SynSAC

Stages

1. 1.
   Synthetic SAC induction for meiotic metaphase arrest(selection)

   This stage exists to obtain synchronized meiotic cell populations that can be collected for downstream characterization, especially of meiosis II events.

   Selection: Chemically inducible dimerization of ectopic Mps1 and Spc105 to generate arrested metaphase I or metaphase II yeast cells.

2. 2.
   Comparative SAC response characterization(functional_characterization)

   This stage exists to characterize how the synthetic checkpoint response differs between meiotic metaphases and to identify a mechanistic determinant of the weaker meiosis I response.

   Selection: Measure and compare SAC response properties in metaphase I and metaphase II synchronized cells, including contribution of the Spc105 PP1-binding site.

3. 3.
   Kinetochore composition and phosphorylation analysis(secondary_characterization)

   This stage exists to demonstrate the utility of the synchronization strategy for downstream molecular characterization of meiotic kinetochores.

   Selection: Analyze kinetochores from metaphase I and metaphase II synchronized cells for composition and phosphorylation differences.

Steps

1. 1.
   Induce dimerization of ectopic Mps1 and Spc105engineered synchronization system

   Trigger a synthetic spindle assembly checkpoint state in yeast.

   This is the initiating manipulation because the method relies on chemically inducible dimerization to create the arrest state needed for all downstream analyses.

2. 2.
   Collect metaphase I or metaphase II arrested yeast cellssynchronization tool

   Obtain synchronized meiotic cell populations for comparative downstream analysis.

   Collection follows synthetic SAC induction because arrested cells are the required material for subsequent SAC and kinetochore characterization.

3. 3.
   Compare SAC response between metaphase I and metaphase IIassay context

   Determine whether synthetic SAC response strength differs between meiotic metaphases.

   This comparison is performed after synchronized populations are obtained because stage-matched arrested cells are needed to assess metaphase-specific SAC behavior.

4. 4.
   Assess contribution of the Spc105 PP1-binding site to meiosis I SAC restraintmechanistic assay context

   Test whether the PP1-binding site within Spc105 contributes to restraining the meiosis I SAC response.

   This mechanistic analysis follows the stage-comparison result to identify a factor contributing to the weaker meiosis I SAC response.

5. 5.
   Analyze kinetochore composition and phosphorylation from metaphase I and metaphase II cellsupstream synchronization tool enabling downstream analysis

   Use synchronized meiotic populations to characterize stage-specific kinetochore molecular state.

   This downstream characterization is possible after synchronized metaphase populations are collected and serves as the paper's utility demonstration for SynSAC.