Biology subjects

Welch, L. G.

Publications and source records attributed to Welch, L. G..

3 recordsLinked to original sources

bioRxiv2024

The FAM114A proteins are adaptors for the recycling of Golgi enzymes

The Golgi apparatus contains many resident enzymes that must remain in place whilst their substrates flow through on their journey from the endoplasmic reticulum to elsewhere in the cell. COPI-coated vesicles bud from the rims of the Golgi stack to recycle Golgi residents to earlier cisternae. Different enzymes are present in different parts of the stack, and at least one COPI adaptor protein, GOLPH3, has been shown to recruit enzymes into vesicles in a specific part of the stack. We have used proximity biotinylation to identify further components of intra-Golgi transport vesicles and found FAM114A2, an uncharacterised cytosolic protein. Affinity chromatography with FAM114A2, and its paralogue FAM114A1 showed that they bind to numerous Golgi resident proteins, with membrane-proximal basic residues in the cytoplasmic tail being sufficient for the interaction. Deletion of both proteins from U2OS cells did not result in substantial defects in Golgi function. However, a Drosophila orthologue of these proteins (CG9590/FAM114A) is also localised to the Golgi and binds directly to COPI. Generation of Drosophila mutants lacking FAM114A revealed defects in glycosylation of glue proteins in the salivary gland. Thus, the FAM114A proteins are COPI vesicle resident proteins that bind to Golgi enzymes and so are candidate adaptors to contribute specificity to COPI vesicle recycling in the Golgi stack.

cell biology↗

bioRxiv2024

A programmable and automated optical electrowetting-on-dielectric (oEWOD) driven platform for massively parallel and sequential processing of single cell assay operations

Recently, there has been an increasing emphasis on single cell profiling for high-throughput screening workflows in drug discovery and life sciences research. However, the biology underpinning these screens is often complex and is insufficiently addressed by singleplex assay screens. Traditional single cell screening technologies have created powerful sets of omic data that allow users to bioinformatically infer biological function, but have as of yet not empowered direct functional analysis at the level of each individual cell. Consequently, screening campaigns often require multiple secondary screens leading to laborious, time-consuming and expensive workflows in which attrition points may not be queried until late in the process. We describe a platform that harnesses droplet microfluidics and optical electrowetting-on-dielectric (oEWOD) to perform highly-controlled sequential and multiplexed single cell assays in massively parallelised workflows to enable complex cell profiling during screening. Soluble reagents or objects, such as cells or assay beads, are encapsulated into droplets of media in fluorous oil and are actively filtered based on size and optical features ensuring only desirable droplets (e.g. single cell droplets) are retained for analysis, thereby overcoming the Poisson probability distribution. Droplets are stored in an array on a temperature-controlled chip and the history of individual droplets is logged from the point of filter until completion of the workflow. On chip, droplets are subject to an automated and flexible suite of operations including the merging of sample droplets and the fluorescent acquisition of assay readouts to enable complex sequential assay workflows. To demonstrate the broad utility of the platform, we present examples of single-cell functional workflows for various applications such as antibody discovery, infectious disease, and cell and gene therapy.

cell biology↗

bioRxiv2021

GOLPH3 and GOLPH3L are broad-spectrum COPI adaptors for sorting into intra-Golgi transport vesicles

Glycosylation is a diverse and abundant modification of proteins, lipids and RNA. The fidelity of glycosylation is, in part, assured by the correct compartmentalisation of Golgi-resident glycosylation enzymes within the Golgi stack. The COPI adaptor GOLPH3 has been shown to interact with the cytoplasmic tails of a subset of Golgi enzymes and direct their retention in the Golgi. However, other mechanisms of retention, and other roles for GOLPH3, have been proposed, and a comprehensive characterisation of the clientele of GOLPH3 and its paralogue GOLPH3L has been lacking. The role of GOLPH3 is of particular interest as it is frequently amplified in several solid tumour types. Here, we combine two orthogonal proteomic analyses to identify a diverse range of GOLPH3+3L clients and find that they act in a wide spectrum of glycosylation pathways, or have other roles in the Golgi. Using binding studies, bioinformatics and an in vivo Golgi retention assay, we show that GOLPH3+3L interact with the cytoplasmic tails of their clients through membrane-proximal positively-charged residues. Furthermore, deletion of GOLPH3+3L causes diverse defects in glycosylation. Thus, GOLPH3+3L are major COPI adaptors that impinge on most, if not all, of the glycosylation pathways of the Golgi.

cell biology↗