Not physical organic chemistry but organic physical chemistry

This post is about physical chemistry with organic characteristics in the sense that capitalism in China is called socialism with Chinese characteristics. A lot of cell biology is also involved.

I remember the first time I heard about Irving Langmuir and the two dimensional gas he created. It even followed a modified perfect gas law (PA = nRT where A is area). He did this by making a monolayer of long chain fatty acids on water, with the carboxyl groups binding to the water, and the hydrocarbon side chain sticking up into the air. I thought this was incredibly neat. It was the first example of organic physical chemistry. He published his work in 1917 and won the Nobel in Chemistry for it in 1932.

Fast forward to our understanding of the membrane encasing our cells (the technical term is plasma membrane to distinguish from the myriad other membranes inside our cells. To a first approximation it’s just two Langmuir films back to back with the hydrocarbon chains of the lipids dissolving in each other, and the hydrophilic parts of the membrane lipids binding to the water on either side. This is why it’s called a lipid bilayer.

Most of the signals going into our cells must pass through the plasma membrane, using proteins spanning it. As a neurologist I spent a lot of time throwing drugs at them — examples include every known receptor for neurotransmitters, reuptake proteins for them (think the dopamine transporter), ion channels. The list goes on and on and includes the over 800 G protein coupled receptors (GPCRs) with their 7 transmembrane segments we have in our genome [ Proc. Natl. Acad. Sci. vol. 111 pp. 1825 – 1830 ’14 ].

Glypiated proteins (you heard right) also known as PIGtailed proteins (you heard that right too) don’t follow this pattern. They are proteins anchored in the outer leaflet of the plasma membrane lipid bilayer by covalently linked phosphatidyl inositol. https://en.wikipedia.org/wiki/Phosphatidylinositol — the picture shows you why — inositol is a sugar, hence crawling with hydroxyl groups, while the phosphatidic acid part has two long hydrocarbon chains which can embed in the outer leaflet. We have 150 of them as of 2009 (probably more now). Examples of PIGtailed proteins include alkaline phosphatase, Thy-1 antigen, acetyl cholinesterase, lipoprotein lipase, and decay accelerating factor. So most of them are enzymes working on stuff outside the cell, so they don’t need to signal.

Enter the lipid raft. [ Cell vol. 161 pp. 433 – 434, 581 – 594 ’15 ] It’s been 18 years since rafts were first proposed, and their existence is still controversial (with zillions of papers saying they exist and more zillions saying they don’t). What are they — definitions vary (particularly about how large they are). Here’s what Molecular Biology of the Cell 4th edition p. 589 had to say about them — Rafts are small (700 Angstroms in diameter). Rafts are rich in sphingolipids, glycolipids and cholesterol. The hydrocarbon chains are longer and straighter than those of most membrane lipids, rafts are thicker than other parts of bilayer. This allows them to better accomodate ‘certain’ membrane proteins, which accumulate there. [ Proc. Natl. Acad. Sci. vol. 100 p. 8055 – 7’03 ] These include glycosylphosphatidylinositol anchored proteins (glypiated proteins), cholesterol linked and palmitoylated proteins such as Hedgehog, Src family kinases and the alpha subunits of G proteins, cytokine receptors and integrins.

Biochemical analysis shows that rafts consist of cholesterol and sphingolipids in the exoplasmic leaflet (outer layer of the plasma membrane) of the lipid bilayer and cholesterol and phospholipids with saturated fatty acids in the endoplasmic leaflet (layer facing the cytoplasm). The raft is less fluid than surrounding areas of the membrane. So if they in fact exist, rafts contain a lot of important cellular players.

The Cell paper introduced synthetic fluorescent glypiated proteins into the outer plasma membrane leaflet of Chinese hamster ovary cells and was able to demonstrate nanoClustering on scales under 1,000 Angstroms (way too small to see with visible light, accounting for a lot of the controversy concerning their existence).

How can the authors make such a statement? The evidence was a decrease in fluorescence anisotropy due to Forster resonance energy transfer effects. Forster energy transfer is interesting in that it doesn’t involve molecule #1 losing energy by emitting a photon which is absorbed by molecule #2 increasing its energy. It works by molecule #1 inducing a dipole in molecule #2 (by a Van der Waals effect). Obviously, to do this, the molecules must be fairly close, and transfer efficiency falls off as the inverse 6th power of the distance between the two molecules.

In Fluorescence Resonance Energy Transfer (FRET), one fluorophore (the donor) transfers its excited state energy to a different fluorophore (the acceptor) which emits fluorescence of a different color. For more details see — https://en.wikipedia.org/wiki/Förster_resonance_energy_transfer — its interesting stuff. Again an example of physical chemistry with organic characteristics (and pretty good evidence for the existence of lipid rafts to boot).

Now it gets even more interesting. Nanoclustering is dependent on the length of the acyl chain forming the GPI anchor (at least 18 carbons must be present). NanoClustering diminishes on cholesterol depletion in actin depleted cell blebs and mutant cell lines deficient in the inner leaflet lipid — phosphatidylserine (PS) — which has two long chain fatty acids hanging off the glycerol. So it looks as if the saturated acyl chains of the glypiated proteins of the outer leaflet interdigitate with those of PS in inner leaflet. The effect is also enhanced on expression of proteins specifically linking PS to the actin cytoskeleton of the cortex. Binding of PS to the cortical actin cytoskeleton determines where and when the clusters will be stabilized. The coupling can work both ways — if something immobilizes and stabilizes the glypiated proteins extracellularly, than PS lipids can form correlated patches.

This might be a mechanism for information transfer across the plasma membrane (and acrossother membranes to boot). This could also serve as a way to couple many outer leaflet membrane lipids such as gangliosides and other sphingolipids with events internal to the cell. Cholesterol can stabilize the local liquid ordered domain over a length scale that is large than the size of the immobilized cluster. A variety of membrane associated proteins inside the cell (spectrin, talin, caldesmon) are able to bind actin. “The formation of the contractile actin clusters then determine when and where the domains may be stabilized, bringing the generation of membrane domains in live cells under control of the actomyosin signaling network.’

So just like the integrins which can signal from outside the cell to inside and from inside the cell to outside, glypiated proteins and the actin cytoskeleton may form a two way network for signaling. No one should have to tell you how important the actomyosin cytoskeleton is in just about everything the cell does. Truly fascinating stuff. Stay tuned.

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