Biology subjects

Keighren, M. A.

Publications and source records attributed to Keighren, M. A..

3 recordsLinked to original sources

bioRxiv2020

A WDR35/IFT121-dependent coatomer transports ciliary membrane proteins from the Golgi to the cilia

Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia and across the diffusion barrier is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the IFT-A peripheral components are degraded and core components accumulate at the transition zone. We reveal deep sequence homology and structural similarity of WDR35 and other IFT-As to the coatomer COPI proteins a and {beta}', and demonstrate an accumulation of coat-less vesicles which fail to fuse with Wdr35 mutant cilia. Our data provides the first in situ evidence of a novel coatomer function for WDR35 likely with other IFT-A proteins in delivering ciliary membrane cargo from the Golgi necessary for cilia elongation.

cell biology↗

bioRxiv2020

Fluorescent in vivo editing reporter (FIVER): A novel multispectral reporter of in vivo genome editing

Advances in genome editing technologies have created opportunities to treat rare genetic diseases, which are often overlooked in terms of therapeutic development. Nonetheless, substantial challenges remain: namely, achieving therapeutically beneficial levels and kinds of editing in the right cell type(s). Here we describe the development of FIVER (fluorescent in vivo editing reporter) -- a modular toolkit for in vivo detection of genome editing with distinct fluorescent read-outs for non-homologous end-joining (NHEJ), homology-directed repair (HDR) and homology-independent targeted integration (HITI). We demonstrate that fluorescent outcomes reliably report genetic changes following editing with diverse genome editors in primary cells, organoids and in vivo. We show the potential of FIVER for high-throughput unbiased screens, from small molecule modulators of genome editing outcomes in primary cells through to genome-wide in vivo CRISPR cancer screens. Importantly, we demonstrate its in vivo application in postnatal organ systems of interest for genetic therapies -- retina and liver. FIVER will broadly help expedite the development of therapeutic genome surgery for many genetic disorders.

genetics↗

bioRxiv2017

ZMYND10 functions in a chaperone relay during axonemal dynein assembly

Molecular chaperones promote the folding and macromolecular assembly of a diverse set of substrate client proteins. How the ubiquitous chaperone machinery directs its activities towards a specific set of substrates and whether this selectivity could be targeted for therapeutic intervention is of intense research. Through the use of mouse genetics, imaging and quantitative proteomics we uncover that ZMYND10 is a novel co-chaperone for the FKBP8-HSP90 chaperone complex during the biosynthesis of axonemal dynein heavy chains required for cilia motility. In the absence of ZMYND10, defects in dynein heavy chains trigger broader dynein motor degradation. We show that FKBP8 inhibition phenocopies dynein motor instability in airway cells, and human disease-causing variants of ZMYND10 disrupt its ability to act as FKBP8-HSP90 co-chaperone. Our study indicates that the motile ciliopathy Primary Ciliary Dyskinesia (PCD) should be considered a cell-type specific protein-misfolding disease and opens the potential for rational drug design that could restore specificity to the ubiquitous chaperone apparatus towards dynein subunits.

cell biology↗