Live cell imaging of outward and inward vesiculation induced by the complement c5b-9 complex.
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.
Moskovich
Oren
O
Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
Fishelson
Zvi
Z
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
C417561:Alexa 488 hydrazide; D018957:CD59 Antigens; D003186:Complement C9; D000939:Epitopes; D005456:Fluorescent Dyes; D006834:Hydrazines; D014168:Transferrin; D050776:Complement C5b