Intercellular transfer of transferrin receptor by a contact-, Rab8-dependent mechanism involving tunneling nanotubes.

Intercellular communication between cancer cells, especially between cancer and stromal cells, plays an important role in disease progression. We examined the intercellular transfer of organelles and proteins in vitro and in vivo and the role of tunneling nanotubes (TNTs) in this process. TNTs are membrane bridges that facilitate intercellular transfer of organelles of unclear origin. Using 3-dimensional quantitative and qualitative confocal microscopy, we showed that TNTs contain green fluorescent protein (GFP)-early endosome antigen (EEA) 1, GFP Rab5, GFP Rab11, GFP Rab8, transferrin (Tf), and Tf receptor (Tf-R) fused to mCherry (Tf-RmCherry). Tf-RmCherry was transferred between cancer cells by a contact-dependent but secretion-independent mechanism. Live cell imaging showed TNT formation preceding the transfer of Tf-RmCherry and involving the function of the small guanosine triphosphatase (GTPase) Rab8, which colocalized with Tf-RmCherry in the TNTs and was cotransferred to acceptor cells. Tf-RmCherry was transferred from cancer cells to fibroblasts, a noteworthy finding that suggests that this process occurs between tumor and stromal cells in vivo. We strengthened this hypothesis in a xenograft model of breast cancer using enhanced (e)GFP-expressing mice. Tf-RmCherry transferred from tumor to stromal cells and this process correlated with an increased opposite transfer of eGFP from stromal to tumor cells, together pointing toward complex intercellular communication at the tumor site.

Role of the AP2 beta-appendage hub in recruiting partners for clathrin-coated vesicle assembly.

Adaptor protein complex 2 alpha and beta-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of beta-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the beta-appendage (the "top" and "side" sites) that bind motifs distinct from those previously identified on the alpha-appendage. We solved the structure of the beta-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor beta-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the beta-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability ("matricity"). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as beta-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.

Contact-dependent transfer of TiO2 nanoparticles between mammalian cells.

Cellular organelles have been shown to shuttle between cells in co-culture. We hereby show that titanium dioxide (TiO2) nanoparticles (NPs) can be transferred in such a manner, between cells in direct contact, along with endosomes and lysosomes. A co-culture system was employed for this purpose and the NP transfer was observed in mammalian cells including normal rat kidney (NRK) and HeLa cells. We found that the small GTPase Arf6 facilitates the intercellular transfer of smaller NPs and agglomerates. Spherical, anatase nano-TiO2 with sizes of 5 (Ti5) and 40 nm (Ti40) were used in this study. Humans are increasingly exposed to TiO2 NPs from external sources such as constituents of foods, cosmetics, and pharmaceuticals, or from internal sources represented by Ti-based implants, which release NPs upon abrasion. Exposure to 5 mg/l of Ti5 and Ti40 for 24 h did not affect cellular viability but modified their ability to communicate with surrounding cells. Altogether, our results have important implications for the design of nanomedicines, drug delivery and toxicity.

Novel microscopy-based screening method reveals regulators of contact-dependent intercellular transfer.

Contact-dependent intercellular transfer (codeIT) of cellular constituents can have functional consequences for recipient cells, such as enhanced survival and drug resistance. Pathogenic viruses, prions and bacteria can also utilize this mechanism to spread to adjacent cells and potentially evade immune detection. However, little is known about the molecular mechanism underlying this intercellular transfer process. Here, we present a novel microscopy-based screening method to identify regulators and cargo of codeIT. Single donor cells, carrying fluorescently labelled endocytic organelles or proteins, are co-cultured with excess acceptor cells. CodeIT is quantified by confocal microscopy and image analysis in 3D, preserving spatial information. An siRNA-based screening using this method revealed the involvement of several myosins and small GTPases as codeIT regulators. Our data indicates that cellular protrusions and tubular recycling endosomes are important for codeIT. We automated image acquisition and analysis to facilitate large-scale chemical and genetic screening efforts to identify key regulators of codeIT.

Study of human RIG-I polymorphisms identifies two variants with an opposite impact on the antiviral immune response.

BACKGROUND: RIG-I is a pivotal receptor that detects numerous RNA and DNA viruses. Thus, its defectiveness may strongly impair the host antiviral immunity. Remarkably, very little information is available on RIG-I single-nucleotide polymorphisms (SNPs) presenting a functional impact on the host response. METHODOLOGY/PRINCIPAL FINDINGS: Here, we studied all non-synonymous SNPs of RIG-I using biochemical and structural modeling approaches. We identified two important variants: (i) a frameshift mutation (P(229)fs) that generates a truncated, constitutively active receptor and (ii) a serine to isoleucine mutation (S(183)I), which drastically inhibits antiviral signaling and exerts a down-regulatory effect, due to unintended stable complexes of RIG-I with itself and with MAVS, a key downstream adapter protein. CONCLUSIONS/SIGNIFICANCE: Hence, this study characterized P(229)fs and S(183)I SNPs as major functional RIG-I variants and potential genetic determinants of viral susceptibility. This work also demonstrated that serine 183 is a residue that critically regulates RIG-I-induced antiviral signaling.

Targeting of beta-arrestin2 to the centrosome and primary cilium: role in cell proliferation control.

BACKGROUND: The primary cilium is a sensory organelle generated from the centrosome in quiescent cells and found at the surface of most cell types, from where it controls important physiological processes. Specific sets of membrane proteins involved in sensing the extracellular milieu are concentrated within cilia, including G protein coupled receptors (GPCRs). Most GPCRs are regulated by beta-arrestins, betaarr1 and betaarr2, which control both their signalling and endocytosis, suggesting that betaarrs may also function at primary cilium. METHODOLOGY/PRINCIPAL FINDINGS: In cycling cells, betaarr2 was observed at the centrosome, at the proximal region of the centrioles, in a microtubule independent manner. However, betaarr2 did not appear to be involved in classical centrosome-associated functions. In quiescent cells, both in vitro and in vivo, betaarr2 was found at the basal body and axoneme of primary cilia. Interestingly, betaarr2 was found to interact and colocalize with 14-3-3 proteins and Kif3A, two proteins known to be involved in ciliogenesis and intraciliary transport. In addition, as suggested for other centrosome or cilia-associated proteins, betaarrs appear to control cell cycle progression. Indeed, cells lacking betaarr2 were unable to properly respond to serum starvation and formed less primary cilia in these conditions. CONCLUSIONS/SIGNIFICANCE: Our results show that betaarr2 is localized to the centrosome in cycling cells and to the primary cilium in quiescent cells, a feature shared with other proteins known to be involved in ciliogenesis or primary cilium function. Within cilia, betaarr2 may participate in the signaling of cilia-associated GPCRs and, therefore, in the sensory functions of this cell "antenna".

Dynamic interaction of HIV-1 Nef with the clathrin-mediated endocytic pathway at the plasma membrane.

The HIV-1 Nef protein perturbs the trafficking of membrane proteins such as CD4 by interacting with clathrin-adaptor complexes. We previously reported that Nef alters early/recycling endosomes, but its role at the plasma membrane is poorly documented. Here, we used total internal reflection fluorescence microscopy, which restricts the analysis to a approximately 100 nm region of the adherent surface of the cells, to focus on the dynamic of Nef at the plasma membrane relative to that of clathrin. Nef colocalized both with clathrin spots (CS) that remained static at the cell surface, corresponding to clathrin-coated pits (CCPs), and with approximately 50% of CS that disappeared from the cell surface, corresponding to forming clathrin-coated vesicles (CCVs). The colocalization of Nef with clathrin required the di-leucine motif essential for Nef binding to AP complexes and was independent of CD4 expression. Furthermore, analysis of Nef mutants showed that the capacity of Nef to induce internalization and downregulation of CD4 in T lymphocytes correlated with its localization into CCPs. In conclusion, this analysis shows that Nef is recruited into CCPs and into forming CCVs at the plasma membrane, in agreement with a model in which Nef uses the clathrin-mediated endocytic pathway to induce internalization of some membrane proteins from the surface of HIV-1-infected T cells.

The conserved isoleucine-valine-phenylalanine motif couples activation state and endocytic functions of beta-arrestins.

Beta-arrestins (betaarrs) play a central role in the regulation of G-protein-coupled receptors (GPCRs). Their binding to phosphorylated activated GPCRs induces a conformational transition to an active state resulting in the release of their flexible C-terminal tail. Binding sites for clathrin and the adaptor protein (AP)-2 clathrin adaptor complex are then unmasked, which drive the recruitment of betaarrs-GPCR complexes into clathrin-coated pits (CCPs). A conserved isoleucine-valine-phenylalanine (IVF) motif of the C-terminal tail controls betaarr activation through intramolecular interactions. Here, we provide structural, biochemical and functional evidence in living cells that the IVF motif also controls binding to AP-2. While the F residue is directly involved in AP-2 binding, substitutions of I and V residues, markedly enhanced affinity for AP-2 resulting in active betaarr mutants, which are constitutively targeted to CCPs in the absence of any GPCR activation. Conformational change and endocytic functions of betaarrs thus appear to be coordinated via the complex molecular interactions established by the IVF motif.

Homo- and hetero-oligomerization of beta-arrestins in living cells.

Arrestins are important proteins, which regulate the function of serpentine heptahelical receptors and contribute to multiple signaling pathways downstream of receptors. The ubiquitous beta-arrestins are believed to function exclusively as monomers, although self-association is assumed to control the activity of visual arrestin in the retina, where this isoform is particularly abundant. Here the oligomerization status of beta-arrestins was investigated using different approaches, including co-immunoprecipitation of epitope-tagged beta-arrestins and resonance energy transfer (BRET and FRET) in living cells. At steady state and at physiological concentrations, beta-arrestins constitutively form both homo- and hetero-oligomers. Co-expression of beta-arrestin2 and beta-arrestin1 prevented beta-arrestin1 accumulation into the nucleus, suggesting that hetero-oligomerization may have functional consequences. Our data clearly indicate that beta-arrestins can exist as homo- and hetero-oligomers in living cells and raise the hypothesis that the oligomeric state may regulate their subcellular distribution and functions.