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bioRxiv · 10.1101/2021.03.07.434292

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

Nowak, R. B.·Alimohamadi, H.·Pestonjamasp, K.·Rangamani, P.·Fowler, V. M.
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Abstract

Red 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.

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Nowak, R. B., Alimohamadi, H., Pestonjamasp, K., Rangamani, P., Fowler, V. M.. 2021-03-08. Nanoscale organization of Actin Filaments in the Red Blood Cell Membrane Skeleton. https://doi.org/10.1101/2021.03.07.434292

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