Pooled multicolour tagging for visualizing subcellular protein dynamics.
Andreas ReicherJiří ReinišMaria CiobanuPavel RůžičkaMonika MalikMarton SiklosVictoria KartyshTatjana TomekAnna KorenAndré F RendeiroStefan KubicekPublished in: Nature cell biology (2024)
Imaging-based methods are widely used for studying the subcellular localization of proteins in living cells. While routine for individual proteins, global monitoring of protein dynamics following perturbation typically relies on arrayed panels of fluorescently tagged cell lines, limiting throughput and scalability. Here, we describe a strategy that combines high-throughput microscopy, computer vision and machine learning to detect perturbation-induced changes in multicolour tagged visual proteomics cell (vpCell) pools. We use genome-wide and cancer-focused intron-targeting sgRNA libraries to generate vpCell pools and a large, arrayed collection of clones each expressing two different endogenously tagged fluorescent proteins. Individual clones can be identified in vpCell pools by image analysis using the localization patterns and expression level of the tagged proteins as visual barcodes, enabling simultaneous live-cell monitoring of large sets of proteins. To demonstrate broad applicability and scale, we test the effects of antiproliferative compounds on a pool with cancer-related proteins, on which we identify widespread protein localization changes and new inhibitors of the nuclear import/export machinery. The time-resolved characterization of changes in subcellular localization and abundance of proteins upon perturbation in a pooled format highlights the power of the vpCell approach for drug discovery and mechanism-of-action studies.
Keyphrases
- living cells
- machine learning
- high throughput
- genome wide
- drug discovery
- high resolution
- deep learning
- single molecule
- dna methylation
- randomized controlled trial
- stem cells
- poor prognosis
- gene expression
- squamous cell carcinoma
- artificial intelligence
- quantum dots
- protein protein
- amino acid
- papillary thyroid
- photodynamic therapy
- long non coding rna
- microbial community
- clinical practice
- big data
- lymph node metastasis
- fluorescence imaging
- study protocol
- optical coherence tomography
- wastewater treatment
- squamous cell
- phase iii