Characterizing SAXO1 Localization and Binding Behavior in vitro

Abstract

Microtubules are dynamic polymers that are an integral part of cytoskeletal structures in cells. Microtubule-based organelles such as sperm cell flagella are tightly regulated by microtubule inner proteins (MIPs) to ensure organelle functionality. Recently, the binding structure of SAXO1 to the lumen of the B-tubule of flagellar axonemal microtubule doublets was uncovered, but not a lot is known about the timing and mechanism of entry of SAXO1 into the lumen of microtubules. Here, we investigate the localization of SAXO1 in in vitro reconstitution assays to prepare for cryo-electron microscopy imaging to uncover SAXO1 localization mechanisms. We find that SAXO1 binds to microtubules in vitro in two separate phenotypic ways: islands and decoration. We found the highest levels of SAXO1 decoration with 15 μM microtubule lattices in combination with 500 nM SAXO1. SAXO1 furthermore showed no clear preference for stabilized microtubules or for either the plus or minus end. Our findings contribute towards future experiments to further understand the role of SAXO1 in flagellar motility and consequently in abnormal sperm function.