ABSTRACT Neutrophils rely on glycolysis for energy production. How mitochondria regulate neutrophil function is not fully understood. Here, we report that mitochondrial outer membrane protein Mitofusin 2 (MFN2) regulates neutrophil homeostasis and chemotaxis in vivo . Mfn2 -deficient neutrophils are released from the hematopoietic tissue, trapped in the vasculature in zebrafish embryos, and not capable of chemotaxis. Consistent with this, human neutrophil-like cells that are deficient for MFN2 fail to arrest on activated endothelium under sheer stress or perform chemotaxis on 2D surfaces. Deletion of MFN2 results in a significant reduction of neutrophil infiltration to the inflamed peritoneal cavity in mice. Mechanistically, MFN2-deficient neutrophil-like cells display disrupted mitochondria–ER interaction, heightened intracellular Ca 2+ levels and elevated Rac activation after chemokine stimulation. Restoring a mitochondria–ER tether rescues the abnormal Ca 2+ levels, Rac hyperactivation and chemotaxis defect resulting from MFN2 depletion. Finally, inhibition of Rac activation restores chemotaxis in MFN2-deficient neutrophils. Taken together, we have identified that MFN2 regulates neutrophil migration via maintaining the mitochondria–ER interaction to suppress Rac activation, and uncovered a previously unrecognized role of MFN2 in regulating cell migration and the actin cytoskeleton. This article has an associated First Person interview with the first authors of the paper.

Studies on the mechanism of integrin inside‐out activation have been focused on the role of β‐integrin cytoplasmic tails, which are relatively conserved and bear binding sites for the intracellular activators including talin and kindlin. Cytoplasmic tails for α‐integrins share a conserved GFFKR motif at the membrane‐proximal region and this forms a specific interface with the β‐integrin membrane‐proximal region to keep the integrin inactive. The α‐integrin membrane‐distal regions, after the GFFKR motif, are diverse both in length and sequence and their roles in integrin activation have not been well‐defined. In this study, we report that the α‐integrin cytoplasmic membrane‐distal region contributes to maintaining integrin in the resting state and to integrin inside‐out activation. Complete deletion of the α‐integrin membrane‐distal region diminished talin‐ and kindlin‐mediated integrin ligand binding and conformational change. A proper length and suitable amino acids in α‐integrin membrane‐distal region was found to be important for integrin inside‐out activation. Our data establish an essential role for the α‐integrin cytoplasmic membrane‐distal region in integrin activation and provide new insights into how talin and kindlin induce the high‐affinity integrin conformation that is required for fully functional integrins.

ABSTRACT Inflammatory cells acquire a polarized phenotype to migrate towards sites of infection or injury. A conserved polarity complex comprising PAR-3, PAR-6 and atypical protein kinase C (aPKC) relays extracellular polarizing cues to control cytoskeletal and signaling networks affecting morphological and functional polarization. However, there is no evidence that myeloid cells use PAR signaling to migrate vectorially in three-dimensional (3D) environments in vivo . Using genetically encoded bioprobes and high-resolution live imaging, we reveal the existence of F-actin oscillations in the trailing edge and constant repositioning of the microtubule organizing center (MTOC) to direct leukocyte migration in wounded medaka fish larvae ( Oryzias latipes ). Genetic manipulation in live myeloid cells demonstrates that the catalytic activity of aPKC and the regulated interaction with PAR-3 and PAR-6 are required for consistent F-actin oscillations, MTOC perinuclear mobility, aPKC repositioning and wound-directed migration upstream of Rho kinase (also known as ROCK or ROK) activation. We propose that the PAR complex coordinately controls cytoskeletal changes affecting both the generation of traction force and the directionality of leukocyte migration to sites of injury.

ABSTRACT Immune cell trafficking requires the frequent breaching of the endothelial barrier either directly through individual cells (‘transcellular’ route) or through the inter-endothelial junctions (‘paracellular’ route). What determines the loci or route of breaching events is an open question with important implications for overall barrier regulation. We hypothesized that basic biomechanical properties of the endothelium might serve as crucial determinants of this process. By altering junctional integrity, cytoskeletal morphology and, consequently, local endothelial cell stiffness of different vascular beds, we could modify the preferred route of diapedesis. In particular, high barrier function was associated with predominantly transcellular migration, whereas negative modulation of junctional integrity resulted in a switch to paracellular diapedesis. Furthermore, we showed that lymphocytes dynamically probe the underlying endothelium by extending invadosome-like protrusions (ILPs) into its surface that deform the nuclear lamina, distort actin filaments and ultimately breach the barrier. Fluorescence imaging and pharmacologic depletion of F-actin demonstrated that lymphocyte barrier breaching efficiency was inversely correlated with local endothelial F-actin density and stiffness. Taken together, these data support the hypothesis that lymphocytes are guided by the mechanical ‘path of least resistance’ as they transverse the endothelium, a process we term ‘tenertaxis’.

ABSTRACT Intercellular adhesion molecule 2 (ICAM-2) is expressed on endothelial cells (ECs) and supports neutrophil extravasation. However, the full details of its role remain unknown, and the present study investigates the functional mechanisms of ICAM-2 in neutrophil–endothelial-cell interactions. Our initial studies showed expression of ICAM-2 at both EC junctions and on the EC body. In line with the observed expression profile analysis of neutrophil–vessel-wall interactions using real-time in vivo confocal microscopy identified numerous functional roles for ICAM-2 within the vascular lumen and at the stage of neutrophil extravasation. Functional or genetic blockade of ICAM-2 significantly reduced neutrophil crawling velocity, increased frequency of crawling with a disrupted stop-start profile, and prolonged interaction of neutrophils with EC junctions prior to transendothelial cell migration (TEM), collectively resulting in significantly reduced extravasation. Pharmacological blockade of the leukocyte integrin MAC-1 indicated that some ICAM-2-dependent functions might be mediated through ligation of this integrin. These findings highlight novel roles for ICAM-2 in mediating luminal neutrophil crawling and the effect on subsequent levels of extravasation.

Summary Phospholipase D2 (PLD2) is a cell-signaling molecule that bears two activities: a guanine-nucleotide exchange factor (GEF) and a lipase that reside in the PX/PH domains and in two HKD domains, respectively. Upon cell stimulation, the GEF activity yields Rac2-GTP and the lipase activity yields phosphatidic acid (PA). In the present study, we show for the first time that these activities regulate one another. Upon cell stimulation, both GEF and lipase activities are quickly (within ∼3 min) elevated. As soon as it is produced, PA positively feeds back on the GEF and further activates it. Rac2-GTP, on the other hand, is inhibitory to the lipase activity. PLD2 would remain downregulated if it were not for the contribution of the tyrosine kinase Janus kinase 3 (JAK3), which restores lipase action (by phosphorylation at Y415). Conversely, the GEF is inhibited upon phosphorylation by JAK3 and is effectively terminated by this action and by the increasing accumulation of PA at >15 min of cell stimulation. This PA interferes with the ability of the GEF to bind to its substrate (Rac2-GTP). Thus, both temporal inter-regulation and phosphorylation-dependent mechanisms are involved in determining a GEF–lipase switch within the same molecule. Human neutrophils stimulated by interleukin-8 follow a biphasic pattern of GEF and lipase activation that can be explained by such an intramolecular switch. This is the first report of a temporal inter-regulation of two enzymatic activities that reside in the same molecule with profound biological consequences in leukocyte cell migration.

Immune-system functions require that blood leukocytes continuously traffic throughout the body and repeatedly cross endothelial barriers (i.e. diapedese) as they enter (intravasate) and exit (extravasate) the circulation. The very earliest studies to characterize diapedesis directly in vivo suggested the coexistence of two distinct migratory pathways of leukocytes: between (paracellular pathway) and directly through (transcellular pathway) individual endothelial cells. In vivo studies over the past 50 years have demonstrated significant use of the transcellular diapedesis pathway in bone marrow, thymus, secondary lymphoid organs, various lymphatic structures and peripheral tissues during inflammation and across the blood-brain barrier and blood-retinal barrier during inflammatory pathology. Recently, the first in vitro reports of transcellular diapedesis have emerged. Together, these in vitro and in vivo observations suggest a model of migratory pathfinding in which dynamic `invadosome-like protrusions' formed by leukocytes have a central role in both identifying and exploiting endothelial locations that are permissive for transcellular diapedesis. Such `probing' activity might have additional roles in this and other settings.

The membrane-associated adhesion molecule JAM-A is required for neutrophil infiltration in inflammatory or ischemic tissues. JAM-A expressed in both endothelial cells and neutrophils has such a role, but the mechanism of action remains elusive. Here we show that JAM-A has a cell-autonomous role in neutrophil chemotaxis both in vivo and in vitro, which is independent of the interaction of neutrophils with endothelial cells. On activated neutrophils, JAM-A concentrates in a polarized fashion at the leading edge and uropod. Surprisingly, a significant amount of this protein is internalized in intracellular endosomal-like vesicles where it codistributes with integrin β1. Clustering of β1 integrin leads to JAM-A co-clustering, whereas clustering of JAM-A does not induce integrin association. Neutrophils derived from JAM-A-null mice are unable to correctly internalize β1 integrins upon chemotactic stimuli and this causes impaired uropod retraction and cell motility. Consistently, inhibition of integrin internalization upon treatment with BAPTA-AM induces a comparable phenotype. These data indicate that JAM-A is required for the correct internalization and recycling of integrins during cell migration and might explain why, in its absence, the directional migration of neutrophils towards an inflammatory stimulus is markedly impaired.

Podosomes, highly dynamic adhesion structures implicated in cell motility and extracellular matrix degradation, are characteristic of certain cells of the myeloid lineage and a limited range of other cell types. The nature and the mechanisms that regulate their high turnover are unknown at present. The cysteine protease calpain is involved in the regulation of cell migration in part by promoting either formation or disassembly of adhesion sites. Despite the fact that many known substrates of calpain are also structural components of the podosome complex, no studies have yet demonstrated that calpain participates in the regulation of podosome dynamics. In the present work, we show that inhibition of calpain in primary mouse dendritic cells leads to enhanced accumulation of actin filaments, the Wiskott Aldrich Syndrome protein (WASP), β 2 integrins, talin, paxillin and vinculin in podosomes. This accumulation of components is associated with stabilisation of podosome turnover, overall reduction in velocity of cell locomotion and impaired transmigration across an endothelial monolayer. We also demonstrate that calpain cleaves the podosome components talin, Pyk2 and WASP in dendritic cells. In summary, our results provide evidence that calpain regulates podosome composition and turnover and that this process is required for efficient migration of dendritic cells.

Leukocytes are recruited from the bloodstream to sites of inflammation by the selectin family of adhesion receptors. In vivo and in vitro studies reveal distinctive rolling velocities of polymorphonuclear leukocytes over E-, P- and L-selectin substrates. The kinetic and mechanical properties of the selectin-ligand bonds responsible for these differences at the single-molecule level are not well understood. Using single-molecule force spectroscopy, we probe in situ the rupture force, unstressed off-rate and reactive compliance of single selectin receptors to single ligands on whole human polymorphonuclear leukocytes (PMNs) under conditions that preserve the proper orientation and post-translational modifications of the selectin ligands. Single L-selectin bonds to PMNs were more labile than either E- or P-selectin in the presence of an applied force. This outcome, along with a higher unstressed off-rate and a higher reactive compliance, explain the faster L-selectin-mediated rolling. By quantifying binding frequency in the presence of a specific blocking monoclonal antibody or following enzyme treatment, we determined that P-selectin glycoprotein ligand-1 is a high-affinity ligand for E-selectin on PMNs under force. The rupture force spectra and corresponding unstressed off-rate and reactive compliance of selectin-ligand bonds provide mechanistic insights that might help to explain the variable rolling of leukocytes over different selectin substrates.

The interaction of leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) is central to the regulation of adhesion in leukocytes. In this report, we investigated the mechanisms by which phorbol myristate acetate (PMA) promotes LFA-1-dependent cell adhesion. The adhesion of PMA-stimulated cells to immobilized ICAM-1 was quantified in direct force measurements acquired by atomic force microscopy (AFM). Enhanced adhesion of PMA-stimulated cells to immobilized ICAM-1 stemmed from an increase in the number of LFA-1–ICAM-1 complexes formed between the two apposing surfaces on contact, rather than by affinity modulation of LFA-1. Single molecule force measurements revealed that the force spectrum of the LFA-1–ICAM-1 complex formed by PMA-stimulated cells is identical to the force spectrum of the complex formed by resting cells. Thus, PMA stimulation does not modify the mechanical strength of the individual LFA-1–ICAM-1 interaction. Instead, the enhanced cell adhesion of PMA-stimulated cells appears to be a complex process that correlates with changes in the mechanical properties of the cell. We estimate that changes in the elasticity of the cell gave rise to a more than 10-fold increase in cell adhesion.