Lecture #10 - Membrane-less organelles and Phase Seperation Flashcards
Question in cell biology
How do molecules find each other and preform important reactions (how are biochemical reactions organized)
Ways that cells compartimentailize Reactions
1 - Membrane bound organelles enclosed by phospholipids (Ex. Lysosomes + ER + mitocondria)
- Membrane bround organelles help organize reactions
- Molecules can’t freely diffuse through
2 - Membrane less organelles (Ex. nucelolus + Cajal bodies)
Discovery of MLO
MLO was found BEFORE membrane bound organelles
Example – Drawing by Ramon y cajal –> looked at the nucleus in neurons and saw electron dense structures in the nucleus (among the electron dense structures is the nucleolus and cajal bodies)
How does MLO form?
How MLO form without enclosure of lipid membrane is NOT completely understood
Membrane less organelles (MLO)
MLO = organelle inside the cell that are not enclosed by membranes
Example - P granuals in C. elegans
- When squeeze C.elegans you can see MLO (P granuals) are flowing = resembles a liquid
Types of MLOs
There are many different MLO in a cell
Diagram on slide = see many kinds of MLO
- Example – Nucleolus inside of nucleus + DNA damage foci in nucleus + stress granusl in cytoplasm + P bodies in cytoplasm
Obervations of MLOs
- MLOs have liquid like properties
- Rapid diffsion of components within MLOs
MLOs have liquid like properties Example #1
Example – germ granuals in C. elegans flow and coalece in response to shear stress
P bodies (germ granuals) in c.elegans can flow on the nucelus when squeeze C.elgans + P bodies can fuse to form larger structures –> MEANS P bodies are NOT solid agregates but instead resemble liquid droplets
MLOs have liquid like properties Example #2
MLOs have liquid like properties in frog egg extra system
2 Spherical nucleoli from Xenopus Laevis fuse and form a larger spherical structure –> means that the nucleolus can ALSO have liquid like properties
Observation #2 – Rapid diffsion of components within MLOs
Used FRAP and watch dynamics of molecules
- FRAP = measures diffusion in cells
Results:
Photobleach a fixed flourecnet sample –> THEN there is no recovery because eveyrthing is statinoary (flourescence never comes back)
FRAP on live sample = see florusnce recovery in bleached area then a stationary end point where it stays filled in
- Can see how quickly things are moving (get a diffision coeficiant)
Example FRAP
FRAP can be used to study the dynamics of material within YAP condensates
- YAP = transcription co-activator ; forms condesates when activly signaling
Experiment - fused GFP to YAP and looked at dynamics to show YAP forms dynamic condensates while actively signaling
- After photobleaching YAP floresnce intensity recovers –> recovery indicates that YAP condesates is a highly dynamic structure where YAP is rapdily diffusing in and out of the condensate (because flouressnce from elsewhere can diffuse into the structure)
END - Shows the condestae is a dynamic structure that is actively and quickly exchanging with the surrounding
FRAP on a solid agregate
FRAP on a solid agregate then the flouresnce recovery would have been much slower
Liquid = faster recovery
Phase of MLO
Based on observation (FRAP and live cell imaging) –> know MLO is liquid like structure
Liquids = have transient bonds that form and break to give dynamic structure –> THIS led to the concept that MLO are formed by weak multi valent interactions
Formation of MLO
Overall – MLO are formed by weak multivalent interactions
What determines how spontenous formation of MLO happens
The balance between entropy and enthalpy determines how spontaneous things happen –> have phase seprpations
Phase separation/MLO formation is probably driven by bond energy (driven by enthalpy that will counteract the increase in entropy)
- Entropy = means there is more space in system
If the MLO rapidly coelece into a define structure then it is probably a bond energy/entropy driven process
What kind of binding/energy interaction takes place in MLO/MLO formation
MLO are NOT high affinity macromolecular complexes that rely on interactions with a fixed ratio of molecules (NO fixed stoichemtry in intrecation)
Interaction with Fixed ratio would be Ribosomes intercation with a 1:1 interaction between the large and small SU to form a defined structure
We know that MLO formation is NOT a interaction with fixed stoichemtry because MLO are NOT stable INSTEAD weak multivalent interactions drives MLO interactions
What drives the weak multi valent interactions
Weak multivalent interactions are driven by:
1. Smaller structure interactions that are repeated many times (multivalent proteins)
2. Interactions between disordered protein domains drives multivalent interactions
- 30% of proteome is constitute of disordered regions
- Disorder regions are implicated in forming MLOs
What drives the weak multi valent interactions #1
Weak multivalent interactions are driven by multivalent proteins (repeated structures interact and form MLO)
Experiment – In a test tube they made synthetic structured domains between SH3-PRM –> mixed repeated domain together
- SH3 and PRM meidate weak interactions in and of itself BUT when mix repeat domain together in test tube they form a droplet like structure
- Formation of droplet structure = indicates weak multivalent interactions can form MLOs
- Droplets ALSO have liquid properties where droplets can fuse with neighboring droplets
What drives the weak multi valent interactions #2
Intrinsically disorder regionsare also implicated in forming MLOs
Intrinsically disordered proteins = proteins that do NOT form defined 3D structures
- Intrinsically disorder domains = noodle like domains
Despite lacking 3D structures these proteins still carry out diverse biologic functions
When mix disordered 3D domains = can form liquid like structures
What interactions might mediate MLO formation (Molecular forces implicated in MLO formation)
Slides = almost all kinds of amino acids interactions might be involved in forming MLOs (DON”T MEMEORIZE)
Example – Cation-pi interactions OR Structured domains such as coil-coil might mediate condensate formation
Looking at different proteins structures –> can allow you to discover new interactions that meidate MLO formation
Implication of MLO having liquid like properties
MLO having liquid like properties implicates there is a phase transition processes occurring
Start -
1. Protein that is diffusely localized in a cell might be a gas like phase (one-phase regime)
2. After gas phase –> when have an increase in concentration of a protein or a specific perterbation put into the cells then you can change the local concentration of the protein –> NOW can have a gas to liquid transition and can now form a liquid MLO (2 phase regime)
- Proteins undergo the gas to liquid transition
- 2 phase regime = co-existing phase of liquid AND gas
3. After 2 phase regime –> IF you further increase the concentration or add a different pertuvation then could enter a liquid like phase (protein enters a liquid like phase?)
Can draw a phase diagram based on in vitro data
Phase diagram
In vitro can make a phase diagram by changing the protein concentration or doing a specific pertuvaion of the protein to cause the protein to go from gas phase a 2 phase regime
- Get 2 phase regime when increase the concentration
Phase seperation helps in thinking about how MLO forms in vitro and in cell (BUT STILL a question if phase sepration is what is forming the MLO inside of a cell)
Answer:
- Free transition of molecules without transporters
- Quick regulation of processes (revrsible)
- STILL have rapid exchange of molecules and if there are reactions you can have fatser export of product in process
Functions of phase separation and forming an MLO
- Bring together enzymes and substrates
- Sequesting
- MLO structures can be an organizational hub that can form a structure that is important for function
- NO role in reactiions ; instead organizational hub
Functions of phase separation and forming an MLO - Bring together enzymes and substrates
Bring togther enzymes and substrates –> Phase seperation compartments can bring toegther enzymes and substrates
There are many examples that shows you can concentrate molecules inside the MLO to be in the same spcace for an accelerated reaction
Functions of phase separation and forming an MLO - Sequestering
MLO/phase seperation can have sequestering function
IF don’t want reaction to happen THEN phase compartment can sequester the molecule in different places away from where the reaction should happen
- Way for MLO to shut down reaction
Bimolecular condensates
Biomolecular Condensates: Micron scale compartment inside of cell that lacks suroudning mebranes
Condensates = formed by assembly of proteins and or nucleic acids with non-fixed ratios (non-stoichemtry)
Bimolecular condensates is a broader term that includes MLOs AND other membrane less entities
Example of other membraneless entities
Example of other membraneless entities = synapses in nuerons
- Synpase has membranless entity (biomolecular condensate) but is is NOT a MLO
At synapse have proteins that form membraneless entities that retain the synaptic vesicle at the pre-synaptic space to prevent their release THEN when have AP the membrane-less entities get phosphorylated and disassble and get the release of the vescule
Affect of dysregulation of phase seperation/MLO #1
Dysregulation of phase seperation/MLO leads to diseases and disorders
Example - Changes in material properties underlies nuerodegenrative diseases
- Change in material properties –> Liquid to solid tranision happens when have modifications/regulation that makes the liquid property decrease (diffusion decreases) and form a solid structure instead of liquid ; IF add ATP then material goes back to liquid
- Ex. in disease TBD43 or Fus can have liquid to solid transition and form agregtaes (underlys plaque formation in nuerodegenerative diseases)
Affect of dysregulation of phase seperation/MLO #2
Dysrgelation of phase seperation can lead to cancer (changes in formation or dissolution)
- Different condensates in cell can be implicated in cancer formation
Example #1 – Some Oncoproteins are involoved in condesate formation inside of nucleus which might drive higher gene activity involved in oncogenesis
Example #2 – Different DNA repair pathways in nucleus might implicate some MLO or lengthening of telomeres can be implicated in some MLO
Examples of MLOs/Biomolecular condesates (overview)
Insude if nucleus: Nucleolus + Nuclear speckles + Transcription Hubs + Paraspeckes + PML bodies + Cajal Bodies
Inside of cytoplasm: Stress granuals
MLOs in non-mammalian systems:
1. In plants – Germination granuals
2. In Insects – P granuals
Membrane bound organelles in nucelus
The nucleus does not contain ANY membrane bound organelles
BUT do have MLO –> Nucleus is a dense environment which organizes distict condensates
- Reactions in nucleus happen in MLOs or other bimolecular condensates
Image – staining of structures in nucleus –> can see MLO in nucleus
Nucleolus
Largest MLO in the nucleus
Function – for rRNA transcription and processing (make ribosomal components)
- Important for sequential processing of nascent rRNA transcripts
Where is the nucleolus found
Formed at genomic sites (Sites = nucleolar organizing regions (NORs))
- Organized around DNA (NOR)
Whole thing is stationed around repeated DNA structure to form large component
What is the nucleolus composed of
Contains sub compartments as immiscible liquid phases (have liquid properties)
- Nucleus is 1 MLO BUT has subphases that are determied by different surface tensions
- Whole nucleolus has 3 layers that are seperate phases from each other
Sub compartments are important for the sequential processing of nascent ribosomal transcripts
- Example - Make the rRNA in 1 compartmemt –> THEN diffuse to sequential sublayers to do processing (do splicing and assmebly with ribsomal protein to make robsome in the sequentoal sublayer)
Nuclear speckes
Location - Nuclear speckeles exist in the interchromasoaml space inside of nucelus
Function - Enrich factors involved in transcription + RNA splicing + RNA export
- Active genes associated with nuclear speckles for faster RNA processing
- Usually considered a storage site for splciing molecules BUT also have actiave genes associated with nuclear speckles so the actve genes can access the stored RNA moldification /splicing components for fasting RNA processing and faster assembly of active genes
Green dots on image
Interchromosomal space
Interchromosomal space = space between DNA
Space = has nucleolus or nuclear speckles among them
- Large structures in the inter chromosomal space
Transcription hubs
Transcription hubs = condensates that concentrate key transcription related factors (Ex. Concentrates DNA + RNA + transcrtion factors + cofactors/co-acivators all in 1 region to lead to high gene expressio of the traget gene)
- Get high expression of the DNA that is in that region
Function – mediates high gene expression
Example:
1. Mediator –> transcrtion co-activator needed for transcription of genes (bright dots in nucleus in image)
2. YAP (Dots in image = conctrated YAP in nucleus)
Transcription hubs + diseases
In disease – can compare actively transcribed genes in healthy vs. Cancer cells
What is found in cytoplasm
Membrane bound organelles are in the cytoplasm BUT cytoplasm also has MLOs
Ex MLO - stress granuals that forms when cells experiments stress
- Dont see stress grauals in normally functioning cell ONLY when there is a stress you can see the granulas rapidly form in the cytoplasm of cells
- Example stress = arsenic (toxins) + hyperosmotic stress + temperature change
Labeling Stress granuals
G3BP1 = labels stress granuals (G3BP1 is a major component of stress granules)
G3BP1 = diffuse at start in cytoplasm (before stress)
- After stress when add arsenite G3BP1 forms distcits compartments in the cytoplasm (formation happens quickly)
Function of stress granuals
Stress granuslas = contains RNA (most RNA in cytoplasm?) + proteins
Function = granules shuts down tranlsation process and protects the cells under stress
- Granuals protects the RNA and proteins in seperate entities during shut down
- Important for maintaining cell function when cell is stressed
Stress granuals and disease
Granuslas are imporant for the pathogenesis of neurodegerative diseases (Ex – ALS + FTD + IBM)
- Important in nuerodegenerative disease because almost all the protein implictaed in diseases ALS and FTD are found within stress granuals
Rasies question about what is stress granuals doing in these nuerodegertaive diseases (is it becuase the normal liquid like dynamicis bevcome more sold when these proteins are implicated in nuerodegreation???)
MLO in non-mammalian systems
Plants – seed germination –> Seeds changed in wet and dry conditions in seed and germination when wet can have changes in condensate formation in flower proteins
- Changes in dynamics leads to different timinig in seed germination
Insects – Have P granuals (germ granuals)
- Condensation in embyro of C.elegans can lead to changes in sepcifcying the fate of different cells (germ cells or somatic cells)
Lots of things blocks the droplets from fusing
Examples of things that block unlimited growth (ALL Regulation of MLO in complex envrinment of the cell):
1. Confinmens
2. ATP dependent processes
3. Post translational modifications
4. Mutations
Regulation of MLO in complex envrinment of the cell - Confinments (overall)
Have cofinmemnts in cell that restricts the growth of MLO (regulation of unlimited MLO growth)
- Confinement’s = major limitation to MLO growth
Have confinment:
Inside nucleus + inside cytoplasm + Membranes
Confinments inside of nucelus
Large volume of the nucleus consists of chromatin (DNA) –> chromatin restricts MLO growth/fusion
Experiment – used light to grow optogenically modulated condensates
- See that when the octodroplets form at heterochromatin region they have a hard time growing (Can’t grow near heterpchromatin and limited to a smaller size )
- Shows DNA has a large influence on the growth of condensates inside nucleus
Confinments in the cytoplasm + membranes
In cytoplasm have compartments that restcits MLO growth + have actin filemants/cytosklaton that restricts growth and fusions of MLOs
Membranes can also confine MLOs
- Have membranes in the cytoplasm that form lysosome/ER/mitochondria –> membranes pose physical constrains for movement and fusion of MLO
Regulation of MLO in complex envrinment of the cell - ATP dependent processes (overall)
ATP dirven processes can form or dissolve MLOs
Example – Microtubulues in the cell cytoskelton is important for the fusion and fission of stress granuals in the cytoplasm
- There are motor proteins (dynemain on microtubules) that can fuse and link with stress granual compoenents and lead to the dissolution of mature stress granulas
END - Stress granule formation is driven by motor protein dynein ; stress granule dissolution is driven by motor protein kinesin (dynein and kinesin use ATP)
- ATP dirven porcesses that drive dynein can lead to the formation for disolution of MLO
Regulation of MLO in complex envrinment of the cell - Post translational modifications (overall)
Many post tranlsational modifications affect MLO formation
- Example post translational modification that affects MLO formation = phosphorylation
Proteins and RNA making the MLO can undergo post tranlatiional modification which can chnage the interaction among residues that chnages the regulation of MLO formation
Affect of phsophorylation on MLO formation
Phosphorylation can have positive and negtaive affects on MLO formation depending on the context
Example:
Phosphorylation can have negtaive affect – dual specifcity Kinase 3 (DYRK3) phosphrylates the major compoennts of the MLO and dissables MLOs during Mitosis
- In mitosis if the cell wants to distrubute MLOs into the two daughter cells evenly then the cell needs to arrange the MLOs evenly or diassble the MLOs and make them diffuse and distribute in the daughter cells and reform the MLO when the daughter cels are made
Positive effect of phosphorylation – Tau phosphorlaytion increases it ability to form protein agregates
- Tau is the major protein involoved in nuerodegeneration
- Phorphorylation of Tau can leads Tau to form more agregates to furtehr push in the phase digram to condensate formation/phase seperation
Regulation of MLO in complex envrinment of the cell - Mutations (overall)
Mutants in components can lead to differential regulation of the formation of MLO
Mutations can map to domains that mediate phase seperation (ex. P53)
- Before – used to only study mutations in structured domains BUT NOW people found mutaions in intrinsically disordered regions (ask if the mutation is shifting the different phases of the protein involoved in diseases)
Change in phase seperation can lead to diseases (ex. cancer)
Example of mutations and condesate formation
Chromatin reader ENL protein can have mutation leads to condesate formation and elevated cancer gene expression
Methods to study MLOs
- In vitro reconstittion
- In cell
- Candidate based approaches
- Proteomic studies of MLOs
Methods to study MLOs - In vitro reconstittion
In vitro reconstittion – used to see if RNA or protein forms condesates
Includes:
1. Biocehmical reconstitution
2. Phase diagram
3. Optical traps
Biochemical reconstitution
Use - See if protein or RNA form condenssates
Overall - Purify the protein in test tube and use biochemical reconstitution
When doing biochemical reconsititution –> see if when chnage protein concetration or preform a pertivasion to the proetin IF the proteins undergo condensation process
IF the proteins do have a concentration dependent concdetartion process and you can darw a phase diagram –> THEN that shows that the protein has this intrinsic property of forming condesates
LIST on slide = things to do to get measuresmnts of phase diagram and concentration in vitro
Optical traps
Use - study the biophysical properties of MLOs
Once you have condetates (droplets) of proteins in a test tube you can do optical traps
In optical traps see if the droplets can grab neighboring droplets and ask if the two drlopes will fuse (what is the biophysical properties of the MLOs)
Methods to study MLOs - In cell
Overall – looking at the organization of proteisn in cell and the function of condensates
- Endogenous protein visualization (immunoflourecence)
- Flourecntley labeled protein
- Mutation studies
- Functinoal studies
How do you know if protein/RNA froms condesates in cell
IF want to know if proteins/RNA forms condensates in a cell you really need to study in a cell
Need to do in a cell because things can vary in a cell (Example – expression of proteins/concertation of proetins which is important for phase separation process)
Endogenous protein visualization (immunoflourecence)
Use - Gives protein organization
To study a protein in endoygenous context in cell you label the protein with immunoflrouneces –> ask if the protein forms visible condetates in cell at the endogenous protein level (NO protein overexpression)
- Uses immunoflourence to stain cells and look at organization inside living cell
Depending on how the protein of interest is organized confocal or super resolution is needed
Why do you need super resolution imaging in Endogenous protein visualization
IF looking at endogenous organization of protein in cell line –> do NOT always see discrete sphriecal structure BUT instead you see formation of small foci like organization = need super resultion imaging to look at them
Shows another reason why it is important to study condensates in the cell and see what kinds of condesates they form because there could be a difference in the function of smaller vs large
Flourecntley labeled protein (In cell)
Use - protein dynamics in MLO (In cell)
To study dyamics – label endogenus proteins with floruence and do FRAP to study dynamics
FRAP shows the liquid properties
- IF have a dynamic liquid property in FRAP THEN move to doing mutational studies
- FRAP = can study dynamics in vitro (in test tube) by labeling purified protein with flourescece OR can do FRAP in living cell
Mutation studies (In cell)
Use - identify the domains that are important for MLO formation
IF have a dynamic liquid like property in FRAP THEN move to doing mutational studies of proteins to see domains that are needed for MLO/condesate formation
- IF find domains that disrupt MLO formation THEN this gives you a tool to perform functional studies
Functional studies (In cell)
IF find domains that disrupt MLO formation THEN this gives you a tool to perform functional studies
- Asking - Do you disrupt function when you disrupt the MLO formation
Ex. Are you changing the ability to form cancers when you disrupt a domain)
Candidate based approaches
One way to study MLOs is to look at what components are bring recruited to the structure
- Candidate based approaches = allow you to study components of MLO
IF you think that a protein of interest interacts with another protein THEN use a candiate based appreach where you label the proteins and see if the two proteins are found in the same MLO
- Need to already have a protein of interest
Proteomic studies of MLOs
2nd way to study components of MLOs –> Used to find novel components of MLO that you don’t know about
- Wouldn’t really use Co-IP INSTEAD Proximity-based labeling proteomics (APEX, BioID, TurboID, etc.) are use to identify the components of MLOs
Why can’t you ise co-IP to find components of MLOs
Usually use co-IP to identify the interactions of a protein of interest (find protein-protein interactions) BUT because a lot of MLOs are formed by weak macromeular intercations you can’t sue Co-IP becuase immunoprecipiating can only see strong interactinos (might not be able to see the weak interaction inside of condeates when using Co-IP)
Instead of Co-iP use proximity labeling proteomics
Proximity labeling proteomics
Uses APEX or bioID or TurboID ; works in living cells
Process – attatch protein of interest to biotin ligase (enzyme like APEX) –> expression the fusion protein in living cell and add ligand (biotin) in situ –> biotin ligase will biotynilate proteins that are close by 9within 20nm) –> NOW aded biotin to the nieghboring proteins
NOW you know the proteins that form the condensate and biotylated proteins nearby protein of interst –> NOW when open cell you can isolate the biotynlated prteins using streptavridin beads and subject the proteins to mass spec analysis to see what proteins are there
2nd use of proximity labeling
Proximity labelling can also be used to studying signaling pathways
Studying function depends on the system
Example – Transcription –> measure function based on transcription
- IF know the Transcription factor transcribes certain genes and when you disrupt domains to form condensates can see if the genes are still transcribed
- Depends on what normal function is
How are most MLOs studied
Usually study MLO in 2D tissue culture BUT in reality have 3D structure in organisms
It is important to go from 2D to 3D and see if the MLO will still form and if they have similar function in 3D tissue
Experiment – used pateint derived cell line to make 3D tumor spheroid and looking to see of the oncoproteins condetates still form in the kidney cancer spheroid
- In spheroid you can see clear foci formed by fusion oncoporotein
What is changed from 2D to 3D?
- Different tissue mechanics
- Different signlaing
- Other environmental factors (immune cells)
Xenograph models
People are making xenograoh models and seeing if you still have condensates in tumor xenograoh and what the condensate functions are there
Used to go from 2D to 3D
Synthetic MLOs
Synthetic MLOs can be light activated or activated by chemicals so you can make them on demand and see if by forming condensates in a specific context if you can achieve certain function
- Synthetic MLOs can be activated by light or chemicals, and control cellular functions.
Issue in deisgning drugs
Most disease-related proteins are hard to target because no pocket good enough for drug binding
- Example – Transcription factors do not have a good enough pocket to target to teat diseases implicating the transcription factor
Solution to drug targeting using MLOs (Theraputic targeting MLOs)
Solution - some drugs can specifically incorporate into MLOs formed by disease-related proteins
- IF we know that MLO is formed by transcription factor is the functioning unit then you can find drugs that perturb the MLO so you can make new drugs to treat disease (IF NEED MLO to function then can target MLO)
Issue – not all MLO formed by certain factors are functioning or they positively function instead of negatively function (MEANS we need a functional understanding of MLOs before targeting MLOs)
- Example – maybe the MLO protects the cells so you don’t want to disassble them
Summary
