Genetic analysis of centrioles and cilia

Project Summary/Abstract
Basal bodies are large protein complexes that template cilia and organize the cytoskeleton. Defects in
basal bodies contribute to both cancer and ciliopathies. Ciliopathies are human syndromic diseases that
include brain malformations, mental retardation, polydactyl, blindness, obesity, and defective kidneys.
Cilia are key for multiple functions that include respiratory function, fertility, and establishment of
laterality. Our long-term goal is to understand the mechanisms that are altered to cause these diseases.
We will study events needed for ciliary function and structure and for assembling basal bodies. From our
studies using single particle cryo-EM, we will explore the function of microtubule inner proteins (MIPs)
that are localized in the lumen of doublet microtubules of the cilia. To understand the roles of MIPs, we
will use genetic, biochemical, proteomic, and quantitative imaging approaches. We have found that at
least four MIPs are needed for the symmetric waveform that is used in sperm and nodal cilia and variants
in these genes are associated with infertility and laterality defects in humans. These MIPs are likely to act
via the outer dynein arms and tektin, a protein first described for its insolubility in cilia. We will also study
a class of MIPs called SAXO proteins that have a Mn-motif, and test if they play a role in the stability of
cilia. To examine basal body assembly, we will study delta, epsilon, and zeta tubulin. Delta-tubulin
mutants assemble doublet microtubules and epsilon-tubulin mutants assemble singlet microtubules.
Suppressors of ciliary mutants provided great insight into the regulation of motility. We will take
advantage of suppressors of a missense epsilon-tubulin mutant to identify proteins that interact with this
tubulin isoform. We will use 4x-expansion microscopy for studying the assembly pathway for triplet
microtubules using genes identified by suppressors. Tubulin undergoes many post-translational
modifications. We are studying a new post-translational modification on α-tubulin that is the addition of
the β-sulfonic amino acid, taurine. The role of taurine-tubulin (τ-tub) is unknown, but taurine itself has
been implicated in aging and cancer. Our early characterization shows that τ-tub is present in cilia, basal
bodies and cytoplasmic microtubules in some tissues. We will use genetics and 4x-expansion
microscopy to understand the role of τ-tub in ciliary and basal body function. τ-tub is likely to have
consequence on other known post-translational modification of tubulin; we will tease these interactions
apart. Our studies will provide excellent training experiences for undergraduate and graduate students as
well as postdoctoral researchers and professional research assistants. We will continue our established
collaborations with labs that use electron microscopy and mechanics to expand the impact of our studies.