Opto-magnetic capture of individual cells based on visual phenotypes

Binan, Loïc; Bart, François; Uriarte, Maxime; Lemay, Jean-François; Koninck, Jean Christophe Pelletier De; Roy, Joannie; Affar, El Bachir; Drobetsky, Elliot; Würtele, Hugo; Costantino, Santiago

doi:10.7554/elife.45239

Cited by 10 publications

(15 citation statements)

References 62 publications

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“…The LIMPLA1 strategy is related to cell labelling via photobleaching (CLaP), 20, 21 a method designed to specifically tagging individual cells based on photobleaching. In CLaP, a laser is used to crosslink fluorescent biotin conjugates to cell membrane of living cells via free radicals generated by photobleaching.…”

Section: Resultsmentioning

confidence: 99%

Organelle specific protein profiling with light mediated proximal labeling in living cells

2019

Preprint

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I have developed a far-red light mediated proximal labeling (LIMPLA) strategy for subcellular protein profiling in live cells. This strategy can facilitate the proteomic profiling of subcellular organelle, such as the mitochondria with no need of transgene. I modified the mitochondria-targeting dye rhodamine 123 with an alkynyl group (Alk-R123). These modifications do not change the mitochondria targeting property and can act as a bioorthogonal 'handle' for subsequently imaging or enrichment. After Alk-R123 are accumulated in the mitochondria, light illumination can activate the dye to label proximal proteins in situ. In order to enhance protein labeling efficiency, I have found that various mitochondria-targeting dyes such as Mitotracker deep red can mediate 2-Propynylamine (PA) to label proximal proteins under illumination. PA tagged proteins with alkynyl group are subsequently derivatized via click chemistry with azido fluorescent dye for imaging or with azido biotin for further enrichment and mass-spec identification. Main textEukaryotic cells are elaborately subdivided into functionally distinct, membraneenclosed compartments or organelles. Each organelle contains its own characteristic set of proteins, which underlie its characteristic structural and functional properties. For example, mitochondria, a double-membrane-bound organelle, are energy center and plays crucial roles in energy conversion and calcium ions storage. Understanding how organelles function appropriately and specifically requires identifications of proteomics in specific organelles.

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Section: Resultsmentioning

confidence: 99%

Organelle specific protein profiling with light mediated proximal labeling in living cells

2019

Preprint

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“…The ability to retrieve specific cells with desired phenotypic profiles is a widespread need, bolstered by enthusiasm for single-cell multiomics technologies and cell atlas projects (39), but remains challenging. Advanced microscopy-based techniques have emerged for singlecell retrieval (8)(9)(10)(11)(12)(13)(14). However, existing approaches are best adapted to specific experimental contexts (e.g., cell type, imaging chamber, assay type and/or experimental preparation), limiting the breadth of their applications.…”

Section: Discussionmentioning

confidence: 99%

Versatile phenotype-activated cell sorting

et al. 2020

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Unraveling the genetic and epigenetic determinants of phenotypes is critical for understanding and re-engineering biology and would benefit from improved methods to separate cells based on phenotypes. Here, we report SPOTlight, a versatile high-throughput technique to isolate individual yeast or human cells with unique spatiotemporal profiles from heterogeneous populations. SPOTlight relies on imaging visual phenotypes by microscopy, precise optical tagging of single target cells, and retrieval of tagged cells by fluorescence-activated cell sorting. To illustrate SPOTlight’s ability to screen cells based on temporal properties, we chose to develop a photostable yellow fluorescent protein for extended imaging experiments. We screened 3 million cells expressing mutagenesis libraries and identified a bright new variant, mGold, that is the most photostable yellow fluorescent protein reported to date. We anticipate that the versatility of SPOTlight will facilitate its deployment to decipher the rules of life, understand diseases, and engineer new molecules and cells.

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“…These methods enable the investigation of protein pathways regulating subcellular organization and positioning in an unbiased manner. In addition to unbiased CRISPR screens linking microscopic phenotypes to genotypes, single-cell images linking microscopic phenotypes to genotypes were established by a new method called single-cell magneto-optical capture ( Binan et al, 2019 ). Although these processes are elegant and will improve genetic studies, they are not well suited for high-throughput large-scale screens.…”

Section: Introductionmentioning

confidence: 99%

Image-based pooled whole-genome CRISPRi screening for subcellular phenotypes

Kanfer

Sarraf

Maman

et al. 2021

Journal of Cell Biology

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Genome-wide CRISPR screens have transformed our ability to systematically interrogate human gene function, but are currently limited to a subset of cellular phenotypes. We report a novel pooled screening approach for a wider range of cellular and subtle subcellular phenotypes. Machine learning and convolutional neural network models are trained on the subcellular phenotype to be queried. Genome-wide screening then utilizes cells stably expressing dCas9-KRAB (CRISPRi), photoactivatable fluorescent protein (PA-mCherry), and a lentiviral guide RNA (gRNA) pool. Cells are screened by using microscopy and classified by artificial intelligence (AI) algorithms, which precisely identify the genetically altered phenotype. Cells with the phenotype of interest are photoactivated and isolated via flow cytometry, and the gRNAs are identified by sequencing. A proof-of-concept screen accurately identified PINK1 as essential for Parkin recruitment to mitochondria. A genome-wide screen identified factors mediating TFEB relocation from the nucleus to the cytosol upon prolonged starvation. Twenty-one of the 64 hits called by the neural network model were independently validated, revealing new effectors of TFEB subcellular localization. This approach, AI-photoswitchable screening (AI-PS), offers a novel screening platform capable of classifying a broad range of mammalian subcellular morphologies, an approach largely unattainable with current methodologies at genome-wide scale.

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Opto-magnetic capture of individual cells based on visual phenotypes

Cited by 10 publications

References 62 publications

Organelle specific protein profiling with light mediated proximal labeling in living cells

Organelle specific protein profiling with light mediated proximal labeling in living cells

Versatile phenotype-activated cell sorting

Image-based pooled whole-genome CRISPRi screening for subcellular phenotypes

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