Membraneless Organelles Overview
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Questions and Answers

What role does the scaffold play in the formation of membraneless organelles?

  • It inhibits the formation of protein aggregates.
  • It serves solely as a storage unit for cellular molecules.
  • It provides structural integrity to solid protein deposits.
  • It mediates the recruitment of client proteins and RNAs to droplets. (correct)

    • Which characteristic of proteins facilitates liquid-liquid phase separation (LLPS)?

  • Presence of a rigid tertiary structure.
  • Possession of intrinsically disordered regions (IDRs). (correct)
  • Absence of post-translational modifications.
  • High binding affinity for water molecules.

    • How do post-translational modifications influence phase separation?

  • They stabilize solid aggregates of proteins.
  • They eliminate the need for RNA during droplet formation.
  • They regulate the interactions that proteins and RNA can participate in. (correct)
  • They simplify the functional roles of proteins.

    • What outcome is associated with liquid-to-solid phase transitions in cells?

    <p>Formation of solid protein deposits linked to diseases.</p> Signup and view all the answers

    What defines a multivalent protein or nucleic acid?

    <p>It can interact with multiple partners due to several sites.</p> Signup and view all the answers

    What is a primary function of membraneless organelles in cellular functions?

    <p>To store molecules and organize reactions.</p> Signup and view all the answers

    What determines the composition and organization of condensates?

    <p>Post-translational modifications of proteins and RNA.</p> Signup and view all the answers

    Which of the following diseases is related to liquid-to-solid phase transitions?

    <p>Motor Neurone Disease.</p> Signup and view all the answers

    What is a defining structural characteristic of membraneless organelles?

    <p>Does not have a phospholipid boundary</p> Signup and view all the answers

    Which mechanism is primarily used to explain the formation of membraneless organelles?

    <p>Liquid-liquid phase separation</p> Signup and view all the answers

    What type of biological roles do membraneless organelles play?

    <p>They concentrate specific components that enter and leave dynamically</p> Signup and view all the answers

    Which type of physiological transition is associated with disease in membraneless organelles?

    <p>Liquid to solid transition</p> Signup and view all the answers

    Which of the following membraneless organelles is involved in mRNA processing and decay?

    <p>P bodies</p> Signup and view all the answers

    How do stress granules function in the cell?

    <p>They store translationally stalled mRNA and proteins</p> Signup and view all the answers

    What is one of the key drivers of liquid-liquid phase separation in membraneless organelles?

    <p>Hydrophobic interactions</p> Signup and view all the answers

    Which of the following contributes to the regulation of phase separation in cells?

    <p>RNA concentration</p> Signup and view all the answers

    What occurs to stress granule (SG) proteins under chronic stress conditions?

    <p>They become gel-like aggregates.</p> Signup and view all the answers

    What is a characteristic feature of the protein TDP-43?

    <p>It contains a pronounced N-terminal domain (NTD).</p> Signup and view all the answers

    What happens to RNA binding proteins (RBPs) during acute stress conditions?

    <p>They aggregate into stress granules.</p> Signup and view all the answers

    Which of the following is a consequence of resolved stress regarding pathological aggregates?

    <p>They persist despite stress resolution.</p> Signup and view all the answers

    How are extracellular factors related to neuronal stress?

    <p>They cause neuronal stress.</p> Signup and view all the answers

    What role does phosphorylation play in relation to TDP-43?

    <p>It is associated with developing pathology.</p> Signup and view all the answers

    During the process of liquid-liquid phase separation, what happens at the molecular level?

    <p>Proteins demix into distinct phases.</p> Signup and view all the answers

    What is one of the proposed hypotheses related to RNA binding proteins (RBPs)?

    <p>RBPs may function in a cascade effect.</p> Signup and view all the answers

    Study Notes

    Membraneless Organelles

    • Membraneless organelles are liquid droplets that act as storage compartments and reaction crucibles within cells, organizing cellular functions.
    • They are formed through liquid-liquid phase separation. This is mechanism through which the formation of membraneless organelles is explained
    • Key components of membraneless organelles include RNA and RNA-binding proteins
    • Liquid-to-solid transitions in membraneless organelles are often associated with diseases
    • Stress granules are membraneless organelles acting as storage compartment for non-translating mRNAs.
    • Many viral proteins also undergo phase separation in cells to form viral factories for promoting virus replication and assembly.
    • Tools like FRAP (Fluorescence recovery after photobleaching) and 1,6-hexanediol can probe membraneless organelles in cells.

    Learning Outcomes

    • The behavior of membraneless organelles
    • The mechanism of liquid-liquid phase separation in formation of membraneless organelles
    • The key drivers of phase separation
    • How phase separation can be modulated in cells
    • The association of liquid-to-solid transitions with disease
    • The function, assembly, and regulation of stress granule formation
    • The contribution of phase separation to virus infection

    Conventional Organelles

    • Membrane-bound, vesicle-like structure
    • Surrounded by a lipid bilayer
    • Aqueous environment inside and out

    P Bodies

    • Involved in mRNA processing and decay

    Stress Granules

    • Store translationally stalled mRNAs and translation machinery proteins
    • Involved in the utilization of mRNA during stress, inflammation, and viral infections
    • Include RNA-binding proteins and non-RNA-binding proteins.
    • Contain mRNAs stalled in pre-initiation complexes and various translation initiation factors
    • Participate in the regulation of mRNA utilization during processes associated with stress, inflammation and viral infections.
    • Important for controlling the utilization of mRNA in periods of stress, inflammation, or viral infections which are implicated in diseases like cancer, neurodegeneration, and inflammatory disorders

    Germ Granules

    • Regulate mRNA translation in cytoplasm
    • Involved during oocyte dormancy

    Balbiani Body

    • Involved in protection of organelles during oocyte dormancy

    Nucleolus

    • Ribosome biogenesis

    Paraspeckle

    • Involved in regulation of gene expression

    Nuclear Speckles

    • Storage of splicing factors

    Cajal Bodies

    • Regulation of snRNP maturation

    PML Bodies

    • Regulation of transcription and protein storage

    Forces Driving Demixing

    • Protein concentration above a critical threshold favors droplet formation
    • Post-translational modifications alter this threshold, as does temperature, and ionic strength
    • Droplets allow for diffusion and exchange of molecules within and between the compartment and dilute phase

    Scaffolds and Clients

    • Key proteins and RNAs drive membraneless organelle formation
    • Scaffold assembly and client recruitment are regulated by post-translational modifications
    • Nucleic acid/proteins are bound to the scaffold

    Regulation of LLPS

    • Multivalent proteins and intrinsically disordered regions (IDRs), are key in driving phase separation
    • Interactions between multivalent protein (ordered domains and IDRs) and RNA drive LLPS.

    Features of Proteins Driving Membraneless Organelle Formation

    • Stereochemistry specific interactions between well-folded domains
    • Specific interaction are between local structures of intrinsically disordered regions
    • Promiscuous interactions between disordered regions exist between (π-π, charge-charge, cation-π)

    Liquid-to-Solid Phase Transitions

    • Liquid-like membraneless organelles (MLOs) can transition to solid forms.
    • Solid forms like amyloid fibers are associated with diseases like motor neuron disease and dementia

    Assembly of Stress Granules

    • mRNA associates with 40S ribosomal particle
    • Separation of the 60S subunit can lead to mRNA complex accumulation
    • Proteins with RNA-binding can nucleate stress granule formation.
    • Puromycin and cycloheximide affect stress granule formation

    Formation of Pre-Initiation Complexes

    • elF2-tRNAi met ternary complex formation, and combining with elF3-40S (43S PIC)
    • elF4F complex recognizes 5' mRNA cap
    • 43S PIC associates with elF4F-mRNA to form 48S-PIC
    • 60S ribosomal particle binds initiating mRNA translation

    Inhibiting Translation Initiation

    • mTOR inhibition reduces 4E-BP phosphorylation and elF4E binding
    • Phosphorylation of elF2 prevents tRNA binding
    • Drugs can block elF4F complex assembly causing displacement of mRNA

    Cycle of Stress Granule Formation

    • Stress granules disperse, soluble RBPs return to the nucleus
    • Solid-like RBPs are disposed via autophagosomes
    • Key RBPs are stored in the nucleus and move to the cytoplasm for SG formation

    Liquid-to-Solid Transition of SG Proteins

    • Even after stress resolution, some proteins form pathological aggregates
    • Under chronic stress, liquid assemblies turn into gel-like aggregates and proteins such as TDP-43 are phosphorylated
    • These transitions are associated with various neurodegenerative diseases

    TDP-43 Key Role

    • Key in neurodegenerative diseases
    • Consists of a folded N-terminal domain (NTD), nuclear localization sequence (NLS), nuclear export sequence (NES), two RNA recognition motifs (RRMs), and an unstructured C-terminal tail (CTD)
    • Phosphorylation is associated with disease

    RBP Cascade Hypothesis

    • A cascade of events involving genetic, environmental, and modifying factors
    • Extracellular factors cause neuronal stress, and intracellular proteins (e.g. pTau, TDP-43) mediate effects.
    • The RBP cascade is associated with different neurological diseases.

    Protein Assembly and Amyloid

    • Protein assembly into amyloid is associated with diseases
    • The environment within a stress granule can accelerate TDP-43 amyloid assembly (enviromental factors)

    TDP-43 Forms Amyloid Fibrils

    • Amyloids are solid aggregates of B-sheet rich fibrils
    • Cryo-EM has been utilized to understand the structure of TDP-43 amyloid fibrils from patient donors, further exploring its role in disease

    Phase Separation in Disease States

    • RNP granule dynamics and function in neurodegeneration
    • Mutations or repeat expansions, abnormal PTMs, and changes in localization or translation initiation efficiency lead to aberrant condensate formation and liquid-to-solid transitions
    • Mutations in signalling receptors alter signalling clusters associated with transcription or DNA damage repair
    • These factors influence disease progression

    Viral Factories

    • Composed of viral proteins, host proteins and RNA
    • Fully assembled virus capsids emerge from viral factories
    • They concentrate elements required for virus replication and evasion of the immune response.
    • Rotavirus is an example of a viral factory in the context of acute gastroenteritis in children.

    Liquid-Like Viral Factories

    • 1,6-hexanediol (1,6-HD) used to distinguish liquid-like and gel-like states of membraneless organelles.
    • Early infection viral factories are usually completely dissolved by the probe
    • Viral factories show a difference in structure over time.

    RSV Replication Blocked

    • RSV Replication is affected/interfered by condensate hardening drugs
    • RSV nucleoprotein (N), phosphoprotein (P) in key for the viral factories formation.
    • Treatments with small molecules that reduce viral factory fluidity successfully decreased viral replication.

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    Description

    This quiz explores the fascinating world of membraneless organelles in cells. It covers their formation through liquid-liquid phase separation, key components, and their role in diseases. Additionally, it examines tools used to study these structures and their implications in viral replication.

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