Force-Velocity Measurements of a Few Growing Actin Filaments

The authors propose a new mechanism for actin-based force generation based on results using chains of actin-grafted magnetic colloids

Actin Polymerization Controls the Organization of WASH Domains at the Surface of Endosomes

Sorting of cargoes in endosomes occurs through their selective enrichment into sorting platforms, where transport intermediates are generated. The WASH complex, which directly binds to lipids, activates the Arp2/3 complex and hence actin polymerization onto such sorting platforms. Here, we analyzed the role of actin polymerization in the physiology of endosomal domains containing WASH using quantitative image analysis. Actin depolymerization is known to enlarge endosomes. Using a novel colocalization method that is insensitive to the heterogeneity of size and shape of endosomes, we further show that preventing the generation of branched actin networks induces endosomal accumulation of the WASH complex. Moreover, we found that actin depolymerization induces a dramatic decrease in the recovery of endosomal WASH after photobleaching. This result suggests a built-in turnover, where the actin network, i.e. the product of the WASH complex, contributes to the dynamic exchange of the WASH complex by promoting its detachment from endosomes. Our experiments also provide evidence for a role of actin polymerization in the lateral compartmentalization of endosomes: several WASH domains exist at the surface of enlarged endosomes, however, the WASH domains coalesce upon actin depolymerization or Arp2/3 depletion. Branched actin networks are thus involved in the regulation of the size of WASH domains. The potential role of this regulation in membrane scission are discussed

Molecular and Mechanobiological Pathways Related to the Physiopathology of FPLD2

International audienceLaminopathies are rare and heterogeneous diseases affecting one to almost all tissues, as in Progeria, and sharing certain features such as metabolic disorders and a predisposition to atherosclerotic cardiovascular diseases. These two features are the main characteristics of the adipose tissue-specific laminopathy called familial partial lipodystrophy type 2 (FPLD2). The only gene that is involved in FPLD2 physiopathology is the LMNA gene, with at least 20 mutations that are considered pathogenic. LMNA encodes the type V intermediate filament lamin A/C, which is incorporated into the lamina meshwork lining the inner membrane of the nuclear envelope. Lamin A/C is involved in the regulation of cellular mechanical properties through the control of nuclear rigidity and deformability, gene modulation and chromatin organization. While recent studies have described new potential signaling pathways dependent on lamin A/C and associated with FPLD2 physiopathology, the whole picture of how the syndrome develops remains unknown. In this review, we summarize the signaling pathways involving lamin A/C that are associated with the progression of FPLD2. We also explore the links between alterations of the cellular mechanical properties and FPLD2 physiopathology. Finally, we introduce potential tools based on the exploration of cellular mechanical properties that could be redirected for FPLD2 diagnosis

Mechanical adaptation of monocytes in model lung capillary networks

International audienceProper circulation of white blood cells (WBCs) in the pulmonary vascular bed is crucial for an effective immune response. In this branched vascular network, WBCs have to strongly deform to pass through the narrowest capillaries and bifurcations. Although it is known that this process depends on the cell mechanical properties, it is still poorly understood due to the lack of a comprehensive model of cell mechanics and of physiologically relevant experiments. Here, using an in-house microfluidic device mimicking the pulmonary capillary bed we show that the dynamics of THP1 monocytes evolves along successive capillary-like channels, from a non-stationary slow motion with hops to a fast and smooth efficient one. We used actin cytoskeleton drugs to modify the traffic dynamics. This led us to propose a simple mechanical model that shows that a very-finely tuned cortical tension combined with a high cell viscosity govern the fast transit through the network while preserving cell integrity. We finally highlight that the cortical tension controls the steady-state cell velocity via the viscous friction between the cell and the channel walls

Force Generation of Curved Actin Gels Characterized by Combined AFM-Epifluorescence Measurements

International audiencePolymerization of actin into branched filaments is the driving force behind active migration of eukaryotic cells and motility of intracellular organelles. The site-directed assembly of a polarized branched array forms an expanding gel that generates the force that pushes the membrane. Here, we use atomic force microscopy to understand the relation between actin poly-merization and the produced force. Functionalized spherical colloidal probes of varying size and curvature are attached to the atomic force microscopy cantilever and initiate the formation of a polarized actin gel in a solution mimicking the in vivo context. The gel growth is recorded by epifluorescence microscopy both against the cantilever and in the perpendicular (lateral) noncon-strained direction. In this configuration, the gel growth stops simultaneously in both directions at the stall force, which corresponds to a pressure of 0.15 nN/µm2. The results show that the growth of the gel is limited laterally, in the absence of external force, by internal mechanical stresses resulting from a combination of the curved geometry and the molecular mechanism of site-directed assembly of a cohesive branched filament array

Dynamics of Individual Red Blood Cells Under Shear Flow: A Way to Discriminate Deformability Alterations

International audienceIn this work, we compared the dynamics of motion in a linear shear flow of individual red blood cells (RBCs) from healthy and pathological donors (Sickle Cell Disease (SCD) or Sickle Cell-β-thalassemia) and of low and high densities, in a suspending medium of higher viscosity. In these conditions, at lower shear rates, biconcave discocyte-shaped RBCs present an unsteady flip-flopping motion, where the cell axis of symmetry rotates in the shear plane, rocking to and fro between an orbital angle ±ϕ observed when the cell is on its edge. We show that the evolution of ϕ depends solely on RBC density for healthy RBCs, with denser RBCs displaying lower ϕ values than the lighter ones. Typically, at a shear stress of 0.08 Pa, ϕ has values of 82 and 72° for RBCs with average densities of 1.097 and 1.115, respectively. Surprisingly, we show that SCD RBCs display the same ϕ-evolution as healthy RBCs of same density, showing that the flip-flopping behavior is unaffected by the SCD pathology. When the shear stress is increased further (above 0.1 Pa), healthy RBCs start going through a transition to a fluid-like motion, called tank-treading, where the RBC has a quasi-constant orientation relatively to the flow and the membrane rotates around the center of mass of the cell. This transition occurs at higher shear stresses (above 0.2 Pa) for denser cells. This shift toward higher stresses is even more remarkable in the case of SCD RBCs, showing that the transition to the tank-treading regime is highly dependent on the SCD pathology. Indeed, at a shear stress of 0.2 Pa, for RBCs with a density of 1.097, 100% of healthy RBCs have transited to the tank-treading regime vs. less than 50% SCD RBCs. We correlate the observed differences in dynamics to the alterations of RBC mechanical properties with regard to density and SCD pathology reported in the literature. Our results suggest that it might be possible to develop simple non-invasive assays for diagnosis purpose based on the RBC motion in shear flow and relying on this millifluidic approach

Fabrication of nano and microstructures for biophysical applications

International audienceThe goal of this poster presentation is to give an overview of nanofabrication techniques commonly used for realization of nanostructures in our micro and nanofabrication facilities. We focus on all types of process equipment available in PLANETE, the CINaM clean room, which is a part of a regional technological platform CT PACA.First, we have applied a technique of microbead assisted lithography for fabrication of large area protein nanocluster arrays [1]. These biofunctionalized arrays were used for manipulating and imaging of living cells in order to study their adhesion and migration on nanostructured surfaces [2]. This method of surface patterning can also be applied for more complex studies of cell-cell interactions, where not only protein films but also lipid bilayer dot arrays need to be nanostructured [1]. Second, we have developed technological steps needed for microfabrication of field-effect-transistors using lipid layers as grid dielectrics [3,4]. The sensors based of these transistors exploit unprecedented mechanical and dielectric properties of ultrathin supported lipid monolayers [4].Chemical engineering of lipids allowed us to fabricate selective sensors for biologically important metal ions in solutions in large concentration range [3].Finally, we will show the fabrication steps necessary for fabrication of high aspect ratio molds of microfluidics. Such types of microcirculation devices have been used for comparative studies of the dynamics of healthy and non-healthy red blood cells by mimicking spleen slits [5].[1] E. Benard, F. Pi, I. Ozerov, A. Charrier, K. Sengupta, “Ligand Nano-cluster Array in Supported Lipid Bilayer”, J. Vis. Exp. 122, e55060 (2017)[2] F. Pi, P. Dillard, R. Alameddine, E. Benard, A. Wahl, I. Ozerov, A. Charrier, L. Limozin, K. Sengupta, “Size-Tunable Organic Nanodot Arrays: a Versatile Platform for Manipulating and Imaging Cells”, Nano Letters 15, 5178-5184 (2015)[3] T.D. Nguyen, A. Labed, R. El Zein, S. Lavandier, F. Bedu, I. Ozerov, H. Dallaporta, J.-M. Raimundo, A. M. Charrier, “A field effect transistor biosensor with a γ-pyrone derivative engineered lipid-sensing layer for ultrasensitive Fe3+ ion detection with low pH interference”, Biosensors and Bioelectronics 54, 571-577 (2014)[4] A. Kenaan, R. El Zein, V. Kilinc, S. Lamant, J.-M. Raimundo, A. M. Charrier, “Ultrathin Supported Lipid Monolayer with Unprecedented Mechanical and Dielectric Properties”, Adv. Func. Mat. 10.1002/adfm.201801024 (2018)[5] P. Gambhire, S. Atwell, C. Iss, F. Bedu, I. Ozerov, C. Badens, E. Helfer, A. Viallat and A. Charrier, “High aspect ratio sub-micron channels using wet etching: Application to the dynamics of red blood cell transiting through biomimetic splenic slits”, Small 13, 1700967 (2017

Fabrication of nano and microstructures for biophysical applications

International audienceThe goal of this poster presentation is to give an overview of nanofabrication techniques commonly used for realization of nanostructures in our micro and nanofabrication facilities. We focus on all types of process equipment available in PLANETE, the CINaM clean room, which is a part of a regional technological platform CT PACA.First, we have applied a technique of microbead assisted lithography for fabrication of large area protein nanocluster arrays [1]. These biofunctionalized arrays were used for manipulating and imaging of living cells in order to study their adhesion and migration on nanostructured surfaces [2]. This method of surface patterning can also be applied for more complex studies of cell-cell interactions, where not only protein films but also lipid bilayer dot arrays need to be nanostructured [1]. Second, we have developed technological steps needed for microfabrication of field-effect-transistors using lipid layers as grid dielectrics [3,4]. The sensors based of these transistors exploit unprecedented mechanical and dielectric properties of ultrathin supported lipid monolayers [4].Chemical engineering of lipids allowed us to fabricate selective sensors for biologically important metal ions in solutions in large concentration range [3].Finally, we will show the fabrication steps necessary for fabrication of high aspect ratio molds of microfluidics. Such types of microcirculation devices have been used for comparative studies of the dynamics of healthy and non-healthy red blood cells by mimicking spleen slits [5].[1] E. Benard, F. Pi, I. Ozerov, A. Charrier, K. Sengupta, “Ligand Nano-cluster Array in Supported Lipid Bilayer”, J. Vis. Exp. 122, e55060 (2017)[2] F. Pi, P. Dillard, R. Alameddine, E. Benard, A. Wahl, I. Ozerov, A. Charrier, L. Limozin, K. Sengupta, “Size-Tunable Organic Nanodot Arrays: a Versatile Platform for Manipulating and Imaging Cells”, Nano Letters 15, 5178-5184 (2015)[3] T.D. Nguyen, A. Labed, R. El Zein, S. Lavandier, F. Bedu, I. Ozerov, H. Dallaporta, J.-M. Raimundo, A. M. Charrier, “A field effect transistor biosensor with a γ-pyrone derivative engineered lipid-sensing layer for ultrasensitive Fe3+ ion detection with low pH interference”, Biosensors and Bioelectronics 54, 571-577 (2014)[4] A. Kenaan, R. El Zein, V. Kilinc, S. Lamant, J.-M. Raimundo, A. M. Charrier, “Ultrathin Supported Lipid Monolayer with Unprecedented Mechanical and Dielectric Properties”, Adv. Func. Mat. 10.1002/adfm.201801024 (2018)[5] P. Gambhire, S. Atwell, C. Iss, F. Bedu, I. Ozerov, C. Badens, E. Helfer, A. Viallat and A. Charrier, “High aspect ratio sub-micron channels using wet etching: Application to the dynamics of red blood cell transiting through biomimetic splenic slits”, Small 13, 1700967 (2017

Fabrication of nano and microstructures for biophysical applications

International audienceThe goal of this poster presentation is to give an overview of nanofabrication techniques commonly used for realization of nanostructures in our micro and nanofabrication facilities. We focus on all types of process equipment available in PLANETE, the CINaM clean room, which is a part of a regional technological platform CT PACA.First, we have applied a technique of microbead assisted lithography for fabrication of large area protein nanocluster arrays [1]. These biofunctionalized arrays were used for manipulating and imaging of living cells in order to study their adhesion and migration on nanostructured surfaces [2]. This method of surface patterning can also be applied for more complex studies of cell-cell interactions, where not only protein films but also lipid bilayer dot arrays need to be nanostructured [1]. Second, we have developed technological steps needed for microfabrication of field-effect-transistors using lipid layers as grid dielectrics [3,4]. The sensors based of these transistors exploit unprecedented mechanical and dielectric properties of ultrathin supported lipid monolayers [4].Chemical engineering of lipids allowed us to fabricate selective sensors for biologically important metal ions in solutions in large concentration range [3].Finally, we will show the fabrication steps necessary for fabrication of high aspect ratio molds of microfluidics. Such types of microcirculation devices have been used for comparative studies of the dynamics of healthy and non-healthy red blood cells by mimicking spleen slits [5].[1] E. Benard, F. Pi, I. Ozerov, A. Charrier, K. Sengupta, “Ligand Nano-cluster Array in Supported Lipid Bilayer”, J. Vis. Exp. 122, e55060 (2017)[2] F. Pi, P. Dillard, R. Alameddine, E. Benard, A. Wahl, I. Ozerov, A. Charrier, L. Limozin, K. Sengupta, “Size-Tunable Organic Nanodot Arrays: a Versatile Platform for Manipulating and Imaging Cells”, Nano Letters 15, 5178-5184 (2015)[3] T.D. Nguyen, A. Labed, R. El Zein, S. Lavandier, F. Bedu, I. Ozerov, H. Dallaporta, J.-M. Raimundo, A. M. Charrier, “A field effect transistor biosensor with a γ-pyrone derivative engineered lipid-sensing layer for ultrasensitive Fe3+ ion detection with low pH interference”, Biosensors and Bioelectronics 54, 571-577 (2014)[4] A. Kenaan, R. El Zein, V. Kilinc, S. Lamant, J.-M. Raimundo, A. M. Charrier, “Ultrathin Supported Lipid Monolayer with Unprecedented Mechanical and Dielectric Properties”, Adv. Func. Mat. 10.1002/adfm.201801024 (2018)[5] P. Gambhire, S. Atwell, C. Iss, F. Bedu, I. Ozerov, C. Badens, E. Helfer, A. Viallat and A. Charrier, “High aspect ratio sub-micron channels using wet etching: Application to the dynamics of red blood cell transiting through biomimetic splenic slits”, Small 13, 1700967 (2017