Files
Abstract
Microtubules are one of the major cytoskeletons in eukaryotes. They are polymersof α- and β-tubulins heterodimers. Microtubules play many important roles in diverse
cellular functions and establishing cellular structures like cilia, neuronal axons, and
centrosomes. Microtubule Posttranslational modifications provide the diversity for
microtubules to carry out these functions. Microtubule acetylation, glycylation,
glutamylation, and detyrosination are the major PTMs in vertebrates. We generated
mutants of enzymes that carry out these modifications to understand their roles in
zebrafish development, ciliary functions, and neuronal functions.
Animal-vegetal (AV) axis formation occurs during oogenesis by forming the Balbiani
body at the future vegetal pole. The formation of chromosomal bouquet during the
zygotene stage is the initial asymmetry-breaking step, and precursors of the Balbianic
body formed at the cytoplasmic site where this chromosomal bouquet is formed. A
zygotene stage-specific cilium plays a critical role in chromosomal bouquet formation. In
our study, we found that mutants that lack both microtubule acetylation and glycylation
show defects in the chromosomal bouquet formation, leading to reduced female fecundity
and fertility. The zygotene cilia of atat1; ttll3 double mutant does not show any
ciliogenesis or ciliary maintenance defect. The loss of these modifications potentially
alters the ciliary function by changing the physical properties of these microtubules.
Additionally, we found that ttll3 exhibits reduced progressive sperm motility and atat1
mutants show altered behavior. Our data show that microtubule acetylation and
glycylation play important roles in zebrafish ciliary and neuronal function, and also these
modifications synergistically regulated oocyte polarization.
In this work, we generated mutants of microtubule detyrosinases vash1 and vash2 using
CRISPR-Cas9 RNP mutagenesis. Single and double mutants of these genes showed
reductions in microtubule detyrosination and vash1; vash2 double mutant showed low
penetrance microcephaly, curved body, and heart edema phenotypes. We also found a
potential alteration in axonal transportation in the double mutant. Additionally, We
generated a mutant of the ttl gene responsible for retyrosination and mutants of
microtubule glutamylation initiases ttll4, ttll5, and ttll7 to study the importance of these
modifications in zebrafish ciliary and neuronal functions.