Controlling cellular processes with light can help elucidate their underlying mechanisms. Here we present zapalog, a small-molecule dimerizer that undergoes photolysis when exposed to blue light. Zapalog dimerizes any two proteins tagged with the FKBP and DHFR domains until exposure to light causes its photolysis. Dimerization can be repeatedly restored with uncleaved zapalog. We implement this method to investigate mitochondrial motility and positioning in cultured neurons. Using zapalog, we tether mitochondria to constitutively active kinesin motors, forcing them down the axon towards microtubule (+) ends until their instantaneous release via blue light, which results in full restoration of their endogenous motility. We find that one-third of stationary mitochondria cannot be pulled away from their position and that these firmly anchored mitochondria preferentially localize to VGLUT1-positive presynapses. Furthermore, inhibition of actin polymerization with latrunculin A reduces this firmly anchored pool. On release from exogenous motors, mitochondria are preferentially recaptured at presynapses.
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The MATLAB code used to analyse the data shown in Fig. 1c(iii) is available for download at https://github.com/ewest11/FTA-Fluorophore-Translocation-Analysis.
The ImageJ macro ‘Kymolyzer’ is available upon request.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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We are grateful for plasmids shared with us by T. Inoue (Johns Hopkins University; Tom20–mCherry–FKBP), C. Hoogenraad (Utrecht University; PEX–mRFP–FKBP) and K. Verhey (U. Michigan; full-length Kif1a). We thank L. Mkhitaryan for technical support and the members of the T. L. Schwarz laboratory for fruitful discussions. We are grateful to S. Vasquez and M. Sahin’s laboratory for assistance with primary hippocampal cultures; D. Tom and L. Ding from Harvard NeuroDiscovery Center’s Enhanced Neuroimaging Core for assistance with live-cell imaging (NINDS P30 Core Center grant no. NS072030); and B. Sabatini (HMS) and G. Pekkurnaz (UCSD) for access to equipment. This research was generously supported by the National Institutes of Health grants R01 GM069808 (NIH/NIGMS) and R21 NS87582 (NIH/NINDS) to T.L.S. as well as a BCH Pilot Study Grant. A.G. was supported by F32 GM110984 (NIH/NIGMS) and T32 NS007484 (NIH/NINDS), and M.R.B. was supported by K99/R00 DA034648 (NIH/NIDA).
Integrated supplementary information
a, Liquid chromatography–mass spectrometry (LC-MS) of zapalog before and after photolysis (above- HPLC chromatogram, below- MS spectrogram of peaks found in HPLC). 1mM of zapalog in DMSO was analyzed by LC-MS and found to be highly pure, exhibiting a single peak at 11.96 min (compound A). Following exposure to 405 nm laser light, compound A is photocleaved into compounds B and C. The expected nitrosyl ketone C was observed along with another photoreaction product of putative structure D. b, Chromatograms of zapalog before and after exposure to 458 nm light. 1mM of zapalog in DMSO was exposed to 458 nm laser light on a confocal microscope (~100 µW for 1 min), then analyzed by HPLC. Zapalog was found to be insensitive to 458 nm light.
A description of each intermediate reaction is presented in Methods.
Supplementary Figure 3 Tethering constitutively active motors to axonal mitochondria with zapalog forces most, but not all, mitochondria towards axon tips.
a, Zapalog dimerizes constitutively active kinesin motor Kif1a(1–489)-DHFR-myc to mitochondria tagged with Tom20-mCherry-FKBP. Representative images of immunocytochemistry with an antibody directed against myc-tagged constitutively active kinesin motor Kif1a(1–489)-DHFR-Myc (green) in DIV10 E18 cultured hippocampal neurons treated for 5 min with 1.5 µM of either DMSO or zapalog, in the dark. Zapalog, but not DMSO, causes the exogenous motors to translocate to the outer surface of all mitochondria. (n = 7 axons). b, A representative kymograph of a section of distal axon of an E18 rat hippocampal neuron that had been transfected with mitochondrial Tom20-mCherry-FKBP and the constitutively active kinesin motor Kif1a(1–489)-DHFR-myc. 1. Endogenous motility consists of mitochondria that are either stationary or motile in the retrograde and anterograde directions. 2. Addition of zapalog induces attachment of motors to mitochondria and subsequently most, but not all mitochondria are dragged in the anterograde direction. 3. Exposure to 405 nm light causes the exogenous motors to detach completely from mitochondria, resulting in immediate stoppage of some dragged mitochondria and resumption of bidirectional motility of others. Representative traces of individual mitochondria that were found to be immovable by zapalog-tethered motors in this assay(red), stationary but movable (yellow) and motile (green). c, Tethering mitochondria to Kinesin motors with zapalog causes them to pile-up at growth cones. Kymograph and sample time-lapsed images of the growth cone of an E18 rat hippocampal neuron that had been transfected with mitochondrial Tom20-mCherry-FKBP and the constitutively active kinesin motor Kif1a(1–489)-DHFR-myc. Addition of zapalog induced attachment of the motors to axonal mitochondria, forced mitochondrial motility towards the (+) end of microtubules, and resulted in the accumulation of mitochondria at axonal growth cones. Photolysis of zapalog released the motors, preventing additional mitochondrial translocation, but the mitochondria piled up at the growth cone do not recover motility and move retrograde. When similar aggregations formed along axons, the aggregates did not disperse upon photolysis of zapalog and the affected axons often degenerated. d, Long term flux analysis of zapalog-mobilized mitochondria reveals depletion of mitochondria from a region of axon. (top) A micrograph depicting a field with two intersecting axons of E18 rat hippocampal neurons, traced in red and blue, that had been transfected with mitochondrial Tom20-mCherry-FKBP and the constitutively active kinesin motor Kif1a(1–489)-DHFR-Myc, in the presence of 1.5 µM zapalog. The arrows indicate the +-end directed flow of mitochondria driven by the Kif1a motor. (bottom) Simultaneous flux analysis of these axons demonstrates that forced mobilization of axonal mitochondria peaks at different times at 40–50 mitochondria per second and then subsequently decreases as movable mitochondria are eventually depleted from the upstream, proximal regions of axon, leaving only the anchored pool. Distal regions, and especially growth cones are correspondingly enriched, as in c. Source data are available online .
Supplementary Figure 4 Latrunculin A eliminates detectable filamentous actin but does not detectably alter axonal tubulin and VGLUT1.
Representative images of axons from DIV10 E18 cultured hippocampal neurons treated for 6 hrs with 2.5 μM of either DMSO or Latrunculin A. Axons were labeled with antibodies directed against VGLUT1 (red), neuron-specific class III β-tubulin (TUJ1, green), and F-actin was visualized by staining with Alexa Fluor 647 phalloidin (greyscale). (scale bar = 10 μm, n = 15 neurons each).
a, Representative micrograph from a time-lapsed Fluorophore Translocation Assay of a COS7 cell transfected with cytoplasmic YFP-DHFR-Myc (cyan) and mitochondrial Tom20-mCherry-FKBP (red). b, Each cell’s outline is thresholded using the cytoplasmic YFP-DHFR-Myc channel before zapalog addition. c, Each cell’s mitochondria are separately thresholded for every timepoint using the mitochondrial Tom20-mCherry-FKBP channel. d, At each timepoint, the mitochondrial fill (c) is subtracted from the total cell fill (b) to calculate the area of the cell that is only cytoplasmic and not mitochondrial. e, The ratio of mitochondrial/cytoplasmic YFP is plotted over time, from immediately following zapalog addition until after zapalog photolysis.
Supplementary Figure 6 Quantification of stationary mitochondria at presynaptic sites and generation of random positions along the axon for use as controls.
a, A representative example of how colocalization of stationary mitochondria and stationary VGLUT1 signals was scored. Stationary mitochondria in kymographs were marked with 5 pixel-wide vertical lines (yellow). The marked lines were then superimposed onto the VGLUT1 channel and scored for coincidence with the stationary VGLUT1-Venus signal. b, A representative example of random position generation along the axon. For each dataset we generated a set of random positions along the axon equal in number to the number of capture events recorded for that dataset. These were plotted as 5 pixel-wide lines (same width as lines drawn to score data in a), overlapped with the dataset and scored similar to the experimental data. To this end, using Apple Numbers, we generated a list of random numbers for each dataset, with each number valued between 1 and the pixel width of the corresponding dataset (in this case 1000 pixels); the length of each list was also set to equal the number of stationary mitochondria found in the corresponding dataset (table 1). We then matched the randomly generated numbers with cells on a 1–1000 table, assigned the corresponding row a value of 100 and plotted as a histogram with 1 pixel-wide bars. We also assigned a value of 100 to the 2 rows before and 2 rows after each random number, widening the bars to 5 pixels in length (table 2). Finally, we superimposed the histogram onto the corresponding kymograph and proceeded to score as shown in a.
Supplementary Figures 1–6, Supplementary Table and Video titles/legends.
Statistics source data.
Zapalog induced translocation of YFP from cytoplasm to mitochondria.
Selective removal of YFP from mitochondria with zapalog and targeted 405 nm light.
Repeated rounds of translocation and removal of YFP on and off of a mitochondrion with zapalog and targeted light.
Repeated rounds of translocation and removal of YFP on and off of peroxisomes with zapalog and targeted light.
Mitochondria are driven towards axon tips by zapalog-dependent tethering to Kif1a motors, then instantaneously released by light.
Mitochondria accumulate at the distal tip of an axon after being tethered to Kif1a motors by zapalog.
Zapalog-induced mobilization of axonal mitochondria.