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Engineering Lipid Vesicles for Cellular Reconstitution

  • Author(s): Richmond, David
  • Advisor(s): Fletcher, Daniel A
  • et al.
Abstract

The reductionist approach to modern cell biology aims to identify the individual molecules and interactions that give rise to complex biological activity. A complementary constructionist approach, known as reconstitution, aims to recapitulate biological structures and functions from basic building blocks in order to show which components are essential and how biophysical constraints, such as membrane boundaries, influence organization and activity. My dissertation research has applied and extended this `bottom-up' approach to study the role of membrane mechanics in the formation of cellular filopodia and to develop new tools for reconstituting processes encapsulated within membranes and for engineering cell-like devices.

We investigated the mechanics of actin-membrane interactions by studying dendritic actin networks grown on the surface of giant unilamellar vesicles. In this minimal system, we observed the formation of parallel filament protrusions arising from the highly branched dendritic actin network, notably in the absence of bundling proteins. We confirmed through a simple theoretical model that a lipid bilayer can drive the emergence of bundled actin filament protrusions from branched actin filament networks, thus playing a role normally attributed to actin-binding proteins. This revealed a critical role for the membrane in organizing actin filaments at the plasma membrane.

This work motivated the development of a technique for encapsulating protein contents in the lumen of lipid vesicles in order to emulate the biophysical boundary conditions of real cells. We demonstrated the use of a microfluidic jet to form lipid vesicles with controlled contents by deforming a planar bilayer. These vesicles mimic an essential organizational feature of cells - encapsulation within a lipid membrane - and provide a platform for more complex cellular reconstitution. Subsequently, we adapted this technique to a pulsed inkjet-based device, enabling greater control of vesicle size and improved throughput. Using this inkjet-based device for vesicle formation, we were able to control membrane properties such as asymmetric lipid composition and insertion of membrane proteins, which are essential for numerous cellular processes. We demonstrated the applicability of this technique by reconstituting SNARE-mediated membrane fusion in a geometry that mimics exocytosis.

In summary, this work has provided new insight into the role of lipid bilayer mechanics on the reconstitution of cellular protrusions and developed a novel technique that enables formation of lipid vesicles with controlled contents and membrane properties. Further development of this technique will enable advanced reconstitution experiments and construction of functional cell-like devices for medical and biomaterials applications.

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