Biology subjects

Cheung, C. H. Y.

Publications and source records attributed to Cheung, C. H. Y..

3 recordsLinked to original sources

bioRxiv2025

Microscopy-Guided Spatial Proteomics Reveals Novel Proteins at the Mitochondria-Lipid Droplet Interface and Their Role in Lipid Metabolism

Mitochondria-lipid droplet (LD) interactions play a critical role in lipid metabolism and the progression of metabolic diseases such as non-alcoholic fatty liver disease (NAFLD). However, the dynamic nature of these interactions has hindered the identification of novel protein constituents and their functional roles. Here, we employed Microscoop Mint, an advanced microscopy-guided spatial proteomics platform, to isolate proteins localized at mitochondria-LD contact sites in oleic acid (OA)-treated HepG2 cells, an in vitro model for studying fatty liver disease. Microscoop Mint integrates high-resolution image analysis and two-photon illumination to achieve precise biotinylation of proteins at subcellular regions of interest. Coupled with mass spectrometry, this approach identified a proteome enriched at the mitochondria-LD interface, including both well-characterized lipid-associated proteins and previously unrecognized candidates. Among the 373 common proteins identified across replicates, five novel candidates with no prior association to lipid metabolism were selected for validation. Immunofluorescence staining confirmed their localization at mitochondria-LD contact sites, with more pronounced association in OA-treated cells. Notably, suppression of one candidate, FHL3, led to reduced LD size, elongated mitochondrial morphology, and diminished mitochondria-LD interactions, suggesting its role in regulating fatty acid {beta}-oxidation. Our findings demonstrate the utility of Microscoop Mint in unveiling novel molecular players at organelle contact sites. This study not only provides insights into the functional dynamics of mitochondria-LD interactions but also highlights potential therapeutic targets for lipid metabolic disorders and NAFLD.

cell biology↗

bioRxiv2023

Microscopy-guided subcellular proteomic discovery by high-speed ultra-content photo-biotinylation

Microscopy-guided proteomics at an organelle-dimension resolution is desired for revealing unknown protein constituents at specific disease- or functional-associated regions at the molecular-molecular interactions level. Here, we achieve protein spatial purification by introducing a firmware-integrated microscopy platform that triggers in situ subcellular photo-biotinylation of proteins at user-defined regions of interest (ROIs) one field of view (FOV) at a time for thousands of FOVs fully automatically. An illumination pattern at the analogous ROIs of each FOV is calculated on the fly by either machine learning or traditional image processing. Photoactivatable amino acid crosslinkers are activated by a two-photon focal light one spot at a time at a sub-millisecond illumination duration per spot. Imaging, pattern generation, targeted illumination, and FOV movement are coordinated and cycled with high-speed mechatronic control to complete illumination on millions of ROI spots within hours. Once enough proteins are biotinylated in a cell or tissue sample, the sample is scraped and lysed, and avidin pulldown is used to enrich proteins to achieve spatial protein scooping at a 240-nm precision. Subsequent LC-MS/MS is implemented to reveal the subcellular proteome in high sensitivity, specificity, and resolution. Using this technology termed optoproteomics, we have revealed novel stress granule-localized and amyloid {beta}-localized proteins validated by immunostaining. Together, spatial purification by ultra-content, high-speed microscopy-targeted photo-biotinylation enables unprecedented subcellular spatial proteomics discovery in any microscopically recognizable regions.

cell biology↗

bioRxiv2020

Redirected tropisms of extracellular vesicles and exomeres yield distinct biodistribution profiles

Extracellular particles (EP) including extracellular vesicles (EVs) and exomeres have been shown to play significant roles in diseases and therapeutic applications. However, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a suitable method. We therefore created a bioluminescence resonance energy transfer (BRET)-based reporter, PalmGRET, to enable pan-EP labelling ranging from exomeres (< 50 nm) to small (< 200 nm) and medium and large (> 200 nm) EVs. PalmGRET emits robust, sustained signals and allows the visualization, tracking and quantification of the EPs from whole-animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, BRET and fluorescence. Using PalmGRET, we show that EPs released by lung metastatic hepatocellular carcinoma (HCC) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. We further demonstrate that gene knockdown of lung-tropic membrane proteins, solute carrier organic anion transporter family member 2A1 (Slco2a1), alanine aminopeptidase (Cd13) and chloride intracellular channel (Clic1) decreases HCC-EP distribution to the lungs and yields distinct biodistribution profiles. We anticipate that EP-specific imaging, quantitative assays and detailed in vivo characterization to be a starting point for more accurate and comprehensive in vivo models of EP biology and therapeutic design.

cell biology↗