Ttll9 −/− mice sperm flagella show shortening of doublet 7, reduction of doublet 5 polyglutamylation and a stall in beating

Abstract

Nine outer doublet microtubules in axonemes of flagella and cilia are heterogeneous in structure and biochemical properties. In mammalian sperm flagella, one of the factors to generate the heterogeneity is tubulin polyglutamylation, although the importance of the heterogeneous modification is unclear. Here, we show that a tubulin polyglutamylase Ttll9 deficiency (Ttll9) causes a unique set of phenotypes related to doublet heterogeneity. Ttll9 −/− sperm axonemes had frequent loss of a doublet and reduced polyglutamylation. Intriguingly, the doublet loss selectively occurred at the distal region of doublet 7, and reduced polyglutamylation was observed preferentially on doublet 5. Ttll9 −/− spermatozoa showed aberrant flagellar beating, characterized by frequent stalls after antihook bending. This abnormal motility could be attributed to the reduction of polyglutamylation on doublet 5, which probably occurred at a position involved in the switching of bending. These results indicate that mammalian Ttll9 plays essential roles in maintaining the normal structure and beating pattern of sperm flagella by establishing normal heterogeneous polyglutamylation patterns.

Glutamylation Regulates Transport, Specializes Function, and Sculpts the Structure of Cilia

“…TTLLs and tubulin glutamylation are required for normal sperm MT structure and motility in mice [46]. TTLL9 [3] and TTLL5 [46] are required for normal mammalian sperm flagella ultrastructure, which supports this hypothesis. We speculate that mutations affecting glutamylation may impact human sperm motility and fertility.…”

“…Although this hypothesis seems unlikely, polyglutamylation can seemingly exert structural effects on different doublets. For example, loss of the glutamylase TTLL9 in mouse sperm flagella causes reduction of glutamylation on doublet 5 but selective loss of doublet 7 [3]. Perhaps even minimal A-tubule glutamylation is sufficient to regulate A-tubule-mediated transport.…”

“…Although virtually all cilia are built by a conserved intraflagellar transport (IFT) process and share a similar architecture, cilia and flagella adopt morphological specializations and serve diverse functions [1]. For example, the rods and cones of the retina are elaborately shaped cilia [2], while sperm have simple whip-like flagella that are variable in length and axoneme structure [3]. C. elegans amphid channel cilia, mammalian olfactory cilia, and mammalian renal primary cilia possess a proximal doublet region followed by a distal A-tubule singlet region [1, 2, 4, 5].…”

Increase in α-tubulin modifications in the neuronal processes of hippocampal neurons in both kainic acid-induced epileptic seizure and Alzheimer’s disease

“…TTLL proteins possess a conserved core catalytic domain, TTL domain16. Eight TTLL proteins, TTLL1, 4, 5, 6, 7, 9, 11, and 13 are involved in tubulin glutamylation16171819. TTLL4 and 5 catalyse the first step, i.e., the initiation of glutamylation, with preferences to α- and β-tubulin, respectively18.…”

Post-translational polymodification of β1 tubulin regulates motor protein localisation in platelet production and function

“…There is some debate as to whether these polymodifications negatively regulate one another (24,25). Mutations in the glutamylases and deglutamylases regulating polymodification can cause male infertility through aberrant spermatogenesis and poor sperm motility, as well as dysfunctions in airway cilia and axonal transport (9,11,(26)(27)(28)(29) To date polyglycylation has not been reported in MKs or platelets. Recently Van Dijke et al report on the polyglutamylation of TUBB1 in a CHO cell line engineered to express TUBB1 downstream of the integrin -2 b -3 , and platelets spread on fibrinogen (30).…”