Hematopoietic Protein-1 Regulates the Actin Membrane Skeleton and Membrane Stability in Murine Erythrocytes

Abstract

Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and β- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1−/− erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1−/− erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A), which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes.

Erythrocyte Stiffness during Morphological Remodeling Induced by Carbon Ion Radiation

“…This assembly configuration not only plays an important role in the maintenance of plasma membrane shape, integrity, and cytoskeletal structure, influence influencing deformability or resulting in damage [90] , [94] , [95] and finally determining biological behavior and functional aspects [96] – [99] ; it can also produce a rapid response and break the tetramers [100] . A dysfunctional underlying membrane skeleton results in the erythrocyte membrane being partially devoid of a spectrin-actin network, no longer existing as a ghost, and causing disruption of the unique arrangement of spectrin, F-actin, protein 4.1 and ankyrin, with direct and indirect connections to the membrane bilayer with its immersed integral proteins [101] ( Figure 11 ). Consequently, the distribution of membrane tension is regulated by signaling molecules through the activated complex signaling transduction pathway to mediate the biochemical reaction between target proteins or molecules [102] – [104] and triggering exquisite changes in cell shape or membrane damage [105] .…”

Loss of Hem1 disrupts macrophage function and impacts on migration, phagocytosis and integrin-mediated adhesion

“…We therefore investigated integrin function in platelets isolated from WT and Hem1 KO mice at approximately 10-12 weeks of age. In blood smear preparations, red blood cells were confirmed to display a more irregular morphology (61). Moreover, significant deviations of the shape of platelets became evident ( Fig.…”

Anatomy of the red cell membrane skeleton: unanswered questions

“…Mice that lack both Rac1 and Rac2 GTPases, 38 or lack Hem-1, a member of the Wiskott-Aldrich syndrome verprolin-homologous (WAVE) protein complex that regulates F-actin polymerization, 39 develop hemolytic anemias with misshaped red cells and clumped or irregular skeletons. This finding suggests that Rac GTPases and Hem-1 help organize or regulate the red cell membrane skeleton, acting through pathways that stimulate actin polymerization in many cell types.…”