Just like balsamic vinegar and olive oil when separated together into distinct phases, the phase separation of sticky droplets in the crowded abdomen of a cell can concentrate certain biomolecules and speed up biochemical reactions. However, it has been difficult to determine the importance of liquid-phase separation (technically named, condensers) in biologically relevant phenomena such as fluctuations in molecular signals.
People generally try to assess the importance of these droplets by chopping or altering the molecules that are an integral part of their composition. They see, as the droplets disintegrate, the cell breaks, and they concluded that suppressors are important, said Liam Holt, Ph.D., associate professor at the Institute of Genetics at NYU Langone Health. “But there are two reasons why a cell breaks — either sticky droplets or a molecule that has been altered or shredded, which is crucial to the cell.”
Liquid droplets in cells are a complex and heterogeneous mixture in which, in addition to other functions, certain molecular components play a critical structural role. The study of simpler synthetic droplets presents an opportunity to better understand general principles through the systematic variations of individual parameters.
Holt believes that building is a better way to discover biological principles than to smash things. A study was published by his team in molecular cell (Intense phase signaling can expand kinase specificity and respond to molecular crowding out.) uses synthetic biology approaches to build simple liquid capacitors to investigate their role in influencing phosphorylation – a trick cells use to coordinate information. Holt’s study shows that capacitors enable new signaling mechanisms and can create sensors that respond to biophysical changes in the cell’s internal environment.
This work revealed several important angles for the potential roles of biological suppressors in cellular signaling. The idea that such intense signals acting on flexible or disorganized scaffolds can act as sensors that respond to changes in the cellular environment, said Vladimir Oversky, PhD, PhD, a biophysicist and professor in the USF Health School of Molecular Medicine. in Tampa, Florida. (Uversky was not involved in the study.)
Sang and colleagues set out an exciting exploration of the poorly understood role of intense phase signaling and the effect of phase separation on intracellular molecular crowding and cellular interaction dynamics. Their innovative research confirms that kinase-substrate contacts are accelerated within capacitors and are regulated in part by phosphorylation,” said physician and evolutionary biologist, William Miller Jr., author of: Bioverse. “The importance of human health is noteworthy. It reveals a link between intracellular hyperphosphorylation and tau neurofibrillary tangles that are thought to be responsible for Alzheimer’s disease.” (Miller was not involved in the study.)
Holt’s team used two droplet condensation systems in their investigations – one fully synthetic and one inspired by a real biological system found in human cells. These systems originated from two collaborations.
“We spent time with Mike Rosen, Ph.D., from US Southwest University, who is a pioneer in the field, and others from around the world including Tony Hyman, Ron Vale and Amy Gladfelter, at Woods Hole Marine Biology Laboratories. This is where we started this project about five years ago,” said Holt.
One of the drops that Holt used in the current study was inspired by a simplified system developed by Michael Rosen, PhD, professor and chair of the Department of Biophysics at US Southwestern University. Rosen’s droplets, in turn, were inspired by a biological system called PML nucleosomes – condensers of RNA and protein involved in splicing, transcription, viral defense and apoptosis. One of the major proteins in PML nucleosomes is an enzyme that labels protein substrates called SUMO (small Ubiquitin-like modifier) which binds to SIMs (SUMO interactive elements) to form a network.
Mike’s hypothesis was that the network of interactions between SUMO and SIM would be sufficient for capacitors. This is true. “Strings of SUMO and SIM domains on peptide-shaped networks form condensers,” Holt said. Holt’s SUMO-SIM-inspired flexible condenser is much simpler than the Rosen condenser with only one protein making up the condenser.
Other, more rigid capacitors used by Holt’s team were more synthetic, generated as a result of an ongoing collaboration with Emanuel Levy, Ph.D., and scientists Meta Heidenreich, Ph.D. at the Weizmann Institute in Israel. They developed a condensed, highly organized system containing dimer, tetragonal, or hexagonal proteins that formed extended solid shapes, similar to crystal lattices.
“These do not work well as triggers and we are investigating the causes in our paper,” Holt said.
Scaffold and client proteins
How do you deal with entities that have no names? While working at Woods Hole Marine Biology Laboratories in the summer of 2017, Holt and colleagues invented a new nomenclature that aided in their discussions about droplets, and they use these new terms in their current research.
They use the term “scaffold” for the protein that forms the condenser. For example, the SUMO-SIM molecular network is the mainstay in flexible Holt capacitors. The scaffold absorbs the components added to the reaction crucible of a drop of liquid.
On the other hand, the “clients” are the proteins that are absorbed into the crucible and whose behaviors within the crucible may change. In Holt’s current study, clients are kinases – enzymes that catalyze phosphate binding to protein substrates. The phosphorylation of lipids and protein kinase-directed proteins is one of the main ways cells compute information.
“[In this study] We’re looking at how this information flows when customers are in the condenser versus when they aren’t,” said Holt.
To understand the factors that increase phosphorylation in repressors, Holt’s team altered the properties of both scaffold proteins and client proteins. This revealed that in addition to increasing client concentration, the availability of more client-binding sites within the capacitors and the flexibility of the scaffolds were factors that significantly affected the rate of phosphorylation within the capacitors.
Reaction rates in capacitors
We generally found increased activity and expanded specificity of the kinase [in condensates]The authors note. Holt’s team found an increased rate of phosphorylation in some synthetic suppressors that could be partially attributed to the increased concentration of kinases and its substrates. However, this is not the whole story.
“Increasing the concentration of the reactants increases the rates of the biochemical reaction, which then become saturated. But that doesn’t explain all the effects we’re seeing,” Holt said.
The presence of two different types of condensers, one rigid and one flexible, was useful in analyzing other possible explanations for the increased rate of phosphorylation in condensers.
“On stiffer capacitors, we can increase customer concentration a lot and not see an increase in certain types of phosphorylation. More flexible and more efficient SUMO-SIM capacitors with the same concentrations,” Holt said.
This indicates that it is not only the concentration of the clients that affects the phosphorylation. Holt’s team found that other factors that affected the phosphorylation of the capacitors included the number of binding sites available and whether they could aggregate.
Increased concentrations and the resulting molecular crowding out in the condensers may increase the rate of phosphorylation by increasing the proximity of the substrate-enzyme pairs that interact but may also mask the enzymatic active sites resulting in decreased activity.
“We found that phosphorylation within capacitors can respond to molecular crowding out, thus creating a biophysical sensor,” the authors note.
In flexible capacitors, molecular crowding tends to increase the rate of phosphorylation, while the opposite occurs in solid capacitors. Holt proposes some hypotheses to explain these observations, which at this point remain speculative because such hypotheses are difficult to test due to the technical limitations of microscopy.
“While we don’t fully understand what’s going on, one possibility is that molecular crowding compresses the elastic capacitors, pushing the enzyme and substrate together. It’s hard to compress solids. They don’t feel that effect very much,” Holt said. Crowding can make things stick together. Under normal conditions in solid capacitors, the substrate and kinase flow freely and can find each other. If you force them to stick more firmly at points in the crucible, it can push them further apart resulting in lower reaction rates.”
New pathogens for Alzheimer’s disease
Finally, Holt’s team studied a natural condenser associated with Alzheimer’s disease, to check whether their observations in synthetic systems are also true in a biological context.
In Alzheimer’s disease, tau proteins acquire excessive phosphate at certain residues causing them to form the frequently observed tangles at autopsy in neurons of Alzheimer’s disease patients. Previous studies showed that tau can be transformed into a liquid phase separated state. In disease, phosphorylated tau accumulates.
Holt said, “We’ve looked at tau when it diffuses into solution versus when it separates into a liquid droplet but not in the aggregate state. It’s not known if tau dissociates into liquid droplets in normal cells, but it’s likely to some extent.” .
To analyze tau in solution and condensate, Holt collaborated with NMR spectrologist Markus Zwicketter, Ph.D., professor at the University of Göttingen in Germany. Nuclear magnetic resonance spectroscopy or NMR is a technique that can provide detailed information about molecular structures, dynamics, reaction states, and chemical environments in solution by analyzing local magnetic fields around atomic nuclei obtained when solutions pulse with radio waves.
“In the intense droplets, the phosphorylation of tau sites associated with Alzheimer’s disease was three times faster,” Holt said.
Holt’s team showed that phosphorylation of tau by CDK2 (cyclin-dependent kinase 2) was increased in the intense phase at sites associated with Alzheimer’s disease. This observation provides an intriguing gateway to a potential new disease mechanism for Alzheimer’s disease.
Holt plans to follow up on the results of this study. The tau experiments presented in this paper were all in vitro. “We would like to use NMR to see if crowding alters tau condensation and whether that affects phosphorylation in the cell and can lead to neuronal death,” Holt said.
“We started looking at neurons in the lab and started studying whether molecular crowding could lead to neurodegeneration,” Holt said.
Holt’s team also hopes that their findings will advance the design of other cellular capacitors that respond to physical forces and lead to the application of these systems as sensors capable of detecting malignancies.