RESUMO
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.
Assuntos
Membrana Celular/metabolismo , Antígeno de Macrófago 1/metabolismo , Macrófagos/metabolismo , Pseudópodes/metabolismo , Quinase Syk/metabolismo , Animais , Células Cultivadas , Humanos , Motivo de Ativação do Imunorreceptor Baseado em Tirosina , Camundongos , Camundongos Knockout , Fagocitose , Transdução de SinaisRESUMO
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.
RESUMO
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.
Assuntos
Sinalização do Cálcio , Quimiotaxia , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Macrófagos/metabolismo , Modelos Biológicos , Receptor da Anafilatoxina C5a/metabolismo , Animais , Proteínas Heterotriméricas de Ligação ao GTP/genética , Camundongos Knockout , Receptor da Anafilatoxina C5a/genéticaRESUMO
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.
Assuntos
Miocárdio/metabolismo , Miosinas/metabolismo , Sarcômeros/metabolismo , Animais , Deleção de Genes , Genes Letais , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Camundongos Knockout , Miosinas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
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.
Assuntos
Proteína Quinase CDC2/fisiologia , Miosinas/fisiologia , Fagocitose , Pseudópodes/metabolismo , Animais , Proteína Quinase CDC2/genética , Quimiotaxia , Deleção de Genes , Genótipo , Proteínas de Fluorescência Verde/metabolismo , Concentração de Íons de Hidrogênio , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Knockout , Microscopia Confocal , Mutação , Miosinas/genética , Miosinas/metabolismo , Fenótipo , Saccharomyces cerevisiae/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Macrófagos Peritoneais/enzimologia , Pseudópodes/patologia , Proteínas ras/genética , Proteínas rho de Ligação ao GTP/genética , Proteína rhoB de Ligação ao GTP/genética , Animais , Polaridade Celular , Células Cultivadas , Quimiotaxia , Feminino , Expressão Gênica , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Knockout , Miosinas/genética , Peritonite/enzimologia , Peritonite/patologia , Pseudópodes/enzimologia , Proteínas ras/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP , Proteína rhoB de Ligação ao GTP/metabolismo , Proteína de Ligação a GTP rhoCRESUMO
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.
Assuntos
Quimiotaxia , Macrófagos/citologia , Imagem com Lapso de Tempo/métodos , Animais , Dictyostelium/citologia , Macrófagos/metabolismo , Camundongos , Monócitos/citologia , Neutrófilos/citologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de SinaisRESUMO
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).
Assuntos
Encéfalo/metabolismo , Miosinas/genética , Animais , Encéfalo/diagnóstico por imagem , Membrana Celular/metabolismo , Células Endoteliais/metabolismo , Genótipo , Células HEK293 , Humanos , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Imageamento por Ressonância Magnética , Camundongos , Camundongos Knockout , Miosinas/química , Miosinas/metabolismo , Fagocitose , Fenótipo , Isoformas de Proteínas/metabolismo , Pseudópodes/metabolismo , Pele/metabolismo , Pele/patologiaRESUMO
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.