Characterization of aggregate load and pattern in living yeast cells by flow cytometry.
Protein aggregation is both a hallmark of and a driving force for a number of diseases. It is therefore important to identify the nature of these aggregates and the mechanism(s) by which the cell counteracts their detrimental properties. Currently, the study of aggregation in vivo is performed primarily using fluorescently tagged versions of proteins and analyzing the aggregates by fluorescence microscopy. While this strategy is considered the gold standard, it has several limitations, particularly with respect to its suitability for high-throughput screening (HTS). Here, using a GFP fusion of the well-characterized yeast prion amyloid protein [PSI+], we demonstrate that flow cytometry, which utilizes the same physical principles as fluorescence microscopy, can be used to determine the aggregate load and pattern in live and fixed yeast cells. Furthermore, our approach can easily be applied to high-throughput analyses such as screenings with a yeast deletion library.
Department of Medicine I and Clinical Chemistry, University of Heidelberg, Heidelberg, Germany.
Itahisa Hernández
IH
Hidalgo
Department of Medicine I and Clinical Chemistry, University of Heidelberg, Heidelberg, Germany.|German Center for Diabetes Research (DZD), München-Neuherberg, Germany.
Thomas
T
Fleming
Department of Medicine V (Haematology), University of Heidelberg, Heidelberg, Germany.
Volker
V
Eckstein
Institute for Diabetes and Cancer IDC, Helmholtz Center Munich, Germany.|Joint Heidelberg-IDC Translational Diabetes Program, Dept. Inner Medicine I, Heidelberg University Hospital, Germany.
Stephan
S
Herzig
Department of Medicine I and Clinical Chemistry, University of Heidelberg, Heidelberg, Germany.|German Center for Diabetes Research (DZD), München-Neuherberg, Germany.|Joint Heidelberg-IDC Translational Diabetes Program, Dept. Inner Medicine I, Heidelberg University Hospital, Germany.
Peter P
PP
Nawroth
Department of Medicine I and Clinical Chemistry, University of Heidelberg, Heidelberg, Germany.
Jens
J
Tyedmers
D005434:Flow Cytometry / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays; D010454:Peptide Termination Factors / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D066329:Protein Aggregates; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D010454:Peptide Termination Factors; D066329:Protein Aggregates; D011993:Recombinant Fusion Proteins; C068068:SUP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins
Saccharomyces cerevisiae; amyloid aggregates; flow cytometry; fluorescence activated cell sorting (FACS); high-throughput screening (HTS); protein aggregation; yeast prion