Dissecting the Axoneme Interactome

Abstract

The axoneme central apparatus is thought to control flagellar/ciliary waveform and maintain the structural integrity of the axoneme, but proteins involved in these processes have not been fully elucidated. Moreover the network of interactions among them that allows these events to take place in a compact space has not been defined. PF6, a component of the Chlamydomonas central apparatus, is localized to the 1a projection of the C1 microtubule. Mutations in the Chlamydomonas PF6 gene result in flagellar paralysis. We characterized human and murine orthologues of PF6. The murine Pf6 gene is expressed in a pattern consistent with a role in flagella and cilia, and the PF6 protein is indeed localized to the central apparatus of the sperm flagellar axoneme. We discovered that a portion of PF6 associates with the mammalian orthologue of Chlamydomonas PF16 (sperm-associated antigen 6 (SPAG6)), another central apparatus protein that is localized to the C1 microtubule in algae. A fragment of PF6 corresponding to the PF6 domain that interacts with SPAG6 in yeast two-hybrid assays and colocalizes with SPAG6 in transfected cells was missing from epididymal sperm of SPAG6-deficient mice. SPAG6 binds to the mammalian orthologue of PF20, which in Chlamydomonas is located in bridges connecting the C2 and C1 microtubules. Thus, PF6, SPAG6, and PF20 form a newly identified network that links together components of the axoneme central apparatus and presumably participates in its dynamic regulation of ciliary and flagellar beat. Molecular & Cellular Proteomics 4:914 -923, 2005.Cell motility and the movement of surface fluids are dependent upon flagella and cilia. The core structure of these organelles, the axoneme, is remarkably conserved across species, having in common nine doublets of microtubules with the associated force-generating dynein arms and radial spokes surrounding two central singlet microtubules (the central apparatus), which contain associated proteins (1-10). The central apparatus is thought to play a key role in translating the microtubule sliding caused by dynein into the flagellar waveform and maintaining structural integrity of the axoneme (9, 10). However, the central apparatus proteins involved in these processes have not been fully elucidated, and the network of interactions among them that allows these events to take place in a compact space has not been defined. PF6, a component of the Chlamydomonas central apparatus, is localized to the 1a projection of the C1 microtubule (11). The Chlamydomonas pf6 mutant has paralyzed flagella and lacks the 1a projection, indicating that PF6 has a critical role in axoneme structure and function. We characterized the human and murine orthologues of PF6 and demonstrated that the murine Pf6 gene is expressed in a pattern consistent with a role in flagella and cilia and that PF6 protein is indeed localized to the central apparatus of the sperm flagellar axoneme. We discovered that murine PF6 directly associates with the mammalian orthologue of Chlamydomonas PF16 (SPAG6) 1 (1...

Next generation massively parallel sequencing of targeted exomes to identify genetic mutations in primary ciliary dyskinesia: Implications for application to clinical testing

“…The SPAG17 gene encodes an orthologue of Chlamydomonas PF6, a component of the flagellar central apparatus. 30 Mutations of the Chlamydomonas ortholog cause flagellar paralysis, but phenotypes have not been reported in humans or mice. Sanger sequencing confirmed the presence of both variants, but their clinical significance is uncertain.…”

A Draft Sequence of the Neandertal Genome

“…We found only five genes with more than one fixed substitution changing the primary structure of the encoded proteins. One of these is SPAG17 , which encodes a protein important for the axoneme, a structure responsible for the beating of the sperm flagellum (58). The second is PCD16 , which encodes fibroblast cadherin-1, a calcium-dependent cell-cell adhesion molecule that may be involved in wound healing (59).…”

A Protein-Protein Interaction Map of the Trypanosoma brucei Paraflagellar Rod

“…Yeast two-hybrid analyses have been extensively used to systematically investigate interactions of proteins in many model organisms [19], [20], [21], [22] at genome scale. Although local studies of flagellar functions have used the yeast two-hybrid approach to interrogate specific protein-protein interactions [23], [24], [25], [26], the utility of this technique for high throughput investigation has yet to be exploited for this organelle. A systematic interrogation of protein interactions in the flagellum is likely to provide valuable insight into its molecular architecture and functions.…”