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 .