Wednesday, April 2, 2025
4/2/2025
Determining the impact of cargo membrane fluidity on motor protein function - Project Summary/Abstract The goal of this renewal application is to determine the impact of the lipid membrane enclosing cellular cargos on the function of the major microtubule-based motor protein kinesin-1. Motor protein-based transport underlies all eukaryotic cell function and survival; understanding the mechanistic basis of motor protein regulation is critical for understanding this fundamental process of intracellular transport. Our central hypothesis is that the fluid nature of the cargo membrane is a key determinant of motor protein function. In cells, motor proteins are typically attached to cargos via a fluid lipid membrane. This membrane is “fluid” in that its constituents, including associated motor proteins, are mobile and diffuse on the cargo surface. Alterations in membrane fluidity are increasingly linked to aging and neurodegeneration, in which dysfunction in motor-based transport is a common early hallmark. Quantitative investigations of cargo membrane effects on motor function have remained limited, with most cargos in current in vitro assays lacking a physiological lipid membrane. Closing this major gap, in prior funding periods, the research team developed a robust optical-trapping assay to directly measure the transport of membrane-enclosed cargos in vitro. Using this assay, the research team demonstrated the first direct support for the central hypothesis of this project. Specifically, a fluid membrane enhances the productive binding of kinesin to microtubules; this enhancement is countered by cholesterol, which reduces membrane fluidity. These membrane effects were established in the presence of tau, an in vivo factor critical for microtubule polymerization and stabilization but that occludes kinesin binding sites on microtubules. Building on this recent work, in the next funding period the research team will test the central hypothesis by pursuing three independent but related aims. Aim 1 will leverage the recently developed assay to test the hypothesis that a reduction in membrane fluidity underlies the inhibitory effect of membrane cholesterol on kinesin binding in the presence of tau. Aim 2 will extend the assay and implement high temporal resolution detection to test the hypothesized membrane effect on kinesin binding in the absence of tau. Aim 3 will develop a stochastic simulation model to quantitatively test the hypothesis that cargo membrane fluidity determines kinesin-microtubule binding by impacting the diffusive search time of kinesin for open binding sites on the microtubule. Accomplishing the proposed project has the potential to establish cargo membrane fluidity as an unexplored physiological determinant of kinesin-microtubule binding, advancing scientific knowledge in the mechanistic basis of motor protein regulation, and providing a controlled experimental and computational platform for quantitative investigations of physiological determinants of motor protein function. Findings could pave the way for future therapeutics that target membrane properties to mitigate dysfunctions in motor protein-based transport early in the progression of diseases including neurodegeneration.
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