Recently, we showed that the putative microtubule-severing protein, KATNAL2, is essential for spermatid differentiation. In mice, Katnal2 loss results in spermatid head-shaping defects due to excessive constriction and elongation of the manchette microtubules, accompanied by a lack of manchette movement and delays in its disassembly. An absence of axoneme generation and, in the few sperm flagella that did form, mitochondrial sheath abnormalities were also observed.
Here, we show, unlike its paralogues, KATNAL2 does not sever α/β tubulin microtubules. Instead, our data suggests KATNAL2 targets multiple microtubule-associated proteins. We identify δ (TUBD1) and ε (TUBE1) tubulin as components of the manchette and as strong candidates for being the mysterious manchette microtubule-microtubule and microtubule-nuclear links that modulate manchette constriction and movement. Importantly, through co-immunoprecipitation and proximity ligation assays, we show TUBD1 and TUBE1 bind KATNAL2 and that these complexes localize to the manchette. To investigate the manchette elongation phenotype, we also characterised the expression of the microtubule nucleator, γ-tubulin, in spermatids. We show, γ-tubulin complexes localise to the caudal tips of the manchette microtubules, and in Katnal2 knockout mice the removal of these γ-tubulin foci is delayed, suggesting KATNAL2 is required for the release of manchette microtubules from the microtubule organising centre. Finally, through a forward yeast two-hybrid screen using Katnal2 as bait against a mouse testis prey library, we identified CPEB2 and CPEB4, two RNA-binding proteins, as KATNAL2 binding partners. Our characterisation of these proteins in spermatogenesis supports a model wherein CPEB2 and CPEB4, in partnership with KATNAL2, facilitate a manchette microtubule-based mRNA transport pathway to deliver components to the site of spermatid flagella assembly. Collectively, our data reveals TUBD1, TUBE1, CPEB2, and CPEB4 as new components of the spermiogenesis machinery, and supports a model wherein KATNAL2 modulates the association of the manchette microtubules with diverse targets to facilitate spermatid remodelling.