Journal: Nature cell biology
Article Title: Reversible amyloids of pyruvate kinase couple cell metabolism and stress granule disassembly
doi: 10.1038/s41556-021-00760-4
Figure Lengend Snippet: (A) Schematic representation of the domains of Cdc19. The four mutated residues located within the LCR in the Cdc19 irrev mutant are indicated (red circles). (B)-(C) Both Cdc19 WT and Cdc19 irrev form ThT- and Congo Red (CR)-positive aggregates upon heat shock in vitro . Purified Cdc19 WT , Cdc19 irrev and a non-aggregating Cdc19 mutant as negative control (Cdc19 ΔPEP ) were incubated with ThT (B) or CR (C) at 4 °C or after heat shock (42 °C, 10 min). Fluorescence emission was measured at 490 nm or 614 nm, respectively. Graphs show mean ± S.E.M. of three independent experiments (two-tailed t-test, ThT: P WT = 0.0000149, P irrev = 0.0091, CR: P WT = 0.0003, P irrev = 0.0128). (D) In vivo -formed Cdc19 WT and Cdc19 irrev aggregates are CR-positive. Cells expressing GFP-tagged Cdc19 WT or Cdc19 irrev were harvested when exponentially growing or after heat shock (42 °C, 30 min) and lysed. Cdc19-GFP was immobilized in a GFP-trap microfluidic chamber and stained with CR. GFP and CR signals were detected by fluorescence microscopy, and merged to visualize co-localization. Arrowheads indicate CR-positive Cdc19-GFP aggregates. Images are representative of three independent experiments. Scale bar: 10 μm. (E) Limited-Proteolysis Mass Spectrometry (LiP-MS) results indicate that Cdc19 WT and Cdc19 irrev undergo comparable structural transitions upon aggregation in vitro and in vivo . Purified soluble or aggregated (42 °C, 10 min) Cdc19 WT or Cdc19 irrev , as well as cell extracts obtained from cells expressing Cdc19 WT -GFP or Cdc19 irrev -GFP harvested during exponential growth or stationary phase (2 days) to induce aggregation were analysed by LiP-MS as described in the (n = 3 independent experiments). Peptides detected in soluble and aggregated Cdc19 WT and Cdc19 irrev are displayed in volcano plots, and upregulated (red) or downregulated (blue) conformation-sensitive peptides are highlighted. Conformation-specific LiP-MS-peptides detected in vitro and in vivo were mapped to the Cdc19 schematic drawing (green). (F) Intracellular ATP levels (mM) were determined in the indicated strains after heat shock (42 °C, 30 min) and recovery (30 °C, 60 min). Mean ± S.E.M. of n = 5 independent experiments is shown (two-tailed Mann-Whitney test, P = 0.0079). Source data for all graphical representations are found in .
Article Snippet: For time-lapse experiments, cells were loaded in microfluidic plates (CellASIC ONIX2, Merck Millipore) at 30 °C [ ].
Techniques: Mutagenesis, In Vitro, Purification, Negative Control, Incubation, Fluorescence, Two Tailed Test, In Vivo, Expressing, Staining, Microscopy, Mass Spectrometry, MANN-WHITNEY
