DOI: 10.5061/dryad.mgqnk9930
title: Strain‐dependent differences in coordination of yeast signalling networks

description: The yeast mitogen activated protein kinase pathways serve as a model
 system for understanding how network interactions affect the way
 in which cells coordinate the response to multiple signals. We have
 quantitatively compared two yeast strain backgrounds YPH499 and Σ1278b
 (both of which have previously been used to study these pathways) and
 found several important differences in how they coordinate the interaction
 between the high osmolarity glycerol (HOG) and mating pathways. In the
 Σ1278b background, in response to simultaneous stimulus, mating pathway
 activation is dampened and delayed in a dose dependent manner. In the
 YPH499 background, only dampening is dose dependent. Further, leakage from
 the HOG pathway into the mating pathway (crosstalk) occurs during
 osmostress alone in the Σ1278b background only. The mitogen activated
 protein kinase Hog1p suppresses crosstalk late in an induction time course
 in both strains but does not affect the early crosstalk seen in the Σ1278b
 background. Finally, the kinase Rck2p plays a greater role suppressing
 late crosstalk in the Σ1278b background than in the YPH499 background. Our
 results demonstrate that comparisons between laboratory yeast strains
 provide an important resource for understanding how signaling network
 interactions are tuned by genetic variation without significant alteration
 to network structure.
description: Data was collected as described in the Materials and Methods
 section of Scott, <em>et al</em>.
description: <strong>AMN1_allele_swap</strong>: Representative
 images of AMN1 wild-type (yMM1174) and amn1 (yMM1584) Sigma S. cerevisiae
 strains. Presented in Figure 8 of Scott, et al. The .tif files can be
 opened in ImageJ.
 <strong>flow_cytometry</strong>: Contains the raw
 Flow Cytometry Standard (.fcs files) for experiments that generated the
 data for Figures 2D, 3B-F, 4A-B, 5,6,7. FlowJo workspace files (.wsp) as
 well as summary analysis files (.xlsx) and supplementary files (e.g. plate
 maps, .xlsx) are also included. The .fcs file can be opened with a variety
 of flow cytometry software, including FlowJo and open-source options such
 as FlowPy. The .wsp files require the propriety software FlowJo but are
 not necessary for analyzing the .fcs files.  The .xlsx files can be opened
 with Excel, Google Sheets or LibreOffice. Subfolders
 are as follows: crosstalk_time_courses:
 Data used to generate Figures 5,6,7
 double_stimulus_time_courses: Data used to generate Figures
 3B-F sorbitol_dose_response: Data used to generate
 Figures 2D, 4A-B 
 <strong>generate_figures</strong>: Summary data and
 code used to generate figures in Scott, et al. Subfolders are as
 follows: data: Summary data for all flow cytometry
 experiments used to make Figures 2D, 3B-F, 4A-B, 5, 6, 7 (folders:
 crosstalk_time_courses, double_stimulus, sorbitol_dose response) and
 summary FISH data (2019-10-03_FISH_dosage.csv) FISH
 images: Representative images of FISH experiments for 0M sorbitol and
 0.75M sorbitol for both YPH499 (yMM736) and Sigma (yMM985) yeast strains.
 Both experiments have 0M alpha-factor. Used to make Figure 4C
 graphs:  Plots generated by code in folder XX and used to make
 Figures in Scott, et al 2022. notebooks: Python
 notebooks used to make Figure3B-C (double_stimulus.ipynb), Figure 4A, 5-7
 (crosstalk_time_courses.ipynb), Figure 4D (FISH.ipynb) and Figure 2D and
 4B (sorbitol_dose_response.ipynb) osmosensitivity: Data
 in this folder was used to generate Figure 2A-C. Files and subfolders are
 as follows: liquid_ypd_data.xlsx: Doubling information
 from liquid culture growth in 0M, 0.75M, and 1.5M sorbitol. Doublings
 calculated as described in the Methods of Scott, et al.  Used to generate
 Figure 2C. ypd_spot_graph.xlsx: Quantification of the
 spot tests in spot_tests. Used to generate Figure 2B.
 spot_tests:  Image files (.scn file generated by Bio-Rad Image
 Lab Software, can be opened in ImageJ) from the top and bottom of each
 plate (0M, 0.25M, 0.5M, 0.75M, 1M sorbitol).  Images were taken at T=24
 hours and T=45 hours for growth. scFISH: Data in this
 folder was used to generate Figure 4C-D. Relevant files and subfolders are
 as follows: yMM736.xlsx: Summary data containing the
 field (image number-xy1, xy2, or xy3), cell label, number of Stl1
 transcripts, number of Stl1 start sites, number of Fus1 transcripts,
 number of Fus1 start sites for each experiment with yMM736
 (YPH499) yMM985.xlsx: Summary data containing the frame
 (image number), cell label, number of Stl1 transcripts, number of Stl1
 start sites, number of Fus1 transcripts, number of Fus1 start sites for
 each experiment with yMM985 (Sigma) Sigma: Folder
 containing data for each individual stimulation experiment (0M alpha +
 varying amounts of sorbitol) for the yMM985 strain. Individual subfolders
 correspond to individual stimulation conditions and are labelled with the
 stimuli (e.g. A0S0 is 0M alpha factor, 0M sorbitol). Each subfolder
 contains: o   Three images of the Quasar 570 probes
 (STL1 transcripts, e.g. CY3_a0s0_xy1.tif) o   Three
 images of the Quasar 670 probes (FUS1 transcripts, e.g.
 CY5_a0s0_xy1.tif) o   Three images of cells stained
 with DAPI (e.g. DAPI_a0s0_xy1.tif) o   Images of the
 identified STL1 and FUS1 transcripts (e.g. CY3_1.jpg, CY5_1.jpg).
 Transcripts spots were identified as described in Scott, et al and
 associated references (McIsaac, et al; Raj, et al)
 o   An excel file (e.g. yMM985A0S0_data.xls) containing a summary
 of the analyzed images, namely the Field (xy1, xy2, or xy3), Cell, channel
 1 particles (FUS1 transcripts), channel 1 starts (FUS1 start sites),
 channel 2 particles (STl1 transcripts), channel 2 starts (STL1 start
 sites).  This excel file also contains the individual data for for FUS1
 (CY5_Data) and STl1 (CY3_Data). YPH499: Same as the
 “Sigma” folder, but for the YPH499 strain (yMM736).
 REFERENCES: McIsaac RS, Silverman
 SJ, Parsons L, Xu P, Briehof R, McClean MN & Botstein D (2013)
 Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ
 Hybridization in Saccharomyces cerevisiae. JoVE (Journal of Visualized
 Experiments), e50382. Scott TD, Xu P, &
 McClean MN (2022) Strain dependent differences in coordination of yeast
 signaling networks bioRxiv doi:
 https://doi.org/10.1101/2022.06.09.495559 Raj A, van
 den Bogaard P, Rifkin SA, van Oudenaarden A & Tyagi S (2008)
 Imaging individual mRNA molecules using multiple singly labeled probes.
 Nat Methods 5, 877–879.