Journal: Nature Communications

Article Title: Loss of cytoplasmic actin filaments raises nuclear actin levels to drive INO80C-dependent chromosome fragmentation

doi: 10.1038/s41467-024-54141-0

Figure Lengend Snippet: a Yeast expressing Las17-GFP (GA-6804) was treated 1.5 h with CMB (1 μM), with Zeocin (100 μg/ml) or both. After fixation, DAPI (blue) and F-actin (Rh-phalloidin, red) were captured by spinning disk confocal microscopy. Images are maximum-intensity projections of focal stacks acquired in each channel (see Methods). Bar = 5 µm. b Yeast expressing Pan1-GFP (GA-6764) was treated and stained as in a . DAPI alone is not shown. Bar = 5 µm. c Scheme of a budding yeast cell illustrating structures of interest. d Las17-GFP expressing cells were treated as in a , but stained with 20 nM Mitotracker Red CMXRos (Thermo Scientific) and DAPI (blue). Colocalization of Las17-GFP and mitochondria is quantified in e . Bar = 5 µm. e Yeast expressing Las17-GFP or Pan1-GFP (see a , b ) were split and treated 1.5 h either with DMSO (Control, blue) or 1 µM CMB and 150 µg/ml Zeocin (YCS, black). Colocalization of Las17-GFP or Pan1-GFP (green) and Mitotracker (red) was quantified by determining the Pearson correlation coefficient in each single plane of an image stack. n = 118 nuclei for Las17-YCS, all others n = 78; line = median. r values show a robust correlation of Las17 with mitochondria. Repeated twice. Quantitations in Source Data file. f Las17-GFP is not enriched in nuclei on Zeocin. Las17-GFP (green) expressing cells were treated ± 500 μg/ml Zeocin for 1 h at 30 °C, fixed and counterstained with DAPI (blue). Image stacks (green and UV) were analyzed for nuclei spanning at least 5 planes. Nuclear GFP intensity was measured per plane and averaged across ≥5 planes for mean intensity per nucleus, plotted as arbitrary GFP units. n = 300 nuclei without Zeo, mean and S.D. = 2861±700; n = 183 with Zeo, mean and S.D. = 3002±805. Unpaired two-tailed T test with Welch’s correction, p = 0.051. See Source Data Files. g Concept of DamID mapping of Las17- and Orc2-Dam fusions, derived from an existing sketch . h Wild-type (GA-1981) cells expressing Dam, Las17-Dam, or Orc2-Dam were treated ±300 μg/ml Zeocin for 1 h at 30 °C. Genomic DNA digested with DpnI (cleaves at G m ATC) or Sau3AI (cleaves GATC, methylation indifferent), was run on 1% agarose gels; stained with SYBR safe or rDNA by Southern blot (see Methods).

Article Snippet: Image acquisition of phalloidin stained and Cof1-RFP (Addgene #37102) expressing yeast cells, or cells treated with 0.25 mM IAA to induce Las17 degradation, as well as with 50 μg/ml Zeocin ± 0.5 μM CMB was carried out as described above .

Techniques: Expressing, Confocal Microscopy, Staining, Control, Two Tailed Test, Derivative Assay, Methylation, Southern Blot

Journal: Nature Communications

Article Title: Loss of cytoplasmic actin filaments raises nuclear actin levels to drive INO80C-dependent chromosome fragmentation

doi: 10.1038/s41467-024-54141-0

Figure Lengend Snippet: a Fraction of budded cells or total cells with F-actin filaments was determined after staining with Rh-phalloidin. Exponentially growing cells (GA-5731, AID control; GA-6840, Las17-AID; GA-1981, WT) were treated as indicated (±0.25 mM IAA for Las17-AID and its control, and 50 μg/ml Zeocin ± 0.5 μM CMB for WT), then fixed and stained with Rh-phalloidin. Image examples in Supplementary Fig. . Both brightfield and fluorescent images were scored for budding index and cells with filaments by three independent operators on blinded images. Number of cells scored per condition is shown in b . Plots show % budded cells with filaments (left) or % total cells with filaments (right). Data presented as mean value (top of bar) ±SEM. Raw numbers in Source Data file. b Budding index (bud presence) was scored for the same cell populations analyzed in a by three operators on blinded images. Total number cells are same for a , b . Plotting Budded cells with filament/Budded cells excludes cell cycle impact on results. c Yeast strains as a were transformed with a Cof1-RFP (internal tag, Addgene #37102) expression plasmid and treated as above. Cof1-RFP intensity monitors both G- and F-actin and was quantified for each condition with a CellProfiler pipeline. Each dot represents one cell and the number of cells scored range from 402 to 1731, as indicated within each bar. All data points are plotted and top of bar indicates median. By Wilcoxon rank sum test actin levels do not vary significantly ± IAA nor under YCS conditions. d Cells of the samples used in a were collected before fixation. Total protein extracts were subjected to SDS-PAGE and western blotting with validated anti-actin (My BioSource, BSS9231831) or anti-tubulin (Abcam, ab6161). e Scoring nuclear actin detected through Cof1-RFP (expressed as in c ) using nuclear masking based on DAPI staining (Fig. ) on WT and an isogenic strain lacking Las17 (LAS17-AID + IAA). Samples visualized as c . Bar = 10 μm. In graph of mean (bar = mean values) of n nuclei ( n = 19, WT; Mean = 0.8339 ± 0.0131; n = 20 Las17-AID; Mean = 0.1316 ± 0.0340). Based on a two-tailed T test p < 0.0001. Data in Data Source file.

Article Snippet: Image acquisition of phalloidin stained and Cof1-RFP (Addgene #37102) expressing yeast cells, or cells treated with 0.25 mM IAA to induce Las17 degradation, as well as with 50 μg/ml Zeocin ± 0.5 μM CMB was carried out as described above .

Techniques: Staining, Control, Transformation Assay, Expressing, Plasmid Preparation, SDS Page, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Loss of cytoplasmic actin filaments raises nuclear actin levels to drive INO80C-dependent chromosome fragmentation

doi: 10.1038/s41467-024-54141-0

Figure Lengend Snippet: a Position of act1-111 (red) and act1-129 (green) mutations on an actin monomer shown in two orientations. Filament formation is intact through pointed and barbed ends. b Actin cytoskeleton staining in act1-111 (GA-8592) ±pACT1. Exponentially growing cells were fixed with PFA, stained Rh-phalloidin for 2 h, and imaged. The phalloidin signal in act1-111 expressing p ACT1 resembles wild-type, while act1-111 has mostly cortical F-actin patches. Quantitation of actin cables was scored by two blinded operators on three isolates of the act1-111 mutant vs act1-111 + pACT1. n total +pACT1 = 1012, n total act1-111 = 2790; data are presented as mean value ±SEM, by two-tailed unpaired T test, p < 0.0001. Scoring is in Source Data file. Scale bar = 10 μm. c Actin levels are constant under YCS conditions in act1-111 strains. Whole protein extracts from strains grown as in b were analyzed by western for a nuclear pore protein (Mab414, Abcam) and actin (My BioSource). Quantitation reflects four blots scanned by the Typhoon scanner; Actin:Mab414 value on YCS is normalized to +pACT1. d The act1-111 mutant ( a ) is hypersensitive to Zeocin. 1:10 dilution series of act1-111 ± pACT1 on SC agar plus glucose with indicated Zeocin concentrations. Colonies grew 3 days at 30 °C; performed in triplicate with similar results. e Las17-GFP act1-111 cells (GA-8592) ± pACT1 plasmid are resistant to YCS. An isogenic wild-type background (GA-9247; BY background) and the act1-111 mutant were treated with 2 µM CMB and 150 µg/ml Zeocin for 1.5 h, and genomic DNA was monitored by CHEF gel analysis. Where indicated, 50 µM LatA was added rather than CMB. Gels and quantitation are as Fig. . B/A values show that act1-111 is resistant to CMB and LatA treatment. Experimental repeats ( n = 3) include panel f. f Wild-type and act1-111 (GA-8592) and act1-129 cells (GA-8590) ± pACT1 plasmid were treated 1.5 h with Zeocin (125 µg/ml), a titration of CMB (0.25–2.5 μM) or Zeocin and CMB as indicated. Genomic DNA was monitored by CHEF gel, stained, and quantified as Fig. . The act1-111 , but not act1-129 cells show less CMB-induced fragmentation. The experiment was repeated three times.

Article Snippet: Image acquisition of phalloidin stained and Cof1-RFP (Addgene #37102) expressing yeast cells, or cells treated with 0.25 mM IAA to induce Las17 degradation, as well as with 50 μg/ml Zeocin ± 0.5 μM CMB was carried out as described above .

Techniques: Staining, Expressing, Quantitation Assay, Mutagenesis, Two Tailed Test, Western Blot, Plasmid Preparation, Titration