EXAM 2 - Session 12: Atypical Organelles and Functional Condensates

Question 1

Explain membrane-less organelle structure/compartment seperation.

Answer 1

liquid within another liquid situation
* doesn’t have cell membrane
* ex. like oil and water seperation

Question 2

What are the function MLOs are associated with?

Answer 2

• cell divison
• chromatin remodeling
• gene transcription
• synapse function
• virus assembly

Question 3

Name the “classic” structures in MLOs.

Answer 3

P-body - concetrates of RNA and proteins without a supporting membrane
* function: RNA breakdown

Nucleolus - domain that is responsible for cell divison (disassembly/reassembly)

Contents within MLO are very dynamic

Question 4

Explain some characteristics of MLOs.

Answer 4

1. Generate new compartments - form/fusion of cytoplasmic granules and create subnuclear compartments.
2. Reorganize existing compartments - nucleoli fusion
3. Vary in time, location, and size

Question 5

What are the similarities of membrane-bound organelles and MLOs?

Answer 5

1. Organization –> optimized function
   lysosome (MB) - acid hydrolases efficiency
   mitochondria (MB) - e- transport, H+ gradients
   p-body (MLO) - RNA decay
2. Vary in size and shape
   nucleus (MB) - 5-10 um diameter
   nucleolus (MLO) - 0.5-2.5 um diameter
   *other MLO (MLO) - <0.5 (frequent) – ~20um (rare)

Question 6

Describe the membrane-bound “organizer”.

Answer 6

Phospholipid bilayer(s) - boundary from aqueous cytoplasm

Question 7

Explain the MLO “organizer”.

Answer 7

Protein biochemistry - dependent on characteristics that are more likely to interact with themselves than interact with aqueous cytoplasm
* similar or partnered proteins that are more likely to associate with specific partners (compatibility) and form membrane-less seperation from cytoplasm.

Question 8

Describe liquid-liquid phase seperation (LLPS).

Answer 8

Dissolved proteins interact with each other (possibly RNA) to “de-mix” from surrounding homogeneous mixtures in the cytoplasm.
* can be reversible depending on the stimulus
* variable in time

Question 9

Give two examples of MLO diversity.

Answer 9

Most MLOs have varied content –> varied functions

Cajal nuclear bodies –> varied content & function
* partially regulate transcription
* process RNA for spliceosome assembly

PML nuclear bodies (green dots in image)
* PML protein - replication suppressor
* ~100 possible partner proteins in different PML bodies for varied function
* apoptosis, telomere elongation

Question 10

What does protein condensation (LLPS) lead to?

Answer 10

• Reaction crucible
• Sequestration
• Organizational hub

Question 11

Explain the reaction crucible after protein condensation (LLPS).

Answer 11

Reaction crucible leads to more efficient processing of reactions
* ex. post-translational modifications –> concentrated subset of molecules enhances reactions
* by increasing the concentration of the processor (red triangle in image) the cell increases the efficiency of the reaction

“liquid” organization allows for easier entry and exit

Question 12

Explain sequestration that occurs after protein condensation.

Answer 12

Proteins are condensed and stored for later processing or secretion
* reduces response time to extracellular signals
* builds a reservoir/deposit

Question 13

Explain the organizational hub that forms after protein condensation.

Answer 13

Normal condensation of proteins (e.g., tau) focuses interaction/polymerization of partner proteins (microtubules)

Question 14

Explain scaffold proteins versus client proteins.

Answer 14

Scaffold proteins - drive LLPS on their own
* enriched for domain repeats (multivalent)
* little 3D structure (disordered)

Client proteins - interact w/ scaffold proteins
* compatible with LLPS of partner proteins
* insufficient for LLPS on their own

Question 15

What does it mean by multivalent?

Answer 15

Multivalent = repeated subdomains –> repeated site for interactions

Question 16

What does it mean by disordered?

Answer 16

• NOT the same as denatured
• no rigid 3D “lock & key-type” conformation
• bc of flexibility –> proteins can interact with many partners
• high content of polar/charged amino acids –> keeps proteins in extended shape
• low content of hydrophobic AA –> less likely to fold up to reduce interaction with water

Question 17

List other extracellular and intracellular conditions that drive phase seperation.

Answer 17

Extracellular: (outside conditions can change intracellular organization)
* pH
* osmolarity
* stressors (toxins)

Intracellular:
* protein concentration
* ion concentration
* partners (other proteins, RNA or DNA)
* ATP (as charged molecule)
* post-translational modifications (e.g, phosphorylation)

Question 18

Explain phase transition.

Answer 18

• occurs abruptly after LLPS
• excessive interactions among components
• uncertain reversibility, uncertain concequences

Question 19

Explain aggregation.

Answer 19

• may occur independently or following LLPT
• disrupts normal cell function
• abnormal, often disease-associated and typically irreversible

Question 20

How does further interaction of proteins beyond LLPS result in dysfunction?

Answer 20

Disperse –> LLPS –> LLPT –> aggregation
* condensation from excess interactions –> generates “solid” structures, insoluble, fibrous amyloids
* nucleation - aggregated proteins are the focal point of condensation for proteins that would otherwise remain in LLPT or return to LLPS (formation of deterimental clumps)

Question 21

Name the conditions that are associated with dysfunctional MLOs.

Answer 21

• Amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease)
• Alzheimer’s disease
• Parkinson’s disease

LLPS/T are located in the nucleus and cytoplasm –> has possible impact on many cell functions –> related to many diseases
* result: diverse pathologies

Question 22

What is the desired outcome of therapeutic interventions for MLOs.

Answer 22

1. Facilitate LLPS for reaction crucible, sequestration, and organizational hub.
2. Reduce LLPS that might be precursor of LLPT or aggregate.
3. Regulate post-translational modification for desired effect.