Online

Hoeprich, Gregory Joseph

Molecular Physiology & Biophysics

2016

Ph. D.

Neurons are specialized cells that transmit information through electrical and chemical signals using structural processes known as dendrites and axons. Dendrites receive information for the cell to interpret while the exceedingly long axon transmits the processed information to its target destination. To ensure the neuron properly carries out its extracellular functions, the orchestration of intracellular cargo (e.g. mitochondria) is critical. This is especially true in the axon, which can be up to a meter in length. There are many challenges involved in the spatial and temporal regulation of cargo over such vast cellular distances. In order to accomplish cargo transport between the cell body and axon terminus the neuron has developed an efficient process to overcome this challenge called axonal transport. Axonal transport utilizes a system of molecular motors coupled to cargo, creating a multi-motor complex, which walks along a set of tracks to position the cargo at the right time and place. One class of molecular motors, called kinesin, are used to traffic cargo away from the cell body and walk along microtubule tracks. These motors work in teams to navigate a complex microtubule landscape that is rich in microtubule-associated proteins (MAPs). One MAP abundantly found within the axon is called Tau and is implicated in a variety of neurodegenerative disorders (e.g. Alzheimer's disease). Much attention has been focused on the kinesin-1 motor while investigating the axonal transport process. However, kinesin-2 plays an equally important role and is not as well characterized as kinesin-1. Previously, it has been demonstrated, in vitro, that Tau disrupts kinesin-1 transport, even below physiological concentrations, however, in vivo evidence suggests the contrary. Given this discrepancy, there are likely other cellular systems in place to provide the necessary navigation of Tau obstacles. One solution may involve multi-motor complexes using two kinesin family members attached to cargo, as both kinesin-1 and kinesin-2 have been observed coupled to cargo. In order to peel away the complex layers of kinesin-1 and kinesin-2 coupled cargo inside the axon, single-molecule imaging techniques were employed to observe the individual behavior of both kinesin-1 and kinesin-2, in vitro. Further, using a combination of genetic engineering, single-molecule analysis and mathematical modeling has helped elucidate differences between these two motors. Kinesin-2 was found to be insensitive to Tau obstacles, unlike kinesin-1, which is in part due to a longer region of the motor called the neck-linker. This region connects the motor domain, which interfaces with the microtubule track, to the coiled-coil stock, which interfaces with the cargo. When the neck-linker lengths were swapped between the motors their behavior in the presence of Tau also switched, and kinesin-2 became sensitive to Tau. To understand kinesin-2's mechanism of navigating Tau obstacles, we looked at the lateral stepping characteristics of both motors. We observed kinesin-2's lateral stepping frequency to be 2-4 fold higher than kinesin-1 and independent of the microtubule obstacle concentration. Thus, kinein-2's longer neck-linker allows a more agile walk along the microtubule surface to navigate obstacles more efficiently than kinesin-1. In a multi-motor complex containing both motors, kinesin-2 is more efficient at maneuvering around MAPs while kinesin-1, which has previously been demonstrated to sustain a higher stall force, is more efficient at towing cargo. This work demonstrates how teams of directionally similar motors may work together to position cargo during axonal transport.