliminate abnormal and aged cells. To date, the process by which 8 {micro}m RBCs pass through 0.3 {micro}m-wide slits remains enigmatic. Does the slit caliber increase during RBC passage as sometimes suggested? Here, we elucidated the mechanisms that govern the RBC retention or passage dynamics in slits by combining multiscale modeling, live imaging, and microfluidic experiments on an original device with sub-micron wide physiologically calibrated slits. We observed that healthy RBCs pass through 0.28 {micro}m-wide rigid slits at 37{degrees}C. To achieve this feat, they must meet two requirements. Geometrically, their surface area-to-volume ratio must be compatible with a shape in two tether-connected equal spheres. Mechanically, the cells with a low surface area-to-volume ratio (28 % of RBCs in a 0.4 {micro}m-wide slit) must locally unfold their spectrin cytoskeleton inside the slit. In contrast, activation of the mechanosensitive PIEZO1 channel is not required. The RBC transit time through the slits follows a -1 and -3 power law with in-slit pressure drop and slip width, respectively. This law is similar to that of a Newtonian fluid in a 2D Poiseuille flow, showing that the dynamics of RBCs is controlled by their cytoplasmic viscosity. Altogether, our results show that filtration through submicron-wide slits is possible without further slit opening. Furthermore, our approach addresses the critical need for in-vitro evaluation of splenic clearance of diseased or engineered RBCs for transfusion and drug delivery. Significance StatementSplenic filtration of red blood cells through narrow interendothelial slits remains poorly understood despite its physiological significance as experiments and imaging of red cells passing through the slits are lacking. Here, we coupled live imaging, biomimetic submicron-fluidics, and multiscale modeling to quantify passage conditions. Remarkably, healthy 8-{micro}m cells can pass through 0.28-{micro}m slits at body temperature. This event is conditioned to cells being able to deform into two tether-connected equal spheres and, in limiting cases, to unfold their spectrin cytoskeleton. We showed that cells behave like a Newtonian fluid and that their dynamics is controlled by the inner fluid viscosity. We thus propose an in-vitro and in-silico approach to quantify splenic clearance of diseased cells and cells engineered for transfusion and drug delivery.

biophysics↗

bioRxiv2022

Talin and kindlin cooperate to control the density of integrin clusters.

Focal adhesions are important mechanosensitive structures, composed of transmembrane integrins, linking the extracellular matrix to the actomyosin cytoskeleton, via cytoplasmic proteins. Cellular adhesion to the extracellular matrix depends on the activation of integrins by intracellular mechanisms. Talin and kindlin are major activators of integrins that are recruited to the inner membrane and bind to {beta}-integrin cytoplasmic tails. Many studies showed the importance of integrin activation and clustering and how the organization of extracellular ligands guides the nanoscale organization of adhesion complexes. However, the roles of talin and kindlin in this process are poorly understood. To determine the contribution of talin, kindlin, lipids and actomyosin in integrin clustering, we performed experiments using a biomimetic in vitro system, made of Giant Unilamellar Vesicles, containing transmembrane integrins, on which purified talin, kindlin, and actomyosin assemble. Here we first show that talin and kindlin individually have the ability to cluster integrins. When added together, talin and kindlin synergize to induce the formation of larger integrin clusters containing the three proteins. Comparison of protein density in the talin-integrin, kindlin-integrin, and talin-kindlin-integrin clusters reveals that kindlin increases talin and integrin density, whereas talin does not affect kindlin and integrin density. Finally, kindlin significantly enhances the segregation of talin-integrin clusters induced by actomyosin contractility, suggesting that it increases the coupling of these clusters to the actin cytoskeleton. Our study unambiguously demonstrates how kindlin and talin cooperate to induce integrin clustering, which is a major parameter for cell adhesion.

biochemistry↗

bioRxiv2022

Enhanced cell viscosity as a marker of premature senescence induced by lamin A/C alterations

Lamin A/C is a well-established key contributor to nuclear stiffness and its role in nucleus mechanical properties has been extensively studied. However, its impact on whole cell mechanics has been poorly addressed, even less so in terms of measurable physical parameters. In the present study, microfluidic experiments combined with theoretical analyses were performed to provide a quantitative estimation of the whole cell mechanical properties. This allowed the characterization of mechanical cell changes induced by lamin A/C alterations resulting from Atazanavir treatment or lipodystrophy-associated LMNA R482W pathogenic variant. Results unveil an increase in the long-time viscosity as a signature of cells affected by lamin A/C alterations. In addition, they show that the whole cell response to mechanical stress is driven not only by the nucleus but also by the nucleo-cytoskeleton links and the microtubule network. This enhanced cell viscosity assessed by our microfluidic device could represent a useful diagnosis marker for lamin-related diseases.

biophysics↗