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1.
Nat Biotechnol ; 40(8): 1231-1240, 2022 08.
Article in English | MEDLINE | ID: mdl-35590073

ABSTRACT

Despite the availabilty of imaging-based and mass-spectrometry-based methods for spatial proteomics, a key challenge remains connecting images with single-cell-resolution protein abundance measurements. Here, we introduce Deep Visual Proteomics (DVP), which combines artificial-intelligence-driven image analysis of cellular phenotypes with automated single-cell or single-nucleus laser microdissection and ultra-high-sensitivity mass spectrometry. DVP links protein abundance to complex cellular or subcellular phenotypes while preserving spatial context. By individually excising nuclei from cell culture, we classified distinct cell states with proteomic profiles defined by known and uncharacterized proteins. In an archived primary melanoma tissue, DVP identified spatially resolved proteome changes as normal melanocytes transition to fully invasive melanoma, revealing pathways that change in a spatial manner as cancer progresses, such as mRNA splicing dysregulation in metastatic vertical growth that coincides with reduced interferon signaling and antigen presentation. The ability of DVP to retain precise spatial proteomic information in the tissue context has implications for the molecular profiling of clinical samples.


Subject(s)
Melanoma , Proteomics , Humans , Laser Capture Microdissection/methods , Mass Spectrometry/methods , Melanoma/genetics , Proteome/chemistry , Proteomics/methods
3.
Sci Rep ; 5: 12879, 2015 Aug 14.
Article in English | MEDLINE | ID: mdl-26271723

ABSTRACT

Contact-dependent intercellular transfer (codeIT) of cellular constituents can have functional consequences for recipient cells, such as enhanced survival and drug resistance. Pathogenic viruses, prions and bacteria can also utilize this mechanism to spread to adjacent cells and potentially evade immune detection. However, little is known about the molecular mechanism underlying this intercellular transfer process. Here, we present a novel microscopy-based screening method to identify regulators and cargo of codeIT. Single donor cells, carrying fluorescently labelled endocytic organelles or proteins, are co-cultured with excess acceptor cells. CodeIT is quantified by confocal microscopy and image analysis in 3D, preserving spatial information. An siRNA-based screening using this method revealed the involvement of several myosins and small GTPases as codeIT regulators. Our data indicates that cellular protrusions and tubular recycling endosomes are important for codeIT. We automated image acquisition and analysis to facilitate large-scale chemical and genetic screening efforts to identify key regulators of codeIT.


Subject(s)
Cell Communication/physiology , Cell Membrane/physiology , GTP Phosphohydrolases/metabolism , Intercellular Junctions/physiology , Microscopy, Confocal/methods , Myosins/metabolism , Cell Membrane/ultrastructure , Cell Tracking/methods , HeLa Cells , Humans , Lab-On-A-Chip Devices , Molecular Imaging/methods
4.
Nat Biotechnol ; 33(4): 384-9, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25774713

ABSTRACT

To understand the function of cellular protein networks, spatial and temporal context is essential. Fluorescence correlation spectroscopy (FCS) is a single-molecule method to study the abundance, mobility and interactions of fluorescence-labeled biomolecules in living cells. However, manual acquisition and analysis procedures have restricted live-cell FCS to short-term experiments of a few proteins. Here, we present high-throughput (HT)-FCS, which automates screening and time-lapse acquisition of FCS data at specific subcellular locations and subsequent data analysis. We demonstrate its utility by studying the dynamics of 53 nuclear proteins. We made 60,000 measurements in 10,000 living human cells, to obtain biophysical parameters that allowed us to classify proteins according to their chromatin binding and complex formation. We also analyzed the cell-cycle-dependent dynamics of the mitotic kinase complex Aurora B/INCENP and showed how a rise in Aurora concentration triggers two-step complex formation. We expect that throughput and robustness will make HT-FCS a broadly applicable technology for characterizing protein network dynamics in cells.


Subject(s)
High-Throughput Screening Assays/methods , Protein Interaction Mapping/methods , Proteome/metabolism , Spectrometry, Fluorescence/methods , Subcellular Fractions/metabolism , Molecular Imaging/methods
5.
Curr Biol ; 24(21): 2598-605, 2014 Nov 03.
Article in English | MEDLINE | ID: mdl-25447992

ABSTRACT

During animal cell division, an actin-based ring cleaves the cell into two. Problems with this process can cause chromosome missegregation and defects in cytoplasmic inheritance and the partitioning of organelles, which in turn are associated with human diseases. Although much is known about how chromosome segregation is coupled to cell division, the way organelles coordinate their inheritance during partitioning to daughter cells is less well understood. Here, using a high-content live-imaging small interfering RNA screen, we identify Myosin-XIX (Myo19) as a novel regulator of cell division. Previously, this actin-based motor was shown to control the interphase movement of mitochondria. Our analysis shows that Myo19 is indeed localized to mitochondria and that its silencing leads to defects in the distribution of mitochondria within cells and in mitochondrial partitioning at division. Furthermore, many Myo19 RNAi cells undergo stochastic division failure--a phenotype that can be mimicked using a treatment that blocks mitochondrial fission and rescued by decreasing mitochondrial fusion, implying that mitochondria can physically interfere with cytokinesis. Strikingly, using live imaging we also observe the inappropriate movement of mitochondria to the poles of spindles in cells depleted for Myo19 as they enter anaphase. Since this phenocopies the results of an acute loss of actin filaments in anaphase, these data support a model whereby the Myo19 actin-based motor helps to control mitochondrial movement to ensure their faithful segregation during division. The presence of DNA within mitochondria makes their inheritance an especially important aspect of symmetrical cell division.


Subject(s)
Cell Division/physiology , Mitochondria/metabolism , Myosins/physiology , Actins/metabolism , Cytokinesis , Gene Knockdown Techniques , HeLa Cells , Humans , Mitochondria/ultrastructure , Mitochondrial Dynamics , Myosins/genetics , Myosins/metabolism
6.
Nat Methods ; 8(3): 246-9, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21258339

ABSTRACT

Quantitative microscopy relies on imaging of large cell numbers but is often hampered by time-consuming manual selection of specific cells. The 'Micropilot' software automatically detects cells of interest and launches complex imaging experiments including three-dimensional multicolor time-lapse or fluorescence recovery after photobleaching in live cells. In three independent experimental setups this allowed us to statistically analyze biological processes in detail and is thus a powerful tool for systems biology.


Subject(s)
Fluorescence Recovery After Photobleaching/methods , Microscopy, Fluorescence/methods , Software , Systems Biology/methods , Chromobox Protein Homolog 5 , Chromosomal Proteins, Non-Histone/analysis , Green Fluorescent Proteins/analysis , HeLa Cells , Humans
7.
Nature ; 464(7289): 721-7, 2010 Apr 01.
Article in English | MEDLINE | ID: mdl-20360735

ABSTRACT

Despite our rapidly growing knowledge about the human genome, we do not know all of the genes required for some of the most basic functions of life. To start to fill this gap we developed a high-throughput phenotypic screening platform combining potent gene silencing by RNA interference, time-lapse microscopy and computational image processing. We carried out a genome-wide phenotypic profiling of each of the approximately 21,000 human protein-coding genes by two-day live imaging of fluorescently labelled chromosomes. Phenotypes were scored quantitatively by computational image processing, which allowed us to identify hundreds of human genes involved in diverse biological functions including cell division, migration and survival. As part of the Mitocheck consortium, this study provides an in-depth analysis of cell division phenotypes and makes the entire high-content data set available as a resource to the community.


Subject(s)
Cell Division/genetics , Genome, Human/genetics , Microscopy, Fluorescence/methods , Phenotype , Animals , Cell Movement/genetics , Cell Survival/genetics , Color , Gene Knockdown Techniques , Genes/genetics , HeLa Cells , Humans , Kinetics , Mice , Mitosis/genetics , RNA Interference , Reproducibility of Results , Spindle Apparatus/genetics , Spindle Apparatus/metabolism , Time Factors
8.
Science ; 328(5978): 593-9, 2010 Apr 30.
Article in English | MEDLINE | ID: mdl-20360068

ABSTRACT

Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization, and tandem-affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or had only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex and the gamma-tubulin ring complex--large complexes that are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high-throughput follow-up analyses of phenotypic screens in mammalian cells.


Subject(s)
Chromosome Segregation , Mitosis , Multiprotein Complexes/metabolism , Spindle Apparatus/metabolism , Tubulin/metabolism , Ubiquitin-Protein Ligase Complexes/metabolism , Anaphase-Promoting Complex-Cyclosome , Centrosome/metabolism , Chromosomes, Artificial, Bacterial , Databases, Genetic , Genomics , Green Fluorescent Proteins , HeLa Cells , Humans , Open Reading Frames , Protein Binding , Protein Interaction Mapping , Protein Subunits/metabolism , RNA Interference
9.
J Biomol Screen ; 13(7): 575-80, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18599879

ABSTRACT

Solid-phase reverse transfection on cell microarrays is a high-throughput method for the parallel transfection of mammalian cells. However, the cells transfected in this way have been restricted so far to microscopy-based analyses. Analysis methods such as reverse transcriptase-polymerase chain reaction (RT-PCR) and access to higher cell numbers for statistical reasons in microscopy-based assays are not possible with solid-phase reverse transfection on cell microarrays. We have developed a quick and reliable protocol for automated solid-phase reverse transfection of human cells with siRNAs in multiwell plates complementing solid-phase reverse transfection on cell microarrays. The method retains all advantages of solid-phase reverse transfection such as long-term storage capacity after fabrication, reduced cytotoxicity, and reduced cost per screen compared with liquid-phase transfection in multiwell plates. The protocol has been tested for the RNAi-mediated knockdown of several genes in different cell lines including U20S, RPE1, A549, and HeLa cells. We show that even 3 months after production of the "ready to transfect" multiwell plates, there is no reduction in their transfection efficiency as assessed by RT-PCR and nuclear phenotyping by fluorescence microscopy. We conclude that solid-phase reverse transfection in multiwell plates is a cost-efficient and flexible tool for multiplexing cellular assays.


Subject(s)
RNA, Small Interfering/genetics , Transfection/methods , Automation , Biological Assay/methods , Cell Line, Tumor , Gene Expression Profiling , Genetic Techniques , HeLa Cells , Humans , Microscopy , Microscopy, Fluorescence , Phenotype , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction
10.
Exp Cell Res ; 312(13): 2605-18, 2006 Aug 01.
Article in English | MEDLINE | ID: mdl-16780834

ABSTRACT

The actin-binding protein (ABP) drebrin, isoform E2, is involved in remodelling of the actin cytoskeleton and in formation of cell processes, but its role in cell migration has not yet been investigated. Therefore, we have studied the organization of drebrin in motile cultured cells such as murine B16F1 melanoma and human SV80 fibroblast cells, using live cell confocal microscopy. In cells overexpressing DNA constructs encoding drebrin linked to EGFP, numerous long, branched cell processes were formed which slowly retracted and extended, whereas forward movement was halted. In contrast, stably transfected B16F1 cells containing drebrin-EGFP at physiological levels displayed lamellipodia and were able to migrate on laminin. Surprisingly, in such cells, drebrin was absent from anterior lamellipodia but was enriched in a specific juxtanuclear zone, the "drebrin-enriched zone" (DZ), and in the tail. In leading edges of SV80 cells, characterized by pronounced actin microspikes, drebrin was specifically enriched along posterior portions of the microspikes, together with tropomyosin. Drebrin knock-down by small interfering RNAs did not impair movements of SV80 cells. Our results confirm the role of drebrin E2 in the formation of branching processes and further indicate that during cell migration, the protein contributes to retraction of the cell body and the tail but not to lamellipodia formation. In particular, the novel, sizable juxtanuclear DZ structure will have to be characterized in future experiments with respect to its molecular assembly and cell biological functions.


Subject(s)
Actins/metabolism , Cell Movement , Cell Nucleus/metabolism , Neuropeptides/metabolism , Animals , DNA, Complementary/genetics , Fibroblasts/cytology , Green Fluorescent Proteins/metabolism , Humans , Mice , Protein Transport , Pseudopodia/metabolism , RNA, Small Interfering/genetics , Recombinant Fusion Proteins/metabolism , Subcellular Fractions/metabolism , Swiss 3T3 Cells , Transfection , Tropomyosin/metabolism , Tumor Cells, Cultured
11.
J Invest Dermatol ; 125(4): 761-74, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16185277

ABSTRACT

Isoform E2 of drebrin, an actin-binding protein originally identified in neuronal cells, has recently been identified in diverse non-neuronal cells, mostly in association with cell processes and intercellular junctions. Here, we report on the presence of drebrin in normal human skin, epithelial skin cancers, and cultured keratinocytes. Keratinocytes of normal epidermis contain almost no drebrin but the protein is readily seen in hair follicles. By immunohistochemistry and immunoblot, basal cell carcinomas (BCC) are rich in drebrin, and confocal laser scanning and immunoelectron microscopy show accumulation at adhering junctions, in co-localization with actin and partially with plaque proteins. In squamous cell carcinomas, keratoacanthomas, and in epidermal precancers, drebrin is heterogeneously distributed, appearing as mosaics. Primary keratinocyte cultures contain significant amounts of drebrin enriched at adhering junctions. When epithelium-derived cells devoid of drebrin are transfected with drebrin-enhanced green fluorescent protein, constructs accumulate in the cell periphery, and immunoprecipitation shows complexes with actin. During epidermal growth factor induced formation of cell processes, drebrin retains this junction association, as observed by live cell microscopy. Our results suggest novel functions of drebrin such as an involvement in cell-cell adhesion and tumorigenesis and a potential value in diagnosis of BCC.


Subject(s)
Keratinocytes/metabolism , Microfilament Proteins/analysis , Neoplasms, Glandular and Epithelial/chemistry , Neuropeptides/analysis , Skin Neoplasms/chemistry , Cell Line, Tumor , Epidermal Growth Factor/pharmacology , Female , Fluorescent Antibody Technique , Humans , Immunoblotting , Microscopy, Confocal , Microscopy, Immunoelectron , Neoplasms, Glandular and Epithelial/metabolism , Neuropeptides/biosynthesis , Skin/chemistry , Skin Neoplasms/metabolism , Transfection
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