Of the three groups of supplements, only minerals could improve growth (i.e. by the time cytokinesis has finished (Sabatinos and Forsburg 2010). Interestingly, this timing can be influenced by manipulating G1 duration by providing the cells with different sources of nitrogen (Carlson mutants have been identified in which septation and/or cytokinesis erroneously take place in the absence of normal sister chromatid separation. This often results in the so-called cut terminal phenotype of undivided nucleus being intersected by the septum (Uemura and Yanagida 1984; Hirano show high incidence of the cut phenotype when grown in YES (P?evorovsky et al. 2009, 2016; Kwon acetyl-coenzyme A carboxylase gene (P?evorovsky et al. 2015, 2016). Cut6 is the rate-limiting enzyme of fatty acid synthesis and the mutant exerts the cut phenotype at restrictive temperature. The precise nature of the mutation is not known (Saitoh cells (P?evorovsky or and cells is largely diminished when cells are grown in the minimal defined EMM medium (P?evorovsky et al. 2015, 2016). Temperature-sensitive mutations in and and mutants, or by growing the cells in EMM medium in the case of (Yamashita and lipid metabolism mutants. MATERIALS AND METHODS Strains, media and cultivations strains used in this study were JB32 (cells were grown at 32C according to standard procedures (Moreno, Klar and Nurse 1991). Temperature-sensitive strains were grown at 25C, or at the semi-permissive temperature of 30C. Cultivation media used in this study included the minimal defined EMM (Formedium, UK), complex YES (0.5% yeast extract, 3% glucose, 50 mg L?1 each of adenine, uracil, L-histidine, L-leucine and L-lysine) and YES variants supplemented with EMM-contained chemical compounds at concentrations listed in Table S1 (Supporting Information) (EMM composition as declared by the manufacturer). For medium shift experiments, exponentially growing cells cultured in EMM were collected by centrifugation (1000??g, 3 min, 25C), resuspended in EPZ011989 the same volume of fresh YES and incubated at 32C. In all other experiments, cultures were grown in the indicated media for the whole duration of the experiment. For growth rate measurements, cells were first grown exponentially in YES. Culture volumes corresponding to 1 1.2??106 cells were collected and centrifuged (1000??g, 3 min, EPZ011989 25C). Supernatants were removed and cell pellets were washed with the appropriate media. The resulting cell suspensions were then centrifuged again (1000??g, 3 min, 25C), supernatants were discarded, and cell pellets were resuspended in 1.5 mL of appropriate media. Aliquots of 1 1.4 mL of resulting cell suspensions were loaded into 12-well plates and introduced into the VarioSkan Flash plate reader (Thermo Scientific). Plates were incubated at 32C with background shaking (180 spm, rotation diameter 20 mm). Optical densities were measured at 10 min intervals EPZ011989 at ?=?595 nm. Doubling times (DT) were calculated according to the formula DT?=?1/k, where k represents the slope of logarithmic phase of growth. Microsoft Excel 2007 was used for data processing and determination of k-value. Microscopy For nuclear staining, exponentially growing cells were collected by centrifugation (1000??g, 3 min, 25C) and fixed by resuspending in 70% ethanol. EPZ011989 Ethanol-fixed cells were centrifuged again (1000??g, 3 min, 25C) and resuspended in deionized H2O. Cells were stained in suspension with 1 g mL?1 4?,6-diamidine-2?-phenylindole dihydrochloride (DAPI). Cell images were taken using the Olympus Cell R and Leica AF 6000LX microscopic systems. Frequency of cut phenotype occurrence was determined by manual counting of cut cells using the ImageJ software, version 1.51j8 (Schneider, Rasband and Eliceiri 2012). At least 200 cells per sample were analyzed. For lipid droplet visualisation in live cells, exponentially growing cells were stained in suspension with 0.1 g mL?1 BODIPYTM 493/503 (Thermo EPZ011989 Fisher Scientific) and briefly mixed by vortexing. MGF No washes or sample dilution/concentration steps were performed to avoid stressing the cells or affecting their metabolism. Cells were centrifuged (1000??g, 3 min, 25C) and promptly imaged on soybean lectin-coated slides using the Olympus Cell R microscope. For imaging Ptl2-GFP, cells were fixed with 10% formaldehyde for 15 min, and washed three times with PBS, followed by microscopy. Fluorescent images were acquired as 16-bit Z-stacks (0.3 m step size, 10 steps) in the.