Journal: bioRxiv

Article Title: The phosphatase Glc7 controls eisosomal response to starvation via posttranslational modification of Pil1

doi: 10.1101/2022.08.09.503340

Figure Lengend Snippet: A) Whole cell lysates of wild-type, pkh1 Δ, pkh2 Δ and pkh3 Δ cells were analysed by immunoblot using α-Pil1 and α-GAPDH antibodies, with Ponceau S stained membrane included. The percentage phosphorylated Pil1 from each yeast strain was quantified (n=3). B) Cells co-expressing Nce102-mCherry and indicated GFP tagged Pkh-kinases were grown to mid-log phase and imaged using confocal microscopy (Airyscan 2). The Pearson’s Correlation Coefficient was measured of colocalisation between Nce102-mCherry with the respective GFP-tagged kinases (n > 40). C) Wild-type and pkh2 Δ cells were transformed with either an empty vector control (-) or a 2μ Pkh2 over-expression plasmid (+). Whole cell lysates were generated from transformants and Pil1 phosphorylation assessed by immunoblot. Levels of GAPDH and Ponceau S are shown as loading controls (left). The percentage of phosphorylated Pil1 was quantified (n=3) and shown (right). Statistical significance was determined using Student’s t -test. Scale bar = 5μm

Article Snippet: Images were captured sequentially to minimise any potential bleedthrough and processed using Zeiss Zen software standard airyscan algorithm and modified for publication using ImageJ software (NIH).

Techniques: Western Blot, Staining, Expressing, Confocal Microscopy, Transformation Assay, Plasmid Preparation, Over Expression, Generated

Journal: bioRxiv

Article Title: The phosphatase Glc7 controls eisosomal response to starvation via posttranslational modification of Pil1

doi: 10.1101/2022.08.09.503340

Figure Lengend Snippet: A) Schematic showing the increased diffusion of nutrient transporters into eisosomes in response to glucose starvation, and their potential exit upon a return to replete nutrient conditions to aid recovery. B) Cells co-expressing Pil1-mCherry and Mup1-GFP were imaged using confocal microscopy (Airyscan 2) with centre and top focus under glucose conditions and following 10 minutes of exchange with raffinose media. Mup1 localised to endosomes (red arrow) and Pil1 marked eisosomes (yellow arrow) after raffinose treatment are indicated. C) Wild-type cells exposed to raffinose media for 1, 5 and 10 minutes prior to lysate generation were immunoblotted using α-Pil1 antibodies and compared to wild-type and pil1 Δ cells grown in glucose replete conditions. GAPDH blot and Ponceau S stained membrane is included as a loading control. Percentage of phosphorylated Pil1 was generated for WT vs 10 minutes of raffinose treatment for wild-type cells was quantified (right).

Article Snippet: Images were captured sequentially to minimise any potential bleedthrough and processed using Zeiss Zen software standard airyscan algorithm and modified for publication using ImageJ software (NIH).

Techniques: Diffusion-based Assay, Expressing, Confocal Microscopy, Staining, Generated

Journal: bioRxiv

Article Title: The phosphatase Glc7 controls eisosomal response to starvation via posttranslational modification of Pil1

doi: 10.1101/2022.08.09.503340

Figure Lengend Snippet: A) Whole cell lysates of wild-type and phosphatase mutant candidates from glucose replete media (left) or following 10 minutes exchange with raffinose media were generated and analysed by immunoblotting using α-Pil1and α-GAPDH antibodies. B) Quantification of percentage of Pil1 phosphorylated in indicated mutants was calculated. Statistical significance was determined using Student’s t -test. C) Yeast expressing GFP tagged phosphatases were cultured to mid-log and stationary phase prior to confocal microscopy (Airyscan). The number of peripheral dots per cell (n > 30) from separate experiments (n = 4) were quantified in each condition (right). Scale bar = 5 μm.

Article Snippet: Images were captured sequentially to minimise any potential bleedthrough and processed using Zeiss Zen software standard airyscan algorithm and modified for publication using ImageJ software (NIH).

Techniques: Mutagenesis, Generated, Western Blot, Expressing, Cell Culture, Confocal Microscopy

Journal: bioRxiv

Article Title: The phosphatase Glc7 controls eisosomal response to starvation via posttranslational modification of Pil1

doi: 10.1101/2022.08.09.503340

Figure Lengend Snippet: A) Cartoon showing Pil1 fusion to mGFP, including its verified phosphosites and BAR domain. Pil1 cassettes that have been mutated to alanine (yellow) or aspartate (red) that were stably integrated at the PIL1 locus are also shown. B) Pil1 or indicated 8A and 8D mutants were expressed from the endogenous locus as mGFP fusions in strains grown to mid-log phase, harvesting and lysate generation for immunoblotting with α-Pil1 and α-GAPDH antibodies. C) Versions of mGFP tagged Pil1 were expressed as the sole chromosomal copy and localised by confocal microscopy coupled to Airyscan 2 detector. D) Pil1 labelled eisosomes were identified by otsu segmentation and number per cell quantified (n = > 37) with p values generated from Student’s t -test comparisons. E) Integrated density for all GFP tagged Pil1 versions localised to eisosomes identified from segmentation performed in D was calculated as a percentage of the total signal, with Student’s t -test comparison generated p values shown. F) Indicated yeast strains were grown to log phase in either YPD rich media or SC minimal media, equivalent cell numbers were estimated by optical density measurements and harvested. 10-fold serial dilutions were generated, and yeast spotted out on YPD and SC plates. Growth was recorded at 24 and 48 hours. G) Cells at mid-log phase were subjected to 2 hours of glucose starvation (raffinose treatment), returned to glucose replete conditions and growth measured over time. H) The growth assay in G was repeated (n = 4) and the growth relative to wild-type was quantified for each indicated timepoint. Statistical significance was determined using a Student’s t -test.

Article Snippet: Images were captured sequentially to minimise any potential bleedthrough and processed using Zeiss Zen software standard airyscan algorithm and modified for publication using ImageJ software (NIH).

Techniques: Stable Transfection, Western Blot, Confocal Microscopy, Generated, Growth Assay