The assembly of signaling nanoterritories at the T cell immunological synapse is controlled by the coordinated trafficking and fusion of specific vesicles containing the signaling molecules Lck, LAT, and TCRζ.

Transcytosis is a widespread pathway for apical targeting in epithelial cells. MAL2, an essential protein of the machinery for apical transcytosis, functions by shuttling in vesicular carriers between the apical zone and the cell periphery. We have identified INF2, an atypical formin with actin polymerization and depolymerization activities, which is a binding partner of MAL2. MAL2-positive vesicular carriers associate with short actin filaments during transcytosis in a process requiring INF2. INF2 binds Cdc42 in a GTP-loaded-dependent manner. Cdc42 and INF2 regulate MAL2 dynamics and are necessary for apical transcytosis and the formation of lateral lumens in hepatoma HepG2 cells. INF2 and MAL2 are also essential for the formation of the central lumen in organotypic cultures of epithelial MDCK cells. Our results reveal a functional mechanism whereby Cdc42, INF2, and MAL2 are sequentially ordered in a pathway dedicated to the regulation of transcytosis and lumen formation.

Post-translational farnesylation or geranylgeranylation at a C-terminal cysteine residue regulates localization and function of over 100 proteins, including the Ras isoforms, and is a therapeutic target in diseases including cancer and infection. Here we report global and selective profiling of prenylated proteins in living cells enabled by development of isoprenoid analogues YnF and YnGG in combination with quantitative chemical proteomics. Eighty prenylated proteins were identified in a single human cell line, 64 for the first time at endogenous abundance without metabolic perturbation. We further demonstrate that YnF and YnGG enable direct identification of post-translationally processed prenylated peptides, proteome-wide quantitative analysis of prenylation dynamics and alternative prenylation in response to four different prenyltransferase inhibitors, and quantification of defective Rab prenylation in a model of the retinal degenerative disease Choroideremia.

Abbreviations used: dSTORM, direct stochastic optical reconstruction microscopy; MAL, myelin and lymphocyte; Syt7, synaptotagmin-7; TIRF, total internal reflection fluorescence.M.-I. Thoulouze and A. Alcover contributed equally to this paper. Fig. 1, D and H). LAT vesicular compartment co-localized with Rab27a (35%) and Rab37 (30%; Fig. 1, M and N), two Rab molecules known to regulate cytokine and cytotoxic granule exocytosis in CD4 + and CD8 + T cells, respectively (Stinchcombe et al., 2001;Huse et al., 2006). TCR peripheral vesicles colocalized to the fast recycling Rab4b compartment (8%), whereas its core co-localized to Rab3d-(25%) and Rab8b-regulated (30%) exocytic compartments (Huse et al., 2006;Fukuda, 2008; Fig. 1, Q-S). Noteworthy, the secretory v-SNARE protein Ti-VAMP (Chaineau et al., 2009) co-localized with both LAT (30%) and TCR (30%) vesicular compartments (Fig. 1, O and W). Regulated vesicle fusion generates signaling nanoterritories that control T cell activation at the immunological synapse LAT and TCR vesicular release is regulated by calcium and synaptotagmin-7The traffic regulators present in LAT and TCR vesicles (Fig. 1) have been reported to be involved in stimulus-induced exocytosis at cytotoxic synapses (Ménager et al., 2007; MarcetPalacios et al., 2008). To pinpoint the molecular mechanism regulating Lck, LAT, and TCR release from vesicular compartments, we assessed the role of calcium, a known regulator of stimulus-dependent exocytosis (Stojilkovic, 2005;Pang and Südhof, 2010). To increase cytosolic calcium levels, we treated CD4 T cells either with thapsigargin or ionomycin. To accurately quantify Lck, LAT, and TCR distribution between intracellular vesicles and the plasma membrane, we stained for surface CD2 and then used it to automatically segment the plasma membrane. The resulting automated mask delimiting the internal and external outlines of the plasma membrane allowed us to discriminate and quantify the subcellular distribution (plasma membrane versus vesicular compartment) of our proteins of interest (materials and methods; unpublished data). We found LAT and TCR distribution to the vesicular compartment to be higher than anticipated by others (Bonello et al., 2004), 75% (Fig. 2, A, B, H, and I). In fact, through plasma membrane segmentation, we were able to distinguish plasma membrane-resident LAT and TCR from their intracellular compartments and subcortical vesicles, within the limits of the confocal microscopy resolution (unpublished data). Lck displayed a higher plasma membrane distribution with 25% in intracellular vesicles (Fig. 2, C and J; and not depicted).Consistent with LAT localization in Rab27a and Rab37 exocytic compartments, increased cytosolic calcium led to a clear release of its vesicular compartment into the plasma membrane (Fig. 2, A, I, and O; and not depicted). In agreement with TCR-segregated distribution into a Rab3d and Rab8b exocytic compartment and also into Rab4b rapid recycling endosomes, cytosolic calcium increase induced an inc...

Exosomes are a particular type of extracellular vesicle, characterized by their endosomal origin as intraluminal vesicles present in large endosomes with a multivesicular structure. After these endosomes fuse with the plasma membrane, exosomes are secreted into the extracellular space. The ability of exosomes to carry and selectively deliver bioactive molecules (e.g., lipids, proteins, and nucleic acids) confers on them the capacity to modulate the activity of receptor cells, even if these cells are located in distant tissues or organs. Since exosomal cargo depends on cell type, a detailed understanding of the mechanisms that regulate the biochemical composition of exosomes is fundamental to a comprehensive view of exosome function. Here, we review the latest advances concerning exosome function and biogenesis in T cells, with particular focus on the mechanism of protein sorting at multivesicular endosomes. Exosomes secreted by specific T-cell subsets can modulate the activity of immune cells, including other T-cell subsets. Ceramide, tetraspanins and MAL have been revealed to be important in exosome biogenesis by T cells. These molecules, therefore, constitute potential molecular targets for artificially modulating exosome production and, hence, the immune response for therapeutic purposes.

SummaryThe coordinated reformation of the nuclear envelope (NE) after mitosis re-establishes the structural integrity and the functionality of the nuclear compartment. The endosomal sorting complex required for transport (ESCRT) machinery, a membrane remodeling pathway that is highly conserved in eukaryotes, has been recently involved in NE resealing by mediating the annular fusion of the nuclear membrane (NM). We show here that CC2D1B, a regulator of ESCRT polymerization, is required to re-establish the nuclear compartmentalization by coordinating endoplasmic reticulum (ER) membrane deposition around chromatin disks with ESCRT-III recruitment to the reforming NE. Accordingly, CC2D1B determines the spatiotemporal distribution of the CHMP7-ESCRT-III axis during NE reformation. Crucially, in CC2D1B-depleted cells, ESCRT activity is uncoupled from Spastin-mediated severing of spindle microtubules, resulting in persisting microtubules that compromise nuclear morphology. Therefore, we reveal CC2D1B as an essential regulatory factor that licenses the formation of ESCRT-III polymers to ensure the orderly reformation of the NE.

Exosomes secreted by T cells play an important role in coordinating the immune response. HIV-1 Nef hijacks the route of exosome secretion of T cells to modulate the functioning of uninfected cells. Despite the importance of the process, the protein machinery involved in exosome biogenesis is yet to be identified. In this study, we show that MAL, a tetraspanning membrane protein expressed in human T cells, is present in endosomes that travel toward the plasma membrane for exosome secretion. In the absence of MAL, the release of exosome particles and markers was greatly impaired. This effect was accompanied by protein sorting defects at multivesicular endosomes that divert the exosomal marker CD63 to autophagic vacuoles. Exosome release induced by HIV-1 Nef was also dependent on MAL expression. Therefore, MAL is a critical element of the machinery for exosome secretion and may constitute a target for modulating exosome secretion by human T cells.

Tyrosine phosphorylation is one of the key covalent modifications that occur in multicellular organisms. Since its discovery more than 30 years ago, tyrosine phosphorylation has come to be understood as a fundamentally important mechanism of signal transduction and regulation in all eukaryotic cells. The tyrosine kinase Lck (lymphocyte-specific protein tyrosine kinase) plays a crucial role in the T-cell response by transducing early activation signals triggered by TCR (T-cell receptor) engagement. These signals result in the phosphorylation of immunoreceptor tyrosine-based activation motifs present within the cytosolic tails of the TCR-associated CD3 subunits that, once phosphorylated, serve as scaffolds for the assembly of a large supramolecular signalling complex responsible for T-cell activation. The existence of membrane nano- or micro-domains or rafts as specialized platforms for protein transport and cell signalling has been proposed. The present review discusses the signals that target Lck to membrane rafts and the importance of these specialized membranes in the transport of Lck to the plasma membrane, the regulation of Lck activity and the phosphorylation of the TCR.