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MBC in Press, published online ahead of print February 1, 2006
Mol. Biol. Cell 10.1091/mbc.E05-12-1140

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Submitted on December 18, 2005
Revised on January 20, 2006
Accepted on January 23, 2006

Differential Requirements for Actin Polymerization, Calmodulin, and Ca2+ Define Distinct Stages of Lysosome/Phagosome Targeting

Walter Stockinger, Shao C. Zhang, Vishal Trivedi, Larissa A. Jarzylo, Eugenie C. Shieh, William S. Lane, Adam B. Castoreno, and Axel Nohturfft

Department of Molecular and Cellular Biology, The Biological Laboratories, Harvard University, Cambridge, MA 02138

Monitoring Editor: Randy Schekman

Fusion of phagosomes with late endocytic organelles is essential for cellular digestion of microbial pathogens, senescent cells, apoptotic bodies and retinal outer segment fragments. To further elucidate the biochemistry of the targeting process we developed a scintillation proximity assay to study the stepwise association of lysosomes and phagosomes in vitro. Incubation of tritium-labeled lysosomes with phagosomes containing scintillant latex beads led to light emission in a reaction requiring cytosol, ATP and low Ca2+ concentrations. The nascent complex was sensitive to disruption by alkaline carbonate, indicating that the organelles had "docked" but not fused. Through inhibitor studies and fluorescence microscopy we show that docking is preceded by a tethering step that requires actin polymerization and calmodulin. In the docked state ongoing actin polymerization and calmodulin are no longer necessary. The tethering/docking activity was purified to near homogeneity from rat liver cytosol. Major proteins in the active fractions included actin, calmodulin and IQGAP2. IQGAPs are known to bind calmodulin and cross-link F-actin, suggesting a key coordinating role during lysosome/phagosome attachment. The current results support the conclusion that lysosome/phagosome interactions proceed through distinct stages and provide a useful new approach for further experimental dissection.

Address correspondence to: Axel Nohturfft (axno{at}mcb.harvard.edu)




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