Biology subjects

Buchan, J. R.

Publications and source records attributed to Buchan, J. R..

3 recordsLinked to original sources

bioRxiv2022

Rsp5/NEDD4 and ESCRT regulate TDP-43 toxicity and turnover via an endolysosomal clearance mechanism

A key pathological hallmark in >97% of all Amyotrophic Lateral Sclerosis (ALS) cases is the cytoplasmic mislocalization and aggregation of a nuclear RNA binding protein, TDP-43. Driving clearance of cytoplasmic TDP-43 reduces toxicity in various ALS models, though how TDP-43 clearance is regulated remains controversial. To address this, we conducted an unbiased yeast genome-wide screen using high-throughput dot blots to identify genes that affect TDP-43 levels. Our screen identified ESCRT complex factors, which induce membrane invagination (particularly at multi-vesicular bodies; MVBs) and K63-linked ubiquitination as key facilitators of TDP-43 endolysosomal clearance. TDP-43 co-localized and bound Rsp5/NEDD4 and ESCRT proteins, and perturbations to either increased TDP-43 aggregation and accumulation. NEDD4 also ubiquitinates TDP-43. Lastly, TDP-43 accumulation caused formation of "giant" MVBs which could reflect a pathological consequence of TDP-43 pertinent to ALS. Our studies shed light on endolysosomal-mediated cytoplasmic protein degradation, which likely impacts multiple substrates, and may be a target for novel ALS therapeutic strategies.

cell biology↗

bioRxiv2021

HARLEY: A Semi-Automated detection of foci in fluorescence images of yeast

Quantification of cellular structures in fluorescence microscopy data is a key means of understanding cellular function. Unfortunately, numerous cellular structures present unique challenges in their ability to be unbiasedly and accurately detected and quantified. In our studies on stress granules in yeast, users displayed a striking variation of up to 3.7-fold in foci calls and were only able to replicate their results with 62-78% correlation, when requantifying the same images. To facilitate consistent results we developed HARLEY (Human Augmented Recognition of LLPS Ensembles in Yeast), a customizable software for detection and quantification of stress granules in S.cerevisiae. After a brief model training on ~20 cells the detection of foci is fully automated and based on closed loops in intensity contours, constrained only by the a-priori known size of the features of interest. Since no shape is implied, this method is not limited to round features, as is often the case with other algorithms. Candidate features are annotated with a set of geometrical and intensity-based properties to train a kernel Support Vector Machine to recognize features of interest. The trained classifier is then used to create consistent results across datasets. HARLEY is aimed at users without technical expertise, allows for batch processing and is freely available, which should be of broad interest to users focused on analysis of microscopy data in yeast.

bioinformatics↗

bioRxiv2019

Cdc48/VCP and endocytosis regulate TDP-43 and FUS toxicity and turnover

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease. TDP-43 (TAR DNA-binding protein 43) and FUS (fused in sarcoma) are aggregation-prone RNA-binding proteins that in ALS can mis-localize to the cytoplasm of affected motor neuron cells, often forming cytoplasmic aggregates in the process. Such mis-localization and aggregation are implicated in ALS pathology, though the mechanisms of TDP-43 and FUS cytoplasmic toxicity remains unclear. Recently, we determined that the endocytic function aids turnover of TDP-43 and reduces TDP-43 toxicity. Here, we identified that Cdc48 and Ubx3, a Cdc48 co-factor implicated in endocytic function, regulates the turnover and toxicity of TDP-43 and FUS expressed in S. cerevisiae. Cdc48 physically interacts and co-localizes with TDP-43, as does VCP in ALS patient tissue. In yeast, FUS toxicity also depends strongly on endocytic function, but not autophagy under normal conditions. FUS expression also impairs endocytic function, as previously observed with TDP-43. Taken together, our data identifies a role for Cdc48/VCP and endocytosis function in regulating TDP-43 and FUS toxicity and turnover. Furthermore, endocytic dysfunction may be a common defect affecting cytoplasmic clearance of ALS aggregation-prone proteins and may represent a novel therapeutic target of promise.

molecular biology↗