Trak adaptors regulate the recruitment and activation of dynein and kinesin in mitochondrial transport.

Abstract

Mitochondria are cellular power plants that generate ATP and buffer cytosolic Ca2+. In neurons, mitochondria are distributed along microtubules where energy and Ca2+ is necessary and damaged mitochondria are transported back to the cell body for degradation. Deficiencies in mitochondrial transport are associated with neurodegenerative disorders. Mitochondrial transport is mediated by mitochondrial Rho GTPases Miro1 and TRAK adaptors that recruit kinesin-1 and dynein-dynactin. To understand how these opposing motors are regulated during mitochondrial transport, we reconstituted the bidirectional transport of Miro1/TRAK along microtubules in vitro.Motor proteins kinesin-1 and dynein, cargo adaptors TRAK1/2, MIRO1, and SNPH were recombinantly expressed and fluorescently labeled after purification. Using in vitro reconstitution, the velocity, run length, and landing rate of the motor proteins in the presence of TRAK1/2 were analyzed. In vitro pull-down assays revealed the interactions between motor proteins and TRAK. Force generation of motor-cargo adaptor complexes was measured by optical trapping. We show that the coiled-coil domain of TRAK activates dynein-dynactin and enhances the motility of kinesin-1 activated by its cofactor MAP7. TRAK adaptors that recruit both motors moved towards kinesin-1's direction, whereas kinesin-1 was excluded from binding TRAK transported by dynein-dynactin, demonstrating that TRAK coordinates the activity of these opposing motors to avoid tug-of-war. Using the reconstitution assay, we also tested whether disruption of the transport machinery or passive anchoring to microtubules stalls mitochondrial transport in regions with elevated Ca2+. Transport of Miro1/TRAK by kinesin-1 was not affected by Ca2+. Instead, the microtubule docking protein syntaphilin induced resistive forces that stall kinesin-1 and dynein-driven motility in vitro, supporting the model that mitochondrial transport stalls by Ca2+-mediated recruitment of syntaphilin to the mitochondrial membrane.