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J. Biol. Chem., Vol. 282, Issue 41, 29977-29986, October 12, 2007

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Live Cell Imaging of Outward and Inward Vesiculation Induced by the Complement C5b-9 Complex^*

From the Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel

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 µm) 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.

Received for publication, May 7, 2007 , and in revised form, July 9, 2007.

^* This work was supported in part by grants from the Israel Science Foundation, the Israel Cancer Association, and the Israel Cancer Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S7 and Movies S1–S3.

¹ To whom correspondence should be addressed: Dept. of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. Tel.: 972-3-6409620; Fax: 972-3-6407432; E-mail: lifish{at}post.tau.ac.il.

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