Abstract Molecular motor-based transport is crucial for cell function, and defects are linked to diseases including neurodegeneration. Through sophisticated technologies such as optical trapping, single-molecule biophysical studies have revealed a great deal about the function of individual motors in vitro. However, in vivo, cargos are often membrane-bound and moved by small groups of molecular motors, a scenario that is not captured by classical in vitro studies. The presence of a fluid cargo membrane is an important consideration for multiple-motor transport, since it enables the motors to cluster near the microtubule and relaxes the force-based negative interference between motors. The proposed work constitutes the first test of the central hypothesis that the presence of a fluid membrane around a cargo critically alters the sensitivity/response of multiple-motor transport to single-motor modifications. The experimental strategy proposed here relies on the use of cargos that are enclosed in a lipid membrane. A silica core ensures compatibility with state-of-the-art optical trapping technology and associated analyses, and the physical properties of the membrane are tuned through various lipid mixtures to promote fluidity (for hypothesis testing) or rigidity (serving as a control). These investigations employ two previously established experimental handles (ATP levels and the decoration of microtubules with the small protein tau) to alter the binding kinetics between individual motors and their microtubule track. Our novel membrane-coated cargos will yield quantitative insights into transport in a context that more faithfully recapitulates the scenario in living cells. The fluid membrane is predicted to synergize or counter these single-motor modifications, depending on the direction of changes in the ability of individual kinesin motors to bind microtubules. Data analysis will be accomplished using previously established precision particle-tracking algorithms. The feasibility of the proposed approach has been validated in preliminary studies. Findings from the proposed study will critically integrate with biochemical investigations of changes in single-motor properties to elucidate the molecular basis of transport regulation and failure in vivo, for example in neurodegeneration. The proposed work is interdisciplinary, and the research team relies on fruitful collaborations both within the home institute of University of California, Merced and across institutional boundaries. Funding will significantly strengthen and improve the research environment at UC Merced, a minority-serving institution that was established in 2005. The diverse nature of UC Merced's student body is strongly reflected in the research team and in the PI's experience in supervising student research. Both graduate students for the proposed work are first-generation college graduates. These cutting-edge investigations will provide a new opportunity for students at UC Merced to apply quantitative technologies and analyses to issues with strong biomedical relevance, positioning them to contribute to future efforts to improve human health.