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Principal Investigator: William Olaf Hancock
Organization: PENNSYLVANIA STATE UNIVERSITY, THE
Fiscal Year: 2020
Award: $318,780
Funding agency: National Institute of General Medical Sciences
PROJECT SUMMARY
Intracellular cargo transport along microtubules is driven by kinesin and dynein motors that work
antagonistically to precisely target cargo to specific cellular locations. Although the mechanisms
underlying bidirectional transport have received considerable attention, a number of very fundamental
questions remain, and a better quantitative understanding of molecular interactions and
mechanochemistry underlying the regulation of bidirectional transport is essential for the field to move
forward. In this work we will use DNA origami to create complexes with defined numbers of kinesin and
dynein motors in vitro, and use Interfoerometric Scattering (iSCAT) microscopy to track them with 1 ms
and 2 nm resolution. In parallel, we will model the transport using Brownian dynamics simulations to
uncover the molecular-level details underlying the experimentally observed behavior. Our goal is to
understand how the contrasting single-motor properties kinesin-1, -2, and -3 family motors manifest
themselves in multi-motor teams, how roadblocks on microtubules alter bidirectional transport, and what
role membrane fluidity plays in altering multi-motor transport properties. Aim 1 focuses on analyzing the
movement of cargo transported by teams of similar and dissimilar kinesin motors to understand how
differences in motor attachment rates and load-dependent detachment alter multi-motor behavior. Aim 2
will extend this to studying cargo functionalized with kinesin and activated mammalian cytoplasmic
dynein complexes containing dynein, dynactin, and the activator BicD2. The switching behavior of
different kinesins against their natural opponent will be of particular interest and will require high-
resolution tracking in conjunction with experimentally constrained models. In Aim 3 we will attach motors
to supported lipid bilayers and vesicles, measure their transport dynamics, and computationally model
the underlying motor diffusion and bilayer deformation. We hypothesize that by acting as a “shock
absorber”, the fluid bilayer will reduce inter-motor forces, and that motor diffusion in the bilayer will lead
to clustering that enhances motor performance in manner similar to integrins in focal adhesions.
Although bidirectional transport can be described as a “tug-of-war” between kinesin and dynein
pulling in opposite directions, this model fails to account for a large body of experimental observations.
This gulf between molecular-level understanding of motors in vitro and observed transport of cargo in
cells must be bridged to understand the role of tau tangles in Alzheimers' disease, the deterioration of
axonal transport in Huntington's and Parkinson's disease, and other neurodegenerative disorders that
involve defects in microtubule-based bidirectional transport.
Terms: <AD dementia><Adhesion Plaques><Alzheimer><Alzheimer Type Dementia><Alzheimer disease><Alzheimer sclerosis><Alzheimer syndrome><Alzheimer's><Alzheimer's Disease><Alzheimer's disease dementia><Alzheimers Dementia><Alzheimers disease><Amyotrophic Lateral Sclerosis><Amyotrophic Lateral Sclerosis Motor Neuron Disease><Assay><Attention><Axonal Transport><Axoplasmic Transport><Behavior><Bilayer Fluidity><Binding><Bioassay><Biologic Assays><Biological Assay><Cell Body><Cell-Matrix Adherens Junctions><Cells><Chemicals><Circulatory Collapse><Complex><Computer Models><Computerized Models><Coupling><DNA><Data><Defect><Degenerative Neurologic Diseases><Degenerative Neurologic Disorders><Deoxyribonucleic Acid><Deterioration><Diffusion><Disease><Disorder><Dynein><Dynein ATPase><Dynein Adenosine Triphosphatase><Dynein Adenosinetriphosphatase><Family><Focal Adhesions><Focal Contacts><Gehrig's Disease><Geometry><Goals><Human><Huntington Chorea><Huntington Disease><Huntington's><Huntington's Disease><Huntington's Disease Pathway><Huntingtons Disease><In Vitro><Integrins><Integrins Extracellular Matrix><Kinesin><Link><Lipid Bilayers><Liquid substance><Location><Locomotor Activity><Lou Gehrig Disease><MT-bound tau><Measures><Mechanics><Membrane Fluidity><Membrane Transport><Micro-tubule><Microscopy><Microtubule-Associated Proteins><Microtubules><Modeling><Modern Man><Molecular><Molecular Interaction><Molecular Motors><Motor><Motor Activity><Movement><Nerve Cells><Nerve Unit><Nervous System Degenerative Diseases><Neural Cell><Neural Degenerative Diseases><Neural degenerative Disorders><Neurocyte><Neurodegenerative Diseases><Neurodegenerative Disorders><Neurofibrillary Tangles><Neurologic Degenerative Conditions><Neurons><Organelles><Organism><Paralysis Agitans><Parkinson><Parkinson Disease><Parkinson's disease><Parkinsons disease><Performance><Play><Post-Translational Modification Protein/Amino Acid Biochemistry><Post-Translational Modifications><Post-Translational Protein Modification><Post-Translational Protein Processing><Posttranslational Modifications><Posttranslational Protein Processing><Primary Parkinsonism><Primary Senile Degenerative Dementia><Property><Protein Modification><Regulation><Regulatory Protein><Resolution><Role><Shock><Testing><Transmembrane Transport><Tubulin><Vesicle><War><Work><Yeasts><anterograde transport><artificial vesicle><base><body movement><circulatory shock><computational modeling><computational models><computer based models><computer based prediction><computerized modeling><degenerative diseases of motor and sensory neurons><degenerative neurological diseases><dementia of the Alzheimer type><density><dynactin><dynein activator protein><experiment><experimental research><experimental study><fluid><genetic regulatory protein><gold nano particle><gold nanoparticle><interest><lipid bilayer membrane><liquid><living system><mechanical><mechanical force><mechanical properties><microtubule bound tau><microtubule-bound tau><molecular scale><motor behavior><movement analysis><nano gold><nano particle><nano-sized particle><nanoGold><nanoparticle><nanosized particle><neurodegenerative illness><neurofibrillary degeneration><neurofibrillary lesion><neurofibrillary pathology><neuronal><particle><prediction model><predictive modeling><primary degenerative dementia><prognostic model><reconstruction><regulatory gene product><retrograde transport><scaffold><scaffolding><senile dementia of the Alzheimer type><simulation><social role><tangle><tau><tau Proteins><tau factor><τ Proteins>