Abstract
Abstract Study question How are microtubules at the endpiece of the mammalian sperm tail stabilized? Summary answer SAX01 and SPACA9 are microtubule inner proteins that binds at the endpieces. In addition, singlet microtubules are capped by a complex of proteins. What is known already At the core of the sperm flagellum is the axoneme, a supramolecular assembly of nine doublet microtubules around a central pair of singlet microtubules. At the endpiece, axoneme structure is lost and only singlet microtubules remain. Using cryo-electron microscopy (cryo-EM) our group showed that the singlet microtubules contain species-specific microtubule inner proteins in a helical arrangement. Furthermore, we observed a protein density capping the microtubules plus-ends in pig, horse, and mouse sperm. Study design, size, duration To elucidate the function of the sperm endpiece plug and its associated proteins, we apply single-particle analysis cryo-EM. Participants/materials, setting, methods - Main results and the role of chance We revealed that the microtubule inner proteins in the singlets are SAX01 and SPACA9. Our high-resolution structure shows that SPACA9 forms a discontinuously intraluminal spiral. It consists of a bundle of alpha helices that binds across the intradimer α/ÎČ-tubulin interface as well as between adjacent protofilaments. Specifically, SPACA9 interacts with both α- and ÎČ-tubulin within a dimer while also interacting with the M-loop of α-tubulin from the neighbouring protofilament. Our structure further shows that SAX01 binds longitudinally along individual protofilaments with its Mn motif, a 5- to 6-residue helix flanked by a Tyr/Phe residue on one end and by a Ser/Thr on the other that binds the shoulder of the α -tubulin S9/S10 loop. We further observed that microtubule termini are capped with yet to be identified protein complexes. Taken together, we show that microtubule inner proteins longitudinally and laterally reinforce the microtubule lattice, while capped in their termini. Limitations, reasons for caution Limitations include species-specific variations as we previously observed between mouse sperm endpieces and porcine or horse. Cryo-EM structures represent averages across singlet microtubules, so individual variation within particles may be lost. Wider implications of the findings Investigating the mechanism behind stabilization of the endpiece singlet microtubules using cryo-EM provides better understanding of sperm tail stabilization that holds potential for progress in understanding male infertility and can be utilized for a development of male contraception. Trial registration number not applicable
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