Proteomics of isolated rat manchettes

The manchette is a transient microtubular structure that plays a vital role in nuclear shaping during spermiogenesis. It comprises thousands of microtubules (MTs) that build a scaffold surrounding the distal half of the nucleus. The manchette distributes proteins and vesicles during spermiogenesis in a process called intra-manchette transport (IMT). The current hypothesis is that IMT shares many similarities with intra-flagellar transport (IFT) and utilizes both MTs and filamentous actin (F-actin). However, IMT is still poorly understood as direct visualization of IMT complexes is missing, and the presence of F-actin has not been experimentally shown. Here, we use cryogenic-electron tomography (cryo-ET) to visualize IMT components in manchettes extracted from rat spermatids. Additionaly, we used proteomics to identify and quantify IMT composition. We find that F-actin is an integral part of the manchette with two different spatial organizations, namely bundles and single filaments, indicating two different functional roles. We further uncover that IMT on MTs is mediated by two distinct transport machineries: dynein mediated transport of soluble cargo and dynein independent transport for vesicles. Our results provide new insights into the manchette's function as a transport scaffold, highlighting its significance for the polarization of spermatids during spermiogenesis.