Here’s an example of the move to nanoscale that I was writing about the other day, but from the other end of the stick. As chemists have been moving up from small molecules to dealing with larger structures, biologists have been moving down from whole-cell studies to more detailed pictures of what’s happening locally. The two end up meeting at a roughly supramolecular scale. The settlers on the Great Plains called the Platte river “too thick to drink, and too thin to plow”, and that’s the sort of in-betweenness that’s kept this area from being explored until recently. Too large for chemistry, and too small for biology – but perhaps not any more.
The authors are looking at the antigen receptors on the surface of B cells, immunoglobulin M (IgM) and immunoglobulin D (IgD). It’s been known for some time that these are coexpressed in B cells, and that they’re localized to clumps on the cell surface, small “protein islands” in the membrane. But beyond that, there are plenty of mysteries. The two receptor types (bound antibodies with signal transduction equipment on the inside of the cell) are structurally very similar, but their signaling behavior can be quite different. Knocking out IgD in mice doesn’t really seem to do much to them, though, which has made some people wonder just how redundant it must be.
How these things move around on the cell surface is also a puzzle. There are probably over a hundred thousand copies of these things out there, but how are the arranged? One model has them more or less as monomers, when then can come together (as dimers or higher-order structures) when stimulated, and another model has them already organized into some sort of grouping, which then rearranges when an antigen comes along. Inside that model, it’s not clear if IgM and IgD are together in these groupings (and if so, in what ratios), separated into clumps of their own kind, or what.
The problem is, the size of these protein islands is below the usual limits for light microscopy. This paper turns to “stochastic optical reconstruction microscopy” as well as electron microscopy and to proximity ligand activation as well. You need all the help you can get at this level:
The currently available super-resolution techniques are, however, still challenging, and each of them is associated with certain technical limitations. For example, the analysis of results obtained by dSTORM must deal with the intrinsic noise of a fluorescence image and the estimation of signal overcounting. On the other hand, TEM requires fixation and extensive sample manipulation, thus reducing the labeling efficiency. In contrast to these two techniques, Fab-PLA does not require the production of altered or fluorophore-tagged proteins in cell lines but can be used in studies of the surface organization of proteins on fixed primary cells. The disadvantage of PLA, however, is that as a sampling method, it can monitor only 10 to 40 interactions per cell. Given these technical problems, the analysis of the nanoscale organization of proteins on the cell membrane must be based on several techniques, as is the case in this study.
STORM, by the way, is an example of the sort of single-molecule fluorescence technique that won the Nobel last year, and lets you beat the diffraction limit. Putting these techniques together has now established that the two types of receptor actually do exist in separate protein islands from each other, each with its own different protein and lipid composition. No one knows yet how this arrangement is produced or maintained, because even the advanced techniques available now don’t have the spatial and temporal resolution to follow these protein islands as they form and move around.
The authors were, though, able to take “snapshots” under different conditions, and they found that activated B cells have changed quite a bit. The two types of protein islands have become noticeably smaller (for reasons that are unknown), and have assembled into closer proximity to each other for some reason (and via mechanisms unknown). It looks as if they may be exchanging some of their original protein partners, but that’s another level of detail to be worked out. That, though, is the level that we’re going to have to take immunology to (in all the various cell types) to really figure out what’s going on.
The excitement, then, over immuno-oncology is only the beginning, because even though immunology itself looks (from the outside) like a mindbending assortment of fine detail, we’re not even down to the fine details of anything yet. What will we be able to do once we get down there?