Journal: Nature cell biology
Article Title: The light-sensitive dimerizer zapalog reveals distinct modes of immobilization for axonal mitochondria.
Figure Lengend Snippet: Zapalog, a photocleavable heterodimerizer, can be used to reversibly translocate cytosolic YFP to mitochondria. a, Schematic illustration of zapalog function: 1. Two proteins of interest are tagged with DHFR and FKBP domains, 2. Addition of zapalog induces dimerization of the tagged proteins, 3. 405nm light photocleaves zapalog, causing rapid dissociation of the dimer, 4. Addition of uncleaved zapalog outcompetes photolyzed zapalog moieties, reestablishing dimerization. b, Chemical structure of zapalog before and after photolysis of the DANB moiety by 405nm light. Bottom - Schematic illustration of zapalog-induced translocation of YFP: zapalog attaches cytoplasmic YFP-DHFR-Myc to FKBP domains tethered to mitochondrial outer membranes. Exposure to 405nm light photocleaves zapalog, releasing the YFP-DHFR-Myc back to the cytoplasm. c, Time-lapsed imaging demonstrates full translocation of YFP-DHFR-Myc onto mitochondria in a COS7 cell, within ~1m after addition of 10μM zapalog to the medium. c’, Quantification of multiple experiments as in (c) using 10μM zapalog. The ratio of mitochondrial to cytoplasmic YFP-DHFR-Myc was derived from automated image analyses of the datasets, normalized, and average YFP intensity was calculated for each timepoint (n= 5 cells / 3 independent repeats, center value = mean, error bars represent SE, source data in Sup. Table 1 ) c”, From assays of YFP translocation to mitochondria, quantified as in (c’), a dose-response curve was generated by plotting time from 10% to 90% of full YFP-DHFR-Myc translocation for each concentration of zapalog administered. (n=98 cells, 6 independent repeats, center value = mean, error bars represent SD, source data in Sup. Table 1 ) d, Prior to the series of images shown, YFP-DHFR-Myc translocation to mitochondria was induced by zapalog, as in (c). After wash-out of free zapalog from HeLa cells, the YFP reporter was released from the mitochondria sequentially in each of the 3 cells by exposing the circled region to a brief (500ms) pulse of 405nm light. d’, Quantification of data shown in (d), demonstrating that photolysis of zapalog is rapid, complete, and spatially localizable. scale bars = 5µm
Article Snippet: Repeated, localized fluorophore-translocation assay on non-networked mitochondria – 18hrs after HeLa cells were transfected with Tom20-mCherry-FKBP and YFP-DHFR-Myc, they were incubated for 6hrs in 10µM CCCP.
Techniques: Translocation Assay, Imaging, Derivative Assay, Generated, Concentration Assay