Biology subjects

Mitchison, H. M.

Publications and source records attributed to Mitchison, H. M..

3 recordsLinked to original sources

bioRxiv2023

ciliR: a new R package for determining ciliary beat frequency using fast-Fourier transformation.

Cilia are motile hair-like structures that play a vital role in our body. Accurate assessment of ciliary beat frequency is pivotal for investigating ciliary dynamics and diagnosing ciliopathies. This study aims to develop software for accurately measuring the beat frequency of cilia captured using high-speed video microscopy. To achieve this, we developed the ciliR package in R, which was validated against manual counting and three other automated methods of counting cilia beat frequency. The results showed that ciliR produced results that were comparable to manual counting. The accuracy of ciliR was defined by its ability to reduce noise, including only counting data in a biologically significant range (0-60 Hz). Our software is a valuable tool for researchers in the field of ciliobiology as it offers a reliable method for detailed ciliary function analysis, thereby contributing to the broader understanding of mechanisms underlying ciliary-related disease. We encourage researchers to try this package and feed-back their findings to the authors. Instructions for use and processes for providing feedback are provided in supplementary material. SummaryciliR is a novel R package designed for analysing ciliary beat frequency (CBF) via ImageJ and RStudio. The advantage of the ciliR system, lies in its integration with the R environment, increasing processing speed and access to data visualization tools and analysis pipelines available in other R packages. The open-source platform invites community feedback to refine functionality, aiming to advance ciliopathy research with an accessible, comprehensive toolkit.

cell biology↗

bioRxiv2020

High-content screening for rare respiratory diseases: readthrough therapy in primary ciliary dyskinesia

Development of therapeutic approaches for rare respiratory diseases is hampered by the lack of systems that allow medium-to-high-throughput screening of fully differentiated respiratory epithelium from affected patients. This is a particular problem for primary ciliary dyskinesia (PCD), a rare genetic disease caused by mutations in genes that adversely affect ciliary movement and consequently mucociliary transport. Primary cell culture of basal epithelial cells from nasal brush biopsies, followed by ciliated differentiation at air-liquid interface (ALI) has proven to be a useful tool in PCD diagnostics but the techniques broader utility, including in pre-clinical PCD research, has been limited by the number of basal cells that it is possible to expand from such biopsies. Here, we describe a high-content, imaging-based screening method, enabled by extensive expansion of PCD patient basal cells and their culture into differentiated human respiratory epithelium in miniaturised 96-well transwell format ALI cultures. Analyses of ciliary beat pattern, beat frequency and ultrastructure indicate that a range of different PCD defects are retained in these cultures. We perform a proof-of-principle personalized investigation in reduced generation of motile cilia (RGMC), a rare and very severe form of PCD, in this case caused by a homozygous nonsense mutation (c.441C>A; p.Cys147*) in the MCIDAS gene. The screening system allowed multiple drugs inducing translational readthrough to be evaluated alone or in combination with inhibitors of nonsense-mediated decay. Restoration of basal body formation in the patients nasal epithelial cells was seen in vitro, suggesting a novel avenue for drug evaluation and development in PCD. SummaryWe describe primary cell culture of nasal epithelial cells from patients with primary ciliary dyskinesia including differentiatiation of these to a ciliary phenotype and high-content screening in miniaturised air-liquid interface cultures.

cell biology↗

bioRxiv2017

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.

genetics↗