section 12 Atypical organelleles like mebraneless ones (MLO) Flashcards
(57 cards)
1
Q
compartment separation without membranes
A
*liquid-liquid phase separation
- lava lamps, vinaigrette salad dressing, nucleoli
- one liquid within another liquid (droplet/plastic conformation + dynamic)
- 2 immiscible liquids = apply the force of shaking and one in the background of the other (cannot dissolve sitting)
- dynamic in size, shape, number, contents, etc.
- some stuff can come in and some stuff can come out
- can occur in the nucleus and cytoplasm
2
Q
nucleolus as an example of compartment separation without membranes
A
- different compartment in the nucleus (liquid-liquid separation)
- no membrane
- phase separated by MLOs (membrane-less organelles)
- during mitosis it disperses and then reorganizes
*more dynamic (contents and traveling in and out) than membrane-bound
3
Q
membrane structuring of Membraneless Organelles (MLO)
A
protein component, metastability, glass/gel, and fiber,
4
Q
protein component
A
can move in and out (more dynamic than membrane organelles)
5
Q
metastability
A
fine and normal (reversible) - come in and come out and change shape
6
Q
glass/gel and fiber
A
pathology when contents move onto higher organization order –> detrimental
- we know that it can go to higher organizations but we are not sure if it can be reversed by drugs or natural cell
- further organization is bad because of protein arrangements rather than soluble proteins
*soluble –> fibrillar bad (positive) but not sure about the reverse - pathology with protein
7
Q
diversity of MLO
A
function, structure, nucleolous, P-body
8
Q
MLO function
A
associated with cell division, chromatin remodeling, gene transcription, synapse function, virus assembly, diverse contents, duration & size (any cellular process probably has MLO associated)
9
Q
MLO structure
A
include P-body and nucleolus (classic examples) but variable in different cells
10
Q
nucleolus (MLO)
A
disassembly/reassembly with cell division
11
Q
P-body
A
RNA breakdown; variable duration depending on growth conditions - may arise or disperse
- P-body more present during RNA breakdown (not present in all cells)
*often sites of catalytic activity (not passive) and dynamic (dispersion)
12
Q
examples of MLO diversity
A
- virus factory = COVID, HPV, etc. assemble progeny virus
- Tp53 aggregation
*variability of what MLOs are in cells (except nucleolus in most)
13
Q
p-body (function)
A
processing/breakdown of RNA
- may arise or disperse in different cell types
14
Q
MLO: generate new compartments
A
- cytoplasmic granules, form & fuse cytoplasm purple; granule green = droplets fuse and grow (conditions drive larger MLOs)
- subnuclear compartments
- vary based on growth, nutrient, time, etc.
- variability based on size, what they do, in their content
15
Q
MLO:reorganize existing compartments
A
separating out components (not nucleoli) for self-association and organization - subnuclear
- nucleoli fusion = dynamic nucleoli fuse (no membrane, only RNA protein composition)
- subnuclear compartments in the nucleolus that are forming because of liquid-liquid
16
Q
MLO: vary in time, location & size
A
- cytoplasm, nucleus, nucleolus (<0.5 frequent - ~20um rare)
- time is taken to disperse or form variable
17
Q
compare and contrast membrane-bound and MLO organization (function)
A
optimized function (specialized subcompartment)
- lysosome = acid hydrolase efficiency (bound)
- mitochondria = electron transport, H+ gradients (bound)
- P-body = (processing body) RNA decay (membrane less)
*share the ability to function within
18
Q
compare and contrast membrane-bound and MLO size and shape
A
- nucleus = 5-10 um diameter
- nucleolus = 0.5-2.5 um diameter
- other MLO = <0.5 frequent - ~20um rare
*what binds the perimeter?
19
Q
membrane-bound “organizer”
A
- phospholipid bilayer(s)
- boundary from aqueous cytoplasm
- for specialization
20
Q
membrane-less “organizer”
A
- protein biochemistry
- characteristics more likely to self-interact than interact with aqueous cytoplasm
- protein phase separate from the environment (not compatible with protein biochemistry)
- partnered proteins associate, leaving out proteins less likely to interact
21
Q
MLO formation
A
*LLPS - liquid-liquid phase separation
- dissolved proteins interact with each other, and possibly RNA to coalesce (de-mix) from surrounding homogeneous mixtures of diverse macromolecules in cytoplasm or nucleoplasm (dispersion into nuclear shadow)
- reversible depending on stimulus = compare to separation (re-mixing oil-water) and very variable time
*nuclear shadow excluded (organized) and dispersed rapidly but can organize back
22
Q
MLO diverse examples for formation
A
cajal nuclear bodies and PML nuclear bodies
23
Q
Cajal nuclear bodies
A
- varied content & function
- partially regulate transcription
- process (association with) RNA for spliceosome assembly
- increase the efficiency of nuclear events like RNA processing
- nuclear events (mRNA)
24
Q
PML nuclear bodies (green dots ~.2um)
A
- PML protein: replication suppressor
- ~100 possible partner proteins in different PML bodies for varied functions (apoptosis, telomere elongation)
- always have PML protein
- nuclear functions differ depending on partner protein but relate back to PML (most varied)
25
LLPS concepts
- protein condensation leading to...
- subfunctions
- reaction crucible
- sequestration
- organizational hub
- 3 variations (different major subtypes, subfunctions of membrane-less organelles)
*associated with happy, healthy cells
26
sequestration
- storage for later processing or secretion (deposit/reservoir)
- reduces response time to extracellular signals (physiological response decreases lag time)
- secreted, processed at later event
- premade proteins in preparation for physiological change
27
organizational hub
*physical association
- normal condensation of proteins (ex. MT, tau) to focus interaction/polymerization of partner proteins (microtubule stability)
- 2 or more proteins physically associate to build a structure to increase cell
- advance cell
- more efficient if physical components inside MLO
28
scaffold vs. client proteins
scaffold proteins
- can drive LLPS on their own
- enriched for domain repeats
(multivalent) & little 3D structure
(disordered)
- sufficient concentration will condense
+ separate from the surrounding
plasma (cyto or nuclear)
client
- proteins can interact with scaffold proteins
- compatible with LLPS of partner proteins
- typically insufficient for LLPS on their own
29
protein characteristics for LLPS - driving phase separation (scaffolds)
multivalent and disordered
30
multivalent
- repeated subdomains = repeated site for interactions (amino acid series, etc.)
- drives condensation of MLO (because of multivalent for another protein)
- scaffold for partner protein
- ex. SOS & Grb2 (PRM & SH3 subdomains)
- protein-protein recognition for partner-building droplet
31
disordered (not the same as denatured)
- no rigid 3D "lock & key-type" conformation
- do not have 1 defined 3D structure (shape-shifting dependent on partners, etc.) - flexibility
- hi content of polar & charged amino acids = keep protein in extended shape
- low content of hydrophobic AA = less likely to fold up to reduce interaction with water
- reconfigurable (not rigid or dimensional)
- flexible, interact with many partners
- floppy end
- interact with nucleic acids (RNA binding protein has flexible disordered region allowing it to interact) - drives the formation of MLO (smaller than nucleolus)- no rigid 3D "lock & key-type" conformation
32
phase separation of LLPS extracellular conditions
- pH, osmolarity, etc.
- stressors like toxins (environmental stress)
- take homogeneous proteins --> cause some to come out and phase separate
- change extracellular to get a response inside the cell
- ex. cytoplasm --> cytoplasm with condensates
33
phase separation of LLPS intracellular conditions
*cause condensation
- protein concentration
- ion concentration
- partners (other proteins, RNA, or DNA)
- ATP (as charged molecule; not as an energy source)
- post-translational modifications (ex. phosphorylation increasing p-tau (increasing separation) --> MT depolymerization)
- for some proteins, possible disease-specific mutation
- more individual protein --> most likely to dissociate
34
t/f) condensates redirect cell activity
true; reaction crucible, sequestration, organization hub
35
phase transition
*LLPT - liquid-liquid phase transition
- different than separation
- may occur abruptly after LLPS
- excessive interactions among components
- uncertain reversibility, uncertain consequences - may intervene with a drug compound (drive reversal)
- possible disease - denser and denser protein assemblies
36
proteins dispersed --> separation
- self associating
- phase separation
- normal cell physiology --> normally reversible (back to dispersed)
37
aggregation
- may occur independently or following LLPT
- disrupts normal cell function
- abnormal, often disease-associated and typically irreversible
- ex. dispersed & aggregated tau
*pathological consequence from transition --> aggregation
38
separation vs. transition vs. aggregation
1. dispersed well
2. MLO from LLPS good (normal cell physiology) = associated MLOs
3. transition unsure (unsure if we can go back)
4. aggregation is bad and known to be irreversible
39
interaction beyond LLPS bad news
- function --> dysfunction
- all neurological examples but can be anywhere (dramatical clinical presentation of aggregated proteins in neuropathology)
- can occur in lots of different cell types in lots of different cells
- aggregation, nucleation
*likely irreversible nature of assembly (amyloid) and insoluble
40
dispersion to aggregation
*disperse --> LLPS --> LLPT --> aggregation
- condensation from excess interactions = generates "solid" structures, insoluble, fibrous "amyloid"
- fibrils, tangles, etc. associated with many degenerative diseases
- characteristics or cause of dysfunction?
41
nucleation
aggregated proteins serve as condensation foci for proteins that would otherwise remain in LLPT or move back to LLPs
42
example pathologies & dysfunctional proteins
*dramatic - several proteins associated with the neuropathologies transition to the aggregation phase
- amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease) = TAR DNA binding protein-43
- Alzheimer's disease (AD) = microtubule-associated tau
- Parkinson's disease (PD) = a-synuclein amyloid fibrils from LLPT --> amyloid clumps
43
a-synuclein normal role
- vesicle delivery to cell termini
- neurotransmitter deliver and release at the synapse
- found in the cytoplasm under the membrane at the synapse (in the nucleus)
- undefined role in the nucleus
- normal day-to-day functioning delivering neurotransmitters
- issue when transitioning to aggregation phase (amyloid fiber clumps - Parkinson's disease)
- several proteins associated with new neuron
44
a-synuclein example
*potential to clump into amyloid fibers
- normal function - self-associating, delivering to the synapse, etc.
- individual proteins --> LLPS (normal function) --> increased self-interaction --> LLPT (forms aggregate) --> aggregates --> amyloid fibrils (disruption of neurotransmitters and dysfunction of the neuron)
- Parkinson's disease
- decreased vesicle trafficking
- disrupted transmitter release
*pathology in phase separation to phase transition
- timing and progression vary
45
(t/f) insufficient amount of protein also causes pathologies
true; not just more protein to transition (excess protein --> phase transition --> dysfunction) but function missing if insufficient amount
46
exploring therapeutic interventions of MLO and LLPS/LLPT dysfunction (locations)
- LLPS/T locations: nucleus and cytoplasm (possible impact on many cell functions & therefore many diseases)
47
Exploring therapeutic interventions of MLOs and LLPS/LLPT (dysfunction)
dysfunction type
- insufficient interactions for LPPS & therefore no "reaction crucible, storage, or hub"
- excess interaction driving LLPT (multivalent with self or partner)
- partner excess or missing
- wrong type or timing of post-
translational modification
48
Exploring therapeutic interventions of MLO's and LLPS/LLPT ( pathologies)
resulting diverse pathologies
- neurodegeneration (TAR43, p-tau, a-synuclein)
- hyperplasia/cancer (p53, myc, p53)
- other tissue degeneration
49
neurodegeneration therapeutic interventions
- protein aggregates driving drug discovery
- variations of ZPD
- small molecule compound (pre-clinical drug) = inhibit nucleation and aggregation (prevent late phase only if you get to it in time)
- inhibits aggregation and promotes disassembly (reverse phase transition)
- simplifying ZPD chemistry - that interacts with a-synuclein protein to make more compatible with clinical use
50
drug interaction with protein 1 (therapeutic interventions)
facilitates LLPS for reaction crucible, sequestration, organizational hub (organization)
51
drug interaction with protein 2 (therapeutic interventions)
reduce LLPS that might be precursor of LLPT and/or aggregate (disperse to more compatible with normal cell physiology)
52
drug regulation of modifier (therapeutic interventions)
regulate post-translational modification for a desired effect (protein dispersion)
- the drug may impact the modifier
*protein molecule some structure, some disorder (foldable + flexible region)
53
gene expression - review material
- DNA gene --> mRNA transcript --> function or structural protein --> cell activity or structure --> organ activity/structure --> organism
- transcription of DNA in the nucleus into RNA
- rRNA ribosomal RNA - polymerase I
- mRNA messenger RNA - polymerase II
- tRNA transfer RNA - polymerase III
54
RNA pol II
*reads 3 nucleotides per codon, with 4 nucleotides available
- more than enough to cover the 20 encoded amino acids
- DNA gene --> mRNA transcript --> protein
- consequences of mutations
55
mutations - review
- mutations change coding sequence
- may occur in somatic or reproductive cells
- can occur spontaneously (DNA polymerase error) or by radiation or chemical exposure
56
mutation example 1
- nucleotide substitution
- may result in replacement of one aa with another
- example - sicke cell anemia allele single base change in DNA results in aa change in hemoglobin
- or no change if new codon codes for same aa (coding redundancy)
57
mutation example 2
- insertion or deletion of bases in DNA sequence
- results in phase shift of three base reading frame
- affects all codons after mutation
- results in different amino acid sequences
- almost always results in a non-functional polypeptide
