Rapid increase in transferrin receptor recycling promotes adhesion during T cell activation.

BACKGROUND: T cell activation leads to increased expression of the receptor for the iron transporter transferrin (TfR) to provide iron required for the cell differentiation and clonal expansion that takes place during the days after encounter with a cognate antigen. However, T cells mobilise TfR to their surface within minutes after activation, although the reason and mechanism driving this process remain unclear. RESULTS: Here we show that T cells transiently increase endocytic uptake and recycling of TfR upon activation, thereby boosting their capacity to import iron. We demonstrate that increased TfR recycling is powered by a fast endocytic sorting pathway relying on the membrane proteins flotillins, Rab5- and Rab11a-positive endosomes. Our data further reveal that iron import is required for a non-canonical signalling pathway involving the kinases Zap70 and PAK, which controls adhesion of the integrin LFA-1 and eventually leads to conjugation with antigen-presenting cells. CONCLUSIONS: Altogether, our data suggest that T cells boost their iron importing capacity immediately upon activation to promote adhesion to antigen-presenting cells.

The C-terminal domain of p53 orchestrates the interplay between non-covalent and covalent poly(ADP-ribosyl)ation of p53 by PARP1.

The post-translational modification poly(ADP-ribosyl)ation (PARylation) plays key roles in genome maintenance and transcription. Both non-covalent poly(ADP-ribose) binding and covalent PARylation control protein functions, however, it is unknown how the two modes of modification crosstalk mechanistically. Employing the tumor suppressor p53 as a model substrate, this study provides detailed insights into the interplay between non-covalent and covalent PARylation and unravels its functional significance in the regulation of p53. We reveal that the multifunctional C-terminal domain (CTD) of p53 acts as the central hub in the PARylation-dependent regulation of p53. Specifically, p53 bound to auto-PARylated PARP1 via highly specific non-covalent PAR-CTD interaction, which conveyed target specificity for its covalent PARylation by PARP1. Strikingly, fusing the p53-CTD to a protein that is normally not PARylated, renders this a target for covalent PARylation as well. Functional studies revealed that the p53-PAR interaction had substantial implications on molecular and cellular levels. Thus, PAR significantly influenced the complex p53-DNA binding properties and controlled p53 functions, with major implications on the p53-dependent interactome, transcription, and replication-associated recombination. Remarkably, this mechanism potentially also applies to other PARylation targets, since a bioinformatics analysis revealed that CTD-like regions are highly enriched in the PARylated proteome.

SNX9-induced membrane tubulation regulates CD28 cluster stability and signalling.

T cell activation requires engagement of a cognate antigen by the T cell receptor (TCR) and the co-stimulatory signal of CD28. Both TCR and CD28 aggregate into clusters at the plasma membrane of activated T cells. While the role of TCR clustering in T cell activation has been extensively investigated, little is known about how CD28 clustering contributes to CD28 signalling. Here, we report that upon CD28 triggering, the BAR-domain protein sorting nexin 9 (SNX9) is recruited to CD28 clusters at the immunological synapse. Using three-dimensional correlative light and electron microscopy, we show that SNX9 generates membrane tubulation out of CD28 clusters. Our data further reveal that CD28 clusters are in fact dynamic structures and that SNX9 regulates their stability as well as CD28 phosphorylation and the resulting production of the cytokine IL-2. In summary, our work suggests a model in which SNX9-mediated tubulation generates a membrane environment that promotes CD28 triggering and downstream signalling events.

Membrane Heterogeneity Controls Cellular Endocytic Trafficking.

Endocytic trafficking relies on highly localized events in cell membranes. Endocytosis involves the gathering of protein (cargo/receptor) at distinct plasma membrane locations defined by specific lipid and protein compositions. Simultaneously, the molecular machinery that drives invagination and eventually scission of the endocytic vesicle assembles at the very same place on the inner leaflet of the membrane. It is membrane heterogeneity - the existence of specific lipid and protein domains in localized regions of membranes - that creates the distinct molecular identity required for an endocytic event to occur precisely when and where it is required rather than at some random location within the plasma membrane. Accumulating evidence leads us to believe that the trafficking fate of internalized proteins is sealed following endocytosis, as this distinct membrane identity is preserved through the endocytic pathway, upon fusion of endocytic vesicles with early and sorting endosomes. In fact, just like at the plasma membrane, multiple domains coexist at the surface of these endosomes, regulating local membrane tubulation, fission and sorting to recycling pathways or to the trans-Golgi network via late endosomes. From here, membrane heterogeneity ensures that fusion events between intracellular vesicles and larger compartments are spatially regulated to promote the transport of cargoes to their intracellular destination.

Cdc42 Couples T Cell Receptor Endocytosis to GRAF1-Mediated Tubular Invaginations of the Plasma Membrane.

: T cell activation is immediately followed by internalization of the T cell receptor (TCR). TCR endocytosis is required for T cell activation, but the mechanisms supporting removal of TCR from the cell surface remain incompletely understood. Here we report that TCR endocytosis is linked to the clathrin-independent carrier (CLIC) and GPI-enriched endocytic compartments (GEEC) endocytic pathway. We show that unlike the canonical clathrin cargo transferrin or the adaptor protein Lat, internalized TCR accumulates in tubules shaped by the small GTPase Cdc42 and the Bin/amphiphysin/Rvs (BAR) domain containing protein GRAF1 in T cells. Preventing GRAF1-positive tubules to mature into endocytic vesicles by expressing a constitutively active Cdc42 impairs the endocytosis of TCR, while having no consequence on the uptake of transferrin. Together, our data reveal a link between TCR internalization and the CLIC/GEEC endocytic route supported by Cdc42 and GRAF1.

PARP1 regulates DNA damage-induced nucleolar-nucleoplasmic shuttling of WRN and XRCC1 in a toxicant and protein-specific manner.

The prime function of nucleoli is ribogenesis, however, several other, non-canonical functions have recently been identified, including a role in genotoxic stress response. Upon DNA damage, numerous proteins shuttle dynamically between the nucleolus and the nucleoplasm, yet the underlying molecular mechanisms are incompletely understood. Here, we demonstrate that PARP1 and PARylation contribute to genotoxic stress-induced nucleolar-nucleoplasmic shuttling of key genome maintenance factors in HeLa cells. Our work revealed that the RECQ helicase, WRN, translocates from nucleoli to the nucleoplasm upon treatment with the oxidizing agent H2O2, the alkylating agent 2-chloroethyl ethyl sulfide (CEES), and the topoisomerase inhibitor camptothecin (CPT). We show that after treatment with H2O2 and CEES, but not CPT, WRN translocation was dependent on PARP1 protein, yet independent of its enzymatic activity. In contrast, nucleolar-nucleoplasmic translocation of the base excision repair protein, XRCC1, was dependent on both PARP1 protein and its enzymatic activity. Furthermore, gossypol, which inhibits PARP1 activity by disruption of PARP1-protein interactions, abolishes nucleolar-nucleoplasmic shuttling of WRN, XRCC1 and PARP1, indicating the involvement of further upstream factors. In conclusion, this study highlights a prominent role of PARP1 in the DNA damage-induced nucleolar-nucleoplasmic shuttling of genome maintenance factors in HeLa cells in a toxicant and protein-specific manner.