Membrane-less Organelles and Phase Separation Flashcards
membrane-bound organelles
-cells have come up with ways to compartmentalize reactions
-ER, mitochondria, lysosome
membrane-less organelles (MLOs)
-discovered early on before membrane-bound organelles
-organelles in cells not enclosed by membranes
–> many different MLOs in the cell
–> nucleolus inside the nculeus, DNA damage repair foci
MLOs have liquid-like properties
-P granules in C. elegans that are able to flow on the nucleus if you squeeze on them
–> fuse and form a larger structure that look like liquid droplets
-in the frog egg extract system, if you see 2 ginormous nucleoli that are typically found in the frog egg and if they’re close to each other, they can fuse and form a larger, spherical structure
–> indicates that these and others with MLO nucleolus have liquid-like properties
rapid diffusion of components within MLOs
-use light to make perturbations and watch dynamics of molecules
-people use FRAP (fluorescence recovery after photobleaching)
FRAP
-bleaching on a fixed sample, you do not see fluorescence recovery since everything is stationary
-if you do FRAP on live sample, you can see how quickly fluorescence recovers from bleach point into stationary endpoint
FRAP to sutdy the dynamics of material within YAP condensates
-YAP is a transcription coactivator that is important for cell differentiation and cancer
-found that when YAP fuses GFP, we can see YAP forms these dynamic condensates while actively signaling
-using FRAP with high intensity light, you can see that seconds after bleaching, YAP fluorescent intensity recovers –> indicates that this is a really dynamic, separate entity b/c fluorescence is able to diffuse into this structure rapidly after bleaching and it’s a dynamic structure
MLOs are formed by weak, multivalent interactions
-when we talk about liquid, we contrast it with gases and solids
-liquids have transient bonds- constantly forming and un-forming to give dynamic structure
-phase separation/MLO formation is likely driven by bond energy-enthalpy that counteracts increase in entropy (one seems to balance the other for balanced effect on overall free energy of the system)
-entropy- more space in this system but if they rapidly coalesce into a defined structure that’s a bond energy (enthalpy) process
-we know that these are NOT high affinity macromolecular complexes that rely on interactions with fixed ratio of molecules
-we know that these are not very stable with fixed stoichiometry
weak, multivalent interactions driven by multivalent proteins
-in a test tube, they made synthetic, structured domains between SH3 and PRM –> SH3 and PRM mediate specific, weak interactions but if you repeat them many times, you can see them forming droplet-like structures
-repeated, structured interactions are able to form MLOs
weak, multivalent interactions driven by disordered proteins
-intrinsically disordered regions inside proteins are heavily implicated in forming MLOs
-these are proteins that do not form uniquely defined 3D structures
-by mixing disordered domains, they’re able to form liquid-like structures
molecular forces implicated in MLO formation
-almost all kinds of AA interactions might be involved in forming MLOs
Ex. cation-pi interactions and coiled-coil interactions are able to mediate condensate formation
-weak, multivalent interaction is overarching theme
phase separation
-might be phase transition occurring
-proteins that are diffusely localized inside a cell, we might imagine this as gas-like phase –> as the [] increases or specific perturbations occur, they undergo gas to liquid transition and form MLOs
-if you further increase your [] or go to a different perturbation, they can also enter a solid-like phase
–> this is how people think MLOs form in vitro and cells
what are potential benefits of forming MLOs by phase separation, compared to membrane-bound organelles that are enclosed by phospholipids?
-helpful for free diffusion of proteins and molecules without worrying about transport through membranes
-diffusion is rapid
–> not all unimpeded diffusion –> still have surface tension that’s trying to keep things inside liquid phase
functions of phase separation
-phase separation compartment- if they can specifically bring together these enzymes and substrates, they [] molecules inside the same space and accelerate these reactions
-can also act as sequestration by putting molecule away from where reaction is occurring
-they might have any role in reactions- structure can be organizational hub to form structure
biomolecular condensates
-compartment inside cell that lacks surrounding membranes and formed by assembly of proteins and or nucleic acids with non-fixed ratios (non-stoichiometry)
-can contain traditional MLOs- nuclear speckles, nucleolus
-other MLOs include synapses with proteins that form membrane-less entities that retain synaptic vesicles in pre-synaptic space and prevent their release but when AP comes, they’ll get phosphorylated and disassemble and release vesicles
dysregulation of phase separation leads to diseases
-liquid –> solid transition happens when liquid-like property decreases and forms solid structure
Ex. neurodegenerative diseases- FOS, TDP-43 undergo this transition and form aggregates and peopel think this underlies plaque formation
Ex. cancer- various condensates in cells might be implicated in cnacer like oncoproteins that are involved in condensate formation inside the nucleus, which might driver higher gene activity involved in cancer oncogenes
intranuclear MLOs
- nucleolus
- nuclear speckles
- transcription hubs
nucleolus
-largest MLO inside the nucleus
-makes ribosomal components and organized around DNA in genomic sites called nucleolar organizing regions (NORs)
-seeded around repeated DNA structure to form large component
-contains sub-compartments- whole nucleolus contains 3 layers that are separate phases from each other
-believed that sub compartment of nucleolus is important for the sequential processing of nascent ribosomal transcripts
-they’re made in the central compartment then diffuse out to sequential sub layers to do their different processing
nuclear speckles
-exist in interchromosomal space- we know the nucleus has a lot of DNA but there’s space between the DNA
-usually considered storage site for splicing molecules but these active genes can come and associate with these compartments –> access the stored RNA modification, splicing compartments
transcription hub
condensates are shown inside the nucleus that are considered transcription factories and are able to [] specific co-activators and TFs in this region –> leads to higher gene expression of genes when DNAs also in this region
cytoplasmic MLO
stress granule
cytoplasmic MLO- stress granule
-usually do not see them in normal functioning cells but when cells experience stress-like toxins or temperature changes
-G3BP1- major component of stress granules- diffuse in cytoplasm prior to stress but after treating with arsonite, they form distinct compartments in the cytoplasm
-they contain most of the RNAs and proteins and shut down translation process and protect them from separate entities
-maintain cell functions when they’re stressed but also essential to pathogenesis of NDs b/c we know that almost all of the proteins implicated in these diseases are resident stress granule components
why are MLOs NOT growing larger and larger inside the cell if they’re formed from phase separation?
assume that the interior of the cell is not an open space –> confinements in cell that restrict the growth of MLOs
what are the regulations of MLOs in the complex environment of the cell?
- confinement
- ATP-dependent processes
- PTMs
- mutations
confinements within a cell
-inside a nucleus- there’s a large volume taken up by chromatin that restricts MLO growth and fusion
Ex. when octodroplets are formed at this heterochromatin region, they have a harder time growing –> DNA has large influence on growth of condensates
-cytoplasm has many compartments that can restrict the growth of condensates and there’s actin filaments with cytoskeletons
ATP-dependent processes
-microtubule-cytoskeleton- important for fusion and fissionn of stress granule
-Dynein on microtubules can fuse and link with stress granule components –> leads to dissolution of mature stress granule
PTMs
-phosphorylation has both negative and positive effects on MLO formation
-negative- dual specificity with kinase 3 (DYRK3)- can disassemble MLOs during mitosis- in mitosis, if the cells want to distribute their MLOs in 2 daughter cells evenly, they could arrange them equally or disassemble them and diffuse them to be re-formed
-positive- tau phosphorylation- major protein in neurodegeneration- phosphorylation of tau can lead to more aggregate-like state- pushes further in phase separation graph
mutations
-can occur in domains mediating phase separation
-mutations in disordered regions- seeing if they shift different phases of proteins involved in diseases and what are the implications of these mutations in forming condensates?
Ex. chromatin reader ENL protein mutation can lead to condensate formation and elevated cancer formation
method to study MLOs: in vitro reconstitution
-biochemical reconstitution
-phase diagram
-optical traps to study the biophysical properties of MLOs
-phase separation assays and droplet fusion
method to study MLOs: in cell study
-many things can vary inside a cell- how much your protein is expressed and [] is important for phase separation process
-label certain proteins with immunofluorescence and see the endogenous protein expression levels without any protein overexpression and do they form visible condensates?
-confocal imaging or superresolution imaging is needed depending on how POI is organized
-to study dynamics: label them with fluorescent proteins and use FRAP to study their dynamics- if they do have dynamic liquid-like properties shown by FRAP, we can do mutational studies to ID the domains important for MLO formation
-we can also do these mutations if they disrupt MLO formation to do functional studies
method to study MLOs: candidate-based approaches
-POI can usually interact with another protein- candidate-based approach where you label the other protein and see if they organize and localize into the same condensate that the POI targets
-if you want to find novel components of condensates, might need proteomics approach
method to study MLOs: proteomics studies
-people usually use IP to ID interactions of POI but since many MLOs are formed by weak, macromolecular interactions, IP may not be sufficient –> people go towards proximity-based labeling proteomics approach
-proximity-based labeling proteomics (APEX, BioID, TurboID) with similar principles –> attach protein in the cell and by either adding some ligands or biotin in situ and ligase can very specifically biotinylate proteins in vicinity of 20 nm, you can attach biotin to all of the neighboring proteins –> you can know your POI is forming condensate and biotinylate all of the proteins surrounding your POI
-after you lyse your cells, you can isolate these biotinylated proteins with streptavidin
therapeutics targeting MLOs
-with the exception of kinases that have precise binding sites, most of the disease-related proteins are difficult to target like TFs
-look at drugs that specifically perturb MLOs- not all MLOs formed by certain factors are functioning or positively functioning –> need to know functioning before targeting
