Biomedical subjects

Victor Faundez

Publications and source records attributed to Victor Faundez.

16 recordsLinked to original sources

PubMed2006

Architecture of the vimentin cytoskeleton is modified by perturbation of the GTPase ARF1.

Intermediate filaments are required for proper membrane protein trafficking. However, it remains unclear whether perturbations in vesicular membrane transport result in changes in the architecture of the vimentin cytoskeleton. We find that treatment of cells with Brefeldin A, an inhibitor of specific stages of membrane transport, causes changes in the organization of vimentin filaments. These changes arise from movement of pre-existing filaments. Brefeldin A treatment also leads to alterations in the microtubule cytoskeleton. However, this effect is not observed in cells lacking intermediate filaments, indicating that microtubule bundling is downstream of perturbations in the vimentin cytoskeleton. Brefeldin A-induced changes in vimentin architecture are probably mediated through its effects on ADP-ribosylation factor 1 (ARF1). Expression of a dominant-negative mutant of ARF1 induces BFA-like modifications in vimentin morphology. The BFA-dependent changes in vimentin architecture occurred concurrently with the release of the ARF1-regulated adaptor complexes AP-3 and AP-1 from membranes and adaptor redistribution to vimentin networks. These observations indicate that perturbation of the vesicular membrane transport machinery lead to reciprocal changes in the architecture of vimentin networks.

ADP-Ribosylation Factor 1↗

PubMed2006

Anthrax toxin receptor 1/tumor endothelium marker 8 mediates cell spreading by coupling extracellular ligands to the actin cytoskeleton.

Tumor endothelial marker 8 (TEM8) is induced in tumor-associated vasculature and acts as a receptor for Protective Antigen (PA), the cell-binding component of the anthrax toxin determinant for toxin entrance into cells. However, the normal function for TEM8 remains unknown. We show that TEM8 functions as an adhesion molecule mediating cell spreading on immobilized PA and collagen I. The mechanism for TEM8 interaction with collagen I was cell type-specific, because binding to collagen I was abrogated by beta1 integrin function blocking antibody in HEK293 cells, but not in primary synovial rabbit fibroblasts. Binding to PA remained unaffected by the addition of beta1 integrin function blocking antibody. Whereas the extracellular and transmembrane domains of TEM8 were sufficient to provide cell attachment, the intracellular domain was critical for spreading. Fusion of the cytosolic domain of TEM8 to the IL-2 receptor, conferred cell-spreading capability on IL-2 receptor antibody substrates. The cytoplasmic domain mediated linkage with the actin cytoskeleton as it co-precipitated actin and determined partitioning of TEM8 to the actin-containing detergent insoluble cellular fraction. TEM8 anchorage to actin was relevant as spreading was inhibited by the cytoskeleton-disrupting drug cytochalasin D, but persisted in the presence of the microtubule-depolymerizing drug nocodazole, and in cells lacking intermediate filaments. Thus, our results indicate that TEM8 is a new adhesion molecule linking collagen I or PA to the actin cytoskeleton.

Actins↗

PubMed2006

A mutation of beta -actin that alters depolymerization dynamics is associated with autosomal dominant developmental malformations, deafness, and dystonia.

Actin, one of the major filamentous cytoskeletal molecules, is involved in a variety of cellular functions. Whereas an association between muscle actin mutations and skeletal and cardiac myopathies has been well documented, reports of human disease arising from mutations of nonmuscle actin genes have been rare. We have identified a missense point mutation in the gene coding for beta -actin that results in an arginine-to-tryptophan substitution at position 183. The disease phenotype includes developmental midline malformations, sensory hearing loss, and a delayed-onset generalized dystonia syndrome in monozygotic twins. Cellular studies of a lymphoblastoid cell line obtained from an affected patient demonstrated morphological abnormalities of the actin cytoskeleton and altered actin depolymerization dynamics in response to latrunculin A, an actin monomer-sequestering drug. Resistance to latrunculin A was also observed in NIH 3T3 cells expressing the mutant actin. These findings suggest that mutations in nonmuscle actins may be associated with a broad spectrum of developmental malformations and/or neurological abnormalities such as dystonia.

Actins↗

PubMed2005

Neuronal zinc stores are modulated by non-steroidal anti-inflammatory drugs: an optical analysis in cultured hippocampal neurons.

Zinc chelation and non-steroidal anti-inflammatory drugs (NSAIDs) have been explored as potential neuroprotective agents. However, it remains unknown whether NSAIDs and zinc chelation may converge on a similar cellular process. Using two-photon microscopy to observe hippocampal neurons labeled with a zinc-sensitive dye, we provide evidence that three chemically unrelated NSAIDs, niflumic acid, ibuprofen, and naproxen, acutely increase intracellular zinc stores from extracellular metal pools. Phospholipase A2 inhibitors triggered similar responses, suggesting that NSAIDs likely control zinc stores by their activity as cyclooxygenase inhibitors. These results provide evidence for a new link between cyclooxygenase metabolites and the mechanisms controlling neuronal zinc pools.

Acetylcysteine↗

PubMed2005

p120-Catenin regulates clathrin-dependent endocytosis of VE-cadherin.

VE-cadherin is an adhesion molecule critical to vascular barrier function and angiogenesis. VE-cadherin expression levels are regulated by p120 catenin, which prevents lysosomal degradation of cadherins by unknown mechanisms. To test whether the VE-cadherin cytoplasmic domain mediates endocytosis, and to elucidate the nature of the endocytic machinery involved, the VE-cadherin tail was fused to the interleukin (IL)-2 receptor (IL-2R) extracellular domain. Internalization assays demonstrated that the VE-cadherin tail dramatically increased endocytosis of the IL-2R in a clathrin-dependent manner. Interestingly, p120 inhibited VE-cadherin endocytosis via a mechanism that required direct interactions between p120 and the VE-cadherin cytoplasmic tail. However, p120 did not inhibit transferrin internalization, demonstrating that p120 selectively regulates cadherin internalization rather than globally inhibiting clathrin-dependent endocytosis. Finally, cell surface labeling experiments in cells expressing green fluorescent protein-tagged p120 indicated that the VE-cadherin-p120 complex dissociates upon internalization. These results support a model in which the VE-cadherin tail mediates interactions with clathrin-dependent endocytic machinery, and this endocytic processing is inhibited by p120 binding to the cadherin tail. These findings suggest a novel mechanism by which a cytoplasmic binding partner for a transmembrane receptor can serve as a selective plasma membrane retention signal, thereby modulating the availability of the protein for endo-lysosomal processing.

Adolescent↗

PubMed2005

Phosphatidylinositol-4-kinase type II alpha is a component of adaptor protein-3-derived vesicles.

A membrane fraction enriched in vesicles containing the adaptor protein (AP) -3 cargo zinc transporter 3 was generated from PC12 cells and was used to identify new components of these organelles by mass spectrometry. Proteins prominently represented in the fraction included AP-3 subunits, synaptic vesicle proteins, and lysosomal proteins known to be sorted in an AP-3-dependent way or to interact genetically with AP-3. A protein enriched in this fraction was phosphatidylinositol-4-kinase type IIalpha (PI4KIIalpha). Biochemical, pharmacological, and morphological analyses supported the presence of PI4KIIalpha in AP-3-positive organelles. Furthermore, the subcellular localization of PI4KIIalpha was altered in cells from AP-3-deficient mocha mutant mice. The PI4KIIalpha normally present both in perinuclear and peripheral organelles was substantially decreased in the peripheral membranes of AP-3-deficient mocha fibroblasts. In addition, as is the case for other proteins sorted in an AP-3-dependent way, PI4KIIalpha content was strongly reduced in nerve terminals of mocha hippocampal mossy fibers. The functional relationship between AP-3 and PI4KIIalpha was further explored by PI4KIIalpha knockdown experiments. Reduction of the cellular content of PI4KIIalpha strongly decreased the punctate distribution of AP-3 observed in PC12 cells. These results indicate that PI4KIIalpha is present on AP-3 organelles where it regulates AP-3 function.

Adaptor Protein Complex 3↗

PubMed2005

Vglut1 and ZnT3 co-targeting mechanisms regulate vesicular zinc stores in PC12 cells.

The lumenal ionic content of an organelle is determined by its complement of channels and transporters. These proteins reach their resident organelles by adaptor-dependent mechanisms. This concept is illustrated in AP-3 deficiencies, in which synaptic vesicle zinc is depleted because the synaptic-vesicle-specific zinc transporter 3 does not reach synaptic vesicles. However, whether zinc transporter 3 is the only membrane protein defining synaptic-vesicle zinc content remains unknown. To address this question, we examined whether zinc transporter 3 and the vesicular glutamate transporter Vglut1 (a transporter that coexists with zinc transporter 3 in brain nerve terminals) were co-targeted to synaptic-like microvesicle fractions in PC12 cells. Deconvolution microscopy and subcellular fractionation demonstrated that these two transporters were present on the same vesicles in PC12 cells. Vglut1 content in synaptic-like microvesicle fractions and brain synaptic vesicles was partially sensitive to pharmacological and genetic perturbation of AP-3 function. Whole-cell flow-cytometry analysis of PC12 cell lines expressing zinc transporter 3, Vglut1 or both showed that vesicular zinc uptake was increased by Vglut1 expression. Conversely, production of zinc transporter 3 increased the vesicular uptake of glutamate in a zinc-dependent fashion. Our results suggest that the coupling of zinc transporter 3 and Vglut1 transport mechanisms regulates neurotransmitter content in secretory vesicles.

Animals↗

PubMed2005

Intermediate filaments and vesicular membrane traffic: the odd couple's first dance?

During the last two decades, much attention has been focused on the regulation of membrane traffic by the actin and microtubule cytoskeletal networks. Their dynamic and polarized behavior and associated motors provide a logical framework from which architectural and movement cues can be communicated to organelles. The study of these cytoskeletal systems has been greatly aided by pharmacological agents. In contrast, intermediate filaments (IFs) have largely been neglected as a potential player in membrane traffic, both because a comprehensive pharmacology to perturb them does not exist and because they lack the intrinsic polarity and specific motors that make the other cytoskeletal systems attractive. In this review, we will discuss evidence suggesting that IFs may play roles in controlling organelle positioning and in membrane protein targeting. Furthermore, we will discuss potential mechanisms by which IFs may regulate the localization and function of organelles.

Adaptor Protein Complex 3↗

PubMed2005

Saccharomyces cerevisiae Npc2p is a functionally conserved homologue of the human Niemann-Pick disease type C 2 protein, hNPC2.

Niemann-Pick Disease Type C (NP-C) is a fatal neurodegenerative disease, which is biochemically distinguished by the lysosomal accumulation of exogenously derived cholesterol. Mutation of either the hNPC1 or hNPC2 gene is causative for NP-C. We report the identification of the yeast homologue of human NPC2, Saccharomyces cerevisiae Npc2p. We demonstrate that scNpc2p is evolutionarily related to the mammalian NPC2 family of proteins. We also show, through colocalization, subcellular fractionation, and secretion analyses, that yeast Npc2p is treated similarly to human NPC2 when expressed in mammalian cells. Importantly, we show that yeast Npc2p can efficiently revert the unesterified cholesterol and GM1 accumulation seen in hNPC2-/- patient fibroblasts demonstrating that it is a functional homologue of human NPC2. The present study reveals that the fundamental process of NPC2-mediated lipid transport has been maintained throughout evolution.

Amino Acid Sequence↗

PubMed2004

The endo-lysosomal sorting machinery interacts with the intermediate filament cytoskeleton.

Cytoskeletal networks control organelle subcellular distribution and function. Herein, we describe a previously unsuspected association between intermediate filament proteins and the adaptor complex AP-3. AP-3 and intermediate filament proteins cosedimented and coimmunoprecipitated as a complex free of microtubule and actin binding proteins. Genetic perturbation of the intermediate filament cytoskeleton triggered changes in the subcellular distribution of the adaptor AP-3 and late endocytic/lysosome compartments. Concomitant with these architectural changes, and similarly to AP-3-null mocha cells, fibroblasts lacking vimentin were compromised in their vesicular zinc uptake, their organellar pH, and their total and surface content of AP-3 cargoes. However, the total content and surface levels, as well as the distribution of the transferrin receptor, a membrane protein whose sorting is AP-3 independent, remained unaltered in both AP-3- and vimentin-null cells. Based on the phenotypic convergence between AP-3 and vimentin deficiencies, we predicted and documented a reduced autophagosome content in mocha cells, a phenotype previously reported in cells with disrupted intermediate filament cytoskeletons. Our results reveal a novel role of the intermediate filament cytoskeleton in organelle/adaptor positioning and in regulation of the adaptor complex AP-3.

Adaptor Protein Complex 3↗

PubMed2004

Teaching resources. Chloride concentration and pH along the endosomal pathway.

This animation depicts three membrane trafficking routes in a mammalian neurosecretory cell, highlighting the changing Cl- concentrations and pH values of the various endosomal subcompartments. The focus is on the endocytotic pathway and the different paths taken by clathrin-coated vesicles: the pathway for receptor recycling to the plasma membrane, the lysosomal pathway, or the pathway for synaptic vesicle recycling (in neurons). Each compartment shown has a distinctive pH and [Cl-], with values for pH indicated in blue and values for Cl- indicated in orange. This animation will be useful for teaching cell biology and highlighting both the dynamic nature of vesicular traffic and the changes in ionic balance associated with different cellular compartments.

Chlorides↗

PubMed2004

Intracellular chloride channels: determinants of function in the endosomal pathway.

Endosomes, and related subcellular compartments, contain various Cl- channels in the ClC family. In this review, we describe the known roles of intracellular Cl- channels and also explore some of the functional implications of transmembrane Cl- flux in these organelles. Cl- influx acts to control intralumenal pH, both by shunting the effects of the proton pump on membrane potential and, possibly, through direct effects of Cl- on the proton pump. Changes in intralumenal pH likely help regulate membrane trafficking. We propose that changes in intralumenal Cl- concentration ([Cl-]) could theoretically play a direct role in regulating membrane trafficking and organellar function through effects on chloride-sensitive proteins in the vesicular membrane, which could transduce information about intralumenal [Cl-] to the outside of the vesicle and thereby recruit various signaling molecules. We present a model in which regulation of cytosolic [Cl-] and vesicular Cl- conductance could help control the amount or type of neurotransmitter stored in a particular population of synaptic vesicles.

Animals↗

PubMed2004

AP-3-dependent mechanisms control the targeting of a chloride channel (ClC-3) in neuronal and non-neuronal cells.

Adaptor protein (AP)-2 and AP-3-dependent mechanisms control the sorting of membrane proteins into synaptic vesicles. Mouse models deficient in AP-3, mocha, develop a neurological phenotype of which the central feature is an alteration of the luminal synaptic vesicle composition. This is caused by a severe reduction of vesicular levels of the zinc transporter 3 (ZnT3). It is presently unknown whether this mocha defect is restricted to ZnT3 or encompasses other synaptic vesicle proteins capable of modifying synaptic vesicle contents, such as transporters or channels. In this study, we identified a chloride channel, ClC-3, whose level in synaptic vesicles and hippocampal mossy fiber terminals was reduced in the context of the mocha AP-3 deficiency. In PC-12 cells, ClC-3 was present in transferrin receptor-positive endosomes, where it was targeted to synaptic-like microvesicles (SLMV) by a mechanism sensitive to brefeldin A, a signature of the AP-3-dependent route of SLMV biogenesis. ClC-3 was packed in SLMV along with the AP-3-targeted synaptic vesicle protein ZnT3. Co-segregation of ClC-3 and ZnT3 to common intracellular compartments was functionally significant as revealed by increased vesicular zinc transport with increased ClC3 expression. Our work has identified a synaptic vesicle protein in which trafficking to synaptic vesicles is regulated by AP-3. In addition, our findings indicate that ClC-3 and ZnT3 reside in a common vesicle population where they functionally interact to determine vesicle luminal composition.

Animals↗

PubMed2003

The zinc transporter ZnT3 interacts with AP-3 and it is preferentially targeted to a distinct synaptic vesicle subpopulation.

Synaptic vesicles (SV) are generated by two different mechanisms, one AP-2 dependent and one AP-3 dependent. It has been uncertain, however, whether these mechanisms generate SV that differ in molecular composition. We explored this hypothesis by analyzing the targeting of ZnT3 and synaptophysin both to PC12 synaptic-like microvesicles (SLMV) as well as SV isolated from wild-type and AP-3-deficient mocha brains. ZnT3 cytosolic tail interacted selectively with AP-3 in cell-free assays. Accordingly, pharmacological disruption of either AP-2- or AP-3-dependent SLMV biogenesis preferentially reduced synaptophysin or ZnT3 targeting, respectively; suggesting that these antigens were concentrated in different vesicles. As predicted, immuno-isolated SLMV revealed that ZnT3 and synaptophysin were enriched in different vesicle populations. Likewise, morphological and biochemical analyses in hippocampal neurons indicated that these two antigens were also present in distinct but overlapping domains. ZnT3 SV content was reduced in AP-3-deficient neurons, but synaptophysin was not altered in the AP-3 null background. Our evidence indicates that neuroendocrine cells assemble molecularly heterogeneous SV and suggests that this diversity could contribute to the functional variety of synapses.

Adaptor Protein Complex 3↗

PubMed2003

Cellular levels of p120 catenin function as a set point for cadherin expression levels in microvascular endothelial cells.

The mechanisms by which catenins regulate cadherin function are not fully understood, and the precise function of p120 catenin (p120ctn) has remained particularly elusive. In microvascular endothelial cells, p120ctn colocalized extensively with cell surface VE-cadherin, but failed to colocalize with VE-cadherin that had entered intracellular degradative compartments. To test the possibility that p120ctn binding to VE-cadherin regulates VE-cadherin internalization, a series of approaches were undertaken to manipulate p120ctn availability to endogenous VE-cadherin. Expression of VE-cadherin mutants that competed for p120ctn binding triggered the degradation of endogenous VE-cadherin. Similarly, reducing levels of p120ctn using siRNA caused a dramatic and dose-related reduction in cellular levels of VE-cadherin. In contrast, overexpression of p120ctn increased VE-cadherin cell surface levels and inhibited entry of cell surface VE-cadherin into degradative compartments. These results demonstrate that cellular levels of p120ctn function as a set point mechanism that regulates cadherin expression levels, and that a major function of p120ctn is to control cadherin internalization and degradation.

Antigens, CD↗

PubMed2003

Mechanisms of VE-cadherin processing and degradation in microvascular endothelial cells.

VE-cadherin is an endothelial-specific cadherin that plays important roles in vascular morphogenesis and growth control. To investigate the mechanisms by which endothelial cells regulate cadherin cell surface levels, a VE-cadherin mutant containing the non-adhesive interleukin-2 (IL-2) receptor extracellular domain and the VE-cadherin cytoplasmic tail (IL-2R-VE-cadcyto) was expressed in microvascular endothelial cells. Expression of the IL-2R-VE-cadcyto mutant resulted in the internalization of endogenous VE-cadherin and in a dramatic decrease in endogenous VE-cadherin levels. The internalized VE-cadherin co-localized with early endosomes, and the lysosomal inhibitor chloroquine dramatically inhibited the down-regulation of VE-cadherin in cells expressing the IL-2R-VE-cadcyto mutant. Chloroquine treatment also resulted in the accumulation of a VE-cadherin fragment lacking the beta-catenin binding domain of the VE-cadherin cytoplasmic tail. The formation of the VE-cadherin fragment could be prevented by treating endothelial cells with proteasome inhibitors. Furthermore, inhibition of the proteasome prevented VE-cadherin internalization and inhibited the disruption of endothelial intercellular junctions by the IL-2RVE-cadcyto mutant. These results provide new insights into the mechanisms of VE-cadherin processing and degradation in microvascular endothelial cells.

Adenoviridae↗