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Vol. 17, Issue 4, 1697-1710, April 2006

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

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

Submitted December 18, 2005; Revised January 20, 2006; Accepted January 23, 2006
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.

Abbreviations used: PVT, polyvinyltoluene.

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

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