Date of Award
Doctor of Philosophy (PhD)
Biomedical and Chemical Engineering
Martin B. Forstner
Membranes, Supported Lipid Bilayers
The phospholipid bilayer is one of the hallmarks of eukaryotic life. This complicated two dimensionally fluid surface is composed of a double layer of lipids which have a region that is hydrophobic and a region that is hydrophilic. The lipid bilayer membranes of a cell act as a barrier that distinguishes the cells and the organelles interiors from the outside environment. In order for the cell to be able to effectively communicate across these impermeable barriers they have evolved many intricate systems of lipid and protein interaction that serve to transmit information from one side of the membrane to the other. The abundance of functionality in the membrane has made them incredibly complicated and intricate structures. This complexity makes the study of any one membrane associated signaling pathway or system more often than not very challenging if not impossible. Because of this a simplified analogue of the biological membrane is necessary for controlled and quantitative studies. The amphiphilic nature of phospholipids allows researcher to create lipid bilayer that can be controlled for composition and placed on a surface that is accessible to many investigative systems. These supported lipid bilayer (SLB) systems have been used for decades to figure out the intricate series of actions and reactions that occur in a natural biomembrane. While there is still a lot to be learned from simple lipid bilayer systems there is also now the need to produce bilayers that incorporate more of the functions of a living cell. These will permit the study of signaling systems involving multiple molecules, the direct observation of cellular responses to surface stimuli, and even the control of cellular behavior. Along with providing greater versatility in the study of cells and membrane mediated systems these enhanced lipid bilayers will have major biomedical applications. Given that a lipid bilayer is the exterior presented by cells in nature there can be no more biocompatible surface than a lipid bilayer, for the surface treatment of medical devices this could have great implications. For instance, if sufficient knowledge is developed about these systems, then all types of medical implants could have a customized lipid bilayer based coating that would help it to integrate perfectly with the tissue to which it is embedded. Joint replacements could have a surface that would promote the growth of osteoblast cells, a cardiac stent could have a coating to prevent clot formation while promoting epithelial cell growth and discouraging smooth muscle cell growth, perhaps even neural implants capable of two way communication for the treatment of paralysis.
It is with this long term vision that we set out to advance the field of lipid bilayer systems to increase the capacity for dynamic control, artificial enhancement, and tissue interface. A series of investigations were undertaken with the goal of promoting each one of these facets.
In the first investigation we studied the dynamic behavior of the PIP2 phospholipid in varying physiological calcium concentrations. This anionic lipid has been hypothesized to have the capacity to organize itself spatially in response to fluctuations in calcium levels. No investigation has been so far carried out that look at PIP2s reaction to physiologically relevant changes in the calcium concentration. We used fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis to look at changes in the dynamics and the brightness of PIP2 in polymer supported lipid bilayers. We found that PIP2 appears to make electrostatic associations with zwitterionic lipids in the bilayer when there is no calcium present which are disrupted with the addition of calcium.
In the second investigation we developed a genetically encoded protein/lipid anchoring system based on an aldehyde. This system allows proteins to be genetically modified to bear a 6 amino acid consensus sequence at, theoretically any position along its amino acid chain. This consensus sequence targets a cysteine within the sequence to a formylglycine generating enzyme (FGE) that converts the cysteine to a formylglycine. Formylglycine bears an aldehyde on one of its residues. Aldehydes are found very rarely in the mammals and are also highly reactive with certain elements such as aminooxy and hydrazides which are also very rare in mammalian biology. This makes an aldehyde an excellent anchoring method for medical applications. We produced aldehyde tagged enhanced green fluorescent proteins (EGFP) and successfully incorporated them with aminooxy modified lipids in a supported lipid bilayer. This showed that the system is a viable and in fact improved alternative to the lipid/protein anchoring systems currently in use.
The third investigation centered on the capacity to culture cells on a SLB surface. Previous studies have found that SLBs have an anti-adhesion property for both proteins and cells. We investigated whether the inclusion of various quantities of both positively and negatively charged lipid into the bilayer would effect the capacity of fibroblast cells to adhere and proliferate. Our system prototyped a novel high throughput technique for bilayer/cell investigations. We found that cells initially had difficulty in adhering to all bilayer surfaces but the inclusion of greater quantities of negatively charged lipids produced a more favorable environment for cell growth. We also looked at how glycolipids, commonly held to be promoters of cell recognition and adhesion, effected fibroblast cells. Such bilayers with sugar elaborations were as suitable for fibroblast growth as the control subjects on collagenated glass. These findings are a significant advancement in the development of bilayer - cell interfaces.
This body of work has provided exciting advances in the capacity to produce and understand biomimetic surfaces. While there is much work to be done yet before fully interfacial surfaces are possible, I have developed some novel tools and unearth some interesting findings that can provide some of the next steps towards this goal
Mc Cabe, Ian, "Observing, Understanding, and Manipulating Biological Membranes" (2015). Dissertations - ALL. 304.