The long-range goal of this research is to determine how actin filament dynamics is regulated in the spectrin-actin lattice of the membrane skeleton to create the red blood cell (RBC) with its specialized membrane, unique biconcave shape and deformability properties. Mutations in membrane skeleton components produce human hereditary hemolytic anemias with fragile RBC membranes, due to defective membrane biogenesis and/or weakened interactions in the membrane skeleton, thereby leading to reduced stability in the circulation. The spectrin-actin lattice consists of spectrin tetramers attached to short actin filaments at junctional complexes (JCs), forming the strands and vertices of a quasi-hexagonal lattice. The short RBC actin filaments are capped by tropomodulin-1 (Tmod1) at their pointed ends and 1/2-adducin at their barbed ends, while tropomyosins (TMs) span along the filament length, binding to Tmod1 at the pointed filament end. Despite the importance of RBC actin filaments as structural nodes in the spectrin latice and JC anchorage sites for membrane proteins, the role of actin monomer-polymer dynamics in membrane skeleton structure and stability, or biogenesis during erythroid terminal differentiation is not understood. This proposal focuses on Tmods, a unique family of TM-regulated, actin filament pointed end capping proteins that control actin dynamics and stability, cell morphology and physiology. Absence of Tmod1 in mouse RBCs leads to a mild compensated sphero-elliptocytic anemia with osmotically fragile RBCs, and a disrupted membrane skeleton with mis- regulated actin filament lengths. In contrast, absence of Tmod3 leads to fetal anemia with embryonic lethality at E14.5-15.5 due to impaired definitive erythropoiesis. Unlike Tmod1, which is a strong actin filament pointed end capper, Tmod3 sequesters actin monomers but is a weak filament capper. We hypothesize that Tmod1 regulates actin dynamics to maintain filament lengths and membrane skeleton structure in mature RBCs, while Tmod3 regulates actin monomer pols to control membrane skeleton assembly during erythroid terminal differentiation. The Specific Aims are: 1) To dissect the molecular and structural basis for Tmod1 and Tmod3 differential regulation of actin dynamics in vitro; 2) To define actin dynamics in normal human RBCs, and in wild-type, Tmod1-null and 1/2-adducin-null mouse RBCs, and to screen as yet undiagnosed human hemolytic anemia patients for defects in TMODs; 3) To determine the cellular basis for the Tmod3-null mouse fetal liver anemia, and test a requirement for Tmod3 in erythroblasts using bone marrow reconstitution and a RBC- specific conditional knockout for Tmod3; 4) To investigate Tmod1 and Tmod3 functions in membrane skeleton assembly during erythroid terminal differentiation from cultured human CD34+ cells isolated from peripheral blood. Our studies of actin dynamics will define a novel control point for membrane skeleton assembly and stability in human and mouse RBCs and their precursors, providing insights into RBC membrane biogenesis and survival in normal contexts and in human hemolytic anemias.