Spermatogenesis is a highly specialised differentiation process driven by a dynamic gene expression program and ending with the production of mature spermatozoa. Whereas hundreds of genes are known to be essential for male germline proliferation and differentiation, the contribution of several genes remains uncharacterized. The predominant expression of the latest globin family member, androglobin (Adgb), in mammalian testis tissue prompted us to assess its physiological function in spermatogenesis. Adgb knockout mice display male infertility, reduced testis weight, impaired maturation of elongating spermatids, abnormal sperm shape and ultrastructural defects in microtubule and mitochondrial organisation. Epididymal sperm from Adgb knockout animals display multiple flagellar malformations including coiled, bifid or shortened flagella, and erratic acrosomal development. Following immunoprecipitation and mass spectrometry, we could identify septin 10 (Sept10) as interactor of Adgb. The Sept10-Adgb interaction was confirmed both in vivo using testis lysates, and in vitro by reciprocal co-immunoprecipitation experiments. Furthermore, absence of Adgb leads to mislocalisation of Sept10 in sperm, indicating defective manchette and sperm annulus formation. Finally, in vitro data suggest that Adgb contributes to Sept10 proteolysis in a calmodulin (CaM)-dependent manner. Collectively, our results provide evidence that Adgb is essential for murine spermatogenesis and further suggest that Adgb is required for sperm head shaping via the manchette and proper flagellum formation.
RNA-sequencing data have been submitted to ENA with accession number PRJEB46499 and is also available as supplemental dataset 1 (excel table).All data generated or analysed during this study are included in the manuscript and supporting files. Source data files are provided for Figures 1, 2, 4B, 4C, 4D, 4E, 6A, 6B, 6C, 6D, 6E, 6F, Fig. 1-fig. suppl. 1, Fig. 4-fig. suppl. 1A, B, C, D, E, F, G, Fig. 4-fig. suppl. 2A, B, C, Fig. 4-fig. suppl. 3A, B, C, D, E, Fig. 4-fig. suppl. 5, Fig. 4-fig. suppl. 6B, Fig. 6-fig. suppl. 1A, Fig. 6-fig. suppl. 4, Fig. 6-fig. suppl. 5D, E.
- David Hoogewijs
- David Hoogewijs
- David Hoogewijs
- Thomas Hankeln
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: All experimental procedures and animal maintenance followed Swiss federal guidelines and the study was revised and approved by the "Service de la sécurité alimentaire et des affaires vétérinaires" (SAAV) of the canton of Fribourg, Switzerland (license number 2017_16_FR).
- T Rajendra Kumar, University of Colorado, United States
- Received: July 21, 2021
- Accepted: June 13, 2022
- Accepted Manuscript published: June 14, 2022 (version 1)
© 2022, Keppner et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Microtubules are dynamic polymers consisting of αβ-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αβ-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, a considerable number of microtubules can polymerize independently of the centrosome in various cell types. Here, we present evidence that the minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation by significantly reducing the nucleation barrier. CAMSAP2 co-condensates with αβ-tubulin via a phase separation process, producing plenty of nucleation intermediates. Microtubules then radiate from the co-condensates, resulting in aster-like structure formation. CAMSAP2 localizes at the co-condensates and decorates the radiating microtubule lattices to some extent. Taken together, these in vitro findings suggest that CAMSAP2 supports microtubule nucleation and growth by organizing a nucleation centre as well as by stabilizing microtubule intermediates and growing microtubules.
Heterozygous, missense mutations in α- or β-tubulin genes are associated with a wide range of human brain malformations, known as tubulinopathies. We seek to understand whether a mutation’s impact at the molecular and cellular levels scale with the severity of brain malformation. Here, we focus on two mutations at the valine 409 residue of TUBA1A, V409I, and V409A, identified in patients with pachygyria or lissencephaly, respectively. We find that ectopic expression of TUBA1A-V409I/A mutants disrupt neuronal migration in mice and promote excessive neurite branching and a decrease in the number of neurite retraction events in primary rat neuronal cultures. These neuronal phenotypes are accompanied by increased microtubule acetylation and polymerization rates. To determine the molecular mechanisms, we modeled the V409I/A mutants in budding yeast and found that they promote intrinsically faster microtubule polymerization rates in cells and in reconstitution experiments with purified tubulin. In addition, V409I/A mutants decrease the recruitment of XMAP215/Stu2 to plus ends in budding yeast and ablate tubulin binding to TOG (tumor overexpressed gene) domains. In each assay tested, the TUBA1A-V409I mutant exhibits an intermediate phenotype between wild type and the more severe TUBA1A-V409A, reflecting the severity observed in brain malformations. Together, our data support a model in which the V409I/A mutations disrupt microtubule regulation typically conferred by XMAP215 proteins during neuronal morphogenesis and migration, and this impact on tubulin activity at the molecular level scales with the impact at the cellular and tissue levels.