Previously, we showed the human erythrocyte cytoplasmic endoskeleton composed of actin filaments and intermediate filaments [

Terasawa K, et al. Arch. Histol. Cytol. 2006;69:326–340
]. The present study aims to determine whether endoskeletal actin filaments contribute to regulate the shape recovery of human erythrocytes. We prepared two types of ghosts: prefixed ghosts which contain endoskeletal network and non-fixed usual ghosts which lack endoskeletal network. To prepare prefixed ghosts, fresh erythrocytes were mildly fixed with 2 % paraformaldehyde and 0.05 % glutaraldehyde in phosphate buffered saline and haemolysed by mild hypotonic Tris buffer with 2 mM Mg-ATP. These prefixed ghosts contained an endoskeletal network which could change their shape quickly by Mg-ATP. In transmission electron microscopy, all the spherical and crenated prefixed ghosts possessed the endoskeletal network as an electron dense matter. Gold particles immuno-labeled for beta actin dispersed over the whole endoskeleton in both crenated and discoid prefixed ghosts. Moreover, cytochalasin B, a blocking agent of fast growing end of long actin polymer, inhibited crenated to discoid shape transition in prefixed ghosts. In non-fixed ghosts, addition of cytochalasin B did not affect recovery to the original shape. Thus, these results suggest that shape transition induced by Mg-ATP is driven through the action of the endoskeletal actin network.

Topics:
buffers, cytochalasin b, cytoplasm, glutaral, gold, hypotonicity, microfilaments, microscopes, transmission electron, phosphates, red cell ghost

Disclosures: No relevant conflicts of interest to declare.

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2008, The American Society of Hematology
2008
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