Metastasis suppressor tetraspanin CD82/KAI1 regulates ubiquitylation of epidermal growth factor receptor.

Ligand-induced ubiquitylation of EGF receptor (EGFR) is an important regulatory mechanism that controls endocytic trafficking of the receptor and its signaling potential. Here we report that tetraspanin CD82/KAI1 specifically suppresses ubiquitylation of EGFR after stimulation with heparin-binding EGF or amphiregulin and alters the rate of recruitment of the activated receptor to EEA1-positive endosomes. The suppressive effect of CD82 is dependent on the heparin-binding domain of the ligand. Deletion of the C-terminal cytoplasmic domain of CD82 (CD82ΔC mutant) inhibits endocytic trafficking of the tetraspanin and compromises its activity toward heparin-binding EGF-activated EGFR. Reduced ubiquitylation of EGFR is accompanied by PKC-dependent increase in serine phosphorylation of c-Cbl in cells expressing elevated levels of CD82. Furthermore, phosphorylation of threonine 654 (PKC phosphorylation site) in the juxtamembrane domain of the receptor is considerably increased in CD82-expressing cells. These results describe previously unsuspected links between tetraspanin proteins and ubiquitylation of their molecular partners (e.g., EGFR). Our data identify CD82 as a new regulator of c-Cbl, which discriminatively controls the activity of this E3 ubiquitin ligase toward heparin-binding ligand-EGFR pairs. Taken together, these observations provide an important new insight into the modulatory role of CD82 in endocytic trafficking of EGF receptor.

Thirty-femtogram detection of iron in mammalian cells.

Inorganic nanomaterials and particles with enhanced optical, mechanical, or magnetic attributes are currently being developed for a wide range of applications. Safety issues have developed however concerning their potential cyto- and genotoxicity. For in vivo and in vitro experimentations, recent developments have heightened the need for simple and facile methods to measure the amount of nanoparticles taken up by cells or tissues. In this work, a rapid and highly sensitive method for quantifying the uptake of iron oxide nanoparticles in mammalian cells is reported. The approach exploits the digestion of incubated cells with concentrated hydrochloric acid reactant and a colorimetric-based UV-visible absorption technique. The technique allows the detection of iron in cells over 4 decades in masses from 0.03 to 300 picograms per cell. Applied on particles of different surface chemistry and sizes, the protocol demonstrates that the coating is the key parameter in the nanoparticle/cell interactions. The data are corroborated by scanning and transmission electron microscopy, and the results stress the importance of resiliently adsorbed nanoparticles at the plasma membrane.

Segregative clustering of Lo and Ld membrane microdomains induced by local pH gradients in GM1-containing giant vesicles: a lipid model for cellular polarization.

Several cell polarization processes are coupled to local pH gradients at the membrane surface. We have investigated the involvement of a lipid-mediated effect in such coupling. The influence of lateral pH gradients along the membrane surface on lipid microdomain dynamics in giant unilamellar vesicles containing phosphatidylcholine, sphingomyelin, cholesterol, and the ganglioside GM1 was studied. Lo/Ld phase separation was generated by photosensitization. A lateral pH gradient was established along the external membrane surface by acid local microinjection. The gradient promotes the segregation of microdomains: Lo domains within an Ld phase move toward the higher pH side, whereas Ld domains within an Lo phase move toward the lower pH side. This results in a polarization of the vesicle membrane into Lo and Ld phases poles in the axis of the proton source. A secondary effect is inward tubulation in the Ld phase. None of these processes occurs without GM1 or with the analog asialo-GM1. These are therefore related to the acidic character of the GM1 headgroup. LAURDAN fluorescence experiments on large unilamellar vesicles indicated that, with GM1, an increase in lipid packing occurs with decreasing pH, attributed to the lowering of repulsion between GM1 molecules. Packing increase is much higher for Ld phase vesicles than for Lo phase vesicles. It is proposed that the driving forces for domain vectorial segregative clustering and vesicle polarization are related to such differences in packing variations with pH decrease between the Lo and Ld phases. Such pH-driven domain clustering might play a role in cellular membrane polarization processes in which local lateral pH gradients are known to be important, such as migrating cells and epithelial cells.

Making a tool of an artifact: the application of photoinduced Lo domains in giant unilamellar vesicles to the study of Lo/Ld phase spinodal decomposition and its modulation by the ganglioside GM1.

Electroformed giant unilamellar vesicles containing liquid-ordered Lo domains are important tools for the modeling of the physicochemical properties and biological functions of lipid rafts. Lo domains are usually imaged using fluorescence microscopy of differentially phase-partionioning membrane-embedded probes. Recently, it has been shown that these probes also have a photosensitizing effect that leads to lipid chemical modification during the fluorescence microscopy experiments. Moreover, the lipid reaction products are able as such to promote Lo microdomain formation, leading to potential artifacts. We show here that this photoinduced effect can also purposely be used as a new approach to study Lo microdomain formation in giant unilamellar vesicles. Photosensitized lipid modification can promote Lo microdomain appearance and growth uniformly and on a faster time scale, thereby yielding new information on such processes. For instance, in egg phosphatidylcholine/egg sphingomyelin/cholesterol 50:30:20 (mol/mol) giant unilamellar vesicles, photoinduced Lo microdomain formation appears to occur by the rarely observed spinodal decomposition process rather than by the common nucleation process usually observed for Lo domain formation in bilayers. Moreover, temperature and the presence of the ganglioside GM1 have a profound effect on the morphological outcome of the photoinduced phase separation, eventually leading to features such as bicontinuous phases, phase percolation inversions, and patterns evoking double phase separations. GM1 also has the effect of destabilizing Lo microdomains. These properties may have consequences for Lo nanodomains stability and therefore for raft dynamics in biomembranes. Our data show that photoinduced Lo microdomains can be used to obtain new data on fast raft-mimicking processes in giant unilamellar vesicles.

A role for CD81 on the late steps of HIV-1 replication in a chronically infected T cell line.

BACKGROUND: HIV-1 uses cellular co-factors for virion formation and release. The virus is able to incorporate into the viral particles host cellular proteins, such as tetraspanins which could serve to facilitate HIV-1 egress. Here, we investigated the implication of several tetraspanins on HIV-1 formation and release in chronically infected T-lymphoblastic cells, a model that permits the study of the late steps of HIV-1 replication. RESULTS: Our data revealed that HIV-1 Gag and Env structural proteins co-localized with tetraspanins in the form of clusters. Co-immunoprecipitation experiments showed that Gag proteins interact, directly or indirectly, with CD81, and less with CD82, in tetraspanin-enriched microdomains composed of CD81/CD82/CD63. In addition, when HIV-1 producing cells were treated with anti-CD81 antibodies, or upon CD81 silencing by RNA interference, HIV-1 release was significantly impaired, and its infectivity was modulated. Finally, CD81 downregulation resulted in Gag redistribution at the cell surface. CONCLUSION: Our findings not only extend the notion that HIV-1 assembly can occur on tetraspanin-enriched microdomains in T cells, but also highlight a critical role for the tetraspanin CD81 on the late steps of HIV replication.

In vitro toxicity of nanoceria: effect of coating and stability in biofluids.

Due to the increasing use of nanometric cerium oxide in applications, concerns about the toxicity of these particles have been raised and have resulted in a large number of studies. We report here on the interactions between 7 nm anionically charged cerium oxide particles and living mammalian cells. By a modification of the particle coating including low-molecular weight ligands and polymers, two generic behaviours are compared: particles coated with citrate ions that precipitate in biofluids and particles coated with poly(acrylic acid) that are stable and remain nanometric. We find that nanoceria covered with both coating agents are taken up by mouse fibroblasts and localized into membrane-bound compartments. However, flow cytometry and electron microscopy reveal that as a result of their precipitation, citrate-coated particles interact more strongly with cells. At cerium concentration above 1 mM, only citrate-coated nanoceria (and not particles coated with poly(acrylic acid)) display toxicity and moderate genotoxicity. The results demonstrate that the control of the surface chemistry of the particles and its ability to prevent aggregation can affect the toxicity of nanomaterials.

Cell sorting by endocytotic capacity in a microfluidic magnetophoresis device.

Magnetically labelled cells are finding a wealth of applications for in vitro analysis as well as in vivo treatments. Sorting of cells into subpopulations based on their magnetite loading is an important step in such procedures. Here, we study the sorting of monocytes and macrophages which internalise nanoparticles to different extents based on their endocytotic capacity. Macrophages featured a high endocytotic activity and were found to internalise between 4 and 60 pg of iron per cell. They were successfully sorted into five subpopulations of narrow iron loading distributions via on-chip free-flow magnetophoresis, thus demonstrating the potential of sorting of relatively similarly loaded cells. Monocytes featured a low endocytotic capacity and took on 1 to 4 pg of iron per cell. Mixtures of monocytes and macrophages were successfully sorted within the free-flow magnetophoresis chip and good purity (>88%), efficacy (>60%) and throughput (from 10 to 100 cells s(-1)) could be achieved. The introduced method constitutes a viable tool for studies of endocytotic capacity and sorting/selection of cells based on this functionality.

Interactions between magnetic nanowires and living cells: uptake, toxicity, and degradation.

We report on the uptake, toxicity, and degradation of magnetic nanowires by NIH/3T3 mouse fibroblasts. Magnetic nanowires of diameters 200 nm and lengths between 1 and 40 µm are fabricated by controlled assembly of iron oxide (γ-Fe(2)O(3)) nanoparticles. Using optical and electron microscopy, we show that after 24 h incubation the wires are internalized by the cells and located either in membrane-bound compartments or dispersed in the cytosol. Using fluorescence microscopy, the membrane-bound compartments were identified as late endosomal/lysosomal endosomes labeled with lysosomal associated membrane protein (Lamp1). Toxicity assays evaluating the mitochondrial activity, cell proliferation, and production of reactive oxygen species show that the wires do not display acute short-term (<100 h) toxicity toward the cells. Interestingly, the cells are able to degrade the wires and to transform them into smaller aggregates, even in short time periods (days). This degradation is likely to occur as a consequence of the internal structure of the wires, which is that of a noncovalently bound aggregate. We anticipate that this degradation should prevent long-term asbestos-like toxicity effects related to high aspect ratio morphologies and that these wires represent a promising class of nanomaterials for cell manipulation and microrheology.

Tetraspanin CD82 controls the association of cholesterol-dependent microdomains with the actin cytoskeleton in T lymphocytes: relevance to co-stimulation.

T-cell activation is initiated by the concerted engagement of the T-cell receptor and different co-stimulatory molecules, and requires cytoskeleton-dependent membrane dynamics. Here, we have studied the relationships between tetraspanins, cytoskeleton and raft microdomains, and their relevance in T-cell signaling. Localization studies and density-gradient flotation experiments indicate that part of tetraspanins localizes in raft microdomains linked to the actin cytoskeleton. First, partial coalescence of lipid raft is triggered by tetraspanin cross-linking and results in large caps in which F-actin also concentrates. Second, the amount of tetraspanins, which are recovered in the cholesterol-dependent insoluble fractions of low and intermediate density, and which appears to be membrane vesicles by electron microscopy, is under cytoskeletal influence. Disruption of actin filaments enhances the amount of tetraspanins recovered in typical raft fractions, whereas F-actin-stabilizing agents induce the opposite effect. Our data also reveal that CD82 constitutes a link between raft domains and the actin cytoskeleton, which is functionally relevant. First, tetraspanin signaling induces a selective translocation of CD82 from detergent-resistant membrane fractions to the cytoskeleton-associated pellet. Second, all functional effects linked to CD82 engagement, such as adhesion to culture plates, formation of actin bundles and early events of tyrosine phosphorylation, are abolished, or strongly reduced, by cholesterol depletion. We also show that dynamic relocalization of CD82 and F-actin at the periphery of the immune synapse is induced upon contact of T cells with antigen-presenting cells. This suggests that the tetraspanin web might participate in the membrane dynamics required for proper T-cell signaling. More generally, the interaction of tetraspanins with raft domains and with the actin cytoskeleton might relate with their role in many cellular functions as membrane organizers.

Rho GTPases link cytoskeletal rearrangements and activation processes induced via the tetraspanin CD82 in T lymphocytes.

Activation of T lymphocytes requires the engagement of the T-cell receptor and costimulation molecules through cell-to-cell contacts. The tetraspanin CD82 has previously been shown to act as a cytoskeleton-dependent costimulation molecule. We show here that CD82 engagement leads to the tyrosine phosphorylation and association of both the Rho GTPases guanosine exchange factor Vav1 and adapter protein SLP76, suggesting that Rho GTPases participate in CD82 signaling. Indeed, broad inactivation of all Rho GTPases, or a specific blockade of RhoA, Rac1 or Cdc42, inhibited the morphological changes linked to CD82 engagement but failed to modulate the inducible association of CD82 with the actin network. Rho GTPase inactivation, as well as actin depolymerization, reduced the ability of CD82 to phosphorylate Vav and SLP76 and to potentiate the phosphorylation of two early TcR signaling intermediates: the tyrosine kinases ZAP70 and membrane adapter LAT. Taken together, this suggests that an amplification loop, via early Vav and SLP76 phosphorylations and Rho-GTPases activation, is initiated by CD82 association with the cytoskeleton, which permits cytoskeletal rearrangements and costimulatory activity. Moreover, the involvement of CD82 in the formation of the immunological synapse is strongly suggested by its accumulation at the site of TcR engagement. This novel link between a tetraspanin and the Rho GTPase cascade could explain why tetraspanins, which are known to form heterocomplexes, are involved in cell activation, adhesion, growth and metastasis.