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

单头、双头和三头轴丝动力蛋白的结构表征

• 批准号: 10657797
• 负责人: Alan Brown
• 金额: $ 35.31万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10657797
• 关键词: ATP phosphohydrolase; Affect; Binding; Biochemical; Bos taurus; Calcium; Cattle; Cerebrospinal Fluid; Chemicals; Chlamydomonas; Chlamydomonas reinhardtii; Chronic; Cilia; Cilium Microtubule; Classification; Complex; Cryoelectron Microscopy; Data; Diagnosis; Docking; Dynein ATPase; Elements; Embryonic Development; Etiology; Evolution; Extracellular Fluid; Family; Fertility; Flagella; Frequencies; Functional disorder; Genetic study; Handedness; Hereditary Disease; Human; Infertility; Location; Locomotion; Mastigophora; Mechanics; Methods; Microtubules; Molecular; Motor; Movement; Mucous body substance; Mutagenesis; Mutation; Natural Immunity; Neonatal Respiratory Distress; Nucleotides; Oocytes; Organelles; Organism; Paralysed; Pattern; Periodicals; Periodicity; Physiology; Play; Positioning Attribute; Power stroke; Radial; Rationalization; Regulation; Resolution; Respiratory Tract Infections; Role; Speed; Structure; Video Microscopy; Work; arm; cell motility; chronic respiratory disease; ciliopathy; cilium motility; driving force; fluid flow; insight; model organism; new therapeutic target; particle; preservation; respiratory; response; sperm cell; targeted treatment

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.

项目总结