Biology subjects

Dutcher, S. K.

Publications and source records attributed to Dutcher, S. K..

8 recordsLinked to original sources

bioRxiv2024

Loss of an extensive ciliary connectome induces proteostasis and cell fate switching in a severe motile ciliopathy

Motile cilia have essential cellular functions in development, reproduction, and homeostasis. Genetic causes for motile ciliopathies have been identified, but the consequences on cellular functions beyond impaired motility remain unknown. Variants in CCDC39 and CCDC40 cause severe disease not explained by loss of motility. Using human cells with pathological variants in these genes, Chlamydomonas genetics, cryo-electron microscopy, single cell RNA transcriptomics, and proteomics, we identified perturbations in multiple cilia-independent pathways. Absence of the axonemal CCDC39/CCDC40 heterodimer results in loss of a connectome of over 90 proteins. The undocked connectome activates cell quality control pathways, switches multiciliated cell fate, impairs microtubule architecture, and creates a defective periciliary barrier. Both cilia-dependent and independent defects are likely responsible for the disease severity. Our findings provide a foundation for reconsidering the broad cellular impact of pathologic variants in ciliopathies and suggest new directions for therapies.

cell biology↗

bioRxiv2024

Ultrastructure expansion microscopy (U-ExM) of mouse and human kidneys for analysis of subcellular structures

Ultrastructure expansion microscopy (U-ExM) involves the physical magnification of specimens embedded in hydrogels, which allows for super-resolution imaging of subcellular structures using a conventional diffraction-limited microscope. Methods for expansion microscopy exist for several organisms, organs, and cell types, and used to analyze cellular organelles and substructures in nanoscale resolution. Here, we describe a simple step-by-step U-ExM protocol for the expansion, immunostaining, imaging, and analysis of cytoskeletal and organellar structures in kidney tissue. We detail the critical modified steps to optimize isotropic kidney tissue expansion, and preservation of the renal cell structures of interest. We demonstrate the utility of the approach using several markers of renal cell types, centrioles, cilia, the extracellular matrix, and other cytoskeletal elements. Finally, we show that the approach works well on mouse and human kidney samples that were preserved using different fixation and storage conditions. Overall, this protocol provides a simple and cost-effective approach to analyze both pre-clinical and clinical renal samples in high detail, using conventional lab supplies and standard widefield or confocal microscopy.

cell biology↗

bioRxiv2023

Reduced dosage of multiple dynein arm preassembly factors limits cilia regeneration in Chlamydomonas reinhardtii

Motile cilia are complex organelles comprised of >800 structural proteins. Variants in 58 of these genes cause primary ciliary dyskinesia (PCD) in humans. We used a second-site non-complementation approach in diploid Chlamydomonas reinhardtii strains to investigate whether reduced dosage of different combinations of motile cilia proteins affects cilia growth / regeneration. Temperature-sensitive mutants in intraflagellar transport genes fail to complement in heterozygous diploids at the restrictive temperature, which may be due to poisonous interactions or a reduction in protein levels. Diploid strains heterozygous for 211 double mutant combinations in 21 genes are phenotypically recessive when unperturbed and do not show SSNC. Consequently, we developed a sensitized screen. When protein synthesis is inhibited, cells utilize the existing cytoplasmic pool of proteins, which is insufficient to build full-length cilia. Six double heterozygous strains in dynein arm preassembly factors (DNAAFs) pf13, pf23, wdr92, and oda8 regrow cilia that are shorter than wild-type diploids, which suggests that double heterozygosity limits the pool of assembled dynein arms further. We isolated a new null allele (pf23-4) that shows a more severe loss of ODAs and IDAs in cilia than the previously studied pf23-1 hypomorphic allele. We also identified a new role for PF23 in cytoplasmic modification of IC138, protein of the I1/f inner dynein arm. In our SSNC assay, the pf23-4 allele also exhibits a more severe phenotype than pf23-1. Whole cell extract immunoblots show a reduction of PF23 protein to one-half of wild-type levels in both single and double heterozygous genotypes. The pf13 mutant shows SSNC with mutants in two outer dynein arm structural proteins, ODA6, and ODA9. We suggest that reduction of multiple dynein preassembly factors limits the pool of assembled dynein arms needed for cilia assembly and regeneration. Our data support PF23 as a scaffolding hub protein involved in dynein arm assembly. Author SummaryMotile cilia are essential for movement of cells and fluids. In humans, motile cilia defects cause primary ciliary dyskinesia (PCD), a rare disease characterized by recurring respiratory infections, left-right asymmetry defects, ear infections, and infertility. Many genes are involved in motile cilia function and assembly. We use Chlamydomonas reinhardtii to study the effects of changes in protein levels in different combinations of proteins needed for motile cilia. Our results show that the proteins that help fold and assemble the dynein motors in the cytoplasm are sensitive to changes in the dosage of these preassembly factors. Reductions in proteins involved in multiple steps of the pathway prevents efficient dynein preassembly and cilia regeneration under stressful cellular conditions.

genetics↗

bioRxiv2023

The effect of Dnaaf5 gene dosage on primary ciliary dyskinesia phenotypes

DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift null deletion in Dnaaf5. Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partial preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. While transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. Together, these findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies. Brief SummaryA mouse model of human DNAAF5 primary ciliary dyskinesia variants reveals gene dosage effects of mutant alleles and tissue-specific molecular requirements for cilia motor assembly.

cell biology↗

bioRxiv2019

Mutation of CFAP57 causes primary ciliary dyskinesia by disrupting theasymmetric targeting of a subset of ciliary inner dynein arms

Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, male infertility, and randomization of the left/right body axis, and is caused by defects of motile cilia and sperm flagella. We screened a cohort of affected individuals that lack an obvious TEM structural phenotype for pathogenic variants using whole exome capture and next generation sequencing. The population sampling probability (PSAP) algorithm identified one subject with a homozygous nonsense variant [(c.1762C>T) p.(Arg588*) exon 11] in the uncharacterized CFAP57 gene. In normal human nasal epithelial cells, CFAP57 localizes throughout the ciliary axoneme. Analysis of cells from the PCD patient shows a loss of CFAP57, reduced beat frequency, and an alteration in the ciliary waveform. Knockdown of CFAP57 in human tracheobronchial epithelial cells (hTECs) recapitulates these findings. Phylogenetic analysis showed that CFAP57 is conserved in organisms that assemble motile cilia, and CFAP57 is allelic with the BOP2 gene identified previously in Chlamydomonas. Two independent, insertional fap57 Chlamydomonas mutant strains show reduced swimming velocity and altered waveforms. Tandem mass spectroscopy showed that CFAP57 is missing, and the \"g\" inner dyneins (DHC7 and DHC3) and the \"d\" inner dynein (DHC2) are reduced. Our data demonstrate that the FAP57 protein is required for the asymmetric assembly of inner dyneins on only a subset of the microtubule doublets, and this asymmetry is essential for the generation of an effective axonemal waveform. Together, our data identifies mutations in CFAP57 as a cause of PCD with a specific defect in the inner dynein arm assembly process.\n\nSignificanceMotile cilia are found throughout eukaryotic organisms and performs essential functions. Primary ciliary dyskinesia (PCD) is a rare disease that affects the function of motile cilia. By applying a novel population sampling probability algorithm (PSAP) that uses large population sequencing databases and pathogenicity prediction algorithms, we identified a variant in an uncharacterized gene, CFAP57. This is the first reported example of PCD caused by a mutation that affects only a subset of the inner dynein arms, which are needed to generate the waveform. CFAP57 identifies an address for specific dynein arms. These findings demonstrate the effectiveness of the PSAP algorithm, expand our understanding of the positioning of dynein arms, and identify mutations in CFAP57 as a cause of PCD.

genetics↗

bioRxiv2018

Mapping and characterization of structural variation in 17,795 deeply sequenced human genomes

A key goal of whole genome sequencing (WGS) for human genetics studies is to interrogate all forms of variation, including single nucleotide variants (SNV), small insertion/deletion (indel) variants and structural variants (SV). However, tools and resources for the study of SV have lagged behind those for smaller variants. Here, we used a cloud-based pipeline to map and characterize SV in 17,795 deeply sequenced human genomes from common disease trait mapping studies. We publicly release site-frequency information to create the largest WGS-based SV resource to date. On average, individuals carry 2.9 rare SVs that alter coding regions, which affect the dosage or structure of 4.2 genes and account for 4.0-11.2% of rare high-impact coding alleles. Based on a computational model, we estimate that SVs account for 17.2% of rare alleles genome-wide whose predicted deleterious effects are equivalent to loss-of-function (LoF) coding alleles; ~90% of such SVs are non-coding deletions (mean 19.1 per genome). We report 158,991 ultra-rare SVs and show that ~2% of individuals carry ultra-rare megabase-scale SVs, nearly half of which are balanced and/or complex rearrangements. Finally, we exploit this resource to infer the dosage sensitivity of genes and non-coding elements, revealing strong trends related to regulatory element class, conservation and cell-type specificity. This work will help guide SV analysis and interpretation in the era of WGS.

genomics↗

bioRxiv2018

How does cilium length affect beating?

The effects of cilium length on the dynamics of cilia motion were investigated by high-speed video microscopy of uniciliate mutants of the swimming alga, Chlamydomonas reinhardtii. Cells with short cilia were obtained by deciliating cells via pH shock and allowing cilia to reassemble for limited times. The frequency of cilia beating was estimated from motion of the cell body and of the cilium. Key features of the ciliary waveform were quantified from polynomial curves fitted to the cilium in each image frame. Most notably, periodic beating did not emerge until the cilium reached a critical length between 2-4 {micro}m. Surprisingly, in cells that exhibited periodic beating, the frequency of beating was similar for all lengths with only a slight decrease in frequency as length increased from 4 {micro}m to the normal length of 10-12 {micro}m. The waveform average curvature (rad/{micro}m) was also conserved as the cilium grew. The mechanical metrics of ciliary propulsion: force, torque, and power all increased in proportion to length. Mechanical efficiency of beating appeared to be maximal at the normal wild-type length of 10-12 m. These quantitative features of ciliary behavior illuminate the biophysics of cilia motion and, in future studies, may help distinguish competing hypotheses of the underlying mechanism of oscillation.

biophysics↗

bioRxiv2018

Exome sequencing identifies high impact trait-associated alleles enriched in Finns

As yet undiscovered rare variants are hypothesized to substantially influence an individuals risk for common diseases and traits, but sequencing studies aiming to identify such variants have generally been underpowered. In isolated populations that have expanded rapidly after a population bottleneck, deleterious alleles that passed through the bottleneck may be maintained at much higher frequencies than in other populations. In an exome sequencing study of nearly 20,000 cohort participants from northern and eastern Finnish populations that exemplify this phenomenon, most novel trait-associated deleterious variants displayed frequencies 10-173 times higher than in other European populations. These enriched alleles underlie 30 novel associations with 20 disease-related quantitative traits and demonstrate a geographical clustering equivalent to that of Mendelian disease mutations characteristic of the Finnish population. Sequencing studies in populations without this unique history would require hundreds of thousands to millions of participants for comparable power.

genomics↗