UC San Diego

Movement is intrinsic to life. Most forms of directed nanoscopic, microscopic and ultimately, macroscopic movement in cells is powered by tiny protein machines known as molecular motors. Microtubule-based motor proteins from the kinesin and dynein superfamilies are essential for many of these transport processes and coordinate to distribute various cellular cargos, including vesicles, organelles, protein complexes, and mRNAs to appropriate destinations within the cell. Due to the extreme compartmentalization of neurons, long range transport is particularly critical and recent advances suggests that these transport systems may fail early in the pathogenesis of a number of neurodegenerative diseases. Although substantial progress has been made to understand the underlying fundamentals, how these two opposite polarity motor protein families cooperate to generate coordinated bidirectional movement is poorly understood. This work reveals a number of novel features of coordinated axonal transport through robust particle tracking and quantitative immunofluorescence for two medically relevant classes of cargos; amyloid precursor protein (APP) and cellular prion protein (PrPC) vesicles. We developed a quantitative approach to analyze the axonal transport of YFP-tagged APP vesicles in Drosophila segmental nerves using heterozygous animals. This allowed us to assess the contribution of individual motor subunits and accessory proteins to coordinated axonal transport. Our approach yielded a novel model for how motor proteins work together to achieve bi-directional transport. We subsequently propose a robust image analysis method to assess relative motor subunit composition of individual endogenous APP vesicles in mouse hippocampal culture. Our data provides new insight on how select motor subunit and cargo attachment protein levels contribute to the overall architecture of these vesicles. Finally, we characterize the intracellular transport and steady-state motor subunit composition of mammalian PrPC vesicles. We suggest a coordination model wherein PrPC vesicles maintain a stable population of associated motors whose activity is modulated by regulatory factors instead of by structural changes to motor-cargo associations. Since disruption of this transport machinery has been implicated in numerous neurodegenerative diseases, such as Alzheimer's, Parkinson's, and ALS, elucidating the underlying fundamentals of this highly coordinated system is an essential part of understanding what happens during the progression of these devastating illnesses