Nanoscale Dynamics of Actin Filaments in the Red Blood Cell Membrane Skeleton

2022 ◽  
Author(s):  
Roberta B. Nowak ◽  
Haleh Alimohamadi ◽  
Kersi Pestonjamasp ◽  
Padmini Rangamani ◽  
Velia M. Fowler
Keyword(s):  
Blood Cell ◽  
Single Molecule ◽  
Red Blood Cell ◽  
Actin Polymerization ◽  
Network Connectivity ◽  
Membrane Skeleton ◽  
Membrane Curvature ◽  
Actin Network ◽  
Node Distribution ◽  
Planar Network

Red blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes (∼37 nm length, 15-18 subunits) interconnected by long spectrin strands at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modeling of forces controlling RBC shape, membrane curvature and deformation, yet the nanoscale organization and dynamics of the F-actin nodes in situ is not well understood. We examined F-actin distribution and dynamics in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence (TIRF) and Zeiss Airyscan confocal microscopy demonstrate that F-actin is concentrated in multiple brightly stained F-actin foci ∼200-300 nm apart interspersed with dimmer F-actin staining regions. Single molecule STORM imaging of Alexa-647-phalloidin-labeled F-actin and computational analysis also indicates an irregular, non-random distribution of F-actin nodes. Treatment of RBCs with LatA and CytoD indicates F-actin foci distribution depends on actin polymerization, while live cell imaging reveals dynamic local motions of F-actin foci, with lateral movements, appearance and disappearance. Regulation of F-actin node distribution and dynamics via actin assembly/disassembly pathways and/or via local extension and retraction of spectrin strands may provide a new mechanism to control spectrin-F-actin network connectivity, RBC shape and membrane deformability.

scholarly journals Nanoscale organization of Actin Filaments in the Red Blood Cell Membrane Skeleton

2021 ◽  
Author(s):  
Roberta B. Nowak ◽  
Haleh Alimohamadi ◽  
Kersi Pestonjamasp ◽  
Padmini Rangamani ◽  
Velia M. Fowler
Keyword(s):  
Blood Cell ◽  
Single Molecule ◽  
Red Blood Cell ◽  
Synthetic Data ◽  
Membrane Skeleton ◽  
Membrane Curvature ◽  
Data Sets ◽  
Actin Network ◽  
Computational Cluster ◽  
Planar Network

AbstractRed blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes interconnected by long spectrin molecules at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modelling of forces controlling RBC shape, membrane curvature and deformation, yet the nanoscale organization of F-actin nodes in the network in situ is not understood. Here, we examined F-actin distribution in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence (TIRF) and Zeiss Airyscan confocal microscopy demonstrate that F-actin is concentrated in multiple brightly stained F-actin foci ∼200-300 nm apart interspersed with dimmer F-actin staining regions. Live cell imaging reveals dynamic lateral movements, appearance and disappearance of F-actin foci. Single molecule STORM imaging and computational cluster analysis of experimental and synthetic data sets indicate that individual filaments are non-randomly distributed, with the majority as multiple filaments, and the remainder sparsely distributed as single filaments. These data indicate that F-actin nodes are non-uniformly distributed in the spectrin-F-actin network and necessitate reconsideration of current models of forces accounting for RBC shape and membrane deformability, predicated upon uniform distribution of F-actin nodes and associated proteins across the micron-scale RBC membrane.

scholarly journals Non-uniform distribution of myosin-mediated forces governs red blood cell membrane curvature through tension modulation

2019 ◽  
Author(s):  
Haleh Alimohamadi ◽  
Alyson S. Smith ◽  
Roberta B. Nowak ◽  
Velia M. Fowler ◽  
Padmini Rangamani
Keyword(s):  
Blood Cell ◽  
Red Blood Cell ◽  
Contractile Activity ◽  
Cell Types ◽  
Membrane Skeleton ◽  
Membrane Curvature ◽  
Force Density ◽  
Additional Contribution ◽  
Rbc Membrane ◽  
Applied Forces

AbstractThe biconcave disk shape of the mammalian red blood cell (RBC) is unique to the RBC and is vital for its circulatory function. Due to the absence of a transcellular cytoskeleton, RBC shape is determined by the membrane skeleton, a network of actin filaments cross-linked by spectrin and attached to membrane proteins. While the physical properties of a uniformly distributed actin network interacting with the lipid bilayer membrane have been assumed to control RBC shape, recent experiments reveal that RBC biconcave shape also depends on the contractile activity of nonmuscle myosin IIA (NMIIA) motor proteins. Here, we use the classical Helfrich-Canham model for the RBC membrane to test the role of heterogeneous force distributions along the membrane and mimic the contractile activity of sparsely distributed NMIIA filaments. By incorporating this additional contribution to the Helfrich-Canham energy, we find that the RBC biconcave shape depends on the ratio of forces per unit volume in the dimple and rim regions of the RBC. Experimental measurements of NMIIA densities at the dimple and rim validate our prediction that (a) membrane forces must be non-uniform along the RBC membrane and (b) the force density must be larger in the dimple than the rim to produce the observed membrane curvatures. Furthermore, we predict that RBC membrane tension and the orientation of the applied forces play important roles in regulating this force-shape landscape. Our findings of heterogeneous force distributions on the plasma membrane for RBC shape maintenance may also have implications for shape maintenance in different cell types.

scholarly journals Direct Measurement of the Area Expansion and Shear Moduli of the Human Red Blood Cell Membrane Skeleton

2001 ◽  
Vol 81 (1) ◽  
pp. 43-56 ◽  
Author(s):  
Guillaume Lenormand ◽  
Sylvie Hénon ◽  
Alain Richert ◽  
Jacqueline Siméon ◽  
François Gallet
Keyword(s):  
Cell Membrane ◽  
Blood Cell ◽  
Direct Measurement ◽  
Red Blood Cell ◽  
Membrane Skeleton ◽  
Shear Moduli ◽  
Red Blood Cell Membrane ◽  
Area Expansion

scholarly journals Contribution of the band 3-ankyrin interaction to erythrocyte membrane mechanical stability

Blood ◽  
1991 ◽  
Vol 77 (7) ◽  
pp. 1581-1586 ◽  
Author(s):  
PS Low ◽  
BM Willardson ◽  
N Mohandas ◽  
M Rossi ◽  
S Shohet
Keyword(s):  
Blood Cell ◽  
Red Blood Cell ◽  
Mechanical Stability ◽  
Membrane Integrity ◽  
Membrane Skeleton ◽  
Band 3 ◽  
Cell Membrane Integrity ◽  
The Face ◽  
Cell Stability

Abstract In an effort to evaluate the role of the band 3-ankyrin linkage in maintenance of red blood cell membrane integrity, solution conditions were sought that would selectively dissociate the band 3-ankyrin linkage, leaving other membrane skeletal interactions intact. For this purpose erythrocytes were equilibrated overnight in nutrient-containing buffers at a range of elevated pHs and then examined for changes in mechanical stability and membrane skeletal composition. Band 3 was found to be released from interaction with the membrane skeleton over a pH range (8.4 to 9.5) that was observed to dissociate the band 3- ankyrin interaction in vitro. In contrast, all other membrane skeletal associations appeared to remain intact up to pH 9.3, after which they were also seen to dissociate. Whereas hemolysis of mechanically unstressed cells did not begin until approximately pH 9.3, where the membrane skeletons began to disintegrate, enhanced fragmentation of shear stressed membranes was seen to begin near pH 8, where band 3 dissociation was first observed. Furthermore, the shear-induced fragmentation rate was found to reach a maximum at pH 9.4, ie, where band 3 dissociation was essentially complete. Based on these correlations, we hypothesize that the band 3-ankyrin linkage of the membrane skeleton to the lipid bilayer is essential for red blood cell stability in the face of mechanical distortion but not for cellular integrity in the absence of mechanical stress.

Comparative Profiling of Red Blood Cells from the Beta-Adducin Knockout Mouse Model of Hemolytic Anemia Using Label Free Differential Proteomics

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3852-3852
Author(s):  
Jason M Wooden ◽  
Greg L Finney ◽  
Michael J Maccoss ◽  
Luanne L. Peters ◽  
Diana M. Gilligan
Keyword(s):  
Blood Cell ◽  
Hemolytic Anemia ◽  
Red Blood Cell ◽  
Membrane Skeleton ◽  
Actin Binding ◽  
Comparative Approach ◽  
Label Free ◽  
Uncharacterized Protein ◽  
Differential Proteomics ◽  
Knock Out

Abstract Inherited hemolytic anemia (spherocytosis or elliptocytosis) is one of the most common inherited diseases. While mild to severe inherited hemolytic anemias can arise from defects in the red blood cell (RBC) membrane skeleton, fundamental questions remain unanswered surrounding the clinical variability of known red blood cell mutations. To identify candidate proteins involved in hemolytic anemia pathophysiology, we utilized a label-free comparative approach to detect differences in RBCs from normal and beta-adducin (ADD2KO) knock-out mice. For each genotype, whole blood was taken from independent biological replicates and RBCs were purified using cellulose acetate chromatography. The isolated RBCs were lysed to generate RBC ghosts whose protein complements were digested with trypsin. For each biological replicate, three replicate runs utilizing 0.5 μg digested protein were performed via microcapillary liquid chromatography coupled with tandem mass spectrometry. Using the recently developed software package CRAWDAD, we detected 7 proteins that were decreased and 31 proteins with a greater abundance in the beta-adducin knock-out RBC ghosts. Differences were detected for previously known membrane skeleton components, including the predicted absence of beta-adducin and decrease in alpha-adducin. The actin binding protein capping protein (actin filament) muscle Z-line alpha 1 was increased along with ankyrin 1, spectrin alpha, tropomyosin, and glycophorin C. Interestingly, differences were also detected for the protein oxidative stress-responsive 1 and the uncharacterized protein C10orf58 which contains a thioredoxin motif. While the protein differences span a broad range of cellular processes, some of the proteins are attractive candidates for modifiers of disease severity. This label free approach is the first demonstration of comprehensively analyzing wild type versus kockout mice that circumvents the need for metabolic or chemical labeling with isotopes. This new approach adds a valuable tool to the list of standard assays for analysis of RBCs in cases of hereditary hemolytic anemia.

Spectrin Properties and the Elasticity of the Red Blood Cell Membrane Skeleton

Biorheology ◽  
1997 ◽  
Vol 34 (4-5) ◽  
pp. 327-348 ◽  
Author(s):  
J.C. Hansen ◽  
R. Skalak ◽  
S. Chien ◽  
A. Hoger
Keyword(s):  
Cell Membrane ◽  
Blood Cell ◽  
Red Blood Cell ◽  
Membrane Skeleton ◽  
Red Blood Cell Membrane
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