Journal: Nature Communications

Article Title: Single-particle imaging of stress-promoters induction reveals the interplay between MAPK signaling, chromatin and transcription factors

doi: 10.1038/s41467-020-16943-w

Figure Lengend Snippet: a Schematics of the transcriptional response induced by the MAPK Hog1 upon osmotic stress. Under normal growth conditions, the genomic locus is repressed by histones set in place by the Ino80 complex and Asf1/Rtt109. In addition, H3K4 methylated histones mediated by Set1 contribute to the further repression of the locus (upper panel). When Hog1 is active (lower panel), it accumulates in the nucleus with the transcription factors Msn2/4. Hog1 binds to the transcription factors Hot1 and Sko1, allowing the remodeling of the chromatin by Rpd3 and the SAGA complex. The polymerases can be recruited to the locus and the RSC and SWR complexes evict nucleosomes on the ORF. b Construction of the transcriptional reporter. The promoter of interest (p PROM ) is cloned in front of 24 stem-loops ( 24xPP7sl ). This construct is transformed in yeast and integrated in the GLT1 locus 5′UTR, replacing the endogenous promoter. Upon induction of the promoter of interest, the mRNA stem-loops are transcribed and recognized by the fluorescently-tagged PP7 phage coat proteins. c Maximum intensity projections of Z-stacks of microscopy images from the p STL1 -PP7sl reporter system in a 0.2 M NaCl osmotic stress time-lapse experiment. The appearance of bright foci (arrow heads) in the nucleus of the cells denotes the active transcription arising from the promoter. Scale bar represents 5 µm. Representative images from at least three biological replicates. d Dynamics of the p STL1 -PP7sl transcription site intensity (20 brightest pixels in the nucleus minus the median fluorescence of the cell) following hyperosmotic stress. The mean of the population is represented by the solid line. The shaded areas represent the s.e.m. Number of cells for each trace: 0.0 M: 313; 0.1 M: 404; 0.2 M: 229; 0.3 M: 201. e Analysis of one representative single-cell trace. The raw trace (gray) is smoothed with a moving average (dark blue) and normalized by subtracting the intensity of the first time point after the stimulus. Multiple quantitative values can be extracted from this trace (see Methods). Source data are provided for d .

Article Snippet: The PP7 stem-loops plasmids are based on the previously published p POL1 24xPP7sl integrative plasmid (Addgene #35196).

Techniques: Methylation, Clone Assay, Construct, Transformation Assay, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Maintenance of neuronal TDP-43 expression requires axonal lysosome transport

doi: 10.1101/2024.09.30.615241

Figure Lengend Snippet: A) Quantification of BORC KD TDP-43 immunofluorescence microscopy. Both BORC genes tested (S2 and S6) show significant decreases in untagged TDP-43 levels compared to a NT guide, indicated by blue dots. N=4 wells per genotype, 9 images per well (small gray dots). Significant p-values indicated on graph. B) Representative images of TDP-43 immunofluorescence imaging with BORCS6 KD. sgRNA plasmids contain a cytoplasmic BFP, enabling identification of cells expressing the sgRNA. Scale bar represents 20 µm. C) Schematic of pLentiCRISPR plasmid. pLentiCRISPR enables lentiviral delivery of a plasmid expressing an sgRNA of interest under a U6 promoter and Cas9 under an Ef1alpha promoter. This enables knockout of a gene of interest targeted by an sgRNA. D) Representative images of BORCS7 KO TDP-43 IF with lysosomes stained with anti-H4A3 antibody. Scale bar is 20 µm. E) Quantification of TDP-43 immunofluorescence on BORCS7 knockout neurons. Both BORCS7 guides significantly reduced the amount of TDP-43 IF signal compared to a NT guide, light blue dots. N=10 wells, 9 images per well (light gray dots). Significant p-values indicated on graph.

Article Snippet: TDP-43 coding sequence and 3’UTR were cloned into a plasmid expressing 24 SunTag repeats under an Ef1alpha promoter followed by 24 PP7 stem loops (modified from Addgene 74928).

Techniques: Immunofluorescence, Microscopy, Imaging, Expressing, Plasmid Preparation, Knock-Out, Staining