ANALYZING MITOCHONDRIAL AGE AND MOTILITY IN DROSOPHILA MOTOR NEURONS USING EOS2 AND MITO-TIMER PROTEIN
Ana Caldaruse, Inna Djagaeva, Bill Saxton.
University of California Santa Cruz, Santa Cruz, CA.
The vast size and asymmetry of neurons has raised questions about how their energy requirements are met by the ATP-producing mitochondria. The majority of mitochondria proteins are encoded by the genomic DNA, synthesized on cytoplasmic ribosomes, and later imported into mitochondria. The classical mitochondria biogenesis hypothesis proposes that new mitochondria are made in the neuron cell body and must be transported great distances to reach energy requiring destinations in the axon. We will use 2 different approaches to test the classical hypothesis. First, we will use mitochondria-targeted photoconvertable fluorescent protein Eos2 that can be permanently converted with a 405 nm laser from green to red, which will help us uniquely mark and trace small groups of mitochondria in axons over prolonged time periods. Second, we will use a different fluorescent protein that spontaneously refolds and changes its emission spectrum from green to red over an ~12 h period and can serve as a mitochondrial fluorescent "timer" that allows us to distinguish the age of mitochondria. Both of these fluorescent proteins will be expressed in motor neurons and will be imaged in real time, using live Drosophila, to give us a better understanding of the behavior of mitochondria. Based on the classical hypothesis we predict that the converted mitochondria will move anterograde the closer they are to the cell body and retrograde/stationary further away. Alternatively, the timer mitochondria are red the older and further they are from the cell body, and green the younger and closer they are to the cell body.