Lipid Pumps and Fatty Shields- Asymmetry in the Plasma Membrane

The two faces constituting eukaryotic cell membranes are asymmetric.

Membranes are one of those incredibly elegant things in biology. Simple chemicals forces are harnessed to create a stable envelope for the cell, with no need to encode the structure in complicated ways. Rather, it self-assembles, using the oil-vs-water forces of surface tension to form a huge structure with virtually no instruction. Eukaryotes decided to take membranes to the max, growing huge cells with an army of internal membrane-bound organelles, individually managed- each with its own life cycle and purposes.

Yet, there are complexities. How do proteins get into this membrane? How do they orient themselves? Does it need to be buttressed against rough physical insult, with some kind of outer wall? How do nutrients get across, while the internal chemistry is maintained as different from the outside? How does it choose which other cells to interact with, preventing fusion with some, but pursuing fusion with others? For all the simplicity of the basic structure, the early history of life had to come up with a lot of solutions to tough problems, before membrane management became such a snap that eukaryotes became possible.

The authors present their model (in atomic simulation) of a plasma membrane. Constituents are cholesterol, sphingomyelin (SM), phosphatidyl choline (PC) phosphatidyl serine (PS), phosphatidyl ethanolamine (PE), and phosphotidyl ethanolamine plasmalogen. Note how in this portrayal, there is far more cholesterol in the outer leaflet (top), facing the outside world, than there is in the inner leaflet (bottom).

The major constituents of the lipid bilayer are cholesterol, phospholipids, and sphingomyelin. The latter two have charged head groups and long lipid (fatty) tails. The head groups keep that side of the molecule (and the bilayer) facing water. The tails hate water and like to arrange themselves in the facing sheets that make up the inner part of the bilayer. Cholesterol, on the other hand, has only a mildly polar hydroxyl group at one end, and a very hydrophobic, stiff, and flat multi-ring body, which keeps strictly with the lipid tails. The lack of a charged head group means that cholesterol can easily flip between the bilayer leaflets- something that the other molecules with charged headgroups find very difficult. It has long been known that our genomes code for flippases and floppases: ATP-driven enzymes that can flip the charged phospholipids and sphingomyelin from one leaflet to the other. Why these enzymes exist, however, has been a conundrum.

Pumps that drive phospholipids against their natural equilibrium distribution, into one or the other leaflet.

It is not immediately apparent why it would be helpful to give up the natural symmetry and fluidity of the natural bilayer, and invest a lot of energy in keeping the compositions of each leaflet different. But that is the way it is. The outer leaflet of the plasma membrane tends to have more sphingomyelin and cholesterol, and the inner leaflet has more phospholipids. Additionally, those phospholipids tend to have unsaturated tails- that is, they have double bonds that break up the straight fatty tails that are typical in sphingomyelin. Membrane asymmetry has a variety of biological effects, especially when it is missing. Cells that lose their asymmetry are marked for cell suicide, intervention of the immune system, and also trigger coagulation in the blood. It is a signal that they have broken open or died. But these are doubtless later (maybe convenient) organismal consequences of universal membrane asymmetry. They do not explain its origin. 

A recent paper delved into the question of how and why this asymmetry happens, particularly in regard to cholesterol. Whether cholesterol even is asymmetric is controversial in the field, since measuring their location is very difficult. Yet these authors carefully show that, by direct measurement, and also by computer simulation, cholesterol, which makes up roughly forty percent of the membrane (its most significant single constituent, actually), is highly asymmetric in human erythrocyte membranes- about three fold more abundant in the outer leaflet than in the cytoplasmic leaflet. 

Cholesterol migrates to the more saturated leaflet. B shows a simulation where a poly-unstaturated (DAPC) phospholipid with 4 double bonds (blue) is contrasted with a saturated phospholipid (DPPC) with staight lipid tails (orange). In this simulation, cholesterol naturally migrates to the DPPC side as more DAPC is loaded, relieving the tension (and extra space) on the inner leaflet. Panel D shows that in real cells, scrambling the leaflet composition leads to greater cholesterol migration to the inner leaflet. This is a complex experiment, where the fluorescent signal (on the right-side graph) comes from a dye in an introduced cholesterol analog, which is FRET-quenched by a second dye that the experimenters introduced which is confined to the outer membrane. In the natural case (purple), signal is more quenched, since more cholesterol is in the outer leaflet, while after phospholipid scrambling, less quenching of the cholesterol signal is seen. Scrambling is verified (left side) by fluorescently marking the erythrocytes for Annexin 5, which binds to phosphatidylcholine, which is generally restricted to the inner leaflet. 

But no cholesterol flippase is known. Indeed, such a thing would be futile, since cholesterol equilibrates between the leaflets so rapidly. (The rate is estimated at milliseconds, in very rough terms.) So what is going on? These authors argue via experiment and chemical simulation that it is the pumped phospholipids that drive the other asymmetries. It is the straight lipid tails of sphingomyelin that attract the cholesterol, as a much more congenial environment than the broken/bent tails of the other phospholipids that are concentrated in the cytoplasmic leaflet. In turn, the cholesterol also facilitates the extreme phospholipid asymmetry. The authors show that without the extra cholesterol in the outer leaflet, bilayers of that extreme phospholipid composition break down into lipid globs.

When treated (time course) with a chemical that scrambles the plasma membrane leaflet lipid compositions, a test protein (top series) that normally (0 minutes) attaches to the inner leaflet floats off and distributes all over the cell. The bottom series shows binding of a protein (from outside these cells) that only binds phosphatidylcholine, showing that scrambling is taking place.

This sets up the major compositional asymmetry between the leaflets that creates marked differences in their properties. For example, the outer leaflet, due to the straight sphingomyelin tails and the cholesterol, is much stiffer, and packed much tighter, than the cytoplasmic leaflet. It forms a kind of shield against the outside world, which goes some way to explain the whole phenomenon. It is also almost twice as impermeable to water. Conversely, the cytoplasmic leaflet is more loosely packed, and indeed frequently suffers gaps (or defects) in its lipid integrity. This has significant consequences because many cellular proteins, especially those involved in signaling from the surface into the rest of the cytoplasm, have small lipid tails or similar anchors that direct them (temporarily) to the plasma membrane. The authors show that such proteins localize to the inner leaflet precisely because that leaflet has this loose, accepting structure, and are bound less well if the leaflets are scrambled / homogenized.

When the fluid mosaic model of biological membranes was first conceived, it didn't enter into anyone's head that the two leaflets could be so different, or that cells would have an interest in making them so. Sure, proteins in those membranes are rigorously oriented, so that they point in the right direction. But the lipids themselves? What for? Well, they do and now there are some glimmerings of reasons why. Whether this has implications for human disease and health is unknown, but just as a matter of understanding biology, it is deeply interesting.

• Who's the deep state now?
• Apparently, everything bad is down to secularism. 

Man is Wolf to Man

The current administration's predatory and corrupt version of capitalism.

What is corruption? Isn't capitalism all about getting as much money as you can? Then doesn't it follow that there can be no such thing as corruption, which is defined as going against the rules? What rules?

We as a country go on a trip with every new president, learning about their nature and values as we accompany them through their brief span of history. Few presidents wear very well after their honeymoon, since the process of getting elected requires some shading of the truth, truth that inevitably comes out later on. The current administration is an odd example, since in his first term, Trump was not allowed (for very good reasons!) to be himself. The second time round has been a different story, and we are getting a deep look at his character. 

The US has always had a double relationship with capitalism, tilting between rampant competition / exploitation and reverence for rules and legal systems. Slavery, obviously, is the foremost example, with slaveholders enshrining in a document dedicated to human freedom their own legal rights to property in comprehensively oppressed people. The founders, on making their constitution, feverishly set to work creating institutions for the common good, such as the treasury, mail system, judicial system, patents, and military. But, at the same time, we have long had an ideology of free enterprise- of land, resource, and human exploitation almost without limit. 

Charles Ponzi was not working in Italy, after all, but in the US, as was Bernie Madoff. Now crypto is the popular mechanism of picking people's pockets, facilitating mundane crime such as money laundering and ransomware attacks at the same time that it provides flourishing vistas of direct fraud, in rug pulls, hacks, and market manipulation. A recent article reviewed the pathetic world of multilevel marketing, another model of predation where ambitious entrepreneurs are sucked into schemes that are engineered both to fail, and to induce the victims to blame themselves.

The administration has clearly made it its mission to celebrate these forms of business- the predators, the grifters, the destructive businessmen among us who think that taxes are for little people, and rules for someone else. Consumer protection agencies have been shuttered, the IRS eviscerated, investigations cancelled. Pardons have been going out, not only to the January 6th conspirators and their militias, but to the money launderers, the corrupt politicians, and crypto bros. It is a sustained campaign of norm and rule-breaking by a grossly tasteless, shockingly greedy and small-minded president, (and sexual predator), who cannot conceive of rational policy, uncorrupted institutions, or fairness, much less civility, as a principle. A person with deep psychological problems. And thus, is incapable of long-term policy that is the bedrock of durable, functional institutions, either commercial or governmental.

Following gold, like a cat following a laser pointer.

We are all worried about fascism, as that seems to be the aesthetic and the model of power the administration is tending towards. But what they have done so far doesn't even come up to the level of fascism, really. The president is not smart enough to have a coherent policy or ideological platform. The weave does not leave room for a program that would be attractive beyond the nihilistic base. There are inclinations, and moods, and tantrums, love for Putin, and a lot of nostalgia for policies of decades, if not centuries, ago. There is hate. But without a program that binds all these ingredients into even a marginally coherent approach to the future, it will inevitably fall apart. True believers don't do policies or reason- conspiracy theories are enough. Thinking, apparently, is for libtards. 

The fact of the matter is that capitalism is not equivalent to the law of the jungle. A legal system, and rules, are required to prevent capitalists from making military forays into each other's empires, and to prevent the workers from taking up their pitchforks, among much else. It is founded on the limited liability company, itself a legal construct, not to mention all the financial, educational, and physical infrastructure that forms its essential background. There is no going Galt here. Indeed, the whole point of captialism is not to screw everyone and make a few people very rich. Rather, it is to diffuse labor, useful products and productive technologies across society in a way that utilizes everyone's talents and supplies everyone's needs.  The point is general prosperity, not inequality. 

Institutions are built on rules, and they can die two ways- either people disregard and lose faith in the rules, (maybe because they are made by corrupt processes), or the rules become so elaborate and sclerotic that the point of the institution is lost. These ways map roughly onto our political divide, which, when it compromises to the middle produces something akin to a functional mean. But the current administration, and its ideology of thorough-going corruption on personal, business, and governmental levels, is, with the connivance of an equally unhinged supreme court, creating a legacy of cruelty and destruction that is surely a sad way to mark next year's anniversary of our institutional founding.

• How the wingnut evangelicals, rightist Catholics, and their funders have bought into the burn-it-all-down program of predation, with a little help from the Russians.
• Being populist means lying, unfortunately.
• We can and should give real help to Ukraine. How about blockading Russian rather than Venezuelan tankers?
• A hiring hellscape, with AI battling on both sides.
• Destroying science, at the NIH.

Mutations That Make Us Human

The ongoing quest to make biologic sense of genomic regions that differentiate us from other apes.

Some people are still, at this late date, taken aback by the fact that we are animals, biologically hardly more than cousins to fellow apes like the chimpanzee, and descendants through billions of years of other life forms far more humble. It has taken a lot of suffering and drama to get to where we are today. But what are those specific genetic endowments that make us different from the other apes? That, like much of genetics and genetic variation, is a tough question to answer.

At the DNA level, we are roughly one percent different from chimpanzees. A recent sequencing of great apes provided a gross overview of these differences. There are inversions, and larger changes in junk DNA that can look like bigger differences, but these have little biological importance, and are not counted in the sequence difference. A difference of one percent is really quite large. For a three gigabyte genome, that works out to 30 million differences. That is plenty of room for big things to happen.

Gross alignment of one chromosome between the great apes. [HSA- human, PTR- chimpanzee, PPA- bonobo, GGO- gorilla, PPY- orangutan (Borneo), PAB- orangutan (Sumatra)]. Fully aligned regions (not showing smaller single nucleotide differences) are shown in blue. Large inversions of DNA order are shown in yellow. Other junk DNA gains and losses are shown in red, pink, purple. One large-scale jump of a DNA segment is show in green. One can see that there has been significant rearrangement of genomes along the way, even as most of this chromosome (and others as well) are easly alignable and traceable through the evolutionary tree.

But most of those differences are totally unimportant. Mutations happen all the time, and most have no effect, since most positions (particularly the most variable ones) in our DNA are junk, like transposons, heterochromatin, telomeres, centromeres, introns, intergenic space, etc. Even in protein-coding genes, a third of the positions are "synonymous", with no effect on the coded amino acid, and even when an amino acid is changed, that protein's function is frequently unaffected. The next biggest group of mutations have bad effects, and are selected against. These make up the tragic pool of genetic syndromes and diseases, from mild to severe. Only a tiny proportion of mutations will have been beneficial at any point in this story. But those mutations have tremendous power. They can drag along their local DNA regions as they are positively selected, and gain "fixation" in the genome, which is to say, they are sufficiently beneficial to their hosts that they outcompete all others, with the ultimate result that mutation becomes universal in the population- the new standard. This process happens in parallel, across all positions of the genome, all at the same time. So a process that seems painfully slow can actually add up to quite a bit of change over evolutionary time, as we see.

So the hunt was on to find "human accelerated regions" (HAR), which are parts of our genome that were conserved in other apes, but suddenly changed on the way to humans. There roughly three thousand such regions, but figuring out what they might be doing is quite difficult, and there is a long tail from strong to weak effects. There are two general rationales for their occurrence. First, selection was lost over a genomic region, if that function became unimportant. That would allow faster mutation and divergence from the progenitors. Or second, some novel beneficial mutation happened there, bringing it under positive selection and to fixation. Some recent work found, interestingly, that clusters of mutations in HAR segments often have countervailing effects, with one major mutation causing one change, and a few other mutations (vs the ancestral sequence) causing opposite changes, in a process hypothesized to amount to evolutionary fine tuning. 

A second property of HARs is that they are overwhelmingly not in coding regions of the genome, but in regulatory areas. They constitute fine tuning adjustments of timing and amount of gene regulation, not so much changes in the proteins produced. That is, our evolution was more about subtle changes in management of processes than of the processes themselves. A recent paper delved in detail into HAR5, one of the strongest such regions, (that is, strongest prior conservation, compared with changes in human sequence), which lies in the regulatory regions upstream of Frizzled8 (FZD8). FZD8 is a cell surface receptor, which receives signals from a class of signaling molecules called WNT (wingless and int). These molecules were originally discovered in flies, where they signal body development programs, allowing cells to know where they are and when they are in the developmental program, in relation to cells next door, and then to grow or migrate as needed. They have central roles in embryonic development, in organ development, and also in cancer, where their function is misused.

For our story, the WNT/FZD8 circuit is important in fetal brain development. Our brains undergo massive cell division and migration during fetal development, and clearly this is one of the most momentous and interesting differences between ourselves and all other animals. The current authors made mutations in mice that reproduce some of the HAR5 sequences, and investigated their effects. 

Two mouse brains at three months of age, one with the human version of the HAR5 region. Hard to see here, but the latter brain is ~7% bigger.

The authors claim that these brains, one with native mouse sequence, and the other with the human sequences from HAR5, have about a seven percent difference in mass. Thus the HAR5 region, all by itself, explains about one fourteenth of the gross difference in brain size between us and chimpanzees. 

HAR5 is a 619 base-pair region with only four sequence differences between ourselves and chimpanzees. It lies 300,000 bases upstream of FZD8, in a vast region of over a million base pairs with no genes. While this region contains many regulatory elements, (generally called enhancers or enhancer modules, only some of which are mapped), it is at the same time an example of junk DNA, where most of the individual positions in this vast sea of DNA are likely of little significance. The multifarious regulation by all these modules is of course important because this receptor participates in so many different developmental programs, and has doubtless been fine-tuned over the millennia not just for brain development, but for every location and time point where it is needed.

Location of the FZD8 gene, in the standard view of the genome at NIH. I have added an arrow that points to the tiny (in relative terms) FZD8 coding region (green), and a star at the location of HAR5, far upstream among a multitude of enhancer sequences. One can see that this upstream region is a vast area (of roughly 1.5 million bases) with no other genes in sight, providing space for extremely complicated and detailed regulation, little of which is as yet characterized.

Diving into the HAR5 functions in more detail, the authors show that it directly increases FZD8 gene expression, (about 2 fold, in very rough terms), while deleting the region from mice strongly decreases expression in mice. Of the four individual base changes in the HAR5 region, two have strong (additive) effects increasing FZD8 expression, while the other two have weaker, but still activating, effects. Thus, no compensatory regulation here.. it is full speed ahead at HAR5 for bigger brain size. Additionally, a variant in human populations that is responsible for autism spectrum disorders also resides in this region, and the authors show that this change decreases FZD8 expression about 20%. Small numbers, sure, but for a process that directs cell division over many cycles in early brain development, this kind of difference can have profound effects.

The HAR5 region causes increased transcription of FZD8, in mice, compared to the native version and a deletion.

The HAR5 region causes increased cell proliferation in embryonic day 14.5 brain areas, stained for neural markers.

"This reveals Hs-HARE5 modifies radial glial progenitor behavior, with increased self-renewal at early developmental stages followed by expanded neurogenic potential. ... Using these orthogonal strategies we show four human-specific variants in HARE5 drive increased enhancer activity which promotes progenitor proliferation. These findings illustrate how small changes in regulatory DNA can directly impact critical signaling pathways and brain development."

So there you have it. The nuts and bolts of evolution, from the molecular to the cellular, the organ, and then the organismal, levels. Humans do not just have bigger brains, but better brains, and countless other subtle differences all over the body. Each of these is directed by genetic differences, as the combined inheritance of the last six million years since our divergence versus chimpanzees. Only with the modern molecular tools can we see Darwin's vision come into concrete focus, as particular, even quantum, changes in the code, and thus biology, of humanity. There is a great deal left to decipher, but the answers are all in there, waiting.

• Who's the poodle?
• We are going totally tin-pot.

Fifty Trillion Dollars Are Missing!

Bernie Sanders says that the last fifty years have seen a massive wealth transfer... to the rich.

Bernie Sanders has a new hook for his discussion of inequality. The Rand (AKA Bland) corporation has a study out that shows that the share of income going to workers over the last fifty years has declined to the point that, cumulatively, fifty to eighty trillion dollars have gone missing. Well, they have not gone to workers, but rather to non-workers: capitalists, shareholders, parasites; generally the 1%. 

The basic mechanism here is that capitalism is built on top of a labor market, where workers are paid only what the competition among them renders suitable to sort them into needed jobs. It is not built to fairly share the gains from their work or anyone else's work. In fact, it is built to skim off all the gains / profits and send them to shareholders, owners, managers ... anyone who has power in the capitalist system, which the workers surely do not. Workers get other rewards, but a share of the profits is not one of them. They are regarded as a necessary expense, like feedstocks and machinery, and their cost is to be minimized assiduously, now with robots and AI where possible. I learned this in dramatic fashion when a company I worked for, where the CEO chummily addressed us a team members and collaborators in the grand adventure of bioinformatics, sold the company and took all the winnings for himself.

With that in mind, it becomes obvious where the 50 trillion has gone to. It has been shared out among those who have real power in the capitalist system. First comes management. Managers hold the keys to the finances and the profits. They tell the board what is going on. They have been theoretically upgraded over the last seventy years from employees to entrepreneurs, and have taken shockingly increased amounts from the till during that time. Second come the shareholders, who have been theoretically upgraded from stakeholders in the corporate enterprise to the sine qua non of the corporation- its very soul and purpose, not as a "company" of people, or or a part of a wider culture, or a service rendering organization, but as a money gathering entity. Money that is raked up from whatever the "business model" might be into the pockets of shareholders, with the management as a necessary, if unfortunate, intermediary, and employees as an afterthought.

Ownership is the medium of power in capitalism. The shareholders are part-owners, and their interests are built into the stock market system, which is a perpetual readout of the value of each share. Analysts are constantly sifting through the value of each enterprise in terms of current assets and future potential. Corporations buy each other based on these valuations, and bankruptcy awaits those who lose control of their business model and fail to send money back to the shareholders. What the company produces, or its quality of work life, are completely irrelevant to its value. Whatever money is made over costs is shipped out to shareholders, in the form of dividends, share buybacks, or re-investment towards future growth and greater value. Managers have some power in this system, and have been able to capture quite a bit of the winnings, but workers have essentially none.

Labor markets tend to settle on what employees need to get by, rather than on the worth of what they produce. No compensation review or salary offer makes any reference to productivity or worth of the product. They are always keyed to what the market will bear.. the job market, that is. And job applicants are always in a weak position, since unemployment is such a catastrophic prospect. The choice at most interviews, especially the crucial first one out of school, is some pay or no pay. Employers work very hard to avoid competing for labor, with all kinds of illicit agreements among each other, and a carefully cleansed information landscape where workers don't know what others are being paid. Especially at the low end, this leads to workers competing for crappy jobs that pay little, because the there is always someone who is unemployed who, in light of their dire plight, can make do with even less. 

So, as the US economy has grown, growing things, making things, and gaining productivity year over year, little of that has trickled down to workers. Look at people's lives on the lower end of the economic ladder, and conditions have not visible improved. Indeed, homelessness and hunger are increasingly, instead of decreasingly, common. The middle classes have some increment of technology, like computers, smart phones, and streaming, but the living standard otherwise has not advanced noticeably, even while population pressures increase and the natural world has degraded. This arises simply from the structure of capitalism, when it is taken seriously and grows into the kind of untouchable gestalt it has become over the recent decades. 

And it is obvious that this is bad. Bernie Sanders is completely correct to point out that Americans across the spectrum could be much better off if the 1% got less of the income and wealth, and the rest of the citizenry got more. Inequality is obviously corrosive socially and politically, quite aside from the misery it causes at the lower end of the economic ladder. The capitalist system as we practice it is a relic of feudalism, when capital was scarce, sword and blood trumped merit, and serfs knew their place.

There are many ways to approach this problem, which is incredibly systemic and entrenched. Obviously we need to start with a bit of a cultural reboot that reconceives corporations as cultural entities with important roles and stakeholders that go beyond just making a dollar. We need a minimum wage that pays not just subsistance, but a normal, civilized living. Unions are another, if quite fraught, way for workers to retake power in the capitalist ecosystem. We need a tax system that values labor income over passive, unproductive income, and which taxes accumulated wealth as well. Employees should have a seat at the table in all companies- the corporate board table. We need serious regulation and enforcement that levels the playing field for companies that play by the rules, and sets rules that not only sustain, but build, society and the environment over the long haul.

Another idea that is quietly gaining revolutionary steam is employee ownership. There is an excellent book that discusses this, as it is currently practiced by hundreds of companies in the US. One example is the Publix supermarket chain, which has been wholly employee owned through five decades of growth, and is by far the largest such company. While the ESOP (employee stock ownership plan) model may start with a company stock buying plan, the serious work comes with majority ownership by employees. There are about 14 million employees in the US with some kind of company stock ownership plan, and 650,000 who majority-own their companies. This is usually arranged by a loan-financed buyout from a founder or other owners. Then the proceeds of the business pay off the loan as the employees accumulate distributed stock. This mode of buyout is an excellent way to transition from the early entrepreneurial phase of a company to a mature business, as it keeps the company culture intact and avoids the many problems of private equity, which may debt-strip a target company and leave it in bankruptcy, or of public stock ownership, which leads to disinterested owners that demand short-term financial performance over long-term health. 

This book is a bit thin on how such companies are managed and how the stock allocations and valuations are made. The stock is partially restricted against outside sale, (though some ESOP companies are publicly traded in part), so companies typically engage experts to value themselves each year. Employees gain stock with time, so it becomes a seniority system, a bit like waiting for a pension or union seniority. That is again not entirely fair to employees who may contribute more despite being young. At any rate, these companies still have professional management, but since it is all employees on the board, and with open financials, there is a track record of fewer layoffs, better morale, more cooperation, and ultimately, better financial performance. And critically, profits are not dispersed to faceless and uninterested "investors", but to the employees.

Imagine if every mature company was not buffeted by stock market analysts and fads, not bled dry by stock buy-backs to fund rich investors, nor haunted by the specter of a private equity strip-and-dump, but was owned wholly by its own employees, who reaped the profits of their collective work and controlled its conditions as well. As increasing numbers of companies become employee-owned, one can imagine a phase transition (analogous to the universality of certain benefits like health insurance) where people just would not want to work anymore for other kinds of companies. Small and startup companies would have to share equity, (as they used to do in Silicon Valley), and would naturally progress as they grew to broad ownership by every employee. That would be amazing! 

• Predation, chaos, and crime are the policy.
• Unitary, schmoonitary.
• Half an hour on how terrible medicare advantage is.
• A new way to heat, together.