Volume 46, Issue 4, Pages 436-448 (May 2012) Cell Differentiation within a Yeast Colony: Metabolic and Regulatory Parallels with a Tumor-Affected Organism  Michal Čáp, Luděk Štěpánek, Karel Harant, Libuše Váchová, Zdena Palková  Molecular Cell  Volume 46, Issue 4, Pages 436-448 (May 2012) DOI: 10.1016/j.molcel.2012.04.001 Copyright © 2012 Elsevier Inc. Terms and Conditions

Molecular Cell 2012 46, 436-448DOI: (10.1016/j.molcel.2012.04.001) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 S. cerevisiae Colony Cells Differentiate into Two Morphologically and Physiologically Distinct Subpopulations (A) Vertical transversal cross-section of a 20-day-old BY4742 colony. (B) Details of U and L cells visualized with Nomarski contrast, fluorescence, and transmission EM. Left panels: Mitochondria were stained with DiOC6(3), vacuoles with FM4-64, and lipid droplets with Nile Red. (C) Details of cells from fractions 1 and 3 visualized with Nomarski contrast and transmission EM. (B and C) n, nucleus; v, vacuole; m, mitochondria; ld, lipid droplet. (D) Center of the colony as shown by the scheme was examined throughout the study. See also Figure S1. Molecular Cell 2012 46, 436-448DOI: (10.1016/j.molcel.2012.04.001) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Location of GFP-Labeled Proteins in U and L Cells (A) Vertical cross-sections of 15-day-old colonies producing GFP-labeled proteins. (B) Typical U and L cells in 7-day-old (9-day-old for Ato3p-GFP) and 20-day-old colonies visualized with fluorescent microscopy and Nomarski contrast. Arrows indicate GFP delivered to vacuoles by autophagy (Met17p-GFP and Ino1p-GFP) or pexophagy (Pox1p-GFP). See also Figure S2. Molecular Cell 2012 46, 436-448DOI: (10.1016/j.molcel.2012.04.001) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Properties of U and L Cells (A) Stress-related features and viability. (B) Development of U and L cell layers. (C) Oxygen consumption by U and L cells. O2 consumption by cells exponentially grown on glycerol or glucose is presented for comparison. (D) Concentration of amino acids and glutamine uptake. Left and right: U and L cells were separated from 15-day-old colonies. (E) Ammonium excreted from U and L cells (left). Induction of U layer formation in a 4-day-old colony evoked by ammonia produced by older adjacent colonies (right). The black bar marks the U cell layer. The mean is shown ± SD (n = 3 for A, B, C, and E; n = 4 for D); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figure S3 and the Supplemental Experimental Procedures. Molecular Cell 2012 46, 436-448DOI: (10.1016/j.molcel.2012.04.001) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Autophagy and TOR Activity in U and L Cells from 15-Day-Old Colonies (A) Atg8p-GFP, Met17p-GFP, and free GFP. (B) Activity of Pho8Δ60p. (C) Morphology of cells localized in the upper and lower regions of colonies formed from autophagy-defective knockout strains. (D) Gat1p-GFP localization in U and L cells and the effect of rapamycin treatment. Confocal microscopy images of colony cross-sections (left) and TOR kinase activity assessed as the proportion of cytoplasmic and nuclear localization of Gat1p-GFP (right). The mean is shown ± SD (n = 4); ∗∗∗p < 0.001. Molecular Cell 2012 46, 436-448DOI: (10.1016/j.molcel.2012.04.001) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Alterations in Cell Morphology and Composition of Colonies Formed from Different Knockout Strains (A) Morphology of cells located in the upper and lower colony regions. Cells in the central sections of 15-day-old colonies were visualized with Nomarski contrast. Typical U and L cells from a parental strain colony (wt) are presented for comparison. (B) Flow cytometry analysis of colony cell composition. PI+, propidium iodide-positive cells. Molecular Cell 2012 46, 436-448DOI: (10.1016/j.molcel.2012.04.001) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 Parallels in the Metabolic Differentiation and Signaling between Yeast Colonies and a Tumor-Affected Mammalian Organism (A) L cells activate degradative processes that could, along with the activation of specific transporters, lead to the export of amino acids, sugars, and other metabolites. These compounds could then be used by U cells. Products of U cell metabolism (ammonia and possibly fusel alcohols, acetate, and pyruvate) are exported from U cells and reused by L cells and/or act as signaling molecules. (B) Tumor cells obtain energy for growth primarily through glycolysis and glutaminolysis. Metabolic waste products are then reused by liver (alanine and lactate) and muscle cells (ammonium) to produce more glucose and glutamine, which can then be reused to the tumor cells. Metabolic cycles in tumor cells and their proposed yeast colony analogs are indicated with green arrows (Cori cycle) and blue arrows (glutamine-ammonia/um cycle). Solid black arrows indicate a metabolic process or transport, dotted red arrows indicate activation and dotted green lines indicate repression. Processes or enzymes in red are induced and those in green are repressed in the particular cell type. AAT, amino acid transporters; GCN, Gcn4 pathway; HXT, hexose transporters; MT, mitochondrion; OXPHOS, oxidative phosphorylation; SPS, SPS sensor; SNF, Snf1p pathway; TCA, tricarboxylic acid cycle. See also Figure S5 and Table S1. Molecular Cell 2012 46, 436-448DOI: (10.1016/j.molcel.2012.04.001) Copyright © 2012 Elsevier Inc. Terms and Conditions