The red cell membrane possesses a spectrin-actin network that imparts the cell with elasticity and resilience. These properties are increasingly measurable at the single molecule level, and we hypothesize that they are strongly influenced by temperature, spectrin association state, and linker structure between spectrin repeats. Among other functions, the spectrin network also appears to anchor - via protein 4.2 - a major fraction of the integral membrane protein CD47, at least in human RBC. This linkage seems lacking in normal mouse RBC where CD47 had been separately shown to mediate adhesion and signal 'Self' to phagocytes. Despite many differences in human vs mouse CD47 as well as associated Rh proteins, we hypothesize that human CD47 engages in similar Self-signaling as in the mouse and that this function is predicated on a critical, dynamic interplay between adhesion to phagocytes versus signaling - processes that may be uniquely influenced in human RBC by CD47's linkage to the network. We propose single molecule investigations of spectrin network flexibility in addition to studies of CD47 membrane integration and function. Atomic force microscopy (AFM) will be applied to individual spectrin constructs and their complexes in order to statistically elaborate pathways of elasticity, unfolding, binding, and thermal stability. Novel simulation methods will allow us to integrate such information into predictive algorithms for molecular response in deformation, membrane network assembly, and receptor diffusion in cell adhesion. To deepen our preliminary understanding of human CD47's association with Rh proteins and the membrane network, mobility and expression level measurements will be made on normal and pathological human RBC (e.g., 4.2-deficient) as well as normal mouse RBC. To study the critical interplay of CD47 adhesion and signaling to phagocytes, heterologous yeast display platforms for human CD47's Ig domain will be created, mutated, and manipulated into contact with phagocytes using an optical trap. Human and mouse RBC's will be analysed in their expected species-specific adhesion strength for surface-bound CD47 counter-receptors, and the role of CD47 to signal against phagocytosis of opsonized human RBC will be separately evaluated. Throughout, expressed fragments of spectrin, CD47 and protein 4.2 will be used to elucidate the broader molecular basis of RBC mechanics, adhesion, and phagocytosis. The results should prove of broad and fundamental significance to hemolytic anemias (e.g., hereditary spherocytosis) and senescence, as well as fleshing out human-mouse comparisons on what is already emerging as a structurally divergent system. ? ?
Showing the most recent 10 out of 56 publications
