Journal: Nucleic Acids Research

Article Title: Mutational analysis of the F plasmid partitioning protein ParA reveals residues required for oligomerization and plasmid maintenance

doi: 10.1093/nar/gkaf537

Figure Lengend Snippet: Residues Q351 and W362 influence nsDNA binding by ParA F . ( A ) Filaments formed by ParA F Q351H–GFP and ParA F W362E–GFP are not nucleoid associated. The ParA F Q351H–GFP and ParA F W362E–GFP filaments appeared to be localized in the space between the nucleoid and the inner membrane, while ParA F –GFP localized over the nucleoids. Nucleoids were stained with DAPI, and the plasma membrane was stained using FM4-64. Scale bars represent 3 μm. ( B ) Super-resolution imaging using 3D-SIM showing that the filaments assembled by (i) ParA F Q351H–GFP and (ii) ParA F W362E–GFP are not associated with the nucleoid. DNA was stained with DAPI and pseudo-coloured red. Cell outlines were obtained from the phase contrast images. Scale bars represent 1 μm. ( C ) Filaments formed by ParA F Q351H–GFP and ParA F W362E–GFP are found in the nucleoid-free spaces in the cytoplasm of E. coli . WT ParA F –GFP localized to the nucleoid. Nucleoids were visualized by staining with DAPI and pseudo-coloured red in the images. Cells were treated with 25 μg/ml cephalexin for 30 min to prevent cell division and 100 μg/ml chloramphenicol for 10 min to condense the nucleoids so that the nucleoid-free space appears pronounced. Scale bars represent 3 μm. Expression of ParA F –GFP or its variants (from pDSW210) for all the above experiments was achieved by adding 100 μM IPTG to the cultures.

Article Snippet: 3D structured illumination microscopy (3D-SIM) imaging was carried out using a DeltaVision OMX-SR Blaze microscope equipped with a PCO Edge 4.2 sCMOS camera, and a ×60, NA 1.42 oil-immersion objective lens was used to acquire raw images.

Techniques: Binding Assay, Membrane, Staining, Clinical Proteomics, Imaging, Expressing

Journal: Nucleic Acids Research

Article Title: RPA interacts with Rad52 to promote meiotic crossover and noncrossover recombination

doi: 10.1093/nar/gkae083

Figure Lengend Snippet: Localization of RPA-single stranded DNA in WT, rfa1-t11 , rad52* and rfa1-t11 rad52* cells. ( A ) Immunofluorescence analysis of a meiotic nuclear spread of WT, rfa1-t11 , rad52* and rfa1-t11 rad52* . The cells were stained for anti-Rfa1 (Red) and anti-HA (Green). The scale bar represents 2.5 μm. (i–iv) The boxed regions at the bottom of each panel are magnified images. The scale bar of magnified images represents 1 μm. ( B ) Representative structured illumination microscopy (SIM) images of WT (4 h), rfa1-t11 (4 h), rad52* (5 h) and rfa1-t11 rad52* (6 h) mutant chromosomes. The cells were stained for Rec8 (Green) and Rfa1 (Red). The scale bar represents 2.5 μm. Enlarged images of the boxed regions are shown to the right of each panel. ( C ) Immunofluorescence analysis of a meiotic nuclear spread in zip1Δ , zip1Δ rfa1-t11 , zip1Δ rad52* and zip1Δ rfa1-t11 rad52* . The cells were stained for Rec8 (Green) and Rfa1 (Red). ( D ) Number of RPA foci per nucleus in the WT, spo11-Y135F , rfa1-t11 , rad52* and rfa1-t11 rad52* strains. Data are presented as the mean ± SD ( N ≥ 20).

Article Snippet: Super-resolution images of chromosome spread were acquired using a Nikon Eclipse Structured Illumination Microscopy (SIM) imaging system equipped with an EM CCD camera iXon897 (100× Plan Apochromat lens, 1.49 NA).

Techniques: Immunofluorescence, Staining, Microscopy, Mutagenesis

Journal: Scientific Reports

Article Title: The autophagy inducer SMER28 attenuates microtubule dynamics mediating neuroprotection

doi: 10.1038/s41598-022-20563-3

Figure Lengend Snippet: SMER28 treatment stabilizes microtubules by α-tubulin acetylation. ( a ) Western Blot analysis of U-2 OS cells treated with 50 and 200 µM of SMER28, 150 nM epothilone B (EpoB), 150 nM paclitaxel (Pacl) or 300 nM rapamycin (Rapa), as indicated, for 4 h. Lysates were analyzed for the protein levels of acetylated (ac)-α-tubulin. GAPDH served as loading control. ( b ) Quantification of relative ratios of ac-α-tubulin to GAPDH as indicated. Data show means of protein levels derived from six independent experiments ± s.e.m. *P < 0.05, ***P < 0.001, Mann–Whitney rank sum test. ( c ) U-2 OS cells were treated with DMSO as vehicle control, 50 µM or 200 µM SMER28, 150 nM epothilone B, 150 nM paclitaxel, 300 nM rapamycin or HBSS, as indicated, for 4 h. Cells were stained for ac-α-tubulin and visualized by SIM. Images show maximum intensity projections. ( d ) 3D object measurement tool of NIS Elements (Nikon) was used to measure the ac-tubulin filament volume of U-2 OS cells treated as indicated. SIM image stacks were used for quantification. Data show means of ac-tubulin filament volume in µm 3 from at least ten cells ± s.e.m. *P < 0.05, **P < 0.01, ****P < 0.0001, one way ANOVA.

Article Snippet: For SIM (3D structured illumination microscopy) images shown in Figs. and , an N-SIM E (Nikon) was used, built on a Ti-Eclipse microscope (Nikon).

Techniques: Western Blot, Derivative Assay, MANN-WHITNEY, Staining