Ca2+-inactivation of the mammalian ryanodine receptor type 1 in a lipidic environment revealed by cryo-EM

Activation of the intracellular Ca2+ channel ryanodine receptor (RyR) triggers a cytosolic Ca2+ surge, while elevated cytosolic Ca2+ inhibits the channel in a negative feedback mechanism. Cryo-EM carried out under partially inactivating Ca2+ conditions revealed two conformations of RyR1, an open state and an inactivated state, resolved at 4.0 and 3.3 Angstroms resolution, respectively. RyR1s were embedded in nanodiscs with two lipids resolved at each inter-subunit crevice. Ca2+ binding to the high affinity site engages the central (CD) and C-terminal domains (CTD) into a quasi-rigid unit, which separates the S6 four-helix bundle and opens the channel. Further out-of-plane rotation of the quasi-rigid unit pushes S6 towards the central axis, closing (inactivating) the channel. The inactivated conformation is characterized by a downward conformation of the cytoplasmic assembly, a tightly-knit subunit interface contributed by a fully occupied and partially remodeled Ca2+ activation site, and two salt bridges between the EF hand domain and the S2-S3 loop of the neighboring subunit validated by naturally-occurring disease-causing mutations. Ca2+ also bound to ATP, mediating a tighter interaction between S6 and CTD. Our study suggests that the closed-inactivated is a distinctive state of the RyR1 and its transition to the closed-activable state is not a simple reverse of the Ca2+ mediated activation pathway.

Defects in the cytoplasmic assembly of axonemal dynein arms cause morphological abnormalities and dysmotility in sperm cells leading to male infertility

Axonemal protein complexes, such as outer (ODA) and inner (IDA) dynein arms, are responsible for the generation and regulation of flagellar and ciliary beating. Studies in various ciliated model organisms have shown that axonemal dynein arms are first assembled in the cell cytoplasm and then delivered into axonemes during ciliogenesis. In humans, mutations in genes encoding for factors involved in this process cause structural and functional defects of motile cilia in various organs such as the airways and result in the hereditary disorder primary ciliary dyskinesia (PCD). Despite extensive knowledge about the cytoplasmic assembly of axonemal dynein arms in respiratory cilia, this process is still poorly understood in sperm flagella. To better define its clinical relevance on sperm structure and function, and thus male fertility, further investigations are required. Here we report the fertility status in different axonemal dynein preassembly mutant males (DNAAF2/ KTU, DNAAF4/ DYX1C1, DNAAF6/ PIH1D3, DNAAF7/ZMYND10, CFAP300/C11orf70 and LRRC6). Besides andrological examinations, we functionally and structurally analyzed sperm flagella of affected individuals by high-speed video- and transmission electron microscopy as well as systematically compared the composition of dynein arms in sperm flagella and respiratory cilia by immunofluorescence microscopy. Furthermore, we analyzed the flagellar length in dynein preassembly mutant sperm. We found that the process of axonemal dynein preassembly is also critical in sperm, by identifying defects of ODAs and IDAs in dysmotile sperm of these individuals. Interestingly, these mutant sperm consistently show a complete loss of ODAs, while some respiratory cilia from the same individual can retain ODAs in the proximal ciliary compartment. This agrees with reports of solely one distinct ODA type in sperm, compared to two different ODA types in proximal and distal respiratory ciliary axonemes. Consistent with observations in model organisms, we also determined a significant reduction of sperm flagellar length in these individuals. These findings are relevant to subsequent studies on the function and composition of sperm flagella in PCD patients and non-syndromic infertile males. Our study contributes to a better understanding of the fertility status in PCD-affected males and should help guide genetic and andrological counselling for affected males and their families.

FAP57/WDR65 targets assembly of a subset of inner arm dyneins and connects to regulatory hubs in cilia

Ciliary motility depends on both the precise spatial organization of multiple dynein motors within the 96 nm axonemal repeat and the highly coordinated interactions between different dyneins and regulatory complexes located at the base of the radial spokes. Mutations in genes encoding cytoplasmic assembly factors, intraflagellar transport factors, docking proteins, dynein subunits, and associated regulatory proteins can all lead to defects in dynein assembly and ciliary motility. Significant progress has been made in the identification of dynein subunits and extrinsic factors required for preassembly of dynein complexes in the cytoplasm, but less is known about the docking factors that specify the unique binding sites for the different dynein isoforms on the surface of the doublet microtubules. We have used insertional mutagenesis to identify a new locus, IDA8/BOP2, required for targeting the assembly of a subset of inner dynein arms (IDAs) to a specific location in the 96 nm repeat. IDA8 encodes flagellar-associated polypeptide (FAP)57/WDR65, a highly conserved WD repeat, coiled coil domain protein. Using high resolution proteomic and structural approaches, we find that FAP57 forms a discrete complex. Cryo-electron tomography coupled with epitope tagging and gold labeling reveal that FAP57 forms an extended structure that interconnects multiple IDAs and regulatory complexes.

C11orf70 mutations causing primary ciliary dyskinesia disrupt a conserved step in the intraflagellar transport-dependent assembly of multiple axonemal dyneins

Primary ciliary dyskinesia (PCD) is a genetically and phenotypically heterogeneous disorder characterized by destructive respiratory disease and laterality abnormalities due to randomised left-right body asymmetry. PCD is mostly caused by mutations affecting components of the core axoneme structure of motile cilia that are essential for cilia movement. In addition, there is a growing group of PCD genes that encode proteins essential for the assembly of the ciliary dynein motors and the active transport process that delivers them from their cytoplasmic assembly site into the axoneme. We screened a cohort of affected individuals for disease-causing mutations using a targeted next generation sequencing panel and identified 2 unrelated families (3 affected children) with mutations in the uncharacterized C11orf70 gene. The affected children share a consistent PCD phenotype from early life with laterality defects and immotile respiratory cilia displaying combined loss of inner and outer dynein arms (IDA+ODA). Phylogenetic analysis shows C11orf70 is highly conserved, distributed across species similarly to proteins involved in the intraflagellar transport (IFT)-dependant assembly of axonemal dyneins. Paramecium C11orf70 RNAi knockdown led to combined loss of ciliary IDA+ODA with reduced cilia beating and swim velocity. Fluorescently tagged C11orf70 in Paramecium and Chlamydomonas localises mainly in the cytoplasm with a small amount in the ciliary component, its abundance in the axoneme being IFT-dependant. During ciliogenesis, C11orf70 accumulates at the ciliary tips in a similar distribution to the IFT-B protein IFT46. In summary, C11orf70 is essential for IFT-dependant assembly of dynein arms and C11orf70 mutations cause defective cilia motility and PCD.