CAMSAP2 and CAMSAP3 localize at microtubule intersections to regulate the spatial distribution of microtubules.

Microtubule networks support many cellular processes and have a highly ordered architecture. However, due to the limited axial resolution of conventional light microscopy, the structural features of these networks cannot be resolved in three-dimensional (3D) space. Here, we use customized ultra-high resolution interferometric single-molecule localization microscopy to characterize the microtubule networks in Caco2 cells. We find that the microtubule minus-ends associated protein CAMSAPs localize at a portion of microtubule intersections. Further investigation shows that depletion of CAMSAP2 and CAMSAP3 leads to the narrowing of the inter-microtubule distance. We find that CAMSAPs recognize microtubule defects, which are often associated with microtubule intersections, and then recruit katanin to remove the damaged microtubules. Therefore, the CAMSAP-katanin complex is a regulating module for the distance between microtubules. Taken together, our results characterize the architecture of the cellular microtubule networks in high resolution and provide molecular insights into how the 3D structure of microtubule networks is controlled.