Lattice Light-Sheet Motor-PAINT: A Method to Map the Orientations of Microtubules in Complex Three-Dimensional Arrays

Microtubules play an essential role in many cellular functions, in part by serving as tracks for intracellular transport by kinesin and dynein. The ability of microtubules to fulfill this role fundamentally depends on the fact that they are polar, with motors moving along them toward either their plus or minus end. Given that the microtubule cytoskeleton adopts a variety of specialized architectures in different cell types, it is important to map precisely how microtubules are oriented and organized in these cells. To this end, motor-PAINT has been developed, but in its current implementation, it relies on total internal reflection fluorescence (TIRF) microscopy and is thus restricted to imaging microtubules in a thin section of the cell immediately adjacent to the coverslip. Here, we report a variant of motor-PAINT that uses lattice light-sheet microscopy to overcome this, allowing for the mapping of microtubule organization and orientation in three-dimensional samples. We describe the necessary steps to purify, label, use, and image kinesin motors for motor-PAINT and outline the analysis pipeline used to visualize the resulting data. The method described here can be used in the future to study the microtubule cytoskeleton in (thick) polarized cells such as intestinal epithelial cells.