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.

Data was collected as described in the Materials and Methods section of Scott, et al.

AMN1_allele_swap: 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.

flow_cytometry: 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

generate_figures: 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.