cell tracker™ blue cmac dye  (Thermo Fisher)

Journal: bioRxiv

Article Title: Alpha-arrestins Aly1 and Aly2 regulate trafficking of the glycerophosphoinositol transporter Git1 and impact phospholipid homeostasis

doi: 10.1101/2021.02.04.429748

Figure Lengend Snippet: A , WT, aly1Δ aly2Δ , or cnb1 Δ cells containing Git1-GFP expressed from the TEF1 promoter were stained with CMAC and imaged by fluorescence microscopy. B , The PM and vacuolar fluorescence intensities from the cells depicted in A were quantified using our automated quantification pipeline and the distributions of PM/vacuolar fluorescence ratios in arbitrary units (a.u.) were plotted as scatter plots. The horizontal midline in black represents the median and the 95% confidence interval is represented by the white error bars. Kruskal-Wallis statistical analyses with Dunn’s post hoc test were performed and statistical significance compared to the wild-type cells is indicated with asterisks. A yellow dashed line is provided that represents the median of wild-type cells to facilitate comparisons. C, Schematic of the sphingolipid biosynthetic pathway demonstrating the points where myriocin and aureobasidin (AureoB) impair the pathway (red lines) and the sites where PI is incorporated into the complex base production in the lower half of the pathway (PI with blue arrows). The intermediates in the pathway are shown but the enzymes are not indicated. Abbreviations in this diagram are as follows: DHS (dihyrdosphingosine), PHS (phytosphingosine), IPC (inositolphosphoryl-ceramide), MIPIC (mannosylinositolphosphoryl-ceramide), and M(IP) 2 C (mannosyl-di-inositolphosphoryl ceramide). For simplicity, some intermediates are not shown and instead, multiple arrowheads are provided for those steps where two or more enzymatic steps have been condensed. D , Growth of serial dilutions of the cells indicated containing either a vector or a LEU2 -marked TEF1pr - GIT1 -GFP plasmid on SC medium lacking leucine and containing the indicated concentrations of GPI, myriocin, or aureobasidin are shown after 2-4 days of incubation at 30°C. E , WT, aly1Δ aly2Δ , cnb1 Δ, or aly1Δ aly2Δ cnb1 Δ cells containing Git1-GFP expressed from the TEF1 promoter were treated with 50μM GPI for 2h, stained with CMAC and imaged by fluorescence microscopy. Automated quantification of the PM and vacuole fluorescence intensities was performed and the median PM/vacuole fluorescence intensity for each is presented in arbitrary units (a.u.) on the right side of the figure.

Article Snippet: To visualize vacuoles, cells were stained with 250 μM Cell Tracker Blue CMAC (7-amino-4-chloromethylcoumarin) dye (Life Technologies, Carlsbad, CA).

Techniques: Staining, Fluorescence, Microscopy, Plasmid Preparation, Incubation

Journal: bioRxiv

Article Title: Alpha-arrestins Aly1 and Aly2 regulate trafficking of the glycerophosphoinositol transporter Git1 and impact phospholipid homeostasis

doi: 10.1101/2021.02.04.429748

Figure Lengend Snippet: A , Growth of serial dilutions of WT or aly1Δ aly2Δ cells containing a LEU2 -marked TEF1pr - GIT1 -GFP plasmid and a pRS316 vector expression nothing or expressing the indicated α-arrestins on SC medium lacking uracil and leucine and containing 50μM GPI is shown after 2 days of incubation at 30°C. B , Cells lacking ALY1 and ALY2 with a plasmid expressing Git1-GFP from the TEF1 promoter and a pRS316-empty vector expressing nothing or the indicated α-arrestins were treated with 50μM GPI for 2 h, stained with CMAC and imaged by fluorescence microscopy. C , The PM and vacuolar fluorescence intensities from the cells depicted in B were quantified using our automated quantification pipeline and the distributions of PM/intracellular fluorescence ratios in arbitrary units (a.u.) were plotted as scatter plots. The horizontal midline in black represents the median and the 95% confidence interval is represented by the white error bars. Kruskal-Wallis statistical analyses with Dunn’s post hoc test were performed and statistical significance compared to the aly1 Δ aly2 Δ cells with vector is indicated with asterisks or as not significant (ns) where applicable. The ††† symbols represent p-values of <0.001 and is used to mark comparisons between aly1 Δ aly2 Δ expressing ALY1 and each of the other conditions. In these conditions, most mutants are not statistically different from wild-type Aly1 (no marker shown); Aly1 5A is the only mutant that is significantly lower PM/vacuole ratios than wild-type Aly1 while the vector control has significantly higher PM/vacuole ratios. In each case, a yellow dashed line is provided that represents the median of the aly1 Δ aly2 Δ cells containing a vector control to facilitate comparisons.

Article Snippet: To visualize vacuoles, cells were stained with 250 μM Cell Tracker Blue CMAC (7-amino-4-chloromethylcoumarin) dye (Life Technologies, Carlsbad, CA).

Techniques: Plasmid Preparation, Expressing, Incubation, Staining, Fluorescence, Microscopy, Marker, Mutagenesis

Journal: bioRxiv

Article Title: Alpha-arrestins Aly1 and Aly2 regulate trafficking of the glycerophosphoinositol transporter Git1 and impact phospholipid homeostasis

doi: 10.1101/2021.02.04.429748

Figure Lengend Snippet: A , The indicated cells were transformed with the plasmid expressing a GFP-fused FYVE domain of Eea1, which marks PI(3)P. Cells were grown to mid-log phase and either untreated (no addition) or treated with 100μM GPI for 2 h, stained with CMAC and imaged by fluorescence microscopy. B , Images of cells captured in A were evenly adjusted. Cells with evident GFP staining of vacuolar limiting membrane were counted in comparison to the total number of vacuoles in the image, as assessed using the CMAC stain. The percentage of cells with limiting membrane fluorescence is presented for each treatment. All cells in at least 3 images per condition were counted for an n of 150 at minimum per condition. C , Radiolabeled 3 H-GPI uptake via the endogenous Git1 transporter and conversion to 3 H-PI was measured in wild-type or aly1 Δ aly2 Δ cells. Cells were grown under low phosphate conditions to induce GIT1 expression in the presence of 5 μM [ 3 H-inositol]-GPI, and intracellular and membrane fractions were isolated. The conversion of 3 H-GPI into 3 H-PI was monitored by using TLC and liquid scintillation counting. The graph presented represents three independent biological replicates that were measured and a Student’s t-test was used to assess significance. A more detailed breakdown of these results is presented in where both the water-soluble and membrane fractions are compared.

Article Snippet: To visualize vacuoles, cells were stained with 250 μM Cell Tracker Blue CMAC (7-amino-4-chloromethylcoumarin) dye (Life Technologies, Carlsbad, CA).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Staining, Fluorescence, Microscopy, Isolation

Journal: bioRxiv

Article Title: Alpha-arrestins Aly1 and Aly2 regulate trafficking of the glycerophosphoinositol transporter Git1 and impact phospholipid homeostasis

doi: 10.1101/2021.02.04.429748

Figure Lengend Snippet: A , The indicated cells were transformed with the plasmid expressing a GFP-fused FYVE domain of Eea1, which marks PI(3)P. Cells were stained with CMAC and imaged by fluorescence microscopy. B , The GFP fluorescence at the limiting membrane of the vacuole from the images in A was quantified by manually drawing a line around the outside of the CMAC stained vacuoles and overlaying this mask onto the GFP channel. The quantified fluorescence intensities (arbitrary units, a.u.) are presented as scatter plots. The horizontal midline in black represents the median and the 95% confidence interval is represented by the black error bars. Kruskal-Wallis statistical analyses with Dunn’s post hoc test were performed and statistical significance compared to the wild-type cells is indicated with asterisks or as not significant (ns) where applicable. A yellow dashed line is provided that represents the median of wild-type cells to facilitate comparisons. C , The indicated cells were transformed with the plasmid expressing a GFP-fused C2 domain of the Lact protein, which marks PS. Cells were stained with CMAC and imaged by fluorescence microscopy. D , The GFP fluorescence at the plasma membrane from the images in C was quantified using our automated pipeline. The quantified fluorescence intensities (arbitrary units, a.u.) are presented as scatter plots. The horizontal midline in black represents the median and the 95% confidence interval is represented by the black error bars. Kruskal-Wallis statistical analyses with Dunn’s post hoc test were performed and statistical significance compared to the wild-type cells is indicated with asterisks or as not significant (ns) where applicable. A yellow dashed line is provided that represents the median of wild-type cells to facilitate comparisons.

Article Snippet: To visualize vacuoles, cells were stained with 250 μM Cell Tracker Blue CMAC (7-amino-4-chloromethylcoumarin) dye (Life Technologies, Carlsbad, CA).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Staining, Fluorescence, Microscopy