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idr0145-study.txt
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# FILL IN AS MUCH INFORMATION AS YOU CAN. HINTS HAVE BEEN PUT IN SOME FIELDS AFTER THE HASH # SYMBOL. REPLACE THE HINT WITH TEXT WHERE APPROPRIATE.
# STUDY DESCRIPTION SECTION
# Section with generic information about the study including title, description, publication details (if applicable) and contact details
Comment[IDR Study Accession] idr0145
Study Title Mec1-Independent Activation of the Rad53 Checkpoint Kinase Revealed by Quantitative Analysis of Protein Localization Dynamics
Study Type high content screen
Study Type Term Source REF EFO
Study Type Term Accession EFO_0007550
Study Description The replication checkpoint is essential for accurate DNA replication and repair, and maintenance of genomic integrity when a cell is challenged with genotoxic stress. Several studies have defined the complement of proteins that change subcellular location in the budding yeast Saccharomyces cerevisiae following chemically-induced DNA replication stress using methyl methanesulfonate (MMS) or hydroxyurea (HU). How these protein movements are regulated remains largely unexplored. We find that the essential checkpoint kinases Mec1 and Rad53 are responsible for regulating the subcellular localization of 159 proteins during MMS-induced replication stress. Unexpectedly, Rad53 regulation of the localization of 52 proteins is independent of its known kinase activators, Mec1, and in some scenarios independent of Tel1, or mediator components, Rad9 and Mrc1. We demonstrate that Rad53 is phosphorylated and active following MMS exposure in cells lacking Mec1 and Tel1. This non-canonical mode of Rad53 activation depends partly on the retrograde signaling transcription factor Rtg3, which also facilitates proper DNA replication dynamics. We conclude that there are biologically important modes of Rad53 protein kinase activation that respond to replication stress and operate in parallel to Mec1 and Tel1.
Study Key Words yeast green-fluorescent protein protein subcellular localization replication stress confocal microscopy
Study Organism Saccharomyces cerevisiae
Study Organism Term Source REF NCBITaxon
Study Organism Term Accession 4932
Study Screens Number 2
Study External URL
Study BioImage Archive Accession
Study Public Release Date 2023-01-17
# Study Publication
Study PubMed ID 37278514
Study Publication Title Mec1-Independent Activation of the Rad53 Checkpoint Kinase Revealed by Quantitative Analysis of Protein Localization Dynamics
Study Author List Ho B, Sanford EJ, Loll-Krippleber R, Torres NP, Smolka MB, Brown GW
Study PMC ID PMC10259420
Study DOI https://doi.org/10.7554/eLife.82483
# Study Contacts
Study Person Last Name Brown
Study Person First Name Grant
Study Person Email [email protected]
Study Person Address Department of Biochemistry, Donnelly CCBR, 160 College Street, Toronto, Ontario, Canada
Study Person ORCID 0000-0002-9002-5003
Study Person Roles submitter
# Study License and Data DOI
Study License CC BY 4.0
Study License URL https://creativecommons.org/licenses/by/4.0/
Study Copyright Ho et al
Study Data Publisher University of Dundee
Study Data DOI https://doi.org/10.17867/10000186
Term Source Name NCBITaxon EFO CMPO Fbbi
Term Source URI http://purl.obolibrary.org/obo/ http://www.ebi.ac.uk/efo/ http://www.ebi.ac.uk/cmpo/ http://purl.obolibrary.org/obo/
# SCREEN SECTION
# Screen Section containing all information relative to each screen in the study including materials used, protocols names and description, phenotype names and description.
# For multiple screens this section should be repeated. Copy and paste the whole section below and fill out for the next screen.
Screen Number 1
Comment[IDR Screen Name] idr0145-ho-replicationstress/screenA
Screen Data DOI https://doi.org/10.17867/10000186a
Screen Sample Type cell
Screen Description High-throughput confocal microscope screen of protein localization screen for 314 yeast strains expressing unique GFP-tagged proteins treated with 0.03% MMS, imaged at 7 time points over 4 hours (0, 30, 60, 90, 120, 180, and 240 minutes), in a Mec1 Sml1 deletion background.
Screen Size Plates:7 5D Images: Planes:1 Average Image Dimension (XYZCT):500 x 500 x 1 x 2 x 1 Total Tb: 0.06127
Screen Example Images
Screen Imaging Method spinning disk confocal microscopy
Screen Imaging Method Term Source REF Fbbi
Screen Imaging Method Term Accession FBbi_00000253
Screen Technology Type protein screen
Screen Technology Type Term Source REF EFO
Screen Technology Type Term Accession EFO_0005398
Screen Type primary screen
Screen Type Term Source REF EFO
Screen Type Term Accession EFO_0007556
Screen Organism
Screen Organism Term Source REF NCBITaxon
Screen Organism Term Accession
Screen Comments
# Library section. The library file should be supplied separately and it should contain the reagents description including, at the absolute minimum: reagent ID, sequences and position in the layout (= plate + position in the plate)
Library File Name idr0145-screenA-annotation
Library File Format tab-delimited text
Library Type GFP protein fusion library
Library Type Term Source REF EFO
Library Type Term Accession EFO_0007566
Library Manufacturer https://yeastgfp.yeastgenome.org/
Library Version
Library Experimental Conditions Cells were treated with 0.03% MMS in low-fluorescent yeast media.
Library Experimental Conditions Term Source REF EFO
Library Experimental Conditions Term Accession
Quality Control Description
# Protocols
Protocol Name growth protocol treatment protocol HCS library protocol HCS image acquisition and feature extraction protocol HCS data analysis protocol
Protocol Type growth protocol treatment protocol HCS library protocol HCS image acquisition and feature extraction protocol HCS data analysis protocol
Protocol Type Term Source REF EFO EFO EFO EFO EFO
Protocol Type Term Accession EFO_0003789 EFO_0003969 EFO_0007571 EFO_0007572 EFO_0007573
Protocol Description The 322 GFP-tagged strains were grown to mid- logarithmic phase in liquid Synthetic Defined (SD) media and imaged on a 384-well imaging plate (PerkinElmer 384 Cell Carrier Ultra, 6057300) in a high-throughput confocal microscope (PerkinElmer Opera High-Content Screening System) as previously described. Briefly, cells were treated with MMS and imaged at 7 timepoints over 4 hours (0, 30, 60, 90, 120, 180, and 240 minutes). The addition of drug to each column of the plate was staggered by 1 minute to compensate for the imaging time of the Opera microscope. For each time point, 4 different regions of the well were imaged. Both GFP and RFP was imaged on the microscope with 800 ms exposure. Cells expressed a constitutively cytoplasmic tdTomato fluorophore, and whole cell image segmentation was conducted using CellProfiler based on tdTomato signal. Dead cells displaying high GFP fluorescence were removed from the analysis. An automated quantification scheme was developed using custom written Python scripts to acquire measurements of GFP pixel intensities for each individual cell from all obtained images.
# Phenotypes
Phenotype Name change in protein localization
Phenotype Description Single cell pixel intensity distributions were normalized by the median GFP intensity measured for each respective cell. For a given protein, the 95th percentile, hereafter referred to as the localization (LOC) value, was determined for each distribution among untreated cells. The median (x) and median absolute distribution (MAD) was calculated for the loc value and were used to determine threshold values for changes in protein localization. If a cell LOC value was greater than 2 MAD from the median (x + 2 MAD), it was considered as an increase in protein relocalization from the untreated condition. Conversely, if a cellular LOC value was less than 2 MAD from the median (x - 2 MAD), it was labeled as decreased localization from the untreated condition. The LOC value was calculated for all cells among all proteins and across all 7 timepoints in our study. Finally, the percentage of cells with a localization change was calculated by dividing the number of proteins with a localization change event (either increased or decreased) by the total number of cells segmented.
Phenotype Score Type automatic
Phenotype Term Source REF CMPO
Phenotype Term Name
Phenotype Term Accession
# Raw Data Files
Raw Image Data Format Evotec/PerkinElmer Opera Flex
Raw Image Organization 7 x 384 well plate. Up to 4 fields per well. Each well contains a different tagged ORF. Each plate represents a different time point imaged
# Feature Level Data Files
Feature Level Data File Name
Feature Level Data File Description
Feature Level Data File Format
Feature Level Data Column Name
Feature Level Data Column Description
# Processed Data Files
Processed Data File Name
Processed Data File Format tab-delimited text
Processed Data File Description
Processed Data Column Name
Processed Data Column Type
Processed Data Column Annotation Level
Processed Data Column Description
Processed Data Column Link To Library File
Screen Number 2
Comment[IDR Screen Name] idr0145-ho-replicationstress/screenB
Screen Data DOI https://doi.org/10.17867/10000186b
Screen Sample Type cell
Screen Description High-throughput confocal microscope screen of protein localization screen for 314 yeast strains expressing unique GFP-tagged proteins treated with 0.03% MMS, imaged at 7 time points over 4 hours (0, 30, 60, 90, 120, 180, and 240 minutes), in a Rad53 Sml1 deletion background.
Screen Size Plates:7 5D Images: Planes:1 Average Image Dimension (XYZCT):500 x 500 x 1 x 2 x 1 Total Tb: 0.06127
Screen Example Images
Screen Imaging Method spinning disk confocal microscopy
Screen Imaging Method Term Source REF Fbbi
Screen Imaging Method Term Accession FBbi_00000253
Screen Technology Type protein screen
Screen Technology Type Term Source REF EFO
Screen Technology Type Term Accession EFO_0005398
Screen Type primary screen
Screen Type Term Source REF EFO
Screen Type Term Accession EFO_0007556
Screen Organism
Screen Organism Term Source REF NCBITaxon
Screen Organism Term Accession
Screen Comments
# Library section. The library file should be supplied separately and it should contain the reagents description including, at the absolute minimum: reagent ID, sequences and position in the layout (= plate + position in the plate)
Library File Name idr0145-screenB-annotation
Library File Format tab-delimited text
Library Type GFP protein fusion library
Library Type Term Source REF EFO
Library Type Term Accession EFO_0007566
Library Manufacturer https://yeastgfp.yeastgenome.org/
Library Version
Library Experimental Conditions Cells were treated with 0.03% MMS in low-fluorescent yeast media.
Library Experimental Conditions Term Source REF EFO
Library Experimental Conditions Term Accession
Quality Control Description
# Protocols
Protocol Name growth protocol treatment protocol HCS library protocol HCS image acquisition and feature extraction protocol HCS data analysis protocol
Protocol Type growth protocol treatment protocol HCS library protocol HCS image acquisition and feature extraction protocol HCS data analysis protocol
Protocol Type Term Source REF EFO EFO EFO EFO EFO
Protocol Type Term Accession EFO_0003789 EFO_0003969 EFO_0007571 EFO_0007572 EFO_0007573
Protocol Description The 322 GFP-tagged strains were grown to mid- logarithmic phase in liquid Synthetic Defined (SD) media and imaged on a 384-well imaging plate (PerkinElmer 384 Cell Carrier Ultra, 6057300) in a high-throughput confocal microscope (PerkinElmer Opera High-Content Screening System) as previously described. Briefly, cells were treated with 0.03% MMS and imaged at 7 timepoints over 4 hours (0, 30, 60, 90, 120, 180, and 240 minutes). The addition of drug to each column of the plate was staggered by 1 minute to compensate for the imaging time of the Opera microscope. For each time point, 4 different regions of the well were imaged. Both GFP and RFP was imaged on the microscope with 800 ms exposure. Cells expressed a constitutively cytoplasmic tdTomato fluorophore, and whole cell image segmentation was conducted using CellProfiler based on tdTomato signal. Dead cells displaying high GFP fluorescence were removed from the analysis. An automated quantification scheme was developed using custom written Python scripts to acquire measurements of GFP pixel intensities for each individual cell from all obtained images.
# Phenotypes
Phenotype Name change in protein localization
Phenotype Description Single cell pixel intensity distributions were normalized by the median GFP intensity measured for each respective cell. For a given protein, the 95th percentile, hereafter referred to as the localization (LOC) value, was determined for each distribution among untreated cells. The median (x) and median absolute distribution (MAD) was calculated for the loc value and were used to determine threshold values for changes in protein localization. If a cell LOC value was greater than 2 MAD from the median (x + 2 MAD), it was considered as an increase in protein relocalization from the untreated condition. Conversely, if a cellular LOC value was less than 2 MAD from the median (x - 2 MAD), it was labeled as decreased localization from the untreated condition. The LOC value was calculated for all cells among all proteins and across all 7 timepoints in our study. Finally, the percentage of cells with a localization change was calculated by dividing the number of proteins with a localization change event (either increased or decreased) by the total number of cells segmented.
Phenotype Score Type automatic
Phenotype Term Source REF CMPO
Phenotype Term Name
Phenotype Term Accession
# Raw Data Files
Raw Image Data Format Evotec/PerkinElmer Opera Flex
Raw Image Organization 7 x 384 well plate. Up to 4 fields per well. Each well contains a different tagged ORF. Each plate represents a different time point imaged
# Feature Level Data Files
Feature Level Data File Name
Feature Level Data File Description
Feature Level Data File Format
Feature Level Data Column Name
Feature Level Data Column Description
# Processed Data Files
Processed Data File Name
Processed Data File Format tab-delimited text
Processed Data File Description
Processed Data Column Name
Processed Data Column Type
Processed Data Column Annotation Level
Processed Data Column Description
Processed Data Column Link To Library File