Lecture #10 - Membrane-less organelles and Phase Seperation

Question 1

Question in cell biology

Answer 1

How do molecules find each other and preform important reactions (how are biochemical reactions organized)

Question 2

Ways that cells compartimentailize Reactions

Answer 2

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)

Question 3

Discovery of MLO

Answer 3

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)

Question 4

How does MLO form?

Answer 4

How MLO form without enclosure of lipid membrane is NOT completely understood

Question 5

Membrane less organelles (MLO)

Answer 5

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

Question 6

Types of MLOs

Answer 6

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

Question 7

Obervations of MLOs

Answer 7

1. MLOs have liquid like properties
2. Rapid diffsion of components within MLOs

Question 8

MLOs have liquid like properties Example #1

Answer 8

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

Question 9

MLOs have liquid like properties Example #2

Answer 9

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

Question 10

Observation #2 – Rapid diffsion of components within MLOs

Answer 10

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)

Question 11

Example FRAP

Answer 11

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

Question 12

FRAP on a solid agregate

Answer 12

FRAP on a solid agregate then the flouresnce recovery would have been much slower

Liquid = faster recovery

Question 13

Phase of MLO

Answer 13

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

Question 14

Formation of MLO

Answer 14

Overall – MLO are formed by weak multivalent interactions

Question 15

What determines how spontenous formation of MLO happens

Answer 15

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

Question 16

What kind of binding/energy interaction takes place in MLO/MLO formation

Answer 16

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

Question 17

What drives the weak multi valent interactions

Answer 17

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

Question 18

What drives the weak multi valent interactions #1

Answer 18

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

Question 19

What drives the weak multi valent interactions #2

Answer 19

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

Question 20

What interactions might mediate MLO formation (Molecular forces implicated in MLO formation)

Answer 20

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

Question 21

Implication of MLO having liquid like properties

Answer 21

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

Question 22

Phase diagram

Answer 22

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)

Question 23

Answer 23

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

Question 24

Functions of phase separation and forming an MLO

Answer 24

1. Bring together enzymes and substrates
2. Sequesting
3. MLO structures can be an organizational hub that can form a structure that is important for function
   
   • NO role in reactiions ; instead organizational hub

Question 25

Functions of phase separation and forming an MLO - Bring together enzymes and substrates

Answer 25

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

Question 26

Functions of phase separation and forming an MLO - Sequestering

Answer 26

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

Question 27

Bimolecular condensates

Answer 27

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

Question 28

Example of other membraneless entities

Answer 28

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

Question 29

Affect of dysregulation of phase seperation/MLO #1

Answer 29

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)

Question 30

Affect of dysregulation of phase seperation/MLO #2

Answer 30

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

Question 31

Examples of MLOs/Biomolecular condesates (overview)

Answer 31

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

Question 32

Membrane bound organelles in nucelus

Answer 32

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

Question 33

Nucleolus

Answer 33

Largest MLO in the nucleus Function – for rRNA transcription and processing (make ribosomal components) - Important for sequential processing of nascent rRNA transcripts

Question 34

Where is the nucleolus found

Answer 34

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

Question 35

What is the nucleolus composed of

Answer 35

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)

Question 36

Nuclear speckes

Answer 36

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

Question 37

Interchromosomal space

Answer 37

Interchromosomal space = space between DNA Space = has nucleolus or nuclear speckles among them - Large structures in the inter chromosomal space

Question 38

Transcription hubs

Answer 38

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)

Question 39

Transcription hubs + diseases

Answer 39

In disease – can compare actively transcribed genes in healthy vs. Cancer cells

Question 40

What is found in cytoplasm

Answer 40

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

Question 41

Labeling Stress granuals

Answer 41

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)

Question 42

Function of stress granuals

Answer 42

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

Question 43

Stress granuals and disease

Answer 43

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???)

Question 44

MLO in non-mammalian systems

Answer 44

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)

Question 45

Answer 45

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

Question 46

Regulation of MLO in complex envrinment of the cell - Confinments (overall)

Answer 46

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

Question 47

Confinments inside of nucelus

Answer 47

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

Question 48

Confinments in the cytoplasm + membranes

Answer 48

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

Question 49

Regulation of MLO in complex envrinment of the cell - ATP dependent processes (overall)

Answer 49

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

Question 50

Regulation of MLO in complex envrinment of the cell - Post translational modifications (overall)

Answer 50

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

Question 51

Affect of phsophorylation on MLO formation

Answer 51

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

Question 52

Regulation of MLO in complex envrinment of the cell - Mutations (overall)

Answer 52

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)

Question 53

Example of mutations and condesate formation

Answer 53

Chromatin reader ENL protein can have mutation leads to condesate formation and elevated cancer gene expression

Question 54

Methods to study MLOs

Answer 54

1. In vitro reconstittion 2. In cell 3. Candidate based approaches 4. Proteomic studies of MLOs

Question 55

Methods to study MLOs - In vitro reconstittion

Answer 55

In vitro reconstittion – used to see if RNA or protein forms condesates Includes: 1. Biocehmical reconstitution 2. Phase diagram 3. Optical traps

Question 56

Biochemical reconstitution

Answer 56

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

Question 57

Optical traps

Answer 57

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)

Question 58

Methods to study MLOs - In cell

Answer 58

Overall – looking at the organization of proteisn in cell and the function of condensates 1. Endogenous protein visualization (immunoflourecence) 2. Flourecntley labeled protein 3. Mutation studies 4. Functinoal studies

Question 59

How do you know if protein/RNA froms condesates in cell

Answer 59

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)

Question 60

Endogenous protein visualization (immunoflourecence)

Answer 60

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

Question 61

Why do you need super resolution imaging in Endogenous protein visualization

Answer 61

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

Question 62

Flourecntley labeled protein (In cell)

Answer 62

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

Question 63

Mutation studies (In cell)

Answer 63

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

Question 64

Functional studies (In cell)

Answer 64

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)

Question 65

Candidate based approaches

Answer 65

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

Question 66

Proteomic studies of MLOs

Answer 66

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

Question 67

Why can't you ise co-IP to find components of MLOs

Answer 67

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

Question 68

Proximity labeling proteomics

Answer 68

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

Question 69

2nd use of proximity labeling

Answer 69

Proximity labelling can also be used to studying signaling pathways

Question 70

Answer 70

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

Question 71

How are most MLOs studied

Answer 71

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

Question 72

What is changed from 2D to 3D?

Answer 72

1. Different tissue mechanics 2. Different signlaing 3. Other environmental factors (immune cells)

Question 73

Xenograph models

Answer 73

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

Question 74

Synthetic MLOs

Answer 74

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.

Question 75

Issue in deisgning drugs

Answer 75

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

Question 76

Solution to drug targeting using MLOs (Theraputic targeting MLOs)

Answer 76

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

Question 77

Summary

Answer 77