Large endosomes (gigantosomes) form in target cells within minutes of triggering CTL degranulation. (A) Large EEA-1⁺ endosomes form in a target cell after CTL degranulation. EGFP–EEA-1–transfected HeLa target cells were incubated with specific CTLs in the presence of EGTA to allow conjugate formation. After 2 minutes, CaCl₂ was added to induce CTL degranulation (bottom; images from supplemental Videos 1-2). Enlarged endosomes form in the target cell within minutes after CTL degranulation, although the size of early endosomes does not change in the absence of calcium (top). Data (deconvolved wide-field images) are representative of 3 independent experiments. (B) Large endosomes formed in target cells after CTL attack contain PFN. Concanavalin A–coated HeLa cells were incubated with LAK cells in the presence of EGTA to allow conjugate formation, and then buffer (top) or CaCl₂ (bottom) was added to induce cytotoxic granule exocytosis 5 minutes before fixation. Data depicted (deconvolved wide-field 3-dimensional images followed by Z projection) are representative of 2 independent experiments. PFN signal was detected using AlexaFluor 488–conjugated secondary antibody and EEA-1 using AlexaFluor 647–conjugated secondary antibody. Color bars and associated numbers indicate fluorescence intensity levels. Scale bars represent 10 μm. Dashed lines indicate plasma membrane.