Unraveling the intricate microtubule inner protein networks that reinforce mammalian sperm flagella

Abstract

To find and fuse with the egg, mammalian sperm must complete an arduous voyage through the female reproductive tract. The sperm cell remarkable odyssey is powered by its flagellum, a microtubule-based molecular machine ornamented with accessory structures that stabilize the sperm tail in viscous media. Recently, cryo-electron tomography (cryo-ET) revealed that mammalian sperm flagella are further reinforced at the molecular scale with sperm-specific microtubule inner proteins (sperm-MIPs), but the identities of these sperm-MIPs are unknown. Here, we use cryo-electron microscopy to resolve structures of native bovine sperm doublet microtubules, thus identifying most sperm-MIPs. In the A-tubule, several copies of testis-specific Tektin-5 contribute to an extended protein network spanning nearly the entire microtubule lumen. Different copies of Tektin-5 adopt a range of conformations and organizations based on their local interactions with other MIPs. The B-tubule is in turn stabilized by sperm-MIPs that bind longitudinally along and laterally across protofilaments. We further resolve structures of endpiece singlet microtubules, revealing MIPs shared between singlets and doublets. Our structures shed light on the molecular diversity of cilia across different cell types of the vertebrate body and provide a structural framework for understanding the molecular underpinnings of male infertility.

Molecular architecture of the ciliary tip revealed by cryo-electron tomography

“…Intriguingly, our subtomogram average of the A-tubule does not show any stabilising protein, raising the question of how these are stabilised. In human, bull, pig and horse sperm cells, SPACA9 provides stability to the A-tubule by forming a helix inside the lumen (Zabeo, Heumann et al 2018, Leung, Roelofs et al 2021, Gui, Croft et al 2022, Leung, Roelofs et al 2022). This difference likely reflects the role of the ciliary tip.…”

“…Using single-particle cryoelectron microscopy (cryo-EM), the structures of the doublet microtubules, the radial spokes, the outer dynein arms and the CP were solved to high resolution (Ichikawa, Liu et al 2017, Ichikawa, Khalifa et al 2019, Ma, Stoyanova et al 2019, Khalifa, Ichikawa et al 2020, Grossman-Haham, Coudray et al 2021, Gui, Farley et al 2021, Gui, Ma et al 2021, Kubo, Yang et al 2021, Rao, Han et al 2021, Walton, Wu et al 2021, Gui, Wang et al 2022, Han, Rao et al 2022. At slightly lower resolution, cryo-ET has been used to study the structures of doublet and singlet microtubules in mammalian sperm and identify proteins de novo (Chen, Shiozaki et al 2022, Gui, Croft et al 2022, Leung, Roelofs et al 2022. Importantly, the tips of cilia have not been studied at high resolution apart from the singlet-A-tubule of sperm cells (Gui, Croft et al 2022, Leung, Roelofs et al 2022.…”

“…At slightly lower resolution, cryo-ET has been used to study the structures of doublet and singlet microtubules in mammalian sperm and identify proteins de novo (Chen, Shiozaki et al 2022, Gui, Croft et al 2022, Leung, Roelofs et al 2022). Importantly, the tips of cilia have not been studied at high resolution apart from the singlet-A-tubule of sperm cells (Gui, Croft et al 2022, Leung, Roelofs et al 2022).…”

Regionalized protein localization domains in the zebrafish hair cell kinocilium

“…SAXO proteins are members of the microtubule associated protein 6 (MAP6) family that stabilize microtubules in axons, cilia, and flagella [75,76]. Recent work shows that MAP6 and SAXO are microtubule inner proteins (MIPs) that can localize to the intraluminal space of microtubules [79][80][81]83]. Full length MAP6 can bind to either the outside or inside of microtubules, and intraluminal localization requires a N-terminal cysteine-rich domain [83].…”

“…SAXO proteins can stabilize microtubules and use their "Mn" repeat domains to associate with basal bodies, centrioles, and cilia [75][76][77][78]. Interestingly, SAXO and MAP6 are MIPs (microtubule inner proteins), meaning they reside in the luminal space of microtubules [79][80][81][82]. Furthermore, luminal MAP6 can stabilize microtubule curvature [83], though it is unknown whether SAXO proteins also share this functionality.…”