Seeing is believing

'Santa Baby - come and fix it for me!'

I have a great interest in the animal cell glycocalyx. This is often described as the fuzzy surface coat, or sugar coat, that surrounds animal cells, and it is also the subject of some very fuzzy thinking. The glycocalyx consists of the protruding carbohydrate portions of integral membrane glycoproteins and glycolipds that interact via numerous charged sites with external long chain acid mucopolysaccharides, and other long-chained molecules like collagen that may be embedded. A number of functions have been put forward for the glycocalyx including an agent of cell recognition, or cell adhesion, inflammatory regulation and a form of mechanochemical buffer. Just how important it is in the life of a cell has probably still not been properly appreciated. The problem can partly be laid at the door of electron microscopists – yes colleagues, hang your heads, a little bit.

Cell fixation and staining is not one-size-fits-all. Different fixatives are better for different cellular components. However, a routine preparation procedure developed for EM, and it was pretty good. This is an initial glutaraldehyde fixation followed by osmium tetroxide postfixation with uranyl acetate and lead citrate staining. This routine delivered an image of what a ‘normal’ animal cell (supposedly) looks like and this then became ‘fixed’ as the consensus view of a cell and so influenced other (functional) thinking. Of course, we microscopists knew the importance of how preparation procedure influences ultrastructure, maybe we just should have shouted louder about this at the time (or more people should have listened).

It just so happens that the routine fixation regime described above does not do much to preserve the glycocalyx and so historically in many ways the glycocalyx has tended to get somewhat overlooked. Seeing is believing after all, or at least a pretty good way getting a handle on something.

By simply adding a tannic acid fixation step (which does ‘tan’ cell surfaces) to your preparation procedure, you actually get a very different view of the glycocalyx and hence a better appreciation of its potential importance. Use the freeze substitution preparation procedure with a really good carbohydrate-fixing agent and in some cases the extracellular space between cells is completely occluded! You cannot see a thing – synaptic clefts between nerve and muscles, for example, are black! How neurotransmitters can ever navigate the space is beyond me!

And so you begin to get a very different ‘feel’ for what goes on between cells. The glycocalyx almost begins to ‘look’ like the sort of cell wall you get round a plant cell. Of course, a plant cell wall is a highly specialised multi-layered structure containing specialised structural sugars and such. What was the glycocalyx again?

One thing for sure, all of that extracellular material is not just hanging around – that’s not the way Nature and evolution tends to work. Sometimes it pays to look at things in as many different ways as possible – seeing is believing after all.

And if this Xmas eve you happen to see a man in a red suit carrying a big bag of presents – well, consider that maybe it’s all to do with your preparation procedure; alcohol is a fixative too.

Happy Christmas and a Merry 2012!