Journal: Nature Communications

Article Title: Junction-based lamellipodia drive endothelial cell rearrangements in vivo via a VE-cadherin-F-actin based oscillatory cell-cell interaction

doi: 10.1038/s41467-018-05851-9

Figure Lengend Snippet: Distinct dynamics of VE-cadherin, F-actin and ZO1 during JBL formation. a , b Still images (Supplementary Movie ) of an embryo showing the DLAV around 32 hpf in an embryo expressing both mRuby2-UCHD and VE-cad-Venus Tg ( fli:Gal4ff ubs3 ;UAS:mRuby2-UCHD ubs20 ;BAC ( cdh5:cdh5-Venus )). b A time series magnification of the inset in a . Individual channels are shown in inversed contrast. Similar observations were made in 11 movies. Open arrow head points to established junctions and black arrowhead to pioneering junction. c and d ) Still images of an embryo showing DLAV around 32 hpf (Supplementary Movie ) in an embryo expressing EGFP-ZO1 and mRuby2-UCHD ( Tg(fli:Gal4ff ubs3 ;UAS:mRuby2-UCHD ubs20 ;UAS:EGFP-hZO1 ubs5 ) ). Imaged at rate of 12 s/stack. Similar observations were made in 9 movies. Open arrow head points to established junctions and black arrowhead to pioneering junction. e Images of endothelial cells in a VE-cad-Venus expressing embryo injected with mCherry-ZO1 encoding plasmid Tg(BAC(cdh5:cdh5-ts)); fli1ep:mCherry-ZO1)) ( n = 7 embryos). f Close-up from panel e . Both channels are shown in inverted contrast. Scale bars 1 µm ( b – d ) and 10 µm ( a , e )

Article Snippet: The EGFP sequence of pT24xnrUAS:EGFP-UCHD was replaced by the sequence of mRuby2 (amplified from pcDNA3-mRuby2 was a gift from Michael Lin; Addgene plasmid #40260) or by the sequence of mClav2 (amplified from pmClavGR2-NT; Allele Biotechnology) to generate the final plasmids pT24xnrUAS:mRuby2-UCHD and pT24xnrUAS:mClav2-UCHD respectively.

Techniques: Expressing, Injection, Plasmid Preparation

Journal: Nature Communications

Article Title: NUP62 localizes to ALS/FTLD pathological assemblies and contributes to TDP-43 insolubility

doi: 10.1038/s41467-022-31098-6

Figure Lengend Snippet: a HEK293 cells were co-transfected with mRuby-NUP62 and eGFP-TDP-43 (wild type). The cells were observed through live-scan confocal microscopy starting 3 h after transfection and images were obtained every 5 min over the course of 15 h. Two populations of cytoplasmic mRuby-NUP62 condensates were observed: reversible or irreversible. Reversible structures exhibit more dynamic activity and appear circular (see arrow). Irreversible structures appear less mobile or more static and have an angular structure (see asterisks). b Schematic depicting characteristics of cytoplasmic mRuby-NUP62 structures is shown at bottom. Representative still images were obtained from the 6–10 h time points of the imaging session. c Quantification of NUP62 area in confocal microscopy images obtained during live imaging (5–15 h timepoints) described in Fig. 5A, B. Irreversible condensates were significantly larger than reversible structures. The size of reversible granules was determined at times point immediately prior to dissipation. Irreversible granule area was calculated at final time point collected during live imaging session. n = 50 (reversible), 20 (irreversible) NUP62 + granules. Statistical significance was determined by two-tailed, unpaired student’s t-test. Data are shown as mean + /- SEM. d The percentage of reversible and irreversible mRuby-NUP62 granules containing eGFP-TDP-43 were calculated for each frame taken throughout the duration of living imaging session (5–15 h timepoints) described in Fig. 5a, b. A greater percentage of irreversible mRuby-NUP62 condensates contained eGFP-TDP43. n = 12 frames per group. Statistical significance was determined by two-tailed, unpaired student’s t-test. Data are shown as mean + /- SEM. e mRuby-NUP62 condensates were characterized for circularity score at the final time point of live image session (5–15 h timepoints) described in Fig. 5a, b. Irreversible mRuby-NUP62 + eGFP-TDP-43 + condensates ( n = 26 condensates) had a significantly reduced circularity score relative to eGFP-TDP-43 - ( n = 17 condensates) and reversible mRuby-NUP62 + eGFP-TDP-43 + condensates ( n = 12 condensates). Statistically differences were calculated by one-way ANOVA with Tukey post hoc analysis. Data are shown as mean + /- SEM. f Representative FRAP analysis images of nuclear eGFP-TDP-43 (reference solubility control) and cytoplasmic eGFP-TDP-43 and mRuby-NUP62 condensates. g Quantification of FRAP analysis shows reduced fluorescence signal recovery in cytoplasmic eGFP-TDP-43 and mRuby-NUP62 condensates relative to nuclear eGFP-TDP-43 control. Data are shown as mean + /- SD. h HEK293 cells were transfected with indicated plasmids for 24 h. Soluble and insoluble biochemical fractionation was then conducted, and Western blot analysis was performed to evaluate TDP-43 and GAPDH (protein loading control). mRuby-NUP62 promotes the formation of increased insoluble TDP-43. Representative western blot image is shown. i HEK293 cells were transfected with mRuby-NUP62 and eGFP-TDP-43 (WT or ΔNLS) for 24 h. Samples were then immunoprecipitated by ChromoTek GFP-Trap Magnetic Agarose affinity beads. Samples were then immunoblotted for NUP62 and TDP-43. * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001 vs control. Scale bar: 10 µm.

Article Snippet: NUP62 (Gift from Akiko Takedo) and mRuby (from Addgene Plasmid# 54614) fragments were PCR-amplified.

Techniques: Transfection, Confocal Microscopy, Activity Assay, Imaging, Two Tailed Test, Solubility, Control, Fluorescence, Fractionation, Western Blot, Immunoprecipitation