The biconcave shape, mechanical strength and remarkable deformability of the human erythrocyte are determined by a filament network underlying the cytoplasmic surface of the membrane that consists of many short actin filaments cross-linked by long spectrin molecules into a strikingly regular hexagonal lattice (the membrane skeleton). The broad, long term objectives of this research are to determine how the spectrin-actin vertices of the membrane skeleton are assembled and how their organization is functionally related to the shape and membrane mechanical properties of normal and abnormal erythrocytes. In this proposal, it is planned to focus on two proteins, tropomyosin and tropomodulin, that are candidates to stabilize and restrict the length of the short actin filaments. Tropomyosin is a rod-like protein that binds along the length of actin filaments and tropomodulin is a new, isoform-specific tropomyosin-binding protein that binds to the end of tropomyosin and blocks tropomyosin head- to-tail association along actin filaments. We will test the hypothesis that tropomyosin and tropomodulin influence membrane deformability and/or mechanical stability by stabilizing and limIting the lengths of the short actin filaments in the membrane skeleton. The presence of tropomyosin and tropomodulin isoforms in muscle, lens and other tissues indicates that this work is likely to have important implications for the regulation of actin filament length and stability in nonerythroid cells. The uniform lengths for erythrocyte actin filaments and the absence of additional complicating effects of actin assembly and disassembly such as occur in other nonmuscle cells makes the human erythrocyte an ideal system to test this hypothesis. Specifically, we will (i) investigate the role of tropomodulin, dematin and other membrane skeleton components in regulating the association of tropomyosin with actin filaments using binding studies with purified proteins and selectively extracted membranes, (2) map the tropomyosin-binding domain on erythrocyte tropomodulin by cDNA deletion analysis and biochemical approaches, (3) Identify the tropomodulin-binding domain on tropomyosin by characterizing tropomodulin interactions with recombinant tropomyosin isoforms and chimeras with alternative N and C terminal sequences, and (4) introduce tropomyosin isoforms and/or recombinant, truncated tropomodulins into resealed ghosts and evaluate their effects on (i) membrane deformability and/or membrane stability using ektacytometry (ii) actin filament length using cytochalasin binding to quantitate numbers of filament ends, and (ill) actin filament stability as measured by susceptibility to depolymerizatlon by deoxyribonuclease I. (5) Finally, we will identify mutants in tropomyosin or tropomodulin function by screening abnormal erythrocytes from patients with hereditary hemolytic anemias (hereditary elliptocytosis, hereditary spherocytosis) for deficiencies or defects in tropomyosin and tropomodulin.
| Smith, Alyson S; Nowak, Roberta B; Zhou, Sitong et al. (2018) Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability. Proc Natl Acad Sci U S A 115:E4377-E4385 |
| Fath, Thomas; Fischer, Robert S; Dehmelt, Leif et al. (2011) Tropomodulins are negative regulators of neurite outgrowth. Eur J Cell Biol 90:291-300 |
| Weber, Kari L; Fischer, Robert S; Fowler, Velia M (2007) Tmod3 regulates polarized epithelial cell morphology. J Cell Sci 120:3625-32 |
| Fischer, Robert S; Yarmola, Elena G; Weber, Kari L et al. (2006) Tropomodulin 3 binds to actin monomers. J Biol Chem 281:36454-65 |
| Gupton, Stephanie L; Anderson, Karen L; Kole, Thomas P et al. (2005) Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin. J Cell Biol 168:619-31 |
| Ehler, Elisabeth; Fowler, Velia M; Perriard, Jean-Claude (2004) Myofibrillogenesis in the developing chicken heart: role of actin isoforms and of the pointed end actin capping protein tropomodulin during thin filament assembly. Dev Dyn 229:745-55 |
| Fowler, Velia M; Greenfield, Norma J; Moyer, Jeannette (2003) Tropomodulin contains two actin filament pointed end-capping domains. J Biol Chem 278:40000-9 |
| Fritz-Six, Kimberly L; Cox, Patrick R; Fischer, Robert S et al. (2003) Aberrant myofibril assembly in tropomodulin1 null mice leads to aborted heart development and embryonic lethality. J Cell Biol 163:1033-44 |
| Fischer, Robert S; Fowler, Velia M (2003) Tropomodulins: life at the slow end. Trends Cell Biol 13:593-601 |
| Fischer, Robert S; Fritz-Six, Kimberly L; Fowler, Velia M (2003) Pointed-end capping by tropomodulin3 negatively regulates endothelial cell motility. J Cell Biol 161:371-80 |
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