Project Summary: The immunological synapse (IS) is the dynamic interface between an immune cell and the cell that it is recognizing. A major function of the IS is directed secretion, which allows specific targeting of immune effector function. Our long-term goal is to use natural killer (NK) cells, which are lymphocytes of the innate immune system critical for host defense, to mechanistically define essential stages involved in formation and function of the secretory IS. Our hypothesis is that critical cytoskeleton-dependent events control sequential formation of the IS in series to allow secretory function and thus promote immune defense. We base this upon our findings that: 1) The cytolytic NK cell IS is dependent upon Wiskott-Aldrich syndrome protein-directed actin polymerization;2) Actin reorganization at the NK cell IS precedes and is required for microtubule function;and 3) Microtubules are needed to translocate lytic granules to the center of the NK cell IS, but not form the actin dependent structures. Based upon our observations, the specific aims of this proposal are twofold. 1) Determine the link between the actin-dependent complex (termed the actinosome) at the IS and the microtubules that is required for lytic granule polarization and directed secretion. Our initial candidate, Cdc42 interacting protein 4 (CIP4) is at least in part responsible for this activity as demonstrated in our preliminary results. CIP4 and others identified through mass spectrometry will be further evaluated for their ability to functionally link the actinosome to microtubules using microscopy, overexpression, dominant negative expression and small-interfering RNA. 2) Determine how the actinosome directs secretion through the IS after lytic granules have been delivered to the IS via microtubules. Preliminary data demonstrate non-muscle myosin proteins within the actinosome are also needed at late time points in the cytolytic process to allow the exocytosis of lytic granules through the IS. Thus, the ability of myosin to shuttle lytic granules as well as inflammatory exosomes though the IS will be determined using microscopy, biochemical, genetic and cell-free approaches. Relevance: We aim to define the mechanism underlying 2 critical checkpoints required to access immunity through secretory function of the IS. This will enable novel strategies for therapeutically manipulating the IS in disease to increase secretion and improve host defense, or decrease secretion and reduce inflammation.