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Membrane-Bound Kinesin Transport and its Applications in Active Matter

  • Author(s): Lopes, Joseph David
  • Advisor(s): Hirst, Linda S
  • et al.
Creative Commons Attribution 4.0 International Public License
Abstract

Understanding the intricacies of biological systems from a first principal approach has provided much insight into the function of cellular systems. The self-assembly of lipid membranes and the far-from-equilibrium nature of protein biochemistry has given rise to a new field of physics, active matter, that considers biological systems as hierarchical far-from-equilibrium phases. In this thesis, I describe experiments that investigate the impact of membrane diffusivity on intracellular transport. The results from these experiments then inspired the development of a novel form of active matter that is capable of altering its environment.

Motor proteins, specifically kinesin, are responsible for the transportation of cellular cargo. This cargo is held inside of a lipid vesicle, and how the vesicle’s membrane affects kinesin-based transport has been a hotly debated topic. Recent in-vitro experiments suggest that transport of lipid vesicles is enhanced by the diffusivity of the membrane cargo itself. In this thesis work, I designed a system in which fluorescently labeled motor proteins are bound to a planar lipid bilayer. This two-dimensional bilayer demonstrates the diffusivity of a spherical vesicle without exhibiting the elastic or geometric effects found in small vesicles that could contribute to transport. When studying individual microtubules, we see several interesting phenomena. We observe that motor proteins are diffusive when bound to a membrane and therefore they can cluster onto a stationary filament. When we add ATP to induce gliding, the motors unbind, and by measuring the bound population density we show that motor protein disassociation from the filament is a diffusion limited process. Surprisingly when gliding on a membrane we also observed that the gliding velocity increases over time before reaching a steady state.

The next phase of the research presented in this thesis was to study if densely packed filaments on a membrane could form an active nematic. Interestingly, when bound to a diffusive membrane, sufficiently large concentrations of gliding filaments can align and move collectively. Furthermore, the results of this experiment are quite striking when the density of filaments is insufficient to form an active nematic phase. Below a certain filament density, we see the formation of a network phase of filament streams that have their own local orientation. These streams can persist for up to an hour and can also exhibit a global rotation. This could be due to the localization of the motor protein, as we see them clustered around streams of filaments. This is quite significant as we have a two-dimensional active matter system that is the product of a diffusive environment, but in turn alters that environment.

This thesis demonstrates the complex interactions motor proteins can have with lipid membranes and their subsequent impact on transport. We take advantage of these properties to design a novel active matter system that restructures its environment to reinforce its steady state.

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