Structural characterization of single, double and triple-headed axonemal dyneins

Project Summary
Motile cilia play essential roles in fertility, innate immunity, and embryonic development. The beat of motile cilia
is powered by a diverse family of ATP-dependent motors called axonemal dyneins. Axonemal dyneins are
tethered in repeating patterns to doublet microtubules within the ciliary axoneme and are classified by the
number of motor domains (or heads) that they contain and their position within the axoneme. Outer dynein
arms (ODA) are either double or triple-headed complexes that repeat every 24 nm, whereas inner dynein arms
(IDA) are either double or single-headed and repeat every 96 nm. Their different positions, periodicities, and
subunit compositions manifest as different activities: the ODA determines the beat frequency, whereas the IDA
determines the amplitude of the waveform. Despite their fundamental importance to ciliary motility and human
physiology, little is known about the structures and mechanisms of the large axonemal dynein family. In this
proposal, we plan to exploit recent advances in single-particle electron cryomicroscopy (cryo-EM) to determine
structures of all major classes of axonemal dynein. To capture axonemal dyneins in their active, microtubule-
bound states we have developed methods to isolate and determine high-resolution structures of native dynein-
bound doublet microtubules from three organisms - the biflagellate model organism Chlamydomonas
reinhardtii, Bos taurus and humans. C. reinhardtii will be used for structural, biochemical, and genetic studies
of a triple-headed ODA (Aim 1) and double and single-headed IDAs (Aim 2). These structures are expected to
reveal the mechanisms that dock axonemal dyneins to the doublet microtubule, the structural rearrangements
they undergo during the powerstroke, their regulation by calcium and microtubule curvature, and the functional
relevance of inter-dynein interactions in generating the ciliary waveform. Structures of single and multi-headed
axonemal dyneins will provide insights into the functions of their idiosyncratic subunits and the general
principles that have guided evolution of the axonemal dynein family. Structures of dynein-bound doublet
microtubules from humans and cows (Aim 3) will reveal their differences with algal axonemal dyneins (for
example the comparison between double and triple-headed ODAs) and help explain the etiology of ciliopathy-
causing mutations. Mutations in axonemal dyneins are the leading cause of primary ciliary dyskinesis (PCD), a
currently incurable inherited disease characterized by neonatal respiratory distress, chronic airway infections,
and infertility. Advances in understanding the structures of axonemal dyneins will therefore have important
implications for the identification of key mechanisms that can targeted for therapy of defective cilia.

Grant Number: 5R01GM143183-04
NIH Institute/Center: NIH
Principal Investigator: Alan Brown