Tether extrusion from red blood cells: integral proteins unbinding from cytoskeleton.
We investigate the mechanical strength of adhesion and the dynamics of detachment of the membrane from the cytoskeleton of red blood cells (RBCs). Using hydrodynamical flows, we extract membrane tethers from RBCs locally attached to the tip of a microneedle. We monitor their extrusion and retraction dynamics versus flow velocity (i.e., extrusion force) over successive extrusion-retraction cycles. Membrane tether extrusion is carried out on healthy RBCs and ATP-depleted or -inhibited RBCs. For healthy RBCs, extrusion is slow, constant in velocity, and reproducible through several extrusion-retraction cycles. For ATP-depleted or -inhibited cells, extrusion dynamics exhibit an aging phenomenon through extrusion-retraction cycles: because the extruded membrane is not able to retract properly onto the cell body, each subsequent extrusion exhibits a loss of resistance to tether growth over the tether length extruded at the previous cycle. In contrast, the additionally extruded tether length follows healthy dynamics. The extrusion velocity L depends on the extrusion force f according to a nonlinear fashion. We interpret this result with a model that includes the dynamical feature of membrane-cytoskeleton association. Tether extrusion leads to a radial membrane flow from the cell body toward the tether. In a distal permeation regime, the flow passes through the integral proteins bound to the cytoskeleton without affecting their binding dynamics. In a proximal sliding regime, where membrane radial velocity is higher, integral proteins can be torn out, leading to the sliding of the membrane over the cytoskeleton. Extrusion dynamics are governed by the more dissipative permeation regime: this leads to an increase of the membrane tension and a narrowing of the tether, which explains the power law behavior of L(f). Our main result is that ATP is necessary for the extruded membrane to retract onto the cell body. Under ATP depletion or inhibition conditions, the aging of the RBC after extrusion is interpreted as a perturbation of membrane-cytoskeleton linkage dynamics.
Borghi
N
N
Laboratoire Physico-Chimie Curie, Centre National de la Recherche Scientifique, UMR168, and Université Paris 6, Institut Curie, F-75231 Paris cedex 05, France. nicolas.borghi@curie.fr
Brochard-Wyart
F
F
D000255:Adenosine Triphosphate / Q000378:metabolism; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism*; D004907:Erythrocyte Deformability; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000502:physiology; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D011485:Protein Binding; D013049:Spectrin / Q000378:metabolism; D013314:Stress, Mechanical; D014783:Viscosity
D008565:Membrane Proteins; D013049:Spectrin; D000255:Adenosine Triphosphate