Immunofluorescence microscopy was carried out essentially as described previously (15 (link)). YAC-1 cells were labeled with CMAC, mixed 1:2 with NK cells, and conjugation was induced by centrifugation for 5 minutes. After an additional 10 minutes, cells were gently resuspended and incubated on Poly-L-lysine coated coverslips for 10 minutes. Cells were then fixed with 3% paraformaldehyde, permeabilized, and labeled with primary antibodies and fluorescent secondary antibodies. Conjugates were imaged using a 63× PlanApo 1.4 NA objective on a spinning disk confocal system (UltraView ERS 6; Perkin Elmer, Waltham MA), equipped with an ORCA-ER camera (Hamamatsu Photonics, Bridgewater NJ) and Volocity software (v6.1.1; PerkinElmer). MTOC to synapse measurements were performed manually as follows: The border of the YAC-1 cell was determined based on CMAC fluorescence intensity, and the center of the MTOC was defined as the pixel with the brightest intensity in the 488 channel (anti-tubulin). The distance from the MTOC center to the nearest site on the YAC-1 border was then measured using Volocity software. Image preparation was performed with ImageJ.