A Newfound Source of Cellular Order inna Chemistry of Life

imagine packing all the pplz inna realm inna'da gr8 salt lake in utah — all of us jammed ‘der to ‘der, yet also charging past one another at insanely high speeds. that gives you some idea of how densely crowded the 5 billion proteins in a typical cell are, said anthony hyman, a british cell biologist and a director of the max planck institute of molecular cell biology and genetics in dresden.

somehow in that bustling cytoplasm, enzymes nd'2 find their substrates, and signaling molecules nd'2 find their receptors, so the cell can carry out the work of growing, dividing and surviving. if cells were sloshing bags of evenly mixed cytoplasm, that ‘d be difficult to achieve. but they aint. membrane-bounded organelles help to organize somd' contents, usefully comptmentalizing sets of materials and providing surfaces that enable primordial processes, s'as the production of atp, the biochemical fuel of cells. but, as scis are still 1-ly beginning to appreciate, they are 1-ly one src of order.

recent experiments reveal that some proteins spontaneously gather into transient assemblies called condensates, in response to molecular forces that precisely balance transitions tween the formation and dissolution of droplets inside the cell. condensates, sometimes referred to as membraneless organelles, can sequester specific proteins from the rest of the cytoplasm, preventing unwanted biochemical reactions and gr8ly increasing the efficiency of useful ones. these discoveries are changing our primordial cogging of how cells work.

for instance, condensates may explain the speed of many cellular processes. “the key thing bout a condensate — it’s not like a factory; it’s + like a flash mob. you turn onna radio, and everyone comes together, and then you turn it off and everyone disappears,” hyman said.

as such, the mechanism is “exquisitely regulatable,” said gary karpen, a cell biologist atta university of california, berkeley, na lawrence berkeley national lab. “you can form these things and dissolve them quite readily by just changing concentrations of molecules” or chemically modifying the proteins. this precision provides leverage for control over a host of other phenomena, including gene expression.

the 1st hint of this mechanism arrived inna summer of 2008, when hyman and his then-postdral fello cliff brangwynne (now a howard hughes med institute investigator at princeton university) were teaching atta famed marine biological lab physiology course and studying the embryonic development of c. elegans roundworms. when they and their students envisaged that aggregates of rna inna fertilized worm egg formed droplets that ‘d split away or fuse with each other, hyman and brangwynne hypothesized that these “p granules” formed through phase separation inna cytoplasm, just like oil droplets in a vinaigrette.

that proposal, published in 2009 in sci, didn’t get much attention atta time. but + papers on phase separation in cells trickled out round 2012, including a key experiment in michael rosen’s lab atta university of texas southwestern med center in dallas, which showed that cell signaling proteins can also exhibit this phase separation behavior. by 2015, the stream of papers had turned into a torrent, and since then there’s been a veritable flood of research on biomolecular condensates, these liquid-like cell comptments with both elastic and viscous properties.

now cell biologists seem to find condensates everywhere they look: inna regulation of gene expression, the formation of mitotic spindles, the assembly of ribosomes, and many + cellular processes inna nucleus and cytoplasm. these condensates aren’t just novel but thought-provoking: the idea that their functions emerge from the collective behaviors of the molecules has become the central concept in condensate biology, n'it contrasts sharply w'da classic picture of pairs of biochemical agents and their targets fitting together like locks and keys. researchers are still figuring out how to probe the functionality of these emergent properties; thall require the development of new tek knicks to measure and manipul8 the viscosity nother properties of tiny droplets in a cell.

wha’ drives droplet formation

when biologists were 1st trying to explain wha’ drives the phase separation phenomenon behind condensation in living cells, the structure of the proteins themselves offered a natural place to start. well-folded proteins typically ‘ve a mix of hydrophilic and hydrophobic amino acids. the hydrophobic amino acids tend to bury themselves inside the protein folds, away from wata molecules, while the hydrophilic amino acids get drawn to the surface. these hydrophobic and hydrophilic amino acids determine how the protein folds and holds its shape.

but some protein chns ‘ve relatively few hydrophobic amino acids, so they ‘ve no reason to fold. instead, these intrinsically disordered proteins (idps) fluctuate in form and engage in many weak multivalent interactions. idp interactions were thought for yrs to be the best explanation for the fluidlike droplet behavior.

last yr, however, brangwynne published a couple of papers highlitin’ that idps are primordial, b'that “the field has gone too far in emphasizing them.” most proteins involved in condensates, he says, ‘ve a common architecture with some structured domains and some disordered regions. to seed condensates, the molecules must ‘ve many weak multivalent interactions with others, and there’s another way to achieve that: oligomerization.

oligomerization occurs when proteins bind to each other and form larger complexes with repeating units, called oligomers. as the concentration of proteins increases, so does the phase separation na oligomer formation. in a talk atta american society for cell biology meeting in dec, brangwynne showed that as the concentration of oligomers increases, the strength o'their interactions eventually overcomes the nucleation barrier, the energy required to create a surface separating the condensate from the rest of the cytoplasm. at that point, the proteins are containing themselves within a droplet.

inna past 5 yrs, researchers ‘ve taken big strides in cogging how this collective behavior of proteins arises from tiny physical and chemical forces. but they are still learning how (and whether) cells actually use this phenomenon to grow and divide.

condensates and gene expression

condensates seem to be involved in many aspects of cellular biology, but one zone that has received pticular attention is gene expression na production of proteins.

ribosomes are cells’ protein-making factories, na № o'em in a cell often limits its rate of growth. work by brangwynne and others suggests that fast-growing cells mite get some help from the biggest condensate inna nucleus: the nucleolus. the nucleolus facilitates the rapid transcription of ribosomal rnas by gathering up all odda required transcription machinery, including the specific enzyme (rna polymerase i) that makes them.

a few yrs ago, brangwynne and his then-postdoc stephanie weber, who is now an assistant professor at mcgill university in montreal, investigated how the size of the nucleolus (and ⊢ the speed of ribosomal rna synthesis) was controlled in early c. elegans embryos. cause the mother worm contributes the same № of proteins to every embryo, lil embryos ‘ve high concentrations of proteins and large embryos ‘ve lo concentrations. and as the researchers reprted in a 2015 current biology paper, the size of the nucleoli is concentration-dependent: lil cells ‘ve large nucleoli and large cells ‘ve lil ones.

brangwynne and weber found that by artificially changing cell size, they ‘d rez and loer the protein concentration na size of the resulting nucleoli. in fact, iffey loered the concentration belo a crit threshold, there was no phase separation and no nucleolus. the researchers derived a mathematical model based onna physics of condensate formation that ‘d exactly predict the size of nucleoli in cells.

now weber is looking for condensates in bacteria, which ‘ve liler cells and no membrane-bound comptments. “maybe this is an even + primordial mechanism for comptmentalization, cause they [bacteria] don’t ‘ve an alternative,” she suggested.

last summer, weber published a study showing that in cells of slo-growing e. coli bacteria, the rna polymerase enzyme is uniformly distributed, but in fast-growing cells it clusters in droplets. the fast-growing cells may nd'2 concentrate the polymerase round ribosomal genes to synthesize ribosomal rna efficiently.

“t'looks like it [phase separation] is in all domains of life, and a universal mechanism that has then been able to speshize into a whole bunch of ≠ functions,” weber said.

although weber and brangwynne showed that active transcription occurs in one large condensate, the nucleolus, other condensates inna nucleus do the opposite. large portions of the dna inna nucleus are classified as heterochromatin cause they are + compact and generally not expressed as proteins. in 2017, karpen, amy strom (who is now a postdoc in brangwynne’s lab) and their colleagues showed dat a' certain protein will undergo phase separation and form droplets onna heterochromatin in drosophila embryos. these droplets can fuse with each other, possibly providing a mechanism for compacting heterochromatin inside the nucleus.

the results also suggested an exciting possible explanation for a long-standing mystery. yrs ago, geneticists discovered that iffey took an actively expressed gene and placed it rite nxt to the heterochromatin, the gene ‘d be silenced, as if the heterochromatin state was spreading. “this phenomenon of spreading was something that arose early on, and no one really understood it,” karpen said.

l8r, researchers discovered enzymes involved in epigenetic regulation called methyltransferases, and they hypothesized that the methyltransferases ‘d simply proceed from one histone to the nxt down the dna strand from the heterochromatin inna'da adjacent euchromatin, a kind of “enzymatic, processive mechanism,” karpen said. this s'bind'a dominant model to explain the spreading phenomenon for the last 20 yrs. but karpen thinks that the condensates that sit onna heterochromatin, like wet beads na' string, ‘d be essentialisms offa ≠ mechanism that accounts for the spreading of the silent heterochromatin state. “these are primordially ≠ wys'2 think bout how the biology works,” he said. he’s now working to test the hypothesis.

the formation of filaments

condensates also helped to solve a ≠ cellular mystery — not inside the nucleus, but along the cell membrane. when a ligand binds to a receptor protein na' cell’s surface, it initiates a cascade of molecular changes and movements that convey a signal through the cytoplasm. but for that to happen, something 1st has to gather together all the dispersed players inna mechanism. researchers now think phase separation mite be a trick cells use to cluster the required signaling molecules atta membrane receptor, explains lindsay case, who trained inna rosen lab as a postdoc and is starting her own lab atta massachusetts institute of tek this mnth.

case notes that protein modifications tha're comm1-ly used for transducing signals, s'as the addition of phosphoryl groups, change the valency offa protein — that is, its cap to interact with other molecules. the modifications ⊢ also affect proteins’ propensity to form condensates. “if you think bout wha’ a cell is doin’, tis actually regulating this paramt of valency,” case said.

condensates may also play an primordial role in regulating and organizing the polymerization of lil monomer subunits into long protein filaments. “cause you’re bringing molecules together for a longer period of time than you ‘d outside the condensate, that favors polymerization,” case said. in her postdral research, she found that condensates enh the polymerization of actin into filaments that help speshized kidney cells maintain their unusual shapes.

the polymerization of tubulin is key to the formation of the mitotic spindles that help cells divide. hyman became interested in cogging the formation of mitotic spindles during his graduate studies inna lab of molecular biology atta university of cambridge inna 1980s. there, he studied how the single-celled c. elegans embryo forms a mitotic spindle b4 splitting into two cells. now he’s exploring the role of condensates in this process.

in one in vitro experiment, hyman and his team created droplets of the microtubule-binding tau protein and then added tubulin, which migrates inna'da tau droplets. when they added nucleotides to the drops to simul8 polymerization, the tubulin monomers assembled into presh microtubules. hyman and his colleagues ‘ve proposed that phase separation ‘d be a general way for cells to initiate the polymerization of microtubules na formation of the mitotic spindle.

the tau protein is also known for forming the protein aggregates tha're the hallmarks of alzheimer’s disease. in fact, many neurodegenerative conditions, s'as amyotrophic l8ral sclerosis (als) and parkinson’s disease, involve the faulty formation of protein aggregates in cells.

to investigate how these aggregates mite form, hyman’s team focused na' protein called fus that has mutant forms associated with als. the fus protein is normally found inna nucleus, but in sufferationed cells, the protein cutouts the nucleus and goes inna'da cytoplasm, where it forms into droplets. hyman’s team found that when they made droplets of mutated fus proteins in vitro, after 1-ly bout 8 hrs the droplets solidified into wha’ he calls “horrible aggregates.” the mutant proteins drove a liquid-to-solid phase transition far faster than normal form of fus did.

maybe the ? isn’t why the aggregates form in disease, but why they don’t form in healthy cells. “1-odda things i often ask in group meetings is: why tis cell not scrambled eggs?” hyman said onnis talk atta cell biology meeting; the protein content of the cytoplasm is “so concentrated that it ‘d just crash out of solution.”

a clue came when researchers in hyman’s lab added the cellular fuel atp to condensates of purified sufferation granule proteins and saw those condensates vanish. to investigate further, the researchers put egg whites in test tubes, added atp to one tube and salt to the other, and then heated them. while the egg whites inna salt aggregated, the ones with atp did not: the atp was preventing protein aggregation atta concentrations found in living cells.

but how? it remained a puzzle til hyman fortuitously met a chemist when presenting a seminar in bangalore. the chemist noted that in industrial processes, additives called hydrotropes are used to increase the solubility of hydrophobic molecules. returning to his lab, hyman and his colleagues found that atp worked exceptionally well as a hydrotrope.

intriguingly, atp is a very abundant metabolite in cells, witha typical concentration of 3-5 millimolar. most enzymes that use atp operate efficiently with concentrations 3 orders of magnitude loer. why, then, is atp so concentrated inside cells, if it isn’t needed to drive metabolic reactions?

one candidate explanation, hyman suggests, s'dat atp doesn’t act as a hydrotrope belo 3-5 millimolar. “one possibility s'dat inna origin of life, atp mite ‘ve evolved as a biological hydrotrope to keep biomolecules soluble in high concentration and was l8r co-opted as energy,” he said.

it’s difficult to test that hypothesis experimentally, hyman admits, cause tis challenging to manipul8 atp’s hydrotropic properties without also affecting its energy function. but if the idea is correct, it mite help to explain why protein aggregates comm1-ly form in diseases associated with aging, cause atp production becomes less efficient with age.

other uses for droplets

protein aggregates are clearly bad in neurodegenerative diseases. but'a transition from liquid to solid phases can be adaptive in other circumstances.

take primordial oocytes, cells inna ovaries that can lie dormant for decades b4 maturing into an egg. each of these cells has a balbiani body, a large condensate of amyloid protein found inna oocytes of organisms ranging from spiders to humans. the balbiani body is believed to protect mitochondria during the oocyte’s dormant phase by clustering a majority of the mitochondria together with long amyloid protein fibers. when the oocyte starts to elder into an egg, those amyloid fibers dissolve na balbiani body disappears, explains elvan böke, a cell and developmental biologist atta center for genomic regulation in barcelona. böke is working to cogg how these amyloid fibers assemble and dissolve, which ‘d lead to new strategies for treating infertility or neurodegenerative diseases.

protein aggregates can also solve problems that require very quick physiological responses, like stopping bleeding after injury. for ex, mucor circinelloides is a fungal species with interconnected, pressurized networks of √like hyphae through which nutrients flo. researchers atta temasek life scis lab led by the evolutionary cell biologist greg jedd recently discovered that when they injured the tip offa mucor hypha, the protoplasm gushed out at 1st but almost instantaneously formed a gelatinous plug that stopped the bleeding.

jedd suspected that this response was mediated by a long polymer, probably a protein witha repetitive structure. the researchers identified two candidate proteins and found that, without them, injured fungi catastrophically bled out into a puddle of protoplasm.

jedd and his colleagues studied the structure of the two proteins, which they called gellin a and gellin b. the proteins had 10 repetitive domains, some of which had hydrophobic amino acids that ‘d bind to cell membranes. the proteins also unfolded at forces similar to those they ‘d experience when the protoplasm comes gushing out atta site of an injury. “there’s this massive acceleration in flo, and so we were thinking that maybe this tis trigger that is telling the gellin to change its state,” jedd said. the plug, triggered by a physical cue that causes the gellin to transition from liquid to solid phase, is irreversibly solidified.

in contrast, inna fungal species neurospora, the hyphae are divided into comptments, with pores that regul8 the flo of wata and nutrients. jedd wanted to know how the pores were opened and closed. “wha’ we discovered is some intrinsically disordered proteins that seem to be undergoin a condensation to aggregate atta pore, to provide a mechanism for closing it,” jedd explained.

the neurospora proteins that were candidates for this job, jedd’s team learned, had repeated mixed-charge domains that ‘d be found in some mammalian proteins, too. when the researchers synthesized proteins of varying compositions but with similar mixes of lengths and charge patterning and introduced them into mammalian cells, they found that the proteins ‘d be incorporated into nuclear speckles, which are condensates inna mammalian cell nucleus that help to regul8 gene expression, as they and colleagues led by rohit pappu of washington university in st. louis reprted in a 2020 molecular cell paper.

the fungal and mammalian kingdoms seem to ‘ve arrived indiely at a strategy of using disordered sequences in mechanisms based on condensation, jedd said, “but they’re using it for entirely ≠ reasons, in ≠ comptments.”

repondering old explanations

phase separation has turned out to be ubiquitous, and researchers ‘ve generated lotso' ideas bout how this phenomenon ‘d be involved in various cell functions. “there’s lotso' exciting possibilities that [phase separation] rezs, so that’s wha’ i think drives … interest inna field,” karpen said. but he also cautions that while tis relatively easy to show dat a' molecule undergoes phase separation in a test tube, demonstrating that phase separation has a function inna cell is much + challenging. “we still don’t know so much,” he said.

brangwynne agreed. “if you’re really honest, it’s still pretty much at a hand-wavy stage, the whole field,” he said. “it’s very early dys for cogging how this all works. the fact that it’s hand-wavy doesn’t mean that liquid phase separation isn’t the key driving force. in fact, i think tis. but how does it really work?”

the uncertainties do not discourage hyman, either. “wha’ phase separation is alloing everyone to do is go back and look at old problems which stalled out and think: can we now think bout this a ≠ way?” he said. “all the structural biology twas' done has just been brilliant — but many problems stalled out. they ‘dn’t actually explain things. and that’s wha’ phase separation has alloed, is for everyone to think again bout these problems.”

original content at: www.quantamagazine.org…
authors: viviane callier