Red Blood Cells

Spectrin, actin filaments and accessory proteins form a highly cross-linked network, termed the membrane skeleton, which underlies the inner surface of plasma membranes of metazoan cells. The membrane skeleton is linked to cell adhesion receptors, ion pumps and channels and organizes sub-domains of the plasma membrane via the network’s long-range connectivity. In addition, the membrane skeleton also influences cell shapes and mechanical stability. The spectrin-based membrane skeleton was first identified in red blood cells (RBCs), which have long provided major new insights into plasma membrane structure and function. In human hereditary hemolytic anemias, defects or deficiencies in assembly or interactions of spectrin and other membrane skeleton components lead to abnormally shaped and fragile RBCs due to impaired biogenesis and/or survival in the circulation. Indeed, hemolytic anemia due to membrane skeleton defects is one of the most common inherited diseases in Northern Europeans (1:2500 to 1:5000).

 

The prototypical membrane skeleton of RBCs is organized as a regular, quasi-hexagonal network in which the vertices are short actin filaments and the strands are long spectrin tetramers. A particularly striking feature is the precise and uniform lengths of the actin filaments, which are all 33-37 nm long, consisting of 15-18 subunits. These short actin filaments are extraordinarily stable, persisting for the lifetime of the RBCs (120 days in humans and 40 days in mice). Tropomodulin (Tmod) caps their pointed ends, adducin caps their barbed ends, and the actin-stabilizing protein, tropomyosin (TM), binds along their lengths. The uniform lengths and stability of RBC actin filaments contrast with the heterogeneity and rapid turnover of actin filaments in the cytoskeletons of motile and proliferating cells. The unusual properties of RBC actin filaments likely require specialized assembly and regulation to confer the unique combination of flexibility and stability on the RBC membrane skeleton. A major goal of our research is to test our long-standing hypothesis that Tmod and TM regulation of actin filaments is critical for membrane skeleton assembly and integrity, thereby controlling RBC biogenesis, survival and function in vivo. We are investigating this hypothesis in RBCs from mouse models with targeted deletions in Tmods and TMs, using biochemistry, confocal fluorescence microscopy, cryo-electron microscopy, biophysics, and physiological approaches.

 

Key questions are:

 

•  What is the role of actin filament length regulation by Tmod and TM in the organization and functional properties of the RBC membrane skeleton?

 

•  What is the 3D molecular structure of the short actin filaments and their accessory proteins in the membrane skeleton network in situ?

 

•  How are Tmods, TM, and actin filaments assembled into the membrane skeleton during RBC differentiation?

 

•  Is RBC actin filament and membrane skeleton organization similar in the large primitive RBCs of early embryos and the small definitive RBCs produced in mid-gestation and in adult animals?

 

•  How does absence of Tmods or TMs affect functional properties of RBCs, including their shape, deformability, mechanical stability and osmotic fragility?

 

•  Can principles of actin regulation discovered in RBCs be translated to the spectrin-actin networks of the membrane skeletons in other cell types, such as epithelial or striated muscle cells?