title: Live cell imaging of outward and inward vesiculation induced by the complement c5b-9 complex.
abstract: Cells resist death induced by the complement membrane attack complex (MAC, C5b-9) by removal of the MAC from their surface by an outward and/or inward vesiculation. To gain an insight into the route of MAC removal, human C9 was tagged with Alexa Fluor 488 and traced within live cells. Tagged C9-AF488 was active in lysis of erythrocytes and K562 cells. Upon treatment of K562 cells with antibody and human serum containing C9-AF488, C9-AF488 containing MAC bound to the cells. Within 5-10 min, the cells started shedding C5b-9-loaded vesicles (0.05-1 mum) by outward vesiculation. Concomitantly, C9-AF488 entered the cells and accumulated in a perinuclear, late recycling compartment, co-localized with endocytosed transferrin-Texas Red. Similar results were obtained with fixed cells in which the MAC was labeled with antibodies directed to a C5b-9 neoepitope. Inhibition of protein kinase C reduced endocytosis of C5b-9. Kinetic analysis demonstrated that peripheral, trypsin-sensitive C5b-9 was cleared from cells at a slower rate relative to fully inserted, trypsin-resistant C5b-9. MAC formation is controlled by CD59, a ubiquitously expressed membrane complement regulator. Analysis at a cell population level showed that the amount of C5b-9-AF488 bound to K562 cells after complement activation was highly heterogeneous and inversely correlated with the CD59 level of expression. Efficient C9-AF488 vesiculation was observed in cells expressing low CD59 levels, suggesting that the protective impact of MAC elimination by vesiculation increases as the level of expression of CD59 decreases.
authors.affiliation: Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Oren
authors.initials: O
authors.lastname: Moskovich
authors.forename: Zvi
authors.initials: Z
authors.lastname: Fishelson
mesh_terms: D018957:CD59 Antigens / Q000096:biosynthesis; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D050776:Complement C5b / Q000737:chemistry*; D003186:Complement C9 / Q000737:chemistry*; D004705:Endocytosis; D000939:Epitopes / Q000737:chemistry; D005089:Exocytosis; D005456:Fluorescent Dyes / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology; D020014:K562 Cells; D007700:Kinetics; D017434:Protein Structure, Tertiary; D014168:Transferrin / Q000737:chemistry
chemical_list: C417561:Alexa 488 hydrazide; D018957:CD59 Antigens; D003186:Complement C9; D000939:Epitopes; D005456:Fluorescent Dyes; D006834:Hydrazines; D014168:Transferrin; D050776:Complement C5b