20: Experiments Remaining to be Done
In the area of immunology, and antigen presentation in particular, this seems to me to remain a wide – open field, ready for many more surprising discoveries. However I am not familiar with current literature in this fast moving field; my only regret is that I did not have the opportunity to study some of these problems with modern spectroscopic techniques. For example, it might be feasible to follow the binding and translocation of individual fluorescently labeled antigenic peptides extracellularly as well as intracellularly. This would be no overnight project; it could require a long dedicated effort since fragmented peptides might be involved.
There remain many relatively simple significant experiments to be carried out in the area of lipid bilayer membranes. To the best of my knowledge there have been few experiments carried out with trans fatty acids, in spite of their notoriety in connection with health issues. One could easily test their role in phase-separated bilayers.
Certainly one of the most interesting problems relating to membranes concerns the regulation of the rate of cholesterol biosynthesis. The question is whether this rate is determined by the chemical activity of cholesterol in a cell? (The implicit assumption is that this activity is approximately constant in all the membranes in a given cell.) My hope is that my former graduate student Arun Radhakrishnan will answer this question in the affirmative.
What are the main factors that determined the chemical activity of cholesterol in complex mixtures?
Model bilayer membranes that show phase separations and have been studied quantitatively are relatively few in number. On the other hand lipids in cell membranes are typically complex mixtures. What can be said about the critical properties of these complex mixtures? A binary mixture can show one or two critical points. A ternary mixture can have a line of critical points. An N-component mixture can doubtless have multiple critical lines in some N+1 dimensional space. Is there any biophysical significance to be found in these multicomponent phase diagrams?
Critical phenomena in three–dimensional liquids have been studied extensively by theoretical and experimental physicists, and physical chemists. Bilayer membranes are two dimensional in most respects but are three- dimensional in terms of hydrodynamics. Sarah Keller and her collaborators have done the majority of the experimental work in dynamic bilayer critical phenomena, and have compared their results with existing theory, with apparently good agreement. See Honerkamp-Smith et al. I have attempted to use the theoretical work by others in order to understand the deuterium NMR of phospholipids as the critical point is approached from above. (See 481). There is a great opportunity for more theoretical work in this area.
In an unpublished work Arun and I observed that binary mixtures of sitosterol and DMPC form three distinct liquid phases in monolayers, and that there is a sharp pressure dependent contrast inversion of the fluorescent probe as a function of pressure. The origin of this interesting result remains a mystery.