Systematic studies of all PIH proteins in zebrafish reveal their distinct roles in axonemal dynein assembly

Abstract

Construction of motile cilia/flagella requires cytoplasmic preassembly of axonemal dyneins before transport into cilia. Axonemal dyneins have various subtypes, but the roles of each dynein subtype and their assembly processes remain elusive in vertebrates. The PIH protein family, consisting of four members, has been implicated in the assembly of different dynein subtypes, although evidence for this idea is sparse. Here, we established zebrafish mutants of all four PIH-protein genes: pih1d1, pih1d2, ktu, and twister, and analyzed the structures of axonemal dyneins in mutant spermatozoa by cryo-electron tomography. Mutations caused the loss of specific dynein subtypes, which was correlated with abnormal sperm motility. We also found organ-specific compositions of dynein subtypes, which could explain the severe motility defects of mutant Kupffer’s vesicle cilia. Our data demonstrate that all vertebrate PIH proteins are differently required for cilia/flagella motions and the assembly of axonemal dyneins, assigning specific dynein subtypes to each PIH protein.Many cells have long, thin structures called cilia on their surface, some types of which can beat back and forth. This beating motion has many roles; for example, cilia on the cells that line the lungs help to sweep out debris, and the tails of sperm beat to move them forward.A structure called the axonemal dynein complex at the core of the cilia generates the beating motion. When the cell makes new cilia, it assembles the complexes in the main body of the cell and then transports them to the right place, like erecting a prefabricated building. Various proteins help to assemble the complexes, of which there are more than eight types. However, the identities of all of these proteins, and their roles in constructing specific axonemal dynein complexes, is not fully known.Studies in algae have suggested that a family of proteins known as PIH (short for protein interacting with Hsp90) helps to construct axonemal dynein complexes. Zebrafish – which share many of the same protein-encoding genes as humans – produce four PIH family proteins. To investigate the roles that each of these proteins play, Yamaguchi et al. used genetic engineering to create four zebrafish mutants that were each unable to produce a different PIH protein.A technique called cryo-electron microscopy enabled the axonemal dynein complexes in the tails of the sperm produced by the zebrafish to be visualized. The sperm from each mutant lacked specific axonemal dynein complexes, revealing that each PIH protein assembles different complexes. The sperm also had difficulties moving. Yamaguchi et al. examined this movement to deduce how specific complexes affect the ability of the sperm to beat their tails.Further work on how PIH proteins interact with the axonemal dynein complexes will help us to understand how cells make cilia, and what happens when this process goes wrong. This could ultimately help us to treat genetic disorders known as ciliopathies, which arise when cilia do not develop normally.