Saturday, March 25, 2017

Centrioles Have Mothers Too

Penetrating the mysteries of one of our most attractive organelles.

The centriole is one of the more glamorous, yet enigmatic, structures of the eukaryotic cell. Yes, it has beautiful ultrastructure. Yes, it serves as the fabulous astral center of the spindle poles that organize mitosis. And yes, it has been studied for well over a century. But does all that mean it is well understood? Absolutely not.

Electron micrographs of centrioles. The inset is a cross-section of one centriole barrel, showing the 9 X 3 microtubule structure as well as the inner cartwheel that seems to be its construction scaffold. The main image shows a side-ways cross-section of a mother-daughter pair of centrioles, (forming a centrosome), showing their length and relationship. Also shown is the nimbus of microtubules coming out of what is called the peri-centriolar material, or PCM. How those relate functionally to the core structure is still not known.

Centrioles are one of the many radical innovations of eukaryotic cells, forming the core(s) of centrosomes, which organize mitosis, and of cilia, which are the novel eukaryotic solution to cellular locomotion. They are are a barrel-shaped quasi-crystalline arrangement of microtubule proteins, grown on a central wheel of template proteins, called the cartwheel. For cilia, these microtubules clearly serve as the foundation stones of microtubule rods that grow out into the long appendage. But things get far more murky at the mitotic spindle core, where a profusion of microtubules emerge from an amorphous zone around the centrioles, but are not templated directly by them. The nature and history of this structure remains quite mysterious, especially as it turns out that some eukaryotic cells can get by without centrioles at all.

Centrosomes are in red, at what are also called the microtubule organizing centers, organizing the green microtubules that  in turn organize the separation of DNA (blue) during mitosis. 

In addition to these starring, if not obligatory, roles, centrioles have another charismatic property, which is that they reproduce in a clearly parental fashion. Every cell cycle is accompanied by the division of the centriole pair into single centriole mothers, which then give birth to new centriole daughters from their sides. While most of the molecular actors in this centriole mini-drama are known, as are some of their roles, a great deal remains to be found out about how the whole process is put together.

Detailed scheme of centriole (green) and centrosome (the whole green + yellow mess) duplication during the cell cycle. The structures are reasonably well known, the molecules and their roles less so. Note the roles of Plk4, Sas6 and colleagues in generation of the nascent cartwheel scaffold at G1/S. Plk4 itself will be disposed of later on, towards M phase.  

A recent paper described some more about how the first steps of this replication process take place. The cooperation of the centrioles with the larger cell cycle is naturally very deep. Some labs have found that the key kinases (which attach regulatory phosphates to other proteins) that regulate the cell cycle operate out of the centrosome- the structure containing the centriole pair. And the centrioles in turn are subject to key kinase steps that license when they can replicate. Key molecules of this process have the name Sas, after a genetic screen for spindle assembly defective mutants, each of which was given a Sas# name. Sas4, Sas5, and Sas6, for instance, are proteins that make up most of the central cartwheel scaffold upon which the centrosomal microtubules are built, along with another protein, Cep135. But the story starts much earlier. Sas6 is brought to the key location on the mother centriole's side by a kinase, Zyg1, which in turn is brought there by Spd2. How did Spd2 get there, and what does it do? Could it be turtles all the way down?

No, the authors identify Sas7 as the protein that binds to Spd2 and gets centriole pregnancy underway. Sas7, finally, is at the centriole all the time, and is activated not by recruitment, but by the cell cycle. One key finding was that all the other known initiating mutations (of Zyg1 and Spd2) depend on Sas7 for their action and localization, but not the reverse. They also find that the centrioles of mutant cells are significantly diminished, missing quite a bit of the outside structure. This suggests that Sas7 is a structural component at just the right position, around the outsides of the mother centriole, to participate in the construction of daughter centrioles.

Complete deletion of Sas7 renders the organism dead. But a partially inactivating mutation (temperature sensitive) allows its function to be observed. Here, the wild-type cells show flamboyant microtubule (green) organization during mitosis. The mutant at the restrictive temperature shows a mess, where centriole duplication has failed and the DNA (orange) is dispersed around one pole instead of being nicely pulled between two poles.

Mutant Sas7 also causes structural problems for centrioles. The outsides of the lower centrioles are severely depleted of material, whatever it is.

But that leaves one last question- what licenses / initiates the beginning of centriole duplcation, given this need for Sas7 and Spd2 interaction and given the need for tight coupling with the cell cycle, and why does it happen at only one position on the side of the mother, rather than all over? This article does not touch on those issues, and one has to revert to other reviews to gain some insight. Zyg1 is known in other species as Plk4, and seems to be the critical link to the cell cycle. Whether its activation is driven in particular ways is not yet known, but its destruction is known to be driven by the SCF complex that funnels many critical cell cycle proteins into the proteasomal trash bin at the the transition between G2 and M phase (with partner betaTrCP). However, how the localization is restricted to one position on the mother is not known at all. The only relevant fact is that supplying an excess of Plk4 can prompt initiation of multiple daughters. Thus one reviewer is reduced to speculating about a concentration-sensitive positive feedback mechanism that forces all the activity to localize in one place.

So, even after all these years, of both macroscopic and molecular study, this beauty still holds quite a bit of mystery. Will resolving it make us happier?

Sunday, March 19, 2017

Why ATP?

Inside cells, the major energy carrier is ATP. Why, and what might have come before?

Two weeks ago, we learned about the intricate mechanism by which organisms transduce energy from a proton gradient into ATP, using a rotating, motor-like enzyme: the ATP synthase. Proton gradients are one important way for organisms to store and distribute their energy, just as we do macroscopically with fossil fuels, and the body does with glucose, distributed through the blood. Inside cells, ATP is the primary short-term form of energy distribution, with longer term stores taking the form of various carbohydrates, lipids, and, in general, all the components of the cell, which can be phagocytosed in times of need (or even entire cells, which can commit suicide and be consumed by others when needed).

But what is so great about ATP? It comes up constantly as the extra ingredient that makes difficult reactions go forward. Drug efflux pumps use ATP. Actin uses ATP to create physical force during its filament assembly and projection, as does myosin, which is the motor that pulls on actin to create muscle action. Likewise, dynein, which moves things along microtubules- another form of locomotion used in cell division and nerve cell organization, among much else. Translation uses a net of four ATP per amino acid incorporated, in the form of one ATP=>AMP (worth two ATP=>ADP), and two GTP, which are close cousins of ATP. ATP fuels our chemical purification factories, such as the kidneys, whose various chemical pumps all depend on a master gradient driven by the ATP-using sodium / potassium antiporter. Countless regulatory pathways use ATP to attach a phosphate to a protein, thus changing its activity, often dramatically. Whenever our enzymes are doing something that is energetically impossible on its own, it is a good bet that ATP is supplying the extra oomph. And life naturally depends on many such impossible steps.

ATP, the molecule. The orange groups are called phosphoanhydride groups, or phosphate groups for short.  Each of those groups carries a negative charge.

The last bond in ATP, which is broken (via hydrolysis with water) to make ADP + phosphate, provides about 30 kJ/mol. In comparison, a hydrogen bond, such as those that hold DNA strands together, are about 8 kJ/mol, and a normal carbon-carbon bond has about 360 kJ/mol. It takes a lot of energy to build up organic molecules, which is why such extraordinary measures like photosynthesis were developed during evolution, (taking about 3,100 kJ/mol worth of ATP and NADPH to produce one sugar molecule). But most other processes that just need a chemical nudge are well within the range of that ADP-P phosphate bond. Breaking the second-to-last instead of the last bond, to release diphosphate and AMP, yields about 41 kJ/mol, for a little extra energy when needed.

Sometimes the phosphate bond is referred to as "high-energy", but that doesn't mean it is a strong bond. One of the strongest bonds is the triple bond between the two atoms of atmospheric nitrogen, which takes 945 kJ/mol to break. Quite the opposite- instead of requiring lots of energy to break, the phosphate bond gives up those 30 kJ/mol energy when hydrolyzed (that is to say, the reaction is exothermic). Those negative charges really want to get away from each other! That makes it an energy store instead of an energy sink. But due to the its high negative charge, it is not easily attacked by water, which means it is kinetically stable and does not spontaneously degrade- again, an important property for an energy store. It takes an small enzymatic nudge to do the job- something our proteins are very good at.

 Indeed, the weakness of the phosphate bond is its strong point, as it makes an excellent "leaving group". That means that it reacts enthusiastically, taking away two electrons when a nucleophilic attack is directed against its neighbor. Hydrolysis by water is the most common mode of attack, or by a target compound or protein that ends up with the phosphate group attached. The phosphate's negative charge is also beneficial, so that chemical intermediates to which it is attached are instantly given a charge, which confines them within the membrane. This can be a significant issue for the many small molecules of metabolic reaction chains.

Table from Westheimer, discussing the utility of phosphate for organic chemistry. Human chemists do not have enzymes at their disposal, so have to use much more caustic and active leaving groups than the chemists of molecular biology can.

But there is a problem from an evolutionary perspective, which is that phosphorous is hard to come by in geological terms. This is a debatable point, but many researchers contend that mineral phosphates are all highly insoluble, and have been since the Earth was formed. Phosphorous could thus be likened to gold- a precious and rare element that changes hands frequently as a medium of exchange, but whose actual abundance is exceedingly low. For example, our bodies are thought to break and reform over 400 pounds of ATP per day, but have only 0.1 pound on hand at any moment.

Whatever the geological case, one research group was inspired to look for possible pre-phosphorous chemistries in the early history of life. They propose that ATP and its phosphate-carrying colleagues may have been a later development in early biotic chemistry, though it was well-entrenched by the time of the last common ancestor of all life, abbreviated LUCA. They took a somewhat round-about route in their analysis, trolling through all the known reactions among existing organisms (KEGG) to find those which do not rely on phosphate. Then they tried to assemble from those as much of a basic metabolism as possible. What they came up with (a phosphate-free metabolism) is interesting.

This metabolism is highly dependent on thioesters, which place sulfur in the place of oxygen in bonds with carbon. The thioester bond is worth, in free energy of hydrolysis terms, about as much as the phosphate bond, but it is not as good a leaving group as phosphate, and does not reliably confer charge on its targets. This chemistry is still used in many biological reactions, however, by way of coenzyme A. With this chemistry, and with numerous metal-containing enzymes, they can access about 315 reactions and 260 metabolites of the roughly 700 metabolites of core metabolism- an impressive achievement, really. The set of reactions they come up with are biased towards those of core metabolism, those using iron and other metal cofactors, and those known to have been key to early life, and to those accessible to shorter genes and proteins with a minimal complement of amino acids.

Naturally, to pull in reactions from all sorts of odd and currently-existing bacteria to stand in for the  possibilities of early evolution is hazardous, but it does indicate that phosphate-free biology is conceivable, if inefficient and incomplete. But it is the geological debate about phosphorous / phosphate availability that needs to be resolved first before this issue becomes pertinant and interesting at all.

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Saturday, March 11, 2017

The Treacherous Invisibility of Sociality

We are dealing with phantasms, which makes drawing a line among them difficult.

It is easy to take potshots at science for its blinkered focus on the measurable and the concrete. How many people have pulled out the famous Shakespeare line about the many more things, poor Horatio, than are dreamt of in your philosophy? How many times does the newest research in social sciences tell us what anyone with common sense already knows? However, on the other side, the quest by softer sciences, like economics and ecology, to man up and drown themselves in math in order to satisfy their envy of the "hard" sciences. There are clearly conflicting emotions on the matter, which occasionally boil over into Trumpism and general anti-elitism.

But understanding by way of careful observation, useful simplification / reduction / schematization is what all scholarship and learning is about. One can't get around learning about something in detail if one wants to master it, either operationally or intellectually. The scientific method was revolutionary development, not only for science per se, but for philosophy and specifically for psychology. It expresses a skepticism of knowledge gained by theoretical, authoritarian, and armchair means, untethered from whatever the object purports to be, whether physics or biblical texts or history. Just as we suspect statements made by our current president when based on nothing, likewise we should suspect other claims lacking evidence of a rigorous, empirical kind.

But there is a deep problem, which is that our most important issues and forms of knowledge are social, not measurable and concrete. While science struggles to grasp social patterns and knowledge from its particular perspective- and not yet terribly successfully- those patterns are at the same time experienced richly in everyday life by everyone and portrayed with great variety and complexity in the arts. It is the core of drama- who knows what, who likes whom, and can I see through layers of deceit.  All of this is invisible in the conventional sense. It may be encoded somewhere in our brains, but the proper level of analysis is clearly not that of the neuron. As scientists, we are left with questionnaires, polls, and, generally, utter blindness when it comes to this most important apparatus of our lives.

Hard to read?

What hides, and what exposes, the social matrix? Language is the premier medium, of course, going far beyond the pheromones, grunts, dancing, and grooming of other animals. Blushing, facial expression, and eye direction, are a few more biological examples of other ways we externalize our social feelings. Yet there is great value in hiding feelings as well, whether out of politeness or deceit. Indirection, subtlety, puns, jokes, allegories, metaphors, a single look. The cues, even when present, are devilishly hard to read, which prompts theories about how sociality drove gains in human intelligence. So even on the social level, let alone the scientific level, it is hard to know what is going on beneath the surface, where sociality truly resides.

An inference of sociality, constructed in typical mid-20th century fashion.

The result of all this is that we are very enthusiastic inferrers and theorists. Conspiracy theories, truthers, birthers are some of the more extreme manifestations, but we all have to do a lot of reading between the lines just to survive as social beings. What is a soap opera but the carefully and gleefully managed reveal of social facts that are not, in timely fashion, apparent to the participants? Some people are more skilled at all this reading and inferring than others are. Extroverts enthusiastically wade into this murky unknown, while introverts regard it as hostile territory, and tend temperamentally to populate the scientific ranks, struggling to find certainty in an uncertain and largely invisible world.

What becomes treacherous about all this is the over-enthusiastic inference of things that are not there. On the social plane this can be over-sensitivity to slights and oversights. But it can also be religion- the natural inference of sociality to inanimate phenomena. Animism seems the most natural human condition, anthropomorphizing everything around us from insects to mountains, and putting ourselves into subtle social relations with it all. The rise of patriarchy seems to have prompted a massive shift from animistic patheism to father-centric monotheism. The theological object is no more real, however, for being consolidated and blown up out of all proportion. It is still an over-enthusiastic inference of sociality / personhood put on the void. Smarter theists have given up trying to explain particular aspects of reality via crackpot theology, such as electricity or evolution. Yet "everything" is still somehow fostered, created, or underpinned by this phantasm, much as prostate health is "supported" by the latest herbal supplement or hemeopathic nostrum.

What's the harm? On the social plane, over-inference leads to a lot of drama, but is quite finely tuned and bounded by actual, empirical, interactions (though our politically partisan echo chambers breake this model). It is how we evolved to deal with each other. On the philosophical plane, it has been disastrous, giving us centuries of bad ideas, intolerant theologies, and mis-directed energies. Think of all the monks and nuns praying away in their cloisters to non-existent deities for undeserving patrons. And today we are still living in a world at war over religious differences, all based on imaginary inferences created out of the template of our social assumptions and desires.

Saturday, March 4, 2017

Round and Round We Go, Making ATP

The mechanism of the proton energy pump that lies deep within, and gives us ATP.

One of the more elegant and dynamic structures in biology is that of the ATP synthase, which lies at the heart of the mitochondrion's conversion of its proton / electromotive gradient into ATP. This large enzyme is not static, but functions like a carousel, whirling around as it lets in groups of H+ ions. Naturally, it has been heavily studied to learn the secrets of why such motion is necessary, and how it works, in detail.

Basic view of ATP synthetase components. The lower complex (a,b,c, epsilon,) is  often called Fo, and is embedded in the membrane bilayer, which otherwise keeps H+ protons out. Top is the internal side (of the bacterium or mitochondrion) and bottom is the outside, where H+ has been pumped by the processes of oxidative phosphorylation. The c subunits comprise the spinning rotor, causing the gamma subunit, which reaches up into the F1 (alpha, beta), to crank the non-spinning F1 proteins through a series of shape changes that prompt them to synthesize ATP.

The primary product of mitochondiral respiration, which burns our food in a controlled way using oxygen, is a transmembrane proton gradient, which is a mechanism that mitochondria inherited from their free-living bacterial ancestors. While it may seem odd that pumping protons out of the cell into the vast outside is a way to efficiently store energy, it was the original battery technology, a charged state that can later be used by many other processes, like transporters that couple the energy-releasing import of H+ with the energy-using import of K+, (a symporter), or with the energy-using export of Na+ (and antiporter). Yes, life is all about chemistry!

ATP quickly became an important chemical currency for life, but only small amounts can be made directly from breaking up food molecules like glucose by glycolysis. Much more can be made by carefully tuning the respiratory chain to export protons (or import electrons) during the stepwise transformations of glucose to smaller molecules, and then later using that electrical / proton gradient for other needs such as making ATP.

Thus the ATP synthetase was born, but it was not born in a vacuum. Rather it seems to have been derived from prior structures that used protons to drive flagellar rotation. The tails of bacteria do not wave side to side, but rather rotate, which, given their particular semi-rigid structure, can drive bacteria forward. At the flagellar base is a rotating motor which lets in H+ ions as its energy source. This structure was evidently married with what seems to have originally been a DNA helicase- a donut-shaped ATP-using enzyme that travels along DNA, prying open the double-helix. Such enzymes are necessary during DNA replication and meiosis, of which at least replication was an ancient process. The ATP-using character of this helicase was reversed to be ATP-generating in its new setting, which is biochemically easier than it seems. Indeed, the whole ATP synthetase can still today run in reverse to use up ATP when needed.

More detailed representation of the ATP synthetase. The plasma membrane is in yellow, inside the cell (or mitochondrion) is above, and outside is below. The outside has a higher concentration of tiny water triads, which represent H3O+, or H2O plus a proton. They dock to the blue transmembrane portion (Fo) of the enzyme, as shown in green and red at the key interface. The protons are handed off to coordinate with portions of the protein in highly regulated fasion. Above, ADP comes into the synthetase part of the enzyme (F1), gets a phosphate group added (yellow and red) to become ATP. 

Two videos of this process are linked above. #2 is accompanied by a great submersible soundtrack, and shows greater detail for the ATP synthesis mechanism. And a video from the paper discussed has even higher detail, showing particularly the extensive structural reshaping that goes on within the F1 subunits that are making ATP. That mechanism could be the subject of another post.

The mechanical details are that H+ is let in only at the interface of the rotating (blue) and stationary (red) parts of the Fo membrane portion of the enzyme. It is allowed in to bind only at one site per segment (green dots), which then rotate around and eventually come back to another part of the stationary part where the H+ is finally let out via a different channel, into the cell. This specific directionality of binding and release is a sort of ratchet which lets the chemical energy in the H+ gradient drive rotational motion.

This energy is then coupled to the second element, the top (F1) complex, where the six red/pink ATP synthase subunits surround the blue shaft which comes up from the rotating Fo component. While those subunits are held stably by the orange stator element at the outside, the blue shaft is like a washing machine rotor that wrenches around, distorting each of the ATP synthase subunits in turn in ways that induce them to carry out the reaction of adding a phosphate group to ADP to form ATP.

A recent paper describes new structural and mutational studies on this enzyme complex, to look for some further mechanistic details. It used primarily cryo-electron micoscopy, which is sufficient to resolve shapes of helices in proteins, though not detailed atomic locations. Yet one can combine this with mutations, prior X-ray structural studies, selective inhibitors, and other modeling to make some interesting inferences. The key one which these authors make is that there is a critical arginine (R) amino acid in the (a) subunit, or the static red part of Fo above (orange in the diagram below), which seems to be the key to H+ conveyance. This amino acid tends to be positively charged, so it would readily bind with an aspartic acids coming along as part of the (c) subunits, popping off their bound hydrogens. It is also genetically essential, as mutations are lethal.

The proposal is that this arginine is specially exposed to the internal side of the membrane, via a channel, (curved arrow leading upward to the cytoplasm in the diagram below), and also positioned such that it can latch onto aspartic acids the blue (c) subunits after rotation. These aspartic acids (D) are carrying the protons that came in via the complementary channel from below (outside) before they started their trip around the carousel. All that time, the membrane has protected those protons from displacement by other chemicals, such as water, other proteins and amino acids, etc.

Image of the proton-conducting interface within the Fo subunit of the ATP synthase. The static (a) subunit is portrayed in orange at rear, while the passing set of c subunits (there are ten of them) are going by in front as a pink band, though dimmed for clarity. The c subunits are progressing from right to left, with hydrogens coordinated on the key aspartate (D) residue #61 on each (c) subunit of the rotating set. As they pass, they are captured by the arginine (R) residue on the static A subunit and released in the only direction possible, up the chute into the cell. Immediately thereafter, the c subunit band passes to another channel where protons can load up again from the external solution.

Those protons are portrayed above by the band containing "c D61", indicating an aspartic acid (D) on a (c) subunit's 61st coded position, whose proton could be displaced by the arginine on the other (a) subunit as it is going by. The gray band of (c) subunits is travelling towards the left, so the idea is that aspartic acid-coordinated protons coming in from the right hit the blue arginine first, where the aspartate and arginine, with their different and complementary charges, bind directly and immediately, releasing the coordinated H+ which then shoots right up the channel to the cytoplasm. At the very next position, protons come in from the outside (periplasm) to bind to the just-vacated D61 spot on the (c) subunit. It is an elegant and very spare, atomic and electrochemical ratchet.

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