The research objective of this award is to predict and detect the formation of lipid nanodomains in giant unilamellar vesicles (GUVs). Lipid nanodomains are believed to play a significant role in a number of critical cellular processes, including replication processes in enveloped viruses such as bird flu and HIV and signaling mechanisms underlying pathological conditions such as cancer. The research approach involves three main activities: 1) developing the first imaging modality suitable to detect and characterize the formation of nanodomains; 2) developing multiscale models and numerical methodologies for nanoraft simulation; and 3) identifying the conditions needed for the formation of nanodomains and image them in GUVs. An important byproduct of these activities concerns addressing the question of whether or not such nanodomains can form at all in the absence of auxiliary stabilizing agents such as proteins.
If successful, the results of this research will provide an imaging modality that can be transferred to bioscientists to observe and detect, and eventually help control, the emergence of rafts in cells. Such knowledge might be used, for example, to guide the development of treatment schemes for cancer involving the control of growth factors and other membrane signaling molecules. Results from the research activities will be disseminated by providing the microscope techniques to manufacturers, making the computer codes freely available online to other scientists and engineers, and by providing images and animations to teachers and science museums. Undergraduate and graduate engineering students will be trained in the experimental, theoretical, and computational techniques involved in this research through a system of lab rotations
How do enveloped viruses such as HIV make their way out of host cells at the end of the viral replication process? Much is unknown about this final step in the replication process, but an important step involves accumulating all of the necessary proteins within a defined patch of cell membrane that can subsequently leave the host cell. If this process could be understood, it would open up pathways for new classes of treatments for HIV and other enveloped viruses such as bird flu. This grant was used to develop fundamental technologies needed to develop an understanding of this process. The question we asked was whether small patches of a specific type of lipid ("membrane rafts") that form spontaneously on a cell membrane could remain stable over time scales that are relevant physiologically. The system we studied was artificial cells that consist of defined populations of lipids enclosing a solvent. These nominally spherical vesicles are termed "giant unilamellar vesicles" or GUVs, but giant in this case means a few tens of micrometers in diameter. Under a change of local conditions such as temperature, the lipids on GUVs can "demix", and nanoscale domains phases can form. The tools we developed were tools to image and predict the behavior of nanoscale domains on GUVs. The challenge of imaging these domains is that physiologically and pathologically relevant domains are smaller than the wavelength of visible light, meaning that they cannot be seen using a microscope. We developed technology to estimate the sizes and concentrations of these domains by embedding flurophors in them and analyzing the statistics of photon emissions from them as they passed through a focussed laser beam. These tools are of broad applicability to the field of fluorescence fluctuation analysis. The challenge in predicting the behavior of these domains was developing a set of mathematical equations that could account for the nanoscale details, but that could be implemented over a much larger region of a GUV. This was accomplished, and new computational tools were developed to enable these equations to be solved accurately. The general outcome of the model is that, in the absence of a mechanism for stabilizing a nanoscale domain, nanoscale domains will coalesce and grow to be too large to be relevant to, say, viral replication. An important future question is whether the growth rates can be sufficiently slow that a virus could use this growing patches when exiting a host cell. However, we observed experimentally a mechanism by which a cell wall may buckle locally, enabling the nano domains to remain stable. We are currently investigating this possibility. The grant also led to new tools for teaching the science of phase transformations to middle school children. We developed, tested, and implemented a series of classroom modules that relate to the results of this work. These modules were delivered in inner city St. Louis middle school classrooms.
