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Vol. 18, Issue 3, 795-805, March 2007

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Asymmetric Localization of Calpain 2 during Neutrophil Chemotaxis

Paul A. Nuzzi^*,, Melissa A. Senetar^*,, and Anna Huttenlocher^*,

Departments of *Pharmacology and Pediatrics, University of Wisconsin, Madison, WI 53706

Submitted October 2, 2006; Revised December 4, 2006; Accepted December 18, 2006
Monitoring Editor: Carole Parent

Chemoattractants induce neutrophil polarization through localized polymerization of F-actin at the leading edge. The suppression of rear and lateral protrusions is required for efficient chemotaxis and involves the temporal and spatial segregation of signaling molecules. We have previously shown that the intracellular calcium-dependent protease calpain is required for cell migration and is involved in regulating neutrophil chemotaxis. Here, we show that primary neutrophils and neutrophil-like HL-60 cells express both calpain 1 and calpain 2 and that chemoattractants induce the asymmetric recruitment of calpain 2, but not calpain 1, to the leading edge of polarized neutrophils and differentiated HL-60 cells. Using time-lapse microscopy, we show that enrichment of calpain 2 at the leading edge occurs during early pseudopod formation and that its localization is sensitive to changes in the chemotactic gradient. We demonstrate that calpain 2 is recruited to lipid rafts and that cholesterol depletion perturbs calpain 2 localization, suggesting that its enrichment at the front requires proper membrane organization. Finally, we show that catalytic activity of calpain is required to limit pseudopod formation in the direction of chemoattractant and for efficient chemotaxis. Together, our findings identify calpain 2 as a novel component of the frontness signal that promotes polarization during chemotaxis.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

These authors contributed equally to this work.

Address correspondence to: Anna Huttenlocher (huttenlocher{at}wisc.edu)

Abbreviations used: C5a, Complement factor 5a; DIC, differential interference contrast; dHL-60, differentiated HL-60; fMLP, N-formyl- L-methionyl-L-leucyl-L-phenylalanine; G_M, ganglioside marker-1; PD, protease dead; PMN, polymorphonuclear cell; PIP₃, phosphatidylinositol-3,4,5-triphosphate; RACK, receptor for activated C kinase.