Atypical Organelles and Condensates

Question 1

Compartment Separation without membranes

Answer 1

liquid-liquid phase separation (lava lamps, vinaigrette salad dressing, nucleoli) - one liquid within another liquid, 2 immiscible liquids = apply force of shaking and one in background of the other (cannot dissolve sitting), dynamic in size, shape, number, can occur in nucleus and cytoplasm

Question 2

Nucleolus as an example of compartment separation without membranes

Answer 2

different compartment in nucleus (liquid-liquid separation), no membrane, phase separated by membrane less organelles, during mitosis it disperses then reorganizes, more dynamic than membrane bound

Question 3

Membrane structuring of membrane less organelles

Answer 3

protein component, metastability, and glass/gel and fiber

Question 4

Protein component of membrane structure

Answer 4

can move in and out (more dynamic than membrane organelles)

Question 5

Metastability

Answer 5

fine and normal (reversible) - come in and out and change shape

Question 6

glass/gel and fiber

Answer 6

pathology when contents move onto higher organization order - detrimental

Question 7

membrane less organelle function

Answer 7

associated with cell division, chromatin remodeling, gene transcription, synapse function, virus assembly

Question 8

membrane less organelle structure

Answer 8

include P-body and nucleolus (disassembly/reassembly with cell division) but variable in different cells

Question 9

membrane less diversity

Answer 9

diverse contents, duration and size (any cellular process probably has membrane less organelles associated)

Question 10

Examples of membrane less organelle diversity

Answer 10

virus factory = COVID, HPV, etc. assemble progeny virus, Tp53 aggregation (variability of what membrane less organelles are in cells)

Question 11

P-body

Answer 11

processing/breakdown of RNA, may arise or disperse in different cell types

Question 12

what do membrane less organelles do?

Answer 12

generate new compartments - variable based on size, what they do, in their content; reorganize existing compartments = separating out components (not nucleoli) for self-association and organization - subnuclear (liquid-liquid); vary in time, location and size

Question 13

nucleoli fusion

Answer 13

dynamic nucleoli fuse (no membrane, only RNA protein composition)

Question 14

compare and contrast membrane-bound and membraneless organization

Answer 14

optimized function (specialized subcompartment), lysosome = acid hydrolase efficiency, mitochondria = electron transport, H+ gradients, P-body = (processing body) RNA decay

Question 15

compare and contrast membrane bound and membrane less organelles size and shape

Answer 15

nucleus = 5-10 um diameter, nucleolus = 0.5-2.5 um diameter, other = less than 0.5 in frequency but are -20 um which is rare

Question 16

membrane bound “organizer”

Answer 16

phospholipid bilayer, boundary from aqueous cytoplasm, for specialization

Question 17

membrane less “organizer”

Answer 17

protein biochemistry, characteristics more likely to self-interact than interact with aqueous cytoplasm, protein phase separate from environment (not compatible protein biochemistry), partnered proteins associate, leaving out proteins less likely to interact

Question 18

Membraneless organelles formation

Answer 18

liquid-liquid phase separation - dissolved protein interact with each other, and possibly RNA to coalesce (de-mix) from surrounding homogeneous pictures of diverse macromolecules in cytoplasm or nucleoplasm (dispersion into nuclear shadow), reversible depending on stimulus = compare to separation (re-mixing oil and water) and vary variable time (nuclear shadow excluded and dispersed rapidly but can organize back)

Question 19

Membrane less organelle diverse examples

Answer 19

cajal nuclear bodies - varied content and function (partially regulate transcription and process RNA for spliceosome assembly which increases efficiency of nuclear events), PML nuclear bodies - replication suppressor, about 100 possible partner proteins in different PML bodies for varied function (apoptosis, telomere elongation)

Question 20

Liquid-liquid phase separation concepts

Answer 20

protein condensation leads to reaction crucible, sequestration, and organizational hub

Question 21

Reaction crucible

Answer 21

more efficient processing (post transitional modifications), concentrated subset of molecules enhances reactions (processing enzyme), liquid organization aids entry/exit, phase separated, processor for precursor to product, increased efficiency

Question 22

Sequestration

Answer 22

storage for later processing or secretion (deposit/reservoir), reduces response time to extracellular signals (physiological response decreases lag time), secreted and processed at later event, premade proteins in preparation for physiological change

Question 23

Organizational hub

Answer 23

normal condensation of proteins to focus interaction/polymerization of partner proteins (microtubule stability), 2 or more proteins physically associate to build structure to increase cell, advance cell, more efficient if physical components inside membrane less organelle, physical association

Question 24

Scaffold proteins

Answer 24

can drive liquid-liquid protein separation on their own, enriched for domain repeats (multivalent) and little 3D structure (disordered), sufficient concentration will condense and separate from surrounding cytoplasm or nuclear plasm

Question 25

Client proteins

Answer 25

proteins can interact with scaffold proteins, compatible with liquid-liquid protein synthesis of partner proteins, typically insufficient for liquid-liquid protein separation on their own

Question 26

protein characteristics for liquid-liquid phase separation

Answer 26

multivalent - repeated subdomains = repeated sites for interactions, drives condensation of membrane less organelles, scaffold for partner protein, SOS and Grb2; disordered - no rigid 3D “lock and key type” conformation, high content of polar and charged amino acids to keep protein in extended shape, low content of hydrophobic AA so less likely to fold up to reduce interaction with water

Question 27

Phase separation of liquid-liquid protein separation extracellular conditions

Answer 27

pH, osmolarity, etc., stressors like toxins (environmental stress), take homogeneous proteins - cause some to come out and phase separate, change extracellular to get response inside cell

Question 28

Phase separation of liquid-liquid protein separation intracellular conditions

Answer 28

protein concentration, ion concentration, partners (other proteins, RNA or DNA), ATP (as charged molecule; not as energy source), post-translational modifications (increasing p-tau that increases separation so more microtubule depolymerization), for some proteins causes possible disease-specific mutation

Question 29

Phase transition

Answer 29

liquid-liquid phase transition = may occur abruptly after liquid-liquid phase separation, excessive interactions among components, uncertain reversibility so uncertain consequences

Question 30

Aggregation

Answer 30

may occur independently or following liquid-liquid phase transition, disrupts normal cell function, abnormal and often disease-associated so typically irreversible

Question 31

Interaction beyond liquid-liquid phase separation bad news

Answer 31

function goes to dysfunction, can occur in lots of different cell types in lots of different cells, aggregation and nucleation, likely irreversible nature of assembly (amyloid) and insoluble

Question 32

Dispersion to aggregation

Answer 32

Disperse to liquid-liquid phase separation to liquid-liquid phase transition to aggregation; condensation from excess interactions = generates “solid” structure, insoluble, fibrous “amyloid”, fibrils and tangles are associated with many degenerative diseases

Question 33

Nucleation

Answer 33

aggregated proteins serve as condensation foci for proteins that would otherwise remain in liquid-liquid protein transition or move back to liquid-liquid protein separation

Question 34

Example pathologies and dysfunctional protein

Answer 34

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

Question 35

A-synuclein normal role

Answer 35

vesicle delivery to cell termini, neurotransmitter deliver and release at synapse, found in cytoplasm under membrane at synapse (in nucleus), undefined role in nucleus, normal day to day functioning delivering neurotransmitters, issue when transition to aggregation phase (amyloid fiber clumps - parkinson’s disease), several proteins associate to new neuron

Question 36

A-synuclein example

Answer 36

potential to clump into amyloid fibers, normal function - self-associating, delivering to synapse, individuals proteins - liquid-liquid protein separation then increased self-interaction then liquid-liquid protein transition (forms aggregate) then aggregates then amyloid fibrils (disruption of neurotransmitters and dysfunction of neuron); Parkinson’s disease, decreased vesicle trafficking, disrupted transmitter release, timing and progression varies

Question 37

Liquid-liquid phase separation and transition dysfunction

Answer 37

nucleus and cytoplasm - possible impact on many cell functions and therefore many diseases

Question 38

Membrane less Organelle and liquid-liquid phase separation and transition dysfunction types

Answer 38

insufficient interaction for liquid liquid phase separation and therefore no “reaction crucible, storage, or hub”, excess interaction driving liquid liquid phase transition (multivalent with self or partner), partner excess or missing, wrong type or timing of post transitional modification

Question 39

Resulting diverse pathologies of membrane less organelles and liquid-liquid phase separation/transition

Answer 39

neurodegeneration (TAR43, p-tau, a-synuclein), hyperplasia/cancer (p53, myc, p53), other tissue degeneration

Question 40

Neurodegeneration therapeutic interventions

Answer 40

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

Question 41

Exploring therapeutic interventions

Answer 41

drug interaction with protein = facilitates liquid-liquid protein separation for reaction crucible, sequestration, organizational hub; reduces liquid-liquid protein separation that might be precursor or liquid-liquid phase transition or aggregate (disperse to more compatible with normal cell physiology), drug regulation of modifier = regulate post-translational modification for desired effect (protein dispersion)