Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling

Inhibitors of Na+/H+ exchange proteins block macropinocytosis by lowering the pH near the plasma membrane, which in turn inhibits actin remodeling by Rho family GTPases

Recycling of the Membrane-anchored Chemokine, CX 3 CL1

CX(3)CL1 (fractalkine) plays an important role in inflammation by acting as both chemoattractant and as an adhesion molecule. As for other chemokines, expression of CX(3)CL1 is known to be regulated at the level of transcription and translation. The unique transmembrane structure of CX(3)CL1 raises the possibility of additional functional regulation by altering its abundance at the cell surface. This could be accomplished in principle by changes in traffic between subcellular compartments. To analyze this possibility we examined the subcellular distribution of CX(3)CL1 in human ECV-304 cells stably expressing untagged or green fluorescent protein-tagged forms of the chemokine. CX(3)CL1 was present in two distinct compartments, diffusely on the plasma membrane and in a punctate juxtanuclear compartment. The latter shared some features with, yet was distinct from the conventional endocytic pathway and may represent a specialized recycling subcompartment. Accordingly, surface CX(3)CL1 was found to be in dynamic equilibrium with the juxtanuclear vesicular compartment. Intracellular CX(3)CL1 co-localized with the SNARE (soluble N-ethylmaleimide factor attachment protein receptor) proteins syntaxin-13 and VAMP-3. Cleavage of VAMP-3 by tetanus toxin or impairment of syntaxin-13 function by expression of a dominant-negative allele inhibited the ability of internalized CX(3)CL1 to traffic back to the plasma membrane. These data demonstrate the existence of a dynamic, SNARE-mediated recycling of CX(3)CL1 from the cell surface to and from an endomembrane storage compartment. The intracellular storage depot may serve as a source of the chemokine that could be rapidly mobilized by stimuli

Quantitative and Dynamic Assessment of the Contribution of the ER to Phagosome Formation

SummaryPhagosomes 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

VSOP/Hv1 proton channels sustain calcium entry, neutrophil migration, and superoxide production by limiting cell depolarization and acidification

Neutrophils kill microbes with reactive oxygen species generated by the NADPH oxidase, an enzyme which moves electrons across membranes. Voltage-gated proton channels (voltage-sensing domain only protein [VSOP]/Hv1) are required for high-level superoxide production by phagocytes, but the mechanism of this effect is not established. We show that neutrophils from VSOP/Hv1−/− mice lack proton currents but have normal electron currents, indicating that these cells have a fully functional oxidase that cannot conduct protons. VSOP/Hv1−/− neutrophils had a more acidic cytosol, were more depolarized, and produced less superoxide and hydrogen peroxide than neutrophils from wild-type mice. Hydrogen peroxide production was rescued by providing an artificial conductance with gramicidin. Loss of VSOP/Hv1 also aborted calcium responses to chemoattractants, increased neutrophil spreading, and decreased neutrophil migration. The migration defect was restored by the addition of a calcium ionophore. Our findings indicate that proton channels extrude the acid and compensate the charge generated by the oxidase, thereby sustaining calcium entry signals that control the adhesion and motility of neutrophils. Loss of proton channels thus aborts superoxide production and causes a severe signaling defect in neutrophils

Etude des relations structure-fonction de l'échangeur Na+ / H+ (NHE-1)

L'isoforme 1 de l'échangeur Na+/H+, qui est exprimée de façon ubiquitaire dans l'organisme, fait partie d'une famille de protéines membranaires qui catalyse, grâce au gradient électrochimique du sodium, l'échange électroneutre d'un proton intracellulaire contre un sodium extracellulaire. Cette protéine est impliquée dans la régulation du pH intracellulaire et du volume cellulaire, mais est aussi responsable de certaines situations pathologiques. Ce transporteur est constitué de deux domaines fonctionnels distincts sur lesquels mes travaux de thèse ont été menés. Le domaine en N-terminal, correspondant à la région transmembranaire de l'échangeur, est suffisant pour catalyser l'échange des cations. L'utilisation de techniques de sélection génétique, basées sur la résistance de cellules à des acidifications intracellulaires, nous a permis d'isoler des clones cellulaires exprimant des formes mutées de cette protéine. Nous avons mis en évidence le rôle important de plusieurs résidus, dans le quatrième segment transmembranaire, engagés dans le site de fixation du sodium. La seconde partie de mon travail concerne le domaine cytoplasmique de l'échangeur qui est impliqué dans la régulation de son activité par des cascades de signalisation. La surexpression de ce domaine cytoplasmique atténue l'activation de l'échangeur Na+/H+ et d'un canal chlorure par les facteurs de croissance. Cette inhibition semble également provoquer un retard dans l'induction du cycle cellulaire par ces facteurs mitogéniques. Cette protéine serait un outil biochimique intéressant pour caractériser ces voies de signalisation et identifier leurs cibles.NICE-BU Sciences (060882101) / SudocSudocFranceF