Abstract
Protocols were developed to automate image analysis and to track the movement of thousands of vesicular compartments in live cells. Algorithms were used to discriminate among different types of movement (e.g. random, caged, and directed). We applied these tools to investigate the steady-state distribution and movement of lytic granules (LG) in live natural killer (NK) cells by high-speed 3-dimensional (3D) spinning disc confocal and 2-dimensional total internal reflection fluorescence microscopy. Both mouse NK cells and a human NK cell line deficient in the small GTPase Rab27a were examined. The unbiased analysis of large datasets led to the following observations and conclusions. The majority of LG in the cytosol and at the plasma membrane of unstimulated NK cells are mobile. The use of inhibitors indicated that movement in the cytosol required microtubules but not actin, whereas movement at the plasma membrane required both. Rab27a deficiency resulted in fewer LG, and in a reduced fraction of mobile LG, at the plasma membrane. In contrast, loss of Rab27a increased the fraction of mobile LG and the extent of their movement in the cytosol. Therefore, in addition to its documented role in LG delivery to the plasma membrane, Rab27a may restrict LG movement in the cytosol.