Dynamics of membrane-associated complement regulatory proteins in red blood cells

Complement activation involves a series of solution-phase and membrane-associated reactions that culminate in opsonizing noxious targets and, for Neisseria species, lysing the bacteria. Normal human cells are protected from autologous complement-mediated damage by the action of regulatory proteins on their plasma membranes. Although the dynamics of these regulatory proteins are likely to have a critical role in regulatory protein function, there is little to no information in the literature concerning the lateral diffusion and membrane domain localization of these molecules. In this thesis, single particle tracking (SPT) was used as the primary approach to examine the lateral diffusion of all three membrane-associated complement regulatory proteins in human red blood cells (RBC): complement receptor 1 (CR1, CD35), decay-accelerating factor (DAF, CD55), and CD59. CR1 is a single-span transmembrane protein, while both DAF and CD59 are glycosylphosphatidylinositol (GPI)-linked proteins.; In both normal and complement-activated RBC, the majority of CR1 molecules showed confined motion, while a minor population exhibited Brownian movement. The confinement of CR1 corresponded to the observation of receptor clustering on the membrane surface, and was due to CR1 interaction with a membrane skeleton-linked Fas-associated phosphatase-1 (FAP-1). CR1 confinement and clustering are likely to be important in the capture and transport of immune complexes by RBC through the circulation. The function of the mobile fraction of CR1 remains unclear.; Most of the DAF molecules exhibited Brownian lateral diffusion in normal RBC. Biochemical studies showed that activation of complement in the fluid phase caused the complement fragment Cab to deposit preferentially onto glycophorin A (GPA) on the RBC membrane surface, and SPT revealed that DAF, Cab, and GPA were all laterally immobilized in the membranes of complement-treated cells. Laser optical tweezers experiments showed that the major fraction of GPA molecules were not associated with the membrane skeleton in the native membrane, but that Cab deposition induced a physical association between GPA and the membrane skeleton. These results are consistent with a model in which complement activation stimulates the formation of a skeleton-linked DAF-Cab-GPA complex on the RBC surface. The accumulation of many such complexes over the life of the RBC may play a role in the removal of senescent RBC from the circulation.; In normal RBC, CD59 was transiently confined in local membrane domains (transient confinements). Within these domains, lateral diffusion of the molecule was significantly hindered. In the presence of C5b-8, CD59 molecules were mostly immobilized. C5b-8 motion was confined as well. Experiments using cholesterol-reduced RBC showed that the CD59 transient confinement and the C5b-8 membrane insertion were sensitive to changes in membrane cholesterol content. These results are consistent with a model in which terminal complement activation induces molecular interaction between CD59 and C5b-8, and in which the lateral mobility of CD59 and the membrane insertion of C5b-8 depend on membrane cholesterol content.