Asymmetric Localization of Calpain 2 during Neutrophil Chemotaxis

Abstract

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. INTRODUCTIONNeutrophils are key components of the innate immune system, representing the first responders to inflammatory stimuli such as bacterial infections and tissue injury. The rapid recruitment of neutrophils to inflammatory sites requires a highly specialized form of cell migration in which polarity and movement are directed by spatial cues from a chemotactic gradient. Although the molecular mechanisms that regulate neutrophil chemotaxis remain poorly understood, recent progress indicates that this process is achieved by two distinct, but functionally linked events, including directional sensing and cell polarization (Devreotes and Janetopoulos, 2003;Franca-Koh and Devreotes, 2004).In professional migratory cells such as neutrophils, G protein-coupled cell surface receptors recognize external chemotactic gradients (Van Haastert and Devreotes, 2004;Huttenlocher, 2005) and are responsible for initiating the translation of spatial information about the chemotactic gradient into an internal gradient of signaling molecules that relay either "front"-or "back"-specific responses. For example, previous work has shown that phosphatidylinositol-3,4,5-trisphosphate (PIP 3 ) is asymmetrically recruited to the membrane adjacent to the highest concentration of chemoattractant (Servant et al., 2000;Kimmel and Parent, 2003) where it stimulates positive feedback mechanisms that promote the frontness signal. Subsequent reinforcement of frontness and backness signals dictate cell polarity, where pseudopod formation is biased according to the direction of the chemotactic gradient, with the leading edge characterized by the asymmetric...

Epac increases melanoma cell migration by a heparan sulfate-related mechanism

“…Syndecan-translocation to lipid rafts plays a role in cell migration (39). Lipid rafts are known to serve as platforms for molecules (42), and translocation of molecules, such as ICAM-1 (30), calpain (32), and integrins (25), to lipid rafts is necessary to activate cell migration. We demonstrated that Epac increased translocation of syndecan-2 to lipid rafts.…”

“…2B) or the expression of the other HSPGs (data not shown). Since syndecan is known to translocate to lipid rafts (40), which serve as platforms for molecules involved in cell migration (26,30,32), we hypothesized that such large particle size indicates accumulation of syndecan-2 in lipid rafts. Indeed, immunocytochemistry showed that both Epac agonist and Epac1 overexpression increased colocalization of syndecan-2 with lipid rafts.…”

Type Iγ PIP Kinase Is a Novel Uropod Component that Regulates Rear Retraction during Neutrophil Chemotaxis

“…HL-60 cells (UCSF tissue culture facility) were cultured and differentiated as previously described (Nuzzi et al, 2007b). Phoenix viral packaging cells were transiently transfected by calcium-phosphate precipitation (Nuzzi et al, 2007a).…”

“…Primary human neutrophils, undifferentiated and differentiated HL-60 cells, and HEK 293 cells were lysed (Nuzzi et al, 2007b), samples were clarified by centrifugation, and protein concentration was determined using a bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). Equal amounts of total protein were denatured in SDS sample buffer, resolved on 4 -20% gradient Representative images of neutrophils demonstrate localization of endogenous PIPKI␥ (P␥; C) and active RhoA (rhotekin; D) to the cell rear upon fMLP stimulation.…”

Regulation of cell polarity by controlled proteolytic systems

“…During neutrophil migration, actin microfi lament polymerization leads to the extension of membrane projections (i.e., protrusions) at the leading edge, including fi lopodia and lamellipodia, which are required for directional migration. In the case of neutrophils, exposure to a chemoattractant gradient induces the rapid enrichment of the protease calpain 2 at the leading edge where it colocalizes with F-actin (Nuzzi et al, 2007). Localization of the protease at the leading edge seems to be causal for neutrophil migration because the expression of an inactive protease variant (Calpain PD) generates multiple lamellipodia surrounding neutrophils without a defi ned leading edge and prevents effi cient chemotaxis (Nuzzi et al, 2007).…”