Analysis of TORC1-body Formation in Budding Yeast

[Abstract] The Target of Rapamycin kinase Complex I (TORC1) is the master regulator of cell growth and metabolism in eukaryotes. In the presence of pro-growth hormones and abundant nutrients, TORC1 is active and drives protein, lipid, and nucleotide synthesis by phosphorylating a wide range of proteins. In contrast, when nitrogen and/or glucose levels fall, TORC1 is inhibited, causing the cell to switch from anabolic to catabolic metabolism, and eventually enter a quiescent state. In the budding yeast Saccharomyces cerevisiae , TORC1 inhibition triggers the movement of TORC1 from its position around the vacuole to a single focus/body on the edge of the vacuolar membrane. This relocalization depends on the activity of numerous key TORC1 regulators and thus analysis of TORC1 localization can be used to follow signaling through the TORC1 pathway. Here we provide a detailed protocol for measuring TORC1 (specifically, Kog1-YFP) relocalization/signaling using fluorescence microscopy. Emphasis is placed on procedures that ensure: (1) TORC1-bodies are identified (and counted) correctly despite their relatively low fluorescence and the accumulation of autofluorescent foci during glucose and nitrogen starvation; (2) Cells are kept in log-phase growth at the start of each experiment so that the dynamics of TORC1-body formation are monitored correctly; (3) The appropriate fluorescent tags are used to avoid examining mislocalized TORC1.

In the presence of pro-growth hormones and abundant nutrients, TORC1 is active and phosphorylates a wide array of proteins to drive protein and ribosome synthesis, activate lipid and nucleotide synthesis, tune nitrogen and amino acid metabolism/transport, and repress autophagy (Kamada et  Sabatini, 2020). In contrast, when cells are exposed to stress or starvation conditions, TORC1 is inactivated to limit cell growth and redirect available resources to the appropriate stress or starvation response (Barbet et al., 1996;Duvel et al., 2010).
Over the last 15 years, detailed analysis of TORC1 signaling has shed light on the protein network that transmits stress and starvation signals to TORC1, but many questions remain about how TORC1 is regulated in the wide range of stimuli that influence cell growth and survival (Loewith and Hall, 2011;Gonzalez and Hall, 2017;Liu and Sabatini, 2020). In the model organism Saccharomyces cerevisiae 2 www.bio-protocol.org/e3975  (where TORC1 was first discovered) (Loewith and Hall, 2011;Gonzalez and Hall, 2017), progress mapping the TORC1 regulatory circuit has been hindered by the absence of a rapid and scalable TORC1 signaling assay. Here we describe a protocol for monitoring the movement of TORC1 (made up of the TOR kinase Tor1, the essential regulatory protein Kog1, and two accessory proteins, Lst8 and Tco89) into a focus/body during glucose and/or nitrogen starvation. Since We generally follow movement of TORC1 on the vacuolar membrane in a standard W303 lab strain (trp1-1;can1-100;leu2-3,112;his3-11,15;ura3;GAL+;ADE+) carrying Kog1 with a yellow fluorescent protein tag at its native locus (Kog1-ECitrine, or Kog1-YFP for short). Tags on other TORC1 subunits (particularly Tco89) can also be used, but Kog1-YFP gives the strongest signal. It is worth noting fluorescent tags on Tor1 disrupt TORC1 localization and activity. 2. Patch out the strains that are going to be examined onto fresh YEPD plates, starting from glycerol stocks (yeast in 15% glycerol and YEPD, stored at -80 °C) using sterile applicator sticks, and then incubate the plates at 30 °C overnight (or up to three days).
3. Transfer approximately 5 μl of each strain/patch into a separate 28 ml tube containing 5 ml of SD medium (again using sterile applicator sticks). 12. Remove the chamber slide from the microscope, aspirate and discard medium from each well.
13. Wash each well three times with synthetic medium missing glucose (S-glucose), or synthetic medium missing nitrogen (S-nitrogen), at 30 °C, using 350 μl, 400 μl, and then 450 μl of medium, by gently pipetting against the same corner of each well and then aspirating and discarding each wash except the last (which is left in the well during imaging).
14. Start a timer after the first wash and capture z-stacks at each time-point in all wells as described in Step 11 (we typically take pictures every 10 min for 1 h), keeping the slide at 30 °C during the entire experiment.
In each experiment, a wild-type (or relevant mutant strain) missing a fluorescent (YFP) tag should also be imaged as a control. We have found that starvation (particularly glucose starvation) can trigger the formation of autofluorescent puncta. The intensity and number of puncta increase over time, and their appearance is highly dependent on the batch of medium being used. We discard data from experiments/time-points where a significant number of autofluorescent foci form in the control strain and rerun the experiment in a fresh batch of medium.

Data analysis
Compress the z-stack for each time-point and strain (composed of all 16 planes) into a maximum intensity projection, using Fiji or other software. Identify in focus cells using the DIC image, and then count the fraction of (in focus) cells that contain a TORC1 (Kog1-YFP) body. A mother cell with an attached bud should be counted as two individual cells due to the observation that a newly formed daughter cell can contain its own TORC1-body. To establish statistical significance, experiments must be completed in triplicate on three different days, and the average fraction of cells with a body at each time-point, and the corresponding standard deviation, were calculated. 5 www.bio-protocol.org/e3975