Nutrient-switch-analysis

Code for analysis and figures in the paper "Single-cell imaging reveals a key role of Bck2 in budding yeast cell size adaptation to nutrient challenges" Includes system for categorising and following single-cells and their progeny after a nutrient switch (downstream of CellACDC). Written in Python using Jupyter notebooks. Details on packages and versions can be found in the environment.yml file. Microsoft Windows 10 Pro was used as an operating system.

General structure

The figures and the corresponding code are grouped into folders based on the underlying experimental datasets used. Within each folder, the dataprep scripts must be run first, in the sequence assigned in their filenames. Once the prepped data is saved, the plotting script for the figures can be run. The location of the prepped data should be updated while importing and saving.

While the functions used for dataprep are common between the different replicates of a particular kind of microscopy experiment, unique scripts have been included for every replicate because they carry information about the experimental setup (eg. data location, what position corresponds to what strain etc.). This also allows for saving analysed data for individual replicates before combining them into one file.

Dataprep for figures based on steady-state microscopy (Fig. 1F) must be performed before plotting figures based on nutrient-switch microscopy (Figs. 4, 5 & 7). Dataprep for steady-state microscopy uses a custom script instead of functions from Cell-ACDC because this data was analysed before the Cell-ACDC downstream analysis notebook was published. However, the steady-state microscopy data has also been analysed using functions from the Cell-ACDC downstream notebook and both methods of dataprep were found to yield the same results. I recommend using the custom script for dataprepping single-channel microscopy files that were analysed with a very early version of Cell-ACDC (<2021), because the custom script is compatible with their nomenclature.

All statistics where performed during plotting time, and results and code can be found within the respective plotting scripts.

Aggregated information on the metadata of the data points can be found in create_file_list.py. And in the file_tables folder.

During revisions, some supplementary figures changed number: S1, S2, S3 -> S1; R1 (S4) -> S2; R2 (S5) -> S3; S6 -> S4. S7, S8 -> S5.

Data availability

The raw microscopy files (.nd2) and the corresponding Cell-ACDC output folders (with manually corrected cell segmentation, cell tracking, cell cycle annotations), as well as the raw microscopy files and the summary tables are available on BioStudies (DOI: 10.6019/S-BIAD4064) For each figure, the dates of the experimental replicates (provided in the dataprep code as well as in this README file) can be used to find the underlying dataset.

Figure-specific information

• Fig1_FigS1_FigS3

  • Fig_1C_S1_smFISH
    • smFISH analysis on datasets 05072021 and 20102022
    • Two scripts for dataprep of individual replicates (dataprep_rep1.ipynb & dataprep_rep2.ipynb) and one script for plotting data from both replicates combined (plots_repscombined.ipynb).
  • Fig_1D_smFISH
    • plotting example images from Position_19 of 05072021 + cell contours and detected spots (smFISH_example_images.ipynb)
  • Fig_1F_1G_S3_SteadyStateMicroscopy
    • Dataprep:
      • 1 script per medium for steady state microscopy experiments:
        • SCD: datasets 20210120 and 20210121, 1script_SCD.ipynb. SCGE: datasets 20210923 and 20230103, 1script_SCGE.ipynb
      • script2 (2script_combined_media.ipynb) and script3 (3script_combined_media.ipynb) for combining the media and further downstream analysis
      • script4 (4script_additionalCcr4rep.ipynb), script5 (5script_additionalCcr4rep.ipynb) and script6 (6script_additionalCcr4rep.ipynb) for integrating an extra replicate of SCD microscopy for the Ccr4 mutant (dataset 20231212)
    • 2 scripts for plotting: plot_Fig1F_and_G.ipynb and plot_FigS3.ipynb
  • Fig. 1B was plotted from qPCR data in Microsoft Excel.

• Fig2

  • Dataprep: example script for dataprep for one replicate (concatenating_timept_files_per_rep.ipynb). This script concatenates coulter counter data from multiple timepoints and cleans it for use in python. Run this script for datasets 04082022, 05092022, 06092022, 07072022, 09062022 and 15062022. Combine the outputs for all the reps and import resulting df into plotting script.
  • 1 script for plotting Fig. 2B, 2C and 2D (plot_Fig2.ipynb)

• Fig4_Fig5_Fig7_FigS4_FigS5 [NOTE: For Fig7, also see Revision section]

  • Dataprep for nutrient-switch experiments - split into three further categories:

    • nutrient_switch_original4strains - containing WT, Δwhi5, Δbck2 and Δwhi5Δbck2

      datasets 09082022, 13102022 and 28032023 - analysed with respective scripts (script1_rep1_09082022.ipynb, script2_rep2_13102022.ipynb, script3_rep3_28032023.ipynb)

      +1 script to combine the replicates script4_repscombined_withsteadystate.ipynb

    • nutrient_switch_cln3deletions - containing WT, Δcln3, Δwhi5Δcln3, Δwhi5Δcln3Δbck2, MK40, MK41 and MK44. MK40, MK41 and MK44 were not used in the paper.

      datasets 06042023 and 12042023 - analysed with respective scripts (script1_rep1_06042023.ipynb, script2_rep2_12042023.ipynb)

      +1 script to combine the replicates script3_repscombined.ipynb

    • nutrient_switch_cln3stabilisations_with_deletion_mutants - containing MK40, Δwhi5_MK40bg, Δbck2_MK40bg and Δwhi5Δbck2_MK40bg, MK44, Δwhi5_MK44bg, Δbck2_MK44bg and Δwhi5Δbck2_MK44bg. These strains were not used in the paper.

      datasets 28092023 and 06102023 - analysed with respective scripts (script1_rep1_28092023.ipynb, script2_rep2_06102023.ipynb)

      +1 script to combine the replicates nutrient_switch_repscombined_Cln3stabs_withMutants.ipynb

  • Plotting script - plot_Fig4_Fig5_Fig7_FigS4_FigS5. NOTE: Please make sure you have run dataprep for Fig_1F_1G_S3_SteadyStateMicroscopy before running this script to include steady-state control cells.

• Fig6_FigS6 [NOTE: For Fig6, also see Revision section]

  • Dataprep for nutrient switch with cell-cycle reporter strains:
    • script1 and script2 for dataprep of each replicate (datasets 13102023 and 19102023): script1_rep1_13102023.ipynb and script2_rep2_19102023.ipynb
    • script3 (script3_fluorescenceanalysis.ipynb), script4 (script4_fluorescenceanalysis_II.ipynb) and script5 (script5.ipynb) for fluorescence analysis and combining it with nutrient-switch analysis
  • Plotting Fig6_FigS6_plots.ipynb

• FigS2

  • 1 script for dataprep and plotting steady-state SCD coulter counter data for WT, Δccr4 and Δwhi5Δccr4 from dataset 04042023 FigS2_dataprep_and_plotting.ipynb

• Revision

  • Revised parts of Fig6 and Fig S6: After running the scripts for the original Fig 6 and S6, run the scripts for the additional replicate performed for revisions, the dataset for which is 11122024. In the folder jupyter_scripts/final_code/Revision/revision_Fig6_CellCycleMarkerdata, run the scripts in the following order: script1_rep3_11122024.ipynb, script3_11122024_fluorescenceanalysis.ipynb, script4_11122024_fluorescenceanalysis_II.ipynb, script5_11122024.ipynb, Revised_Fig6_FigS6_plots.ipynb. The updated panels of Fig6 and Fig S6 must be formatted externally in Inkscape and merged with the original figures.
  • Revised parts of Fig7: After running the scripts for the original Fig 7, run the scripts for the new METprCLN3-11A/METprCLN3 strains, the datasets for which are 07032025 and 27032025. In the folder jupyter_scripts/final_code/Revision/revision_Fig7_Metpr_cln311a_Exps, run the scripts in the following order: script1_rep1_07032025.ipynb, script2_rep2_27032025.ipynb, script3_repscombined.ipynb, Revised_Fig7_basedon_plot_Fig4_Fig5_Fig7_FigS4_FigS5.ipynb. The updated panels of Fig7 must be formatted externally in Inkscape and merged with the original figures.
  • Revision plan: For figures in the revision proposal and their updates, please run scripts in the jupyter_scripts/final_code/Revision/revision_plan_plots folder. old_revision_plan_Fig4_Fig5_Fig7_FigS4_FigS5-Copy1.ipynb is for plots in the original revision proposal. revision_plan_plot_Fig4_Fig5_Fig7_FigS4_FigS5-Copy1.ipynb is for updated versions of these revision proposal figures.