Journal: STAR Protocols

Article Title: Single-Molecule Fluorescence Imaging in Living Saccharomyces cerevisiae Cells

doi: 10.1016/j.xpro.2020.100142

Figure Lengend Snippet:

Article Snippet: Yeast extract powder (YEP) , VWR international , Cat# 212750.

Techniques: Recombinant, Cell Culture, Microscopy

Journal: Journal of Cell Science

Article Title: Distinct TORC1 signalling branches regulate Adc17 proteasome assembly chaperone expression

doi: 10.1242/jcs.261892

Figure Lengend Snippet: Mpk1 is activated and actin depolarised via the Wsc1, Wsc3 and Wsc4 cell wall integrity pathway sensors in response to rapamycin treatment. (A) Proteasome assembly chaperone translation regulation overview. Upon rapamycin treatment, TORC1 is inhibited, leading to activation of Mpk1 and ADC17 mRNA relocalisation to cortical actin patches, together driving increased Adc17 production. (B) Drop assay for WT and CWI sensor mutants on YEPD and YEPD plus rapamycin after 3 days of growth. Representative of three experiments. (C) Mpk1 activation (P-Mpk1) and Adc17 levels following 4 h rapamycin treatment in WT and wsc1Δ cells. Representative of three experiments. (D) Drop assay of WT and wsc1/3/4Δ cells on YEPD and YEPD plus rapamycin after 3 days of growth. Representative of three experiments. (E) Mpk1 activation (P-Mpk1) and Adc17 levels following 4 h rapamycin treatment in WT and wsc1/3/4Δ cells. Representative of three experiments. (F) Quantification of the blots shown in E (mean±s.e.m., n =3, two-way ANOVA followed by a Tukey's multiple comparisons test). UT, untreated; Rapa, rapamycin treated.

Article Snippet: For drop assays, cells were adjusted to 0.2 OD 600 nm in YEPD medium, and serially diluted 1/5 (such that each spot was with one fifth the cells of the previous spot) before being spotted on YEPD plates with or without 20 ng/ml rapamycin (LC Laboratories, R5000).

Techniques: Activation Assay

Journal: Journal of Cell Science

Article Title: Distinct TORC1 signalling branches regulate Adc17 proteasome assembly chaperone expression

doi: 10.1242/jcs.261892

Figure Lengend Snippet: Rapamycin treatment activates the CWL pathway by altering cell wall properties. (A) Time courses of zymolyase resistance in WT cells growing exponentially in YEPD (mean with s.e.m., n =5, one-way ANOVA followed by a Tukey's multiple comparisons test). (B) WT cell resistance to 45 min zymolyase treatment after treatment with rapamycin (mean±s.e.m., n =4, one-way ANOVA followed by a Tukey's multiple comparisons test). (C) Resistance to 45 min zymolyase treatment by WT and wsc1/3/4Δ cells that were either untreated or after 1 h rapamycin treatment (mean±s.e.m., n =4, two-way ANOVA followed by a Tukey's multiple comparisons test). (D) Updated scheme from Fig. 1A , showing role of cell wall alterations, which increase zymolyase resistance in ADC17 translation. UT, untreated; Rapa, rapamycin treated.

Article Snippet: For drop assays, cells were adjusted to 0.2 OD 600 nm in YEPD medium, and serially diluted 1/5 (such that each spot was with one fifth the cells of the previous spot) before being spotted on YEPD plates with or without 20 ng/ml rapamycin (LC Laboratories, R5000).

Techniques: