Endogenous GSK-3/shaggy regulates bidirectional axonal transport of the amyloid precursor protein.

Neurons rely on microtubule (MT) motor proteins such as kinesin-1 and dynein to transport essential cargos between the cell body and axon terminus. Defective axonal transport causes abnormal axonal cargo accumulations and is connected to neurodegenerative diseases, including Alzheimer's disease (AD). Glycogen synthase kinase 3 (GSK-3) has been proposed to be a central player in AD and to regulate axonal transport by the MT motor protein kinesin-1. Using genetic, biochemical and biophysical approaches in Drosophila melanogaster, we find that endogenous GSK-3 is a required negative regulator of both kinesin-1-mediated and dynein-mediated axonal transport of the amyloid precursor protein (APP), a key contributor to AD pathology. GSK-3 also regulates transport of an unrelated cargo, embryonic lipid droplets. By measuring the forces motors generate in vivo, we find that GSK-3 regulates transport by altering the activity of kinesin-1 motors but not their binding to the cargo. These findings reveal a new relationship between GSK-3 and APP, and demonstrate that endogenous GSK-3 is an essential in vivo regulator of bidirectional APP transport in axons and lipid droplets in embryos. Furthermore, they point to a new regulatory mechanism in which GSK-3 controls the number of active motors that are moving a cargo.

Measuring molecular motor forces in vivo: implications for tug-of-war models of bidirectional transport.

Molecular motor proteins use the energy released from ATP hydrolysis to generate force and haul cargoes along cytoskeletal filaments. Thus, measuring the force motors generate amounts to directly probing their function. We report on optical trapping methodology capable of making precise in vivo stall-force measurements of individual cargoes hauled by molecular motors in their native environment. Despite routine measurement of motor forces in vitro, performing and calibrating such measurements in vivo has been challenging. We describe the methodology recently developed to overcome these difficulties, and used to measure stall forces of both kinesin-1 and cytoplasmic dynein-driven lipid droplets in Drosophila embryos. Critically, by measuring the cargo dynamics in the optical trap, we find that there is memory: it is more likely for a cargo to resume motion in the same direction-rather than reverse direction-after the motors transporting it detach from the microtubule under the force of the optical trap. This suggests that only motors of one polarity are active on the cargo at any instant in time and is not consistent with the tug-of-war models of bidirectional transport where both polarity motors can bind the microtubules at all times. We further use the optical trap to measure in vivo the detachment rates from microtubules of kinesin-1 and dynein-driven lipid droplets. Unlike what is commonly assumed, we find that dynein's but not kinesin's detachment time in vivo increases with opposing load. This suggests that dynein's interaction with microtubules behaves like a catch bond.