Image analysis protocols have previously been described (29, 70, 71). Using MetaMorph image analysis software (Molecular Devices), we identified CTLs forming conjugates with Renca target cells using the DIC reference images. Tight cell couple formation was defined as the first time point at which a maximally spread immune synapse formed, or two frames after initial cell contact, whichever occurred first. To assess CTL and TIL morphology, every DIC frame after tight cell couple formation was assessed for the presence of off-synapse lamellae, defined as transient membrane protrusions pointing away from the immune synapse, followed by retraction. For each cell couple, the initial position of the immune synapse on the Renca+HA target cell was compared to the position in the final frame. If the T cell had migrated by a distance greater than the diameter of the immune synapse, this was classed as a translocation event. To analyze protein spatiotemporal distribution, we used an improved version of the method described by Roybal et al. (30) as implemented in version 2.8 of the open source CellOrganizer software ( (70, 73, 74). The first improvement is referred to as synapse tracking. The previous method required annotation of the cell couple synapse position for each time point, whereas the modified method requires only annotation at the initial time point. The fluorescence image for the initial frame is segmented by thresholding using the method described by Otsu (75), and an ellipse is fitted to the segmented object containing the manually annotated synapse position (the T cell). A window of 61 by 61 around the synapse position is chosen, and a circle containing the target cell is found by Hough transformation of the DIC image (76). The coordinates of the endpoints of the synapse are found as the intersection points of the T cell ellipse and the target cell circle. This process is repeated for preceding or subsequent frames with the T cell location information in the previous frame used to find the T cell in the current frame. The coordinates of the two synapse endpoints are saved across time. The second improvement is referred to as alignment refinement. To correct for inadvertent rotation during the initial alignment of all of the cells within the population to be analyzed, we implemented two modifications. The first step is to infer a rotational angle around the z axis with a regression model using as features the intensities of evenly distributed coordinates in the cylinder originating from the synapse center. The second step is to decide whether to flip the cell by 180° around the y axis using a binary classifier with the total intensity of the first and second half of all slices as features. The regressor and classifier were trained with images that had been manually annotated. Last, we fit a line through the synapse region in the central slice and perform a fine correction of the rotation around the z axis using the angle of that line. As a first measurement of protein enrichment, we defined the 10% of the cell volume with the highest fluorescence intensity across the average of the entire population of analyzed cells and determined enrichment in this volume relative to the entire cell, as established previously (30). Additional regions of interest were defined by geometrical criteria. For the synapse region in cofilin-GFP–expressing T cells, we used a cylinder normal to and one voxel distant from the synapse plane, with its axis centered on the synapse region, a radius of 8 voxels, and a height of 4 voxels. For the region defining the interface center in F-tractin–GFP–expressing cells, we used a cylinder similar to the one in the cofilin analysis but with a radius of 8/3 voxels. The computational analysis of cofilin-GFP distributions required additional adaptations because cofilin-GFP accumulated prominently in the nucleus. Enrichment in the 10% of the cell volume with the highest fluorescence intensity did not reliably reflect interface enrichment. Because the T cell nucleus often closely approaches the cellular interface, a cylindrical interface measurement region captured more of the interface accumulation but still not exclusively so. Therefore, we also sorted time points into those with >135% fluorescence intensity in regions of interface accumulation above cellular background and without accumulation, computationally determined enrichment in the 10% of the cell volume with the highest fluorescence intensity at only the time points with accumulation, and multiplied the enrichment intensity by the fraction of cell couples with cofilin accumulation.

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