The ability of a T cell to be activated is critically regulated by the number of TCRs expressed on the plasma membrane. Cell surface TCR expression is influenced by dynamic processes such as synthesis and transport of newly assembled receptors, endocytosis of surface TCR, and recycling to the plasma membrane of internalized receptors. In this study, the internalization of fluorescently labeled anti-TCR Abs was used to analyze constitutive endocytosis of TCRs on T cells, and to investigate the role of the ζ-chain in this process. We found that cell surface TCRs lacking ζ were endocytosed more rapidly than completely assembled receptors, and that reexpression of full-length ζ led to a dose-dependent decrease in the rate of TCR internalization. Rapid TCR internalization was also observed with CD4+CD8+ thymocytes from ζ-deficient mice, whereas TCR internalization on thymocytes from CD3-δ deficient animals was slow, similar to that of wild-type thymocytes. This identifies a specific role for ζ in the regulation of constitutive receptor internalization. Furthermore, chimeric ζ molecules containing non-native intracellular amino acid sequences also led to high levels of TCR expression and reduced TCR cycling. These effects were dependent solely on the length of the intracellular tail, ruling out a role for intracellular ζ-specific interactions with other molecules as a mechanism for regulating TCR internalization. Rather, these findings strongly support a model in which the ζ-chain stabilizes TCR residency on the cell surface, and functions to maintain cell surface receptor expression by sterically blocking internalization sequences in other TCR components.

The MYC oncogene has been studied for decades, yet there is still intense debate over how this transcription factor controls gene expression. Here, we seek to answer these questions with an in vivo readout of discrete events of gene expression in single cells. We engineered an optogenetic variant of MYC (Pi-MYC) and combined this tool with single-molecule RNA and protein imaging techniques to investigate the role of MYC in modulating transcriptional bursting and transcription factor binding dynamics in human cells. We find that the immediate consequence of MYC overexpression is an increase in the duration rather than in the frequency of bursts, a functional role that is different from the majority of human transcription factors. We further propose that the mechanism by which MYC exerts global effects on the active period of genes is by altering the binding dynamics of transcription factors involved in RNA polymerase II complex assembly and productive elongation.

CD8 T cells are essential for adaptive immunity against viral infections. Protease activated receptor 1 (PAR1) is expressed by CD8 T cells; however, its role in T cell effector function is not well defined. Here we show that in human CD8 T cells, PAR1 stimulation accelerates calcium mobilization. Furthermore, PAR1 is involved in cytotoxic T cell function by facilitating granule trafficking via actin polymerization and repositioning of the microtubule organizing center (MTOC) toward the immunological synapse. In vivo, PAR1 À/À mice have reduced cytokine-producing T cells in response to a lymphocytic choriomeningitis virus (LCMV) infection and fail to efficiently control the virus. Specific deletion of PAR1 in LCMV GP33-specific CD8 T cells results in reduced expansion and diminished effector function. These data demonstrate that PAR1 plays a role in T cell activation and function, and this pathway could represent a new therapeutic strategy to modulate CD8 T cell effector function.

Mutations conferring synthetic lethality in combination with null mutations in CAP2, the gene encoding the beta subunit of capping protein of Saccharomyces cerevisiae, were obtained in a colony color assay. Monogenic inheritance was found for four mutations, which were attributed to three genetic loci. One mutation, sac6-69, is in the gene encoding fimbrin, another actin-binding protein, which was expected because null mutations in SAC6 and CAP2 are known to be synthetic-lethal. The other two loci were designated slc for synthetic lethality with cap2. These loci include the mutations slc1-66, slc1-87 and slc2-107. The slc mutations are semi-dominant, as shown by incomplete complementation in slc/SLC cap2/cap2 heterozygotes. The slc mutations and sac6-69 interact with each other, as shown by enhanced phenotypes in diheterozygotes. Moreover, the haploid slc2-107 sac6-69 double mutant is inviable. In a CAP2 background, the slc mutations lead to temperature and osmotic sensitivity. They alter the distribution of the actin cytoskeleton, including deficits in the presence of actin cables and the polarization of cortical actin patches. The slc mutations also lead to a pseudomycelial growth pattern. Together these results suggest that slc1 and slc2 encode components of the actin cytoskeleton in yeast and that the actin cytoskeleton can regulate the patterns of growth.

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