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This behavior was seen in unpublished observations by P. L. Beech on live cells of M. splendens labeled with Mitotrackers Green FM or CMXRos (Molecular Probes) according to the manufacturer's instructions.
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65) isoform. S. cerevisiae cells were transformed with the MsFtsZ-mt/GFP fusion as described [R. George et al., Proc. Natl. Acad. Sci. U.S.A. 95, 2296 (1998)]. Mitochondria were isolated and subfractionated as previously described [B. S. Glick et al., Cell 69, 809 (1992)].
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Cells were examined on a Leica TCS 4D laser scanning confocal microscope under a ×100 1.3 numerical aperture objective. Signals from green (GFP or fluorescein isothiocyanate) and red (Mitotracker) channels were collected simultaneously and merged with Adobe Photoshop.
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note
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For immunofluorescence, a nonsynchronously dividing culture of M. splendens was labeled with 100 nM MitoTracker CMX-Ros (Molecular Probes) for 2 hours; cells were washed in growth medium, then fixed in 2% paraformaldehyde in methanol at -20°C for 10 min, washed in phosphate-buffered saline (PBS), and labeled with primary antibody diluted in blocking buffer (PBS, 0.05% Tween-20, and 1% bovine serum albumin) overnight at 4°C. Primary antibodies were detected with FITC-conjugated goat anti-rabbit IgG (Selenius, Hawthorn, Victoria, Australia), and cells were observed as described (17). Seemingly identical labeling with anti-MsFtsZ-mt was achieved both with and without MitoTracker labeling. Controls for antibody labeling were performed with preimmune sera.
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note
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We thank W. Margolin for the S. meliloti FtsZ probe, C. Cobbett for Arabidopsis RNA, J. Pickett-Heaps and R. Wetherbee for materials and encouragement, and T. Spurck for help with confocal microscopy. P.L.B. is funded by the Australian Research Council and is a Queen Elizabeth II fellow.
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