title: Dynamic actin filaments control the mechanical behavior of the human red blood cell membrane.
abstract: Short, uniform-length actin filaments function as structural nodes in the spectrin-actin membrane skeleton to optimize the biomechanical properties of red blood cells (RBCs). Despite the widespread assumption that RBC actin filaments are not dynamic (i.e., do not exchange subunits with G-actin in the cytosol), this assumption has never been rigorously tested. Here we show that a subpopulation of human RBC actin filaments is indeed dynamic, based on rhodamine-actin incorporation into filaments in resealed ghosts and fluorescence recovery after photobleaching (FRAP) analysis of actin filament mobility in intact RBCs (~25-30% of total filaments). Cytochalasin-D inhibition of barbed-end exchange reduces rhodamine-actin incorporation and partially attenuates FRAP recovery, indicating functional interaction between actin subunit turnover at the single-filament level and mobility at the membrane-skeleton level. Moreover, perturbation of RBC actin filament assembly/disassembly with latrunculin-A or jasplakinolide induces an approximately twofold increase or ~60% decrease, respectively, in soluble actin, resulting in altered membrane deformability, as determined by alterations in RBC transit time in a microfluidic channel assay, as well as by abnormalities in spontaneous membrane oscillations (flickering). These experiments identify a heretofore-unrecognized but functionally important subpopulation of RBC actin filaments, whose properties and architecture directly control the biomechanical properties of the RBC membrane.
authors.affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037.
authors.forename: David S
authors.initials: DS
authors.lastname: Gokhin
authors.affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037.
authors.forename: Roberta B
authors.initials: RB
authors.lastname: Nowak
authors.affiliation: Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02115.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Khoory
authors.affiliation: Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02115.
authors.forename: Alfonso de la
authors.initials: Ade L
authors.lastname: Piedra
authors.affiliation: Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02115.
authors.forename: Ionita C
authors.initials: IC
authors.lastname: Ghiran
authors.affiliation: Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037 velia@scripps.edu.
authors.forename: Velia M
authors.initials: VM
authors.lastname: Fowler
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D001696:Biomechanical Phenomena; D002462:Cell Membrane / Q000502:physiology*; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D009996:Osmotic Fragility; D055503:Protein Multimerization
chemical_list: D000199:Actins