Abstract
Cilia are evolutionarily highly conserved organelles with important functions in many organs. The extracellular component of the cilium protruding from the plasma membrane comprises an axoneme composed of microtubule doublets, arranged in a 9 + 0 conformation in primary cilia or 9 + 2 in motile cilia. These microtubules facilitate transport of intraflagellar cargoes along the axoneme. They also provide structural stability to the cilium, which may play an important role in sensory cilia, where signals are received from the movement of extracellular fluid. Post-translational modification of microtubules in cilia is a well-studied phenomenon, and acetylation on lysine 40 (K40) of alpha tubulin is prominent in cilia. It is believed that this modification contributes to the stabilization of cilia. Two classes of enzymes, histone acetyltransferases and histone deacetylases, mediate regulation of tubulin acetylation. Here we use a genetic approach, immunocytochemistry and behavioral tests to investigate the function of tubulin deacetylases in cilia in a zebrafish model. By mutating three histone deacetylase genes (Sirt2, Hdac6, and Hdac10), we identify an unforeseen role for Hdac6 and Sirt2 in cilia. As expected, mutation of these genes leads to increased acetylation of cytoplasmic tubulin, however, surprisingly it caused decreased tubulin acetylation in cilia in the developing eye, ear, brain and kidney. Cilia in the ear and eye showed elevated levels of mono-glycylated tubulin suggesting a compensatory mechanism. These changes did not affect the length or morphology of cilia, however, functional defects in balance was observed, suggesting that the level of tubulin acetylation may affect function of the cilium.
Highlights
Cilia are microscopic microtubule-based structures necessary for the function of many signal transduction cascades, including those involved in vision, hearing, olfaction, and embryonic patterning (Omori and Malicki, 2006; Burcklé et al, 2011; Malicki, 2012)
Treatment at 6 h post fertilization resulted in the hypoacetylated phenotype in crista kinocilia, and fish treated at 24 and 48 hpf do not display this phenotype (Supplementary Figure 3). This is interesting as cilia in the ear are formed around 18 hpf, which would suggest that the observed hypoacetylation of axoneme occurs during ciliogenesis rather than being the result of later modifications (Whitfield, 2020). To determine whether this phenotype is consistently observed in cilia of other tissues, we investigated kinocilia in inner ear macule, the connecting cilium of photoreceptor cells in the eye, and cilia in the nasal duct Tubulin acetylation in the kinocilia of inner ear macule was reduced in hdac6, hdac10, sirt2 single mutants, and hdac6; hdac10 double mutants
In keeping with existing mouse models (Fukada et al, 2012), we find that inactivation of hdac6 or sirt2 does not cause a visible external phenotype in D. rerio
Summary
Cilia are microscopic microtubule-based structures necessary for the function of many signal transduction cascades, including those involved in vision, hearing, olfaction, and embryonic patterning (Omori and Malicki, 2006; Burcklé et al, 2011; Malicki, 2012). Acetylation is a dynamic and reversible process regulated by two classes of competing enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs). Despite their nomenclature, these enzymes act on many substrates other than histones, including tubulin. Deacetylation of tubulin in the axoneme was shown to be one of the events initiating cilia disassembly in adipocytes, hTERT-RPE1, IMCD-3 murine, and Caki-1 human renal cell lines, suggesting it has a critical role in ciliary dynamics (Pugacheva et al, 2007; Loktev et al, 2008; Forcioli-Conti et al, 2016)
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