In T cells, PI3K activation in the periphery of the immune synapse leads to PIP3 accumulation that promotes actin polymerization in a pathway important for cytotoxic function.

Polarization of the T cell microtubule-organizing center (MTOC) to the immunological synapse maintains the specificity of effector responses by enabling directional secretion toward the antigen-presenting cell. MTOC reorientation is guided by a sharp gradient of diacylglycerol that is centered at the synapse. Here, we used a single cell photoactivation approach to demonstrate that diacylglycerol kinase-α (DGK-α) controls T cell polarity by limiting the diffusion of diacylglycerol. DGK-α deficient T cells exhibited enlarged synaptic diacylglycerol accumulations and impaired MTOC reorientation. By contrast, T cells lacking the related isoform DGK-ζ did not display polarization defects. We also found that DGK-α localized preferentially to the periphery of the synapse, suggesting that it constrains the scope of diacylglycerol accumulation from the outside. Phosphoinositide 3-kinase activity was required for this peripheral localization pattern, establishing an intriguing link between diacylglycerol and phosphatidylinositol signaling during T cell activation. These results reveal a previously unappreciated function of DGK-α and provide insight into the mechanisms of lymphocyte polarity.

The marsupial blastocyst forms in an entirely different manner from its eutherian counterpart, involving cell-zona rather than cell-cell adhesion during the 8- to-16-cell transition. While the eutherian blastocyst consists of a spherical trophoblast completely enveloping a pluripotent inner cell mass, or pluriblast, the marsupial blastocyst forms initially as a bowl-shaped monolayer of cells lining the zona pellucida at the embryonic pole (ep). This monolayer contains a small patch of centrally positioned pluriblast cells edged with trophoblast cells that later coalesce at the abembryonic pole. Using immunocytochemistry, we examined the localization of the proteins Oct4, Cdx2, Tead4, Sox2, and Yap1 in opossum embryos to determine if their temporal expression pattern differed from that in the mouse, given the important differences in cell behavior preceding blastocyst formation in these mammals. Our results indicate that these proteins are expressed in similar temporal patterns despite the topological differences between mouse and opossum cleavage-stage embryos and blastocysts. That the Hippo-pathway protein Yap1 localized specifically around the approximately 128-cell stage to opossum trophoblast nuclei but remained in the cytoplasm of pluriblast cells suggests that this transcriptional regulator participates in allocating cells to the trophoblast lineage, as it does in mouse. Interestingly, in both mouse and opossum embryos, expression of the pluripotency marker Oct4 persisted after Cdx2, which signals trophoblast specification, began to be expressed in trophoblast cells. This and the observation that Cdx2 is present in opossum embryos well before blastomere-zona adhesion even occurs suggests that the proteins studied may have other roles in early mammalian embryonic development.

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The immunological synapse formed by a T lymphocyte on the surface of a target cell contains a peripheral ring of filamentous actin (F-actin) that promotes adhesion and facilitates the directional secretion of cytokines and cytolytic factors. We show that growth and maintenance of this F-actin ring is dictated by the annular accumulation of phosphatidylinositol trisphosphate (PIP 3 ) in the synaptic membrane. PIP 3 functions in this context by recruiting the exchange factor Dock2 to the periphery of the synapse, where it drives actin polymerization through the Rho-family GTPase Rac. We also show that synaptic PIP 3 is generated by class IA phosphoinositide 3-kinases that associate with T cell receptor microclusters and are activated by the GTPase Ras. Perturbations that inhibit or promote PIP 3 -dependent F-actin remodeling dramatically affect T cell cytotoxicity, demonstrating the functional importance of this pathway. These results reveal how T cells use lipid-based signaling to control synaptic architecture and modulate effector responses.