PI(3)Kγ has an important context-dependent role in neutrophil chemokinesis

Abstract

The directional movement of cells in a gradient of external stimulus is termed chemotaxis and is important in many aspects of development and differentiated cell function. Phophoinositide 3-kinases (PI(3)Ks) are thought to have critical roles within the gradient-sensing machinery of a variety of highly motile cells, such as mammalian phagocytes, allowing these cells to respond quickly and efficiently to shallow gradients of soluble stimuli. Our analysis of mammalian neutrophil migration towards ligands such as fMLP shows that, although PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) accumulate in a PI(3)Kgamma-dependent fashion at the up-gradient leading-edge, this signal is not required for efficient gradient-sensing and gradient-biased movement. PI(3)Kgamma activity is however, a critical determinant of the proportion of cells that can move, that is, respond chemokinetically, in reaction to fMLP. Furthermore, this dependence of chemokinesis on PI(3)Kgamma activity is context dependent, both with respect to the state of priming of the neutrophils and the type of surface on which they are migrating. We propose this effect of PI(3)Kgamma is through roles in the regulation of some aspects of neutrophil polarization that are relevant to movement, such as integrin-based adhesion and the accumulation of polymerized (F)-actin at the leading-edge.

Leukocyte transmigration is modulated by chemokine‐mediated PI3Kγ‐dependent phosphorylation of vimentin

“…7C). On the other hand, hVimS4,6,7,8,9A expression in PI3Kg À/À cells did not As a further proof, PI3Kg-null BMDM were infected with a lentivirus expressing another vimentin mutant, in which Ser4,6,7,8,9 residues were replaced with aspartate (hVimS4,6,7,8,9D), hence mimicking negatively charged phosphorylated residues. Interestingly, PI3Kg-null BMDM expressing hVimS4,6,7,8,9D showed higher transmigration than the same cells expressing hVim (41.7 and 33.4% increase, 30 min and 4 h after stimulation, respectively; Fig.…”

“…Indeed, PI3Kg-null granulocytes show a significant reduction in chemotaxis toward chemokines and severely impaired bacteria-elicited peritoneal recruitment [5,6]. This migration defect is thought to be mainly caused by abnormal chemokinesis, adhesion and polarization [7,8].PI3K-dependent signaling is essential in driving the cytoskeletal reorganization necessary for leukocyte migration [9] and chemoattractant stimulation of leukocytes is thought to require remodeling of all three cytoskeletal filament systems: microfilaments, microtubules and intermediate filaments. Among these, the class three intermediate filament vimentin is abundantly expressed by leukocytes and plays multiple roles in cell shape organization.…”

“…These phosphorylation events are triggered by a wide range of protein kinases, such as PKA and PKC [15,19] [25] and involve a large number of serine residues (e.g. Ser 4,[6][7][8][9]25,33,38,41, 50, 55, 65, 71, 72, 82) with a complex pattern of overlapping specificities [15,26,27].Although vimentin appears involved in multiple cellular processes, mice lacking this protein are viable and do not show overt phenotypes [28]. Nonetheless, fibroblasts from vimentindeficient mice show mechanical instability and impaired ability to migrate and to contract a 3-D collagen network [29], thus providing a genetic evidence for the involvement of vimentin in cell plasticity and motility [30,31].…”

“…Protein samples were finally resolved by electrophoresis on a 10% polyacrylamide gel in the presence of SDS and in reducing conditions. and PI3Kg À/À cells infected with lentivirus carrying GFP (GFP), human vimentin (hVim), human mutant vimentin S4,6,7,8,9A (hVimS4,6,7,8,9A) or human mutant vimentin S4,6,7,8,9D (hVimS4,6,7,8,9D). Vinc, vinculin used as loading control.…”

“…Indeed, PI3Kg-null granulocytes show a significant reduction in chemotaxis toward chemokines and severely impaired bacteria-elicited peritoneal recruitment [5,6]. This migration defect is thought to be mainly caused by abnormal chemokinesis, adhesion and polarization [7,8].…”