PubMed Health⌕ Search

Biomedical subjects

Sergio Grinstein

Publications and source records attributed to Sergio Grinstein.

At least 19 recordsLinked to original sources
PubMed2007

Membrane curvature alters the activation kinetics of the epithelial Na+/H+ exchanger, NHE3.

The epithelial Na(+)/H(+) exchanger, NHE3, was found to activate slowly following an acute cytosolic acidification. The sigmoidal course of activation could not be explained by the conventional two-state model, which postulates that activation results from protonation of an allosteric modifier site. Instead, mathematical modeling predicted the existence of three distinct states of the exchanger: two different inactive states plus an active form. The interconversion of the inactive states is rapid and dependent on pH, whereas the conversion between the second inactive state and the active conformation is slow and pH-independent but subject to regulation by other stimuli. Accordingly, exposure of epithelial cells to hypoosmolar solutions activated NHE3 by accelerating this latter transition. The number of surface-exposed exchangers and their association with the cytoskeleton were not affected by hypoosmolarity. Instead, NHE3 is activated by the membrane deformation, a result of cell swelling. This was suggested by the stimulatory effects of amphiphiles that induce a comparable positive (convex) deformation of the membrane. We conclude that NHE3 exists in multiple states and that different physiological parameters control the transitions between them.

Animals↗
PubMed2006

High-throughput assays of phagocytosis, phagosome maturation, and bacterial invasion.

Ingestion of foreign particles by macrophages and neutrophils and the fate of the vacuole that contains the ingested material are generally monitored by optical microscopy. Invasion of host cells by pathogenic bacteria and their intracellular proliferation are similarly studied by microscopy or by plating assays. These labor-intensive and time-consuming methods limit the number of assays that can be performed. The effort required to test multiple reagents or conditions can be prohibitive. We describe high-throughput assays of phagocytosis and of phagosomal maturation. An automated fluorescence microscope-based platform and associated analysis software were used to study Fcgamma receptor-mediated phagocytosis of IgG-opsonized particles by cultured murine macrophages. Phagosomal acidification was measured as an index of maturation. The same platform was similarly used to implement high-throughput assays of invasion of mammalian cells by pathogenic bacteria. The invasion of HeLa cells by Salmonella and the subsequent intracellular proliferation of the bacteria were measured rapidly and reliably in large populations of cells. These high-throughput methods are ideally suited for the efficient screening of chemical libraries to select potential drugs and of small interference RNA libraries to identify essential molecules involved in critical steps of the immune response.

Animals↗
PubMed2006

Phosphorylation-independent ubiquitylation and endocytosis of Fc gammaRIIA.

Endocytosis of the Fc receptor Fc gammaRIIA depends on a functional ubiquitin conjugation system, and the receptor becomes ubiquitylated upon ligand binding. Phosphorylation of tyrosines in Fc gammaRIIA by Src family kinases is thought to be the initiating event in its signaling. However, although the Src family kinase inhibitor PP1 inhibited both ligand-induced phosphorylation of Fc gammaRIIA and phagocytosis in ts20 cells expressing Fc gammaRIIA, it did not inhibit receptor ubiquitylation or endocytosis of soluble ligands. Conversely, genistein and the proteasomal inhibitor MG132 did not inhibit receptor phosphorylation but strongly inhibited both receptor ubiquitylation and endocytosis. A region of the receptor lying within the immunoreceptor tyrosine-based activation motif was found to be necessary for both ubiquitylation and endocytosis. Ubiquitylation occurs at the plasma membrane before internalization. Endocytosis of Fc gammaRIIA is dependent on clathrin but independent of the adaptor protein AP-2. These findings point to a novel mechanism for ubiquitylation and endocytosis of this immunoreceptor.

Adaptor Protein Complex 2↗
PubMed2006

The phosphoinositide-binding protein p40phox activates the NADPH oxidase during FcgammaIIA receptor-induced phagocytosis.

Superoxide produced by the phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is essential for host defense. Enzyme activation requires translocation of p67(phox), p47(phox), and Rac-GTP to flavocytochrome b558 in phagocyte membranes. To examine the regulation of phagocytosis-induced superoxide production, flavocytochrome b558, p47(phox), p67(phox), and the FcgammaIIA receptor were expressed from stable transgenes in COS7 cells. The resulting COS(phox)FcgammaR cells produce high levels of superoxide when stimulated with phorbol ester and efficiently ingest immunoglobulin (Ig)G-coated erythrocytes, but phagocytosis did not activate the NADPH oxidase. COS7 cells lack p40(phox), whose role in the NADPH oxidase is poorly understood. p40(phox) contains SH3 and phagocyte oxidase and Bem1p (PB1) domains that can mediate binding to p47(phox) and p67(phox), respectively, along with a PX domain that binds to phosphatidylinositol-3-phosphate (PI(3)P), which is generated in phagosomal membranes. Expression of p40(phox) was sufficient to activate superoxide production in COS(phox)FcgammaR phagosomes. FcgammaIIA-stimulated NADPH oxidase activity was abrogated by point mutations in p40(phox) that disrupt PI(3)P binding, or by simultaneous mutations in the SH3 and PB1 domains. Consistent with an essential role for PI(3)P in regulating the oxidase complex, phagosome NADPH oxidase activation in primary macrophages ingesting IgG-coated beads was inhibited by phosphatidylinositol 3 kinase inhibitors to a much greater extent than phagocytosis itself. Hence, this study identifies a role for p40(phox) and PI(3)P in coupling FcgammaR-mediated phagocytosis to activation of the NADPH oxidase.

Animals↗
PubMed2006

Intracellular survival of Burkholderia cenocepacia in macrophages is associated with a delay in the maturation of bacteria-containing vacuoles.

Strains of the Burkholderia cepacia complex (Bcc) are opportunistic bacteria that can cause life-threatening infections in patients with cystic fibrosis and chronic granulomatous disease. Previous work has shown that Bcc isolates can persist in membrane-bound vacuoles within amoeba and macrophages without bacterial replication, but the detailed mechanism of bacterial persistence is unknown. In this study, we have investigated the survival of the Burkholderia cenocepacia strain J2315 within RAW264.7 murine macrophages. Strain J2315 is a prototypic isolate of the widespread and transmissible ET12 clone. Unlike heat-inactivated bacteria, which reach lysosomes shortly after internalization, vacuoles containing live B. cenocepacia J2315 accumulate the late endosome/lysosome marker LAMP-1 and start fusing with lysosomal compartments only after 6 h post internalization. Using fluorescent fluid-phase probes, we also demonstrated that B. cenocepacia-containing vacuoles continued to interact with newly formed endosomes, and maintained a luminal pH of 6.4 +/- 0.12. In contrast, vacuoles containing heat-inactivated bacteria had an average pH of 4.8 +/- 0.03 and rapidly merged with lysosomes. Additional experiments using concanamycin A, a specific inhibitor of the vacuolar H+-ATPase, revealed that vacuoles containing live bacteria did not exclude the H+-ATPase. This mode of bacterial survival did not require type III secretion, as no differences were found between wild type and a type III secretion mutant strain. Collectively, our results suggest that intracellular B. cenocepacia cause a delay in the maturation of the phagosome, which may contribute to facilitate bacterial escape from the microbicidal activities of the host cell.

Animals↗
PubMed2006

Receptor activation alters inner surface potential during phagocytosis.

The surface potential of biological membranes varies according to their lipid composition. We devised genetically encoded probes to assess surface potential in intact cells. These probes revealed marked, localized alterations in the charge of the inner surface of the plasma membrane of macrophages during the course of phagocytosis. Hydrolysis of phosphoinositides and displacement of phosphatidylserine accounted for the change in surface potential at the phagosomal cup. Signaling molecules such as K-Ras, Rac1, and c-Src that are targeted to the membrane by electrostatic interactions were rapidly released from membrane subdomains where the surface charge was altered by lipid remodeling during phagocytosis.

Animals↗
PubMed2006

Signaling-dependent immobilization of acylated proteins in the inner monolayer of the plasma membrane.

Phospholipids play a critical role in the recruitment and activation of several adaptors and effectors during phagocytosis. Changes in lipid metabolism during phagocytosis are restricted to the phagocytic cup, the area of the plasmalemma lining the target particle. It is unclear how specific lipids and lipid-associated molecules are prevented from diffusing away from the cup during the course of phagocytosis, a process that often requires several minutes. We studied the mobility of lipid-associated proteins at the phagocytic cup by measuring fluorescence recovery after photobleaching. Lipid-anchored (diacylated) fluorescent proteins were freely mobile in the unstimulated membrane, but their mobility was severely restricted at sites of phagocytosis. Only probes anchored to the inner monolayer displayed reduced mobility, whereas those attached to the outer monolayer were unaffected. The immobilization persisted after depletion of plasmalemmal cholesterol, ruling out a role of conventional "rafts." Corralling of the probes by the actin cytoskeleton was similarly discounted. Instead, the change in mobility required activation of tyrosine kinases. We suggest that signaling-dependent recruitment of adaptors and effectors with lipid binding domains generates an annulus of lipids with restricted mobility.

Actins↗
PubMed2006

Lipid metabolism and dynamics during phagocytosis.

Phagocytosis, the engulfment of particles, mediates the elimination of invading pathogens as well as the clearance of apoptotic cells. Ingested particles reside within a vacuole or phagosome, where they are eventually destroyed and digested. The phagosomal lumen acquires microbicidal and digestive properties through interaction with various components of the endocytic pathway, a process known as maturation. Lipids are known to have numerous roles in phagosome formation and maturation; recent developments in the design of lipid-specific probes and in high-resolution imaging have revealed that lipids, notably phosphoinositides, are involved in signaling, actin assembly and the recruitment of molecular motors to sites of ingestion. In addition, phosphoinositides and other lipids also regulate multiple membrane budding, fission and fusion events required for maturation.

Animals↗
PubMed2006

CD44 is a phagocytic receptor.

CD44, a transmembrane adhesion molecule involved in binding and metabolism of hyaluronan, has additional functions in inflammatory and immune responses, contributing to the ingestion and clearance of particles and apoptotic cells. Our goal was to determine the specific role of CD44 in phagocytosis and whether it functions as a primary or accessory phagocytic receptor. Using hyaluronan-coated beads and erythrocytes coated with antiCD44 antibodies as the phagocytic prey, we determined that CD44 mediates efficient phagocytosis in primary murine peritoneal macrophages and in the murine macrophage cell line RAW 264.7. In RAW cells, the phagocytic index for anti-CD44-coated erythrocytes was 25 +/- 3 (mean +/- SEM) compared with less than 1 for erythrocytes coated with isotype-matched control antibodies. Uptake of anti-CD44-coated erythrocytes was abrogated by pretreatment with a blocking antibody to CD44 and was absent in primary cultures of CD44-deficient murine macrophages. Down-regulation of Fc receptors by aggregated IgG-induced internalization, which blocks uptake of IgG-coated particles, had no effect on CD44-mediated particle engulfment. Using a combination of immunoprecipitation, pharmacologic inhibition, and genetic deletion, we determined that CD44-mediated phagocytosis involves Syk, Rac1, and phosphatidylinositol 3-kinase and induced activation of the phagocyte oxidase. We conclude that CD44 is a competent phagocytic receptor that efficiently mediates internalization of large particles.

Animals↗
PubMed2006

The Unc93b1 mutation 3d disrupts exogenous antigen presentation and signaling via Toll-like receptors 3, 7 and 9.

Here we have identified 'triple D' (3d), a recessive N-ethyl-N-nitrosourea-induced mutation and phenotype in which no signaling occurs via the intracellular Toll-like receptors 3, 7 and 9 (sensors for double-stranded RNA, single-stranded RNA and unmethylated DNA, respectively). The 3d mutation also prevented cross-presentation and diminished major histocompatibility complex class II presentation of exogenous antigen; it also caused hypersusceptibility to infection by mouse cytomegalovirus and other microbes. By positional identification, we found 3d to be a missense allele of Unc93b1, which encodes the 12-membrane-spanning protein UNC-93B, a highly conserved molecule found in the endoplasmic reticulum with multiple paralogs in mammals. Innate responses to nucleic acids and exogenous antigen presentation, which both initiate in endosomes, thus seem to depend on an endoplasmic reticulum-resident protein, which suggests communication between these organellar systems.

Animals↗
PubMed2006

The ESAT-6/CFP-10 secretion system of Mycobacterium marinum modulates phagosome maturation.

Virulence of Mycobacterium tuberculosis and related pathogenic mycobacteria requires the secretion of early secretory antigenic 6 kDa (ESAT-6) and culture filtrate protein 10 (CFP-10), two small proteins that lack traditional signal sequences and are exported through an alternative secretion pathway encoded primarily by the RD1 genetic locus. Mutations affecting the synthesis or secretion of ESAT-6 or CFP-10 attenuate the virulence of M. tuberculosis in murine models of infection. However, the specific functions of these proteins and of their secretion system are currently unclear. In this study, we isolated a mutant of Mycobacterium marinum defective in the secretion of ESAT-6 and CFP-10. The mutation was localized within MM5446, which is orthologous to Rv3871 of M. tuberculosis H37Rv and encodes an ATPase that is a component of the ESAT-6/CFP-10 secretion system. The mutant bacteria were unable to replicate within J774 macrophages although their growth in 7H9 medium was equivalent to the parental strain. Phagosome maturation and acidification were analysed in infected macrophages by confocal and electron microscopy using the late endosome/lysosome marker LAMP-1, along with various fluid-phase markers such as rhodamine-dextran and ferritin and the acidotropic dye LysoTracker Red. These studies demonstrated that while the wild-type parental strain of M. marinum primarily resides in a poorly acidified, non-lysosomal compartment, a significantly higher percentage of the MM5446 mutant organisms are in acidified compartments. These results suggest that the ESAT-6/CFP-10 secretion system plays a role in preventing phagolysosomal fusion, a novel function that accounts for the ability of bacteria to survive inside host cells. This finding provides a mechanism by which the ESAT-6/CFP-10 secretion system potentiates the virulence of pathogenic mycobacteria.

Amines↗
PubMed2006

Activation of kinases upon volume changes: role in cellular homeostasis.

The cell is constantly encountering stimuli or conditions that can induce alterations to cell volume. Despite these challenges, cell volume remains relatively constant, by virtue of a series of complex and often redundant regulatory mechanisms. Acutely, an efflux of ions and organic osmolytes counteracts the tendency of cells to swell. Conversely when cells are exposed to perturbations that cause shrinkage, ions are rapidly imported. Chronic hypertonic stimulation increases the transcription of multiple genes whose products promote the intracellular accumulation of organic osmolytes. How the cell perceives these acute alterations in volume in order to signal the activation of specific channels, transporters and transcription factors has been the focus of a large body of research. In this article, we describe the central role that kinases play in mediating the cellular responses to alterations in cell volume. Although incompletely understood, these fundamental processes are key to cellular homeostasis and have obvious clinical implications.

Adaptation, Physiological↗
PubMed2005

Abnormalities in the pulmonary innate immune system in cystic fibrosis.

Pulmonary infection is the dominant clinical feature of cystic fibrosis (CF), but the basis for this susceptibility remains incompletely understood. One hypothesis is that CF airway surface liquid (ASL) is abnormal and interferes with neutrophil function. To study this possibility, we developed an in vitro system in which we collected ASL from primary cultures of normal and CF airway epithelial cells. Microbial killing was less efficient when bacteria were incubated with neutrophils in the presence of ASL from CF epithelia compared with normal ASL. Antimicrobial functions of human neutrophils were assessed in ASL from CF and normal epithelia using a combination of quantitative bacterial culture, flow cytometry, and microfluorescence imaging. The results of these assays of neutrophil function were indistinguishable in CF and normal ASL. In contrast, the direct bactericidal activity of ASL to Escherichia coli and to clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa was substantially less in CF than in normal ASL, even when highly diluted in media of identical ionic strength. Together, these observations indicate that the antimicrobial properties of ASL in CF are compromised in a manner independent of ionic strength of the ASL, and that this effect is not mediated through a direct effect of the ASL on phagocyte function.

Bronchi↗
PubMed2005

Visualization of vacuolar acidification-induced transcription of genes of pathogens inside macrophages.

The objective of these studies was to analyze the role of the ionic environment of phagosomal vacuoles in the control of pathogens by macrophages. Digital imaging and flow cytometry were used to follow the induction of the phoP promoter of Salmonella enterica Typhimurium within live macrophages. Manipulating the Mg2+ concentration within the Salmonella-containing vacuole (SCV) was without effect on the early induction of PhoPQ. Moreover, direct measurement of [Mg2+] within the SCV using nanosensor particles showed that, during this initial period of phoP activation, the concentration of the divalent cation is rapidly regulated and stabilizes around 1 mm. Extrusion of other divalent cations via the Nramp1 efflux pump was similarly ruled out as an important contributor to the activation of the regulon. By contrast, induction of PhoP was greatly attenuated when the pH gradient across the SCV membrane was dissipated. A second, more modest pH-independent component of PhoP induction was unmasked by inhibition of the vacuolar proton pump. This second component was eliminated by pretreatment of cells with IFNgamma, even though the cytokine augmented the overall PhoP response. These findings demonstrate the existence of at least three separate activators of phoP transcription: resting and IFNgamma-stimulated pH-sensitive components, plus a pH-independent component.

Acids↗
PubMed2005

Quantitative and dynamic assessment of the contribution of the ER to phagosome formation.

Phagosomes were traditionally thought to originate from an invagination and scission of the plasma membrane to form a distinct intracellular vacuole. An alternative model implicating the endoplasmic reticulum (ER) as a major component of nascent and maturing phagosomes was recently proposed (Gagnon et al., 2002). To reconcile these seemingly disparate hypotheses, we used a combination of biochemical, fluorescence imaging, and electron microscopy techniques to quantitatively and dynamically assess the contribution of the plasmalemma and of the ER to phagosome formation and maturation. We could not verify even a transient physical continuity between the ER and the plasma membrane, nor were we able to detect a significant contribution of the ER to forming or maturing phagosomes in either macrophages or dendritic cells. Instead, our data indicate that the plasma membrane is the main constituent of nascent and newly formed phagosomes, which are progressively remodeled by fusion with endosomal and eventually lysosomal compartments as phagosomes mature into acidic, degradative organelles.

Animals↗
PubMed2005

Carboxyl-terminus determinants of the iron transporter DMT1/SLC11A2 isoform II (-IRE/1B) mediate internalization from the plasma membrane into recycling endosomes.

Mutations in DMT1 (Nramp2 and Slc11a2) impair iron metabolism and cause microcytic anemia. DMT1 is expressed at the duodenal brush border where it controls uptake of dietary iron and is present at the plasma membrane and in recycling endosomes of most cells, where it is necessary for acquisition of transferrin-associated iron. The goal of this study was to identify signal(s) in the cytoplasmic segments of DMT1 responsible for its subcellular targeting and internalization from the plasma membrane into recycling endosomes. We introduced mutations in the amino terminus (DeltaNT), carboxyl terminus (DeltaCT), as well as in NPAY28-31, YSCF62-65, and YLLNT555-559 motifs of a DMT1 construct bearing an exofacial epitope tag, which allowed labeling of the transporter at the cell surface for kinetic studies. Mutants were stably expressed in LLC-PK1 kidney cells and were studied for transport activity, subcellular localization, cell-surface and recycling pool distribution, and internalization from the plasma membrane. Kinetic studies showed that carboxyl-terminus mutants (DeltaCT and DeltaYLLNT) had an increased fraction of the "recycling pool" that was expressed at the cell surface because of impaired internalization from the plasma membrane. Further cell-surface-labeling and immunofluorescence studies in intact cells showed that the DeltaYLLNT and DeltaCT mutants were targeted to the lysosomal compartment upon internalization. These results suggest that the major signal for internalization and recycling of DMT1 isoform II (-IRE/1B) resides in its carboxyl terminus and that removal of this signal leads to a default lysosomal targeting.

Amino Acid Sequence↗
PubMed2005

Rho GTPases dictate the mobility of the Na/H exchanger NHE3 in epithelia: role in apical retention and targeting.

Proximal tubular reabsorption of filtered sodium by the sodium/hydrogen exchanger isoform 3 (NHE3), located on the apical membrane, is fundamental to the maintenance of systemic volume and pH homeostasis. NHE3 is finely regulated by a variety of hormones and by changes in ionic composition and volume, likely requiring redistribution of the exchangers. We analyzed the subcellular distribution and dynamics of the exchangers by generating an epithelial line expressing NHE3 tagged with an exofacial epitope, which enabled us to monitor exchanger mobility and traffic in intact cells. Using determinations of fluorescence recovery after photobleaching in combination with dynamic measurements of subcellular distribution, we found that, in renal epithelial cells, NHE3 exists in four distinct subcompartments: a virtually immobile subpopulation that is retained on the apical membrane by interaction with the actin cytoskeleton in a manner that depends on the sustained activity of Rho GTPases; a mobile subpopulation on the apical membrane, which can be readily internalized; and two intracellular compartments that can be differentiated by their rate of exchange with the apical pool of NHE3. We provide evidence that detachment of the immobile fraction from its cytoskeletal anchorage leads to rapid internalization. These observations suggest that modulation of the mobile fraction of NHE3 on the apical membrane can alter the number of functional exchangers on the cell surface and, consequently, the rate of transepithelial ion transport. Regulation of the interaction of NHE3 with the actin cytoskeleton can therefore provide a new mode of regulation of sodium and hydrogen transport.

Amiloride↗