Are the class 18 myosins Myo18A and Myo18B specialist sarcomeric proteins?

Initially, the two members of class 18 myosins, Myo18A and Myo18B, appeared to exhibit highly divergent functions, complicating the assignment of class-specific functions. However, the identification of a striated muscle-specific isoform of Myo18A, Myo18Aγ, suggests that class 18 myosins may have evolved to complement the functions of conventional class 2 myosins in sarcomeres. Indeed, both genes, Myo18a and Myo18b, are predominantly expressed in the heart and somites, precursors of skeletal muscle, of developing mouse embryos. Genetic deletion of either gene in mice is embryonic lethal and is associated with the disorganization of cardiac sarcomeres. Moreover, Myo18Aγ and Myo18B localize to sarcomeric A-bands, albeit the motor (head) domains of these unconventional myosins have been both deduced and biochemically demonstrated to exhibit negligible ATPase activity, a hallmark of motor proteins. Instead, Myo18Aγ and Myo18B presumably coassemble with thick filaments and provide structural integrity and/or internal resistance through interactions with F-actin and/or other proteins. In addition, Myo18Aγ and Myo18B may play distinct roles in the assembly of myofibrils, which may arise from actin stress fibers containing the α-isoform of Myo18A, Myo18Aα. The ß-isoform of Myo18A, Myo18Aß, is similar to Myo18Aα, except that it lacks the N-terminal extension, and may serve as a negative regulator through heterodimerization with either Myo18Aα or Myo18Aγ. In this review, we contend that Myo18Aγ and Myo18B are essential for myofibril structure and function in striated muscle cells, while α- and ß-isoforms of Myo18A play diverse roles in nonmuscle cells.

Elusive physiological role of prostatic acid phosphatase (PAP): generation of choline for sperm motility via auto-and paracrine cholinergic signaling.

Prostatic acid phosphatase (PAP) exists as two splice variants, secreted PAP and transmembrane PAP, the latter of which is implicated in antinociceptive signaling in dorsal root ganglia. However, PAP is predominantly expressed in the prostate gland and the physiological role of seminal PAP, first identified in 1938, is largely unknown. Here, the author proposes that PAP, following ejaculation, functions to hydrolyze phosphocholine (PC) in seminal fluid and generate choline, which is imported by sperm via a choline transporter and converted to acetylcholine (ACh) by choline acetyltransferase. Auto- and paracrine cholinergic signaling, or choline directly, may subsequently stimulate sperm motility via α7 nicotinic ACh receptors (nAChRs) and contractility of the female reproductive tract through muscarinic ACh receptors (mAChRs). Consistent with a role of PAP in cholinergic signaling, 1) seminal vesicles secrete PC, 2) the prostate gland secretes PAP, 3) PAP specifically catalyzes the hydrolysis of PC into inorganic phosphate and choline, 4) seminal choline levels increase post-ejaculation, 5) pharmacological inhibition of choline acetyltransferase inhibits sperm motility, 6) inhibition or genetic deletion of α7 nAChRs impairs sperm motility, and 7) mAChRs are expressed in the uterus and oviduct (fallopian tube). Notably, PAP does not degrade glycerophosphocholine (GPC), the predominant choline source in the semen of rats and other mammals. Instead, uterine GPC phosphodiesterases may liberate choline from seminal GPC. In summary, the author deduces that PAP in humans, and uterine GPC phosphodiesterases in other mammals, function to generate choline for sperm cholinergic signaling, which promotes sperm motility and possibly contractility of the female reproductive tract.

Alarming and Calming: Opposing Roles of S100A8/S100A9 Dimers and Tetramers on Monocytes.

Mechanisms keeping leukocytes distant of local inflammatory processes in a resting state despite systemic release of inflammatory triggers are a pivotal requirement for avoidance of overwhelming inflammation but are ill defined. Dimers of the alarmin S100A8/S100A9 activate Toll-like receptor-4 (TLR4) but extracellular calcium concentrations induce S100A8/S100A9-tetramers preventing TLR4-binding and limiting their inflammatory activity. So far, only antimicrobial functions of released S100A8/S100A9-tetramers (calprotectin) are described. It is demonstrated that extracellular S100A8/S100A9 tetramers significantly dampen monocyte dynamics as adhesion, migration, and traction force generation in vitro and immigration of monocytes in a cutaneous granuloma model and inflammatory activity in a model of irritant contact dermatitis in vivo. Interestingly, these effects are not mediated by the well-known binding of S100A8/S100A9-dimers to TLR-4 but specifically mediated by S100A8/S100A9-tetramer interaction with CD69. Thus, the quaternary structure of these S100-proteins determines distinct and even antagonistic effects mediated by different receptors. As S100A8/S100A9 are released primarily as dimers and subsequently associate to tetramers in the high extracellular calcium milieu, the same molecules promote inflammation locally (S100-dimer/TLR4) but simultaneously protect the wider environment from overwhelming inflammation (S100-tetramer/CD69).

Complement receptor 3 mediates both sinking phagocytosis and phagocytic cup formation via distinct mechanisms.

A long-standing hypothesis is that complement receptors (CRs), especially CR3, mediate sinking phagocytosis, but evidence is lacking. Alternatively, CRs have been reported to induce membrane ruffles or phagocytic cups, akin to those induced by Fcγ receptors (FcγRs), but the details of these events are unclear. Here we used real-time 3D imaging and KO mouse models to clarify how particles (human red blood cells) are internalized by resident peritoneal F4/80+ cells (macrophages) via CRs and/or FcγRs. We first show that FcγRs mediate highly efficient, rapid (2-3 min) phagocytic cup formation, which is completely abolished by deletion or mutation of the FcR γ chain or conditional deletion of the signal transducer Syk. FcγR-mediated phagocytic cups robustly arise from any point of cell-particle contact, including filopodia. In the absence of CR3, FcγR-mediated phagocytic cups exhibit delayed closure and become aberrantly elongated. Independent of FcγRs, CR3 mediates sporadic ingestion of complement-opsonized particles by rapid phagocytic cup-like structures, typically emanating from membrane ruffles and largely prevented by deletion of the immunoreceptor tyrosine-based activation motif (ITAM) adaptors FcR γ chain and DAP12 or Syk. Deletion of ITAM adaptors or Syk clearly revealed that there is a slow (10-25 min) sinking mode of phagocytosis via a restricted orifice. In summary, we show that (1) CR3 indeed mediates a slow sinking mode of phagocytosis, which is accentuated by deletion of ITAM adaptors or Syk, (2) CR3 induces phagocytic cup-like structures, driven by ITAM adaptors and Syk, and (3) CR3 is involved in forming and closing FcγR-mediated phagocytic cups.

Complement receptor 3 mediates both sinking phagocytosis and phagocytic cup formation via distinct mechanisms.

A long-standing hypothesis is that complement receptors (CRs), especially CR3, mediate sinking phagocytosis, but evidence is lacking. Alternatively, CRs have been reported to induce membrane ruffles or phagocytic cups, akin to those induced by Fcγ receptors (FcγRs), but the details of these events are unclear. Here we used real-time 3D imaging and knockout mouse models to clarify how particles (human red blood cells) are internalized by resident peritoneal F4/80+ cells (macrophages) via CRs and/or FcγRs. We first show that FcγRs mediate highly efficient, rapid (2-3 min) phagocytic cup formation, which is completely abolished by deletion or mutation of the FcR γ-chain or conditional deletion of the signal transducer Syk. FcγR-mediated phagocytic cups robustly arise from any point of cell-particle contact, including filopodia. In the absence of CR3, FcγR-mediated phagocytic cups exhibit delayed closure and become aberrantly elongated. Independent of FcgRs, CR3 mediates sporadic ingestion of complement-opsonized particles by rapid phagocytic cup-like structures, typically emanating from membrane ruffles and largely prevented by deletion of the immunoreceptor tyrosine-based activation motif (ITAM) adaptors FcR γ-chain and DAP12 or Syk. Deletion of ITAM adaptors or Syk clearly revealed that there is a slow (10-25 min) sinking mode of phagocytosis via a restricted orifice. In summary, we show that (1) CR3 indeed mediates a slow sinking mode of phagocytosis, which is accentuated by deletion of ITAM adaptors or Syk, (2) CR3 induces phagocytic cup-like structures, driven by ITAM adaptors and Syk, and (3) CR3 is involved in forming and closing FcγR-mediated phagocytic cups.

Class IX Myosins: Motorized RhoGAP Signaling Molecules.

Class IX myosins are simultaneously motor and signaling molecules. In addition to myosin class-specific functions of the tail region, they feature unique motor properties. Within their motor region they contain a long insertion with a calmodulin- and a F-actin-binding site. The rate-limiting step in the ATPase cycle is ATP hydrolysis rather than, typical for other myosins, the release of either product. This means that class IX myosins spend a large fraction of their cycle time in the ATP-bound state, which is typically a low F-actin affinity state. Nevertheless, class IX myosins in the ATP-bound state stochastically switch between a low and a high F-actin affinity state. Single motor domains even show characteristics of processive movement towards the plus end of actin filaments. The insertion thereby acts as an actin tether. The motor domain transports as intramolecular cargo a signaling Rho GTPase-activating protein domain located in the tail region. Rho GTPase-activating proteins catalyze the conversion of active GTP-bound Rho to inactive GDP-bound Rho by stimulating GTP hydrolysis. In cells, Rho activity regulates actin cytoskeleton organization and actomyosin II contractility. Thus, class IX myosins regulate cell morphology, cell migration, cell-cell junctions and membrane trafficking. These cellular functions affect embryonic development, adult organ homeostasis and immune responses. Human diseases associated with mutations in the two class IX myosins, Myo9a and Myo9b, have been identified, including hydrocephalus and congenital myasthenic syndrome in connection with Myo9a and autoimmune diseases in connection with Myo9b.

Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor-mediated chemotaxis.

G protein-coupled receptor signaling is required for the navigation of immune cells along chemoattractant gradients. However, chemoattractant receptors may couple to more than one type of heterotrimeric G protein, each of which consists of a Gα, Gß, and Gγ subunit, making it difficult to delineate the critical signaling pathways. Here, we used knockout mouse models and time-lapse microscopy to elucidate Gα and Gß subunits contributing to complement C5a receptor-mediated chemotaxis. Complement C5a-mediated chemokinesis and chemotaxis were almost completely abolished in macrophages lacking Gnai2 (encoding Gαi2), consistent with a reduced leukocyte recruitment previously observed in Gnai2-/- mice, whereas cells lacking Gnai3 (Gαi3) exhibited only a slight decrease in cell velocity. Surprisingly, C5a-induced Ca2+ transients and lamellipodial membrane spreading were persistent in Gnai2-/- macrophages. Macrophages lacking both Gnaq (Gαq) and Gna11 (Gα11) or both Gna12 (Gα12) and Gna13 (Gα13) had essentially normal chemotaxis, Ca2+ signaling, and cell spreading, except Gna12/Gna13-deficient macrophages had increased cell velocity and elongated trailing ends. Moreover, Gnaq/Gna11-deficient cells did not respond to purinergic receptor P2Y2 stimulation. Genetic deletion of Gna15 (Gα15) virtually abolished C5a-induced Ca2+ transients, but chemotaxis and cell spreading were preserved. Homozygous Gnb1 (Gß1) deletion was lethal, but mice lacking Gnb2 (Gß2) were viable. Gnb2-/- macrophages exhibited robust Ca2+ transients and cell spreading, albeit decreased cell velocity and impaired chemotaxis. In summary, complement C5a-mediated chemotaxis requires Gαi2 and Gß2, but not Ca2+ signaling, and membrane protrusive activity is promoted by G proteins that deplete phosphatidylinositol 4,5-bisphosphate.

Time-lapse Imaging of Mouse Macrophage Chemotaxis.

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a chemical. Chemokinesis and chemotaxis are fundamental for the mobilization and deployment of immune cells. For example, chemokines (chemotactic cytokines) can rapidly recruit circulating neutrophils and monocytes to extravascular sites of inflammation. Chemoattractant receptors belong to the large family of G protein-coupled receptors. How chemoattractant (i.e., ligand) gradients direct cell migration via G protein-coupled receptor signaling is not yet fully understood. In the field of immunology, neutrophils are popular model cells for studying chemotaxis in vitro. Here we describe a real-time two-dimensional (2D) chemotaxis assay tailored for mouse resident macrophages, which have traditionally been more difficult to study. Macrophages move at a slow pace of ~1 µm/min on a 2D surface and are less well suited for point-source migration assays (e.g., migration towards the tip of a micropipette filled with chemoattractant) than neutrophils or Dictyostelium discoideum, which move an order of magnitude faster. Widely used Transwell assays are useful for studying the chemotactic activity of different substances, but do not provide information on cell morphology, velocity, or chemotactic navigation. Here we describe a time-lapse microscopy-based macrophage chemotaxis assay that allows quantification of cell velocity and chemotactic efficiency and provides a platform to delineate the transducers, signal pathways, and effectors of chemotaxis.

A novel isoform of myosin 18A (Myo18Aγ) is an essential sarcomeric protein in mouse heart.

Whereas myosin 18B (Myo18B) is known to be a critical sarcomeric protein, the function of myosin 18A (Myo18A) is unclear, although it has been implicated in cell motility and Golgi shape. Here, we show that homozygous deletion (homozygous tm1a, tm1b, or tm1d alleles) of Myo18a in mouse is embryonic lethal. Reminiscent of Myo18b, Myo18a was highly expressed in the embryo heart, and cardiac-restricted Myo18a deletion in mice was embryonic lethal. Surprisingly, using Western blot analysis, we were unable to detect the known isoforms of Myo18A, Myo18Aα and Myo18Aß, in mouse heart using a custom C-terminal antibody. However, alternative anti-Myo18A antibodies detected a larger than expected protein, and RNA-Seq analysis indicated that a novel Myo18A transcript is expressed in mouse ventricular myocytes (and human heart). Cloning and sequencing revealed that this cardiac isoform, denoted Myo18Aγ, lacks the PDZ-containing N terminus of Myo18Aα but includes an alternative N-terminal extension and a long serine-rich C terminus. EGFP-tagged Myo18Aγ expressed in ventricular myocytes localized to the level of A-bands in sarcomeres, and Myo18a knockout embryos at day 10.5 exhibited disorganized sarcomeres with wavy thick filaments. We additionally generated myeloid-restricted Myo18a knockout mice to investigate the role of Myo18A in nonmuscle cells, exemplified by macrophages, which express more Myo18Aß than Myo18Aα, but no defects in cell shape, motility, or Golgi shape were detected. In summary, we have identified a previously unrecognized sarcomere component, a large novel isoform (denoted Myo18Aγ) of Myo18A. Thus, both members of class XVIII myosins are critical components of cardiac sarcomeres.

Phenotypic analysis of Myo10 knockout (Myo10tm2/tm2) mice lacking full-length (motorized) but not brain-specific headless myosin X.

We investigated the physiological functions of Myo10 (myosin X) using Myo10 reporter knockout (Myo10tm2) mice. Full-length (motorized) Myo10 protein was deleted, but the brain-specific headless (Hdl) isoform (Hdl-Myo10) was still expressed in homozygous mutants. In vitro, we confirmed that Hdl-Myo10 does not induce filopodia, but it strongly localized to the plasma membrane independent of the MyTH4-FERM domain. Filopodia-inducing Myo10 is implicated in axon guidance and mice lacking the Myo10 cargo protein DCC (deleted in colorectal cancer) have severe commissural defects, whereas MRI (magnetic resonance imaging) of isolated brains revealed intact commissures in Myo10tm2/tm2 mice. However, reminiscent of Waardenburg syndrome, a neural crest disorder, Myo10tm2/tm2 mice exhibited pigmentation defects (white belly spots) and simple syndactyly with high penetrance (>95%), and 24% of mutant embryos developed exencephalus, a neural tube closure defect. Furthermore, Myo10tm2/tm2 mice consistently displayed bilateral persistence of the hyaloid vasculature, revealed by MRI and retinal whole-mount preparations. In principle, impaired tissue clearance could contribute to persistence of hyaloid vasculature and syndactyly. However, Myo10-deficient macrophages exhibited no defects in the phagocytosis of apoptotic or IgG-opsonized cells. RNA sequence analysis showed that Myo10 was the most strongly expressed unconventional myosin in retinal vascular endothelial cells and expression levels increased 4-fold between P6 and P15, when vertical sprouting angiogenesis gives rise to deeper layers. Nevertheless, imaging of isolated adult mutant retinas did not reveal vascularization defects. In summary, Myo10 is important for both prenatal (neural tube closure and digit formation) and postnatal development (hyaloid regression, but not retinal vascularization).

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages.

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fcγ receptor (FcγR)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to FcγRs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fcγ receptor- or complement receptor-mediated phagocytosis.

Low density lipoprotein interferes with intracellular signaling of monocytes resulting in impaired chemotaxis and enhanced chemokinesis.

BACKGROUND: Hypercholesterolemia (HC) is an important cardiovascular risk factor characterized by elevated low density lipoprotein-cholesterol (LDL-C) plasma levels. HC negatively affects monocyte function by reducing their chemotactic response towards different growth factors. We aimed to elucidate the molecular mechanisms by which LDL induces monocyte dysfunction. METHODS AND RESULTS: Human monocytes exposed to either native (nLDL) or oxidized LDL (oxLDL) in vitro showed reduced chemotactic responses towards vascular endothelial growth factor A (VEGFA) and monocyte chemotactic protein-1 (MCP-1), but displayed enhanced random migration (chemokinesis). Mechanistically, the exposure to LDL resulted in the activation of p38 mitogen-activated protein kinase (MAPK) and modulated MCP-1 and VEGFA-induced signaling in human monocytes. Furthermore, the aberrant p38 activation induced by oxLDL is due to the functional impairment of Dual Specificity Phosphatase-1 (DUSP-1). In the absence of LDL, the pharmacological inhibition of DUSP-1 alone was sufficient to recapitulate the accelerated chemokinetic and blunted chemotactic phenotype of monocytes. Finally, p38 MAPK inhibition in monocytes isolated from hyperlipidemic mice prevented the aberrant chemokinetic phenotype. CONCLUSIONS: Our data demonstrate that LDL induces monocyte chemokinesis of human monocytes by inducing mononuclear cell activation through the aberrant modulation of DUSP-1-p38/MAPK signaling axis. Moreover, our findings suggest that MCP-1/VEGFA-induced chemotaxis is reduced by LDL secondary to the impairment of ligand-induced signaling. These findings provide novel insight into hypercholesterolemia-associated vascular dysfunction and its potential involvement in the pathogenesis of atherosclerosis.

Multiple roles of filopodial dynamics in particle capture and phagocytosis and phenotypes of Cdc42 and Myo10 deletion.

Macrophage filopodia, finger-like membrane protrusions, were first implicated in phagocytosis more than 100 years ago, but little is still known about the involvement of these actin-dependent structures in particle clearance. Using spinning disk confocal microscopy to image filopodial dynamics in mouse resident Lifeact-EGFP macrophages, we show that filopodia, or filopodia-like structures, support pathogen clearance by multiple means. Filopodia supported the phagocytic uptake of bacterial (Escherichia coli) particles by (i) capturing along the filopodial shaft and surfing toward the cell body, the most common mode of capture; (ii) capturing via the tip followed by retraction; (iii) combinations of surfing and retraction; or (iv) sweeping actions. In addition, filopodia supported the uptake of zymosan (Saccharomyces cerevisiae) particles by (i) providing fixation, (ii) capturing at the tip and filopodia-guided actin anterograde flow with phagocytic cup formation, and (iii) the rapid growth of new protrusions. To explore the role of filopodia-inducing Cdc42, we generated myeloid-restricted Cdc42 knock-out mice. Cdc42-deficient macrophages exhibited rapid phagocytic cup kinetics, but reduced particle clearance, which could be explained by the marked rounded-up morphology of these cells. Macrophages lacking Myo10, thought to act downstream of Cdc42, had normal morphology, motility, and phagocytic cup formation, but displayed markedly reduced filopodia formation. In conclusion, live-cell imaging revealed multiple mechanisms involving macrophage filopodia in particle capture and engulfment. Cdc42 is not critical for filopodia or phagocytic cup formation, but plays a key role in driving macrophage lamellipodial spreading.

Real-time two- and three-dimensional imaging of monocyte motility and navigation on planar surfaces and in collagen matrices: roles of Rho.

We recently found that macrophages from RhoA/RhoB double knockout mice had increased motility of the cell body, but severely impaired retraction of the tail and membrane extensions, whereas RhoA- or RhoB-deficient cells exhibited mild phenotypes. Here we extended this work and investigated the roles of Rho signaling in primary human blood monocytes migrating in chemotactic gradients and in various settings. Monocyte velocity, but not chemotactic navigation, was modestly dependent on Rho-ROCK-myosin II signaling on a 2D substrate or in a loose collagen type I matrix. Viewed by time-lapse epi-fluorescence microscopy, monocytes appeared to flutter rather than crawl, such that the 3D surface topology of individual cells was difficult to predict. Spinning disk confocal microscopy and 3D reconstruction revealed that cells move on planar surfaces and in a loose collagen matrix using prominent, curved planar protrusions, which are rapidly remodeled and reoriented, as well as resorbed. In a dense collagen type I matrix, there is insufficient space for this mode and cells adopt a highly Rho-dependent, lobular mode of motility. Thus, in addition to its role in tail retraction on 2D surfaces, Rho is critical for movement in confined spaces, but is largely redundant for motility and chemotaxis in loose matrices.

Mouse macrophages completely lacking Rho subfamily GTPases (RhoA, RhoB, and RhoC) have severe lamellipodial retraction defects, but robust chemotactic navigation and altered motility.

RhoA is thought to be essential for coordination of the membrane protrusions and retractions required for immune cell motility and directed migration. Whether the subfamily of Rho (Ras homolog) GTPases (RhoA, RhoB, and RhoC) is actually required for the directed migration of primary cells is difficult to predict. Macrophages isolated from myeloid-restricted RhoA/RhoB (conditional) double knock-out (dKO) mice did not express RhoC and were essentially "pan-Rho"-deficient. Using real-time chemotaxis assays, we found that retraction of the trailing edge was dissociated from the advance of the cell body in dKO cells, which developed extremely elongated tails. Surprisingly, velocity (of the cell body) was increased, whereas chemotactic efficiency was preserved, when compared with WT macrophages. Randomly migrating RhoA/RhoB dKO macrophages exhibited multiple small protrusions and developed large "branches" due to impaired lamellipodial retraction. A mouse model of peritonitis indicated that monocyte/macrophage recruitment was, surprisingly, more rapid in RhoA/RhoB dKO mice than in WT mice. In comparison with dKO cells, the phenotypes of single RhoA- or RhoB-deficient macrophages were mild due to mutual compensation. Furthermore, genetic deletion of RhoB partially reversed the motility defect of macrophages lacking the RhoGAP (Rho GTPase-activating protein) myosin IXb (Myo9b). In conclusion, the Rho subfamily is not required for "front end" functions (motility and chemotaxis), although both RhoA and RhoB are involved in pulling up the "back end" and resorbing lamellipodial membrane protrusions. Macrophages lacking Rho proteins migrate faster in vitro, which, in the case of the peritoneum, translates to more rapid in vivo monocyte/macrophage recruitment.

Dendritic cell motility and T cell activation requires regulation of Rho-cofilin signaling by the Rho-GTPase activating protein myosin IXb.

Directed migration of stimulated dendritic cells (DCs) to secondary lymphoid organs and their interaction with Ag-specific T cells is a prerequisite for the induction of primary immune responses. In this article, we show that murine DCs that lack myosin IXB (Myo9b), a motorized negative regulator of RhoA signaling, exhibit increased Rho signaling activity and downstream acto-myosin contractility, and inactivation of the Rho target protein cofilin, an actin-depolymerizing factor. On a functional level, Myo9b(-/-) DCs showed impaired directed migratory activity both in vitro and in vivo. Moreover, despite unaltered Ag presentation and costimulatory capabilities, Myo9b(-/-) DCs were poor T cell stimulators in vitro in a three-dimensional collagen matrix and in vivo, associated with altered DC-T cell contact dynamics and T cell polarization. Accordingly, Myo9b(-/-) mice showed an attenuated ear-swelling response in a model of contact hypersensitivity. The impaired migratory and T cell stimulatory capacity of Myo9b(-/-) DCs was restored in large part by pharmacological activation of cofilin. Taken together, these results identify Myo9b as a negative key regulator of the Rho/RhoA effector Rho-kinase [Rho-associated coiled-coil-forming kinase (ROCK)]/LIM domain kinase signaling pathway in DCs, which controls cofilin inactivation and myosin II activation and, therefore may control, in part, the induction of adaptive immune responses.

TRPC6 regulates CXCR2-mediated chemotaxis of murine neutrophils.

Unraveling the mechanisms involved in chemotactic navigation of immune cells is of particular interest for the development of new immunoregulatory therapies. It is generally agreed upon that members of the classical transient receptor potential channel family (TRPC) are involved in chemotaxis. However, the regulatory role of TRPC channels in chemoattractant receptor-mediated signaling has not yet been clarified in detail. In this study, we demonstrate that the TRPC6 channels play a pronounced role in CXCR2-mediated intermediary chemotaxis, whereas N-formyl-methionine-leucine-phenylalanine receptor-mediated end-target chemotaxis is TRPC6 independent. The knockout of TRPC6 channels in murine neutrophils led to a strongly impaired intermediary chemotaxis after CXCR2 activation which is not further reinforced by CXCR2, PI3K, or p38 MAPK inhibition. Furthermore, CXCR2-mediated Ca(2+) influx but not Ca(2+) store release was attenuated in TRPC6(-/-) neutrophils. We demonstrate that the TRPC6 deficiency affected phosphorylation of AKT and MAPK downstream of CXCR2 receptor activation and led to altered remodeling of actin. The relevance of this TRPC6-depending defect in neutrophil chemotaxis is underscored by our in vivo findings. A nonseptic peritoneal inflammation revealed an attenuated recruitment of neutrophils in the peritoneal cavity of TRPC6(-/-) mice. In summary, this paper defines a specific role of TRPC6 channels in CXCR2-induced intermediary chemotaxis. In particular, TRPC6-mediated supply of calcium appears to be critical for activation of downstream signaling components.

Roles of ion transport in control of cell motility.

Cell motility is an essential feature of life. It is essential for reproduction, propagation, embryonic development, and healing processes such as wound closure and a successful immune defense. If out of control, cell motility can become life-threatening as, for example, in metastasis or autoimmune diseases. Regardless of whether ciliary/flagellar or amoeboid movement, controlled motility always requires a concerted action of ion channels and transporters, cytoskeletal elements, and signaling cascades. Ion transport across the plasma membrane contributes to cell motility by affecting the membrane potential and voltage-sensitive ion channels, by inducing local volume changes with the help of aquaporins and by modulating cytosolic Ca(2+) and H(+) concentrations. Voltage-sensitive ion channels serve as voltage detectors in electric fields thus enabling galvanotaxis; local swelling facilitates the outgrowth of protrusions at the leading edge while local shrinkage accompanies the retraction of the cell rear; the cytosolic Ca(2+) concentration exerts its main effect on cytoskeletal dynamics via motor proteins such as myosin or dynein; and both, the intracellular and the extracellular H(+) concentration modulate cell migration and adhesion by tuning the activity of enzymes and signaling molecules in the cytosol as well as the activation state of adhesion molecules at the cell surface. In addition to the actual process of ion transport, both, channels and transporters contribute to cell migration by being part of focal adhesion complexes and/or physically interacting with components of the cytoskeleton. The present article provides an overview of how the numerous ion-transport mechanisms contribute to the various modes of cell motility.

Role of ion channels and transporters in cell migration.

Cell motility is central to tissue homeostasis in health and disease, and there is hardly any cell in the body that is not motile at a given point in its life cycle. Important physiological processes intimately related to the ability of the respective cells to migrate include embryogenesis, immune defense, angiogenesis, and wound healing. On the other side, migration is associated with life-threatening pathologies such as tumor metastases and atherosclerosis. Research from the last ≈ 15 years revealed that ion channels and transporters are indispensable components of the cellular migration apparatus. After presenting general principles by which transport proteins affect cell migration, we will discuss systematically the role of channels and transporters involved in cell migration.

Transient P2X7 receptor activation triggers macrophage death independent of Toll-like receptors 2 and 4, caspase-1, and pannexin-1 proteins.

The function of P2X(7) receptors (ATP-gated ion channels) in innate immune cells is unclear. In the setting of Toll-like receptor (TLR) stimulation, secondary activation of P2X(7) ion channels has been linked to pro-caspase-1 cleavage and cell death. Here we show that cell death is a surprisingly early triggered event. We show using live-cell imaging that transient (1-4 min) stimulation of mouse macrophages with high extracellular ATP ([ATP]e) triggers delayed (hours) cell death, indexed as DEVDase (caspase-3 and caspase-7) activity. Continuous or transient high [ATP]e did not induce cell death in P2X(7)-deficient (P2X(7)(-/-)) macrophages or neutrophils (in which P2X(7) could not be detected). Blocking sustained Ca(2+) influx, a signature of P2X(7) ligation, was highly protective, whereas no protection was conferred in macrophages lacking caspase-1 or TLR2 and TLR4. Furthermore, pannexin-1 (Panx1) deficiency had no effect on transient ATP-induced delayed cell death or ATP-induced Yo-Pro-1 uptake (an index of large pore pathway formation). Thus, "transient" P2X(7) receptor activation and Ca(2+) overload act as a death trigger for native mouse macrophages independent of Panx1 and pro-inflammatory caspase-1 and TLR signaling.