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. 2014 Jun;88(11):6329–6344. doi: 10.1128/JVI.03840-13

FIG 3.

FIG 3

Relocalization of Vps23 from the cytosol and late endosomes to mitochondria in BY-2 cells coexpressing CIRV p95 and/or p36. (A to C) Representative epifluorescence micrographs of BY-2 cells (co)transformed (as indicated by panel labels) with either full-length CIRV, Rep (both p95 and p36 together), p95, p36, Myc-Vps23, or GFP-Syp21, or mock transformed with empty plasmid DNA and then immunostained for the endogenous mitochondrial protein β-ATPase (top right-hand photo in panel A). All cells shown in Fig. 3 were formaldehyde fixed and processed for immunofluorescence microscopy as described in Materials and Methods. Note that p95 and p36 were immunodetected using primary antibodies raised against a synthetic peptide that corresponds to an amino acid sequence in both proteins. p95 is produced by the translational read-through of the p36 amber stop codon (8). Selected transformed cells were also immunostained (as indicated) for endogenous mitochondrial β-ATPase or CoxII. Also shown are the corresponding merge and differential interference contrast (DIC) images. The yellow color in the merged images indicates colocalization, and the white arrowheads in panel A indicate obvious examples of Myc-Vp23 and GFP-Syp21 colocalization at late endosomes. Bar = 10 μm. (D) Representative confocal micrographs of BY-2 cells cotransformed with either p36 and Myc-Vps23 (top and middle rows), or GFP-Syp21 and Myc-Vps23 (bottom row), which were immunostained for Myc-Vps23 and endogenous CoxII (GFP fluorescence is not shown). All images are medial (midcell) optical sections of cells, and boxes represent the portions of the cells shown at higher magnification in the panels on the right. The Pearson's correlation coefficient r values were as follows: r = 0.81 for p36 and Myc-Vps23, r = 0.71 for Myc-Vps23 and CoxII, and r = 0.21 for Myc-Vps23 and CoxII. (E to H) Representative epifluorescence micrographs of BY-2 cells cotransformed (as indicated) with either p36 and either N-terminal HA epitope- or GFP-tagged Vps23 (E); p36 and a Myc-tagged N. tabacum Vps23 (F), p36 and Myc-tagged Vps28 or Vps25 (G), or p36 and various organelle marker fusion proteins, including Cherry-PTS1 (peroxisome), GFP-Syp52 (early endosome/trans-Golgi network), GFP-Syp21 (late endosome), TIC40-RFP (plastid), PAP26-Cherry (lytic vacuole) (H). Bar = 10 μm.