Journal: bioRxiv
Article Title: Live-cell imaging reveals nutrient-dependent dynamics of ER-mitochondria contact formation via PDZD8
doi: 10.1101/2025.04.17.649323
Figure Lengend Snippet: (A) Representative images from time-lapse imaging shown as Supplementary movie 1. Images were obtained at 0.5 Hz for 32 seconds (17 frames). MERCdRED-positive areas on mitochondria were shown in magenta, and Tomm20-iRFP (cyan) was used as a mitochondrial marker. Yellow arrowheads indicate newly emerging MERCdRED signals that appeared in the subsequent time frame relative to the previous representative frame. Data are representative of 20 cells from three independent experiments. (B) Representative tracking images of MERCdRED-positive puncta shown in Supplementary movie 1. Tracking images were generated using TrackMate (ImageJ plugin) across the images obtained in (A). The colors of trajectories indicate the speeds of MERCdRED-positive puncta (shown in white). (C) Relationship between the area and the speed of each MERCdRED punctum. The average speed and area of each punctum over the entire observation period were calculated from tracking images obtained in (B). (D) The average speed of small puncta (area < 0.05 μm 2 ; left side of the dotted line in (C)) and large puncta (area > 0.05 μm 2 ; right side of the dotted line in (C)) was calculated. Data are means ± s.e.m. of 762 small puncta and 300 large puncta in 20 cells from three independent experiments. Statistical analysis was performed using the two-tailed Mann-Whitney test. (E) Schematic representation depicting the nutritional starvation in the MERCdRED cells. The cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin in fed condition, whereas they were incubated in EBSS for 5 hours in starved condition. (F) Representative images from live-cell imaging of MERCdRED cells in the fed or starved condition. GFP-Sec61β (yellow) or Tomm20-iRFP (cyan) was used as an ER marker or a mitochondrial marker, respectively. The boxed regions of the top panels are shown at higher magnification in the corresponding lower panels. (G) Quantification of the mitochondrial area in images obtained as described (F). The data are presented as individual points on box plots, with the center indicating the median, and the 25th and 75th percentiles represented by the box. Whiskers extend to the minimum and maximum values. n = 14, 13, 12, 12 cells for fed + control, starved + control, fed + Pdzd8 cKO, and starved + Pdzd8 cKO, respectively, from two independent experiments. Statistical analysis was performed using Tukey’s multiple comparisons test. n.s., not significant. (H) Quantification of the MERCdRED intensity on mitochondria in images obtained as described (F). The data are presented as individual points on box plots, with the center indicating the median, and the 25th and 75th percentiles represented by the box. Whiskers extend to the minimum and maximum values. n = 14, 13, 12, 12 cells for fed + control, starved + control, fed + Pdzd8 cKO, and starved + Pdzd8 cKO, respectively, from two independent experiments. Statistical analysis was performed using Tukey’s multiple comparisons test. ****P < 0.0001, ***P < 0.001, **P < 0.01.
Article Snippet: The RA-Sec61β sequence was amplified by PCR from RA-NES (Addgene, catalog no. #61019) and pAc-GFP-Sec61β (Addgene, catalog no. #62008) with the pair of primers 5’-cga gct caa gct tcg aat tca tgg tga gca aga gcg agg a -3’ and 5’-tct gag gct agc ctt gta cag ctc gtc cat gc -3’, 5’-caa ggc tag cct cag atc tat gcc tgg tcc -3’ and 5’-gga gtg aat tgc ggc cgc cta cga acg agt gta ctt gcc c -3’, respectively, and subcloned into the EcoRI and NotI sites of pCIG vector with In-Fusion HD cloning kit, resulting in pCAG-RA-Sec61β.
Techniques: Imaging, Marker, Generated, Two Tailed Test, MANN-WHITNEY, Incubation, Live Cell Imaging, Control
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