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

Abstract

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.

Decoding the molecular interplay of endogenous CD20 and Rituximab with fast volumetric nanoscopy

“…For realization of fast, whole-cell 3D TDI-DNA-PAINT we used a commercially available inverted LLS microscope equipped with high-power lasers. Using a light-sheet thickness of 1.8 µm and a new astigmatism approach 15 enabled us to perform 3D TDI-DNA-PAINT of whole COS7 cells immunolabeled for microtubules (Fig. 2a-c and Fig.…”

“…2A-C, fig. S4 and movie S14) (14). Using a lower TDI-DNA-PAINT imager probe concentration of 2.5 nM to accommodate for the width of astigmatic patterns, we detected 21.3 million localizations for Ox2-R4 and 17.5 million localizations for Ox2-P3 within a 4-hour acquisition time from an acquired volume of 87 µm × 72 µm × 8.8 µm (movies S15-S17).…”