Membraneless Organelle Lecture PDF

Membraneless organelles Dr Anton Calabrese [email protected] @anton_calabrese  Learning Outcomes Understand the behavior of membraneless organelles. Understand the mechanism of liquid-liquid phase separation that is used to explain the formation of mem...

Membraneless organelles Dr Anton Calabrese [email protected] @anton_calabrese  Learning Outcomes Understand the behavior of membraneless organelles. Understand the mechanism of liquid-liquid phase separation that is used to explain the formation of membranless organelles. Knowledge of the key drivers of phase separation. Understand how phase separation can be modulated in cells. Understand that liquid-to-solid transitions are associated with disease. Understand the function, assembly and regulation of stress granule formation. Understand how phase separation contributes to virus infection.  Conventional organelles Membrane bound, vesicle like Surrounded by a lipid bilayer Aqueous environment in and out Liposome and lipid bilayer structure Wikipedia  Membraneless organelles/condensates/RNP granules Do not have a phospholipid boundary. Comprise protein and RNA Concentrate specific components that enter and leave (sec timescale) Dynamic, coherent assemblies that play key roles in biology. Akin to oily droplets in water Banani et al. (2017) Nat Rev Mol Cell Biol, 18, 285  Membraneless organelles You do not need to remember this list! Nucleolus – Ribosome biogenesis Paraspeckle – Regulation of gene expression Nuclear speckle – Storage of splicing factors Cajal bodies – Regulation of snRNP maturation PML bodies – Regulation of transcription and protein storage Banani et al. (2017) Nat Rev Mol Cell Biol, 18, 285  Membraneless organelles You do not need to remember this list! P bodies – mRNA processing and decay Stress granules – storage of translationally stalled mRNA and translation machinery proteins Germ granules – Regulation of mRNA translation in cytoplasm of germ cells Balbiani body – protection of organelles during oocyte dormancy. Banani et al. (2017) Nat Rev Mol Cell Biol, 18, 285  What is their structure? How do they form? How do they contribute to cell function/dysfunction?  Membraneless organelles behave like liquid droplets P granules “drip” around the nucleus in germ line cells. somatic progenitor cell germ cell Brangwynne et al., (2009) Science, 324, 1729  Membraneless organelles behave like liquid droplets Flow and coalesce Grow in size over time Fluorescence Molecules recovery after dynamically photobleaching reorganise (FRAP) Shin and Brangwynne (2017) Science, 6357, eaaf4382  Why do molecules form droplets? Shin and Brangwynne (2017) Science, 6357, eaaf4382  Membraneless organelles form by Liquid-liquid phase separation (LLPS) Protein molecules concentrated in droplets If homotypic interaction energies are more favourable than heterotypic interaction energies then demixing occurs. High entropy Low entropy Gomes and Shorter (2019) JBC, 294, 7115-7127. Hyman et al. (2014) Annu. Rev. Cell Dev. Biol. 30, 39 Brangwynne et al., (2015) Nature Physics, 11, 899  Forces that drive demixing At concentrations above Ccritical, a protein will form droplets. Droplets Posttranslational modifications, temperature and ionic strength can No droplets modulate Ccritical Droplets allow diffusion within the compartment and exchange of molecules with the dilute phase. Phase diagrams are used to show conditions Alberti (2017) Current Biol., 27, R1097 where phase separation does/does not occur  Scaffolds and clients A key set of proteins and RNA drive membraneless organelle formation. This “scaffold” specifically recruits client proteins/RNAs Scaffold assembly and client recruitment is regulated by posttranslational modifications Scaffold RNA Scaffold protein Ditlev et al. (2019) JMB, 23, 4666  Regulation of LLPS Most proteins that drive LLPS are multivalent and have intrinsically disordered regions (IDRs) Multivalent interactions between proteins (ordered domains and IDRs) and RNA drive LLPS Multivalent: a protein/nucleic acid with several interaction sites. IDR: A functional protein region without a unique structure. Pancsa et al. (2019) BBA Proteins Proteom., 1867, 970  Features of proteins that drive membraneless organelle formation IDRs Post-translational modifications of proteins and post-transcriptional modifications of RNA affect the types of interactions they can participate in and thereby regulate phase separation. Van Treeck and Parker, (2018) Cell, 174,791  Liquid-to-solid phase transitions are associated with disease Examples of solid deposits of proteins Liquid-like MLOs can “mature” to form solids associated with Motor Neurone and Huntington’s disease Motor Neurone Disease Dementia Shin and Brangwynne (2017) Science, 6357, eaaf4382  Summary Membraneless organelles are liquid droplets. Act to store molecules, form reaction crucibles and organize cellular functions. A scaffold of protein and RNA mediate droplet formation. Proteins with intrinsically disordered regions predominate the proteomes of membraneless organelles. The scaffold recruits client proteins to the droplets. Protein and RNA modifications can be used to tune the composition and assembly of condensates. Liquid-to-solid phase transitions in cells are associated with disease.  Stress granules You do not need to remember this list of Contain: genes! mRNAs stalled in translation initiation (pre-initiation complexes) Various translation initiation factors RNA-binding proteins, and many non-RNA-binding proteins RBPs associated G3BP (SG marker) is with SGs normally dispersed Highlighted RBPs in cells, but clusters are disease- in SGs in response to linked stress Control the utilization of mRNA during stress Implicated in diseases, e.g. cancer, neurodegeneration, inflammatory disorders and viral infections Jain et al. (2016) Cell, 164, 487  Assembly of stress granules mRNA associates with 40S Separation of the 60S ribosomal particle to form the subunit from the assembly pre-initiation complex (PIC). leads to accumulation of Normally, the 60S ribosomal 40S-mRNA complexes that particle binds leading to mRNA associates with RNA binding translation. proteins that nucleate SG formation Wolozin, Ivanov (2019) Nat Rev Neurosci, 20, 649  Assembly of stress granules Puromycin promotes SGs by disrupting the translating ribosome. Cyclohexamide stalls translation, prevents ribosome disruption and prevents SG formation. Wolozin, Ivanov (2019) Nat Rev Neurosci, 20, 649  Formation of pre-initiation complexes and mRNA translation under basal conditions 2. eIF2 binds initiator tRNA to form a ternary complex 3. This complex combines with eIF3-40S to form the 43S PIC 5. 60S ribosomal particle binds to initiate mRNA translation 4. 43S PIC associates with eIF4F- 1. eIF4F complex recognizes mRNA to form the 48S-PIC the 5′ cap on mRNA Wolozin, Ivanov (2019) Nat Rev Neurosci, 20, 649  Inhibiting translation initiation promotes SG formation 1. mTOR inhibition reduces phosphorylation of eIF4E-binding protein (4E-BP). 4E-BP is now available to bind eIF4E displacing it from its complex with mRNA. 2. Phosphorylation of eIF2 prevents binding to tRNAi 3. Drugs can interfere with eIF4F complex assembly, The incomplete PIC binds RBPs that displacing it from mRNA nucleate SGs, triggering a cascade that results in mature SGs Wolozin, Ivanov (2019) Nat Rev Neurosci, 20, 649  Cycle of stress granule formation 5. Stress granules disperse, Soluble RBPs return to nucleus, solid-like RBPs are 1. Basal conditions of RNA disposed via autophagosomes metabolism. Nuclear RBPs perform functions in nucleus (e.g. splicing) Nucleus 4. Stress granules mature, 2. Nuclear RBPs (red/orange) recruiting other RBPs Cytoplasm translocated to cytoplasm Many of the key RBPs are You do not need to remember stored in the nucleus and this list of proteins! are shuttled to the 3. Stress granule nucleation cytoplasm during SG (e.g. via TIA1, G3BP1, FMRP) Wolozin, Ivanov (2019) Nat Rev Neurosci, 20, 649 formation  Liquid-to-solid Liquid-to-solid transition transition ofproteins of SG SG proteins are associated are associated withwith disease disease 4. Even when stress is resolved, 1. Basal conditions of RNA pathological aggregates remain. metabolism. Nuclear RBPs perform functions in nucleus (e.g. splicing) Associated with Alzheimer’s, some dementia’s and MND. 3. Under chronic stress, liquid assemblies become gel-like aggregates, including the protein TDP-43 which becomes phosphorylated. 2. In acute stress conditions RBPs assemble into SGs. Aggregates associated with disease can seed the formation of these assemblies. Wolozin, Ivanov (2019) Nat Rev Neurosci, 20, 649  TDP-43 is a key player in pathology of neurodegenerative diseases Comprises a folded N-terminal domain (NTD) nuclear localisation sequence (NLS), nuclear export sequence (NES), 2x RNA recognition motifs (RRMs) and an unstructured C-terminal tail. Phosphorylation is associated with disease. TDP-43 NTD RRM RRM CTD NLS NES Gao et al. (2019) J Neurochem, 146, 7  RBP cascade hypothesis You do not need Extracellular factors to remember cause neuronal stress specific details of this cascade! Intracellular proteins such as pTau and TDP-43 mediate the effects of extracellular stresses Translational stress response → SGs RBPs mediate neurodegenerative disease Wolozin, Ivanov (2019) Nat Rev Neurosci, 20, 649 Protein assembly into amyloid is associated with disease The environment within a stress granule, and chemical changes to TDP-43 may accelerate amyloid assembly  TDP-43 forms amyloid fibrils Amyloids are solid aggregates of 𝝱-sheet rich fibrils. Many (or all) proteins can form amyloid fibrils and deposition of these fibrils is associated with > 50 diseases. Cryo-EM has been used to solve the structure of TDP-43 amyloid fibrils from patient donors to understand its role in disease. Motor Neurone Disease Frontotemporal Lobar dementia Li et al. (2021) Nat Commun, 12, 1620; Arseni et al (2023) Nature 620, 898  Phase separation plays a role in many disease states Mutations in signalling receptors alter signalling These factors can lead to clusters/condensates at aberrant condensates sites of transcription or and liquid-to-solid DNA damage repair transitions Liquid-like condensates termed viral factories promote virus replication and modulate antiviral immune responses  Viral factories Comprises viral proteins, host proteins and RNA Fully assembled virus capsids emerge from the viral factories. Concentrate the components required for virus replication, allows immune response evasion Rotavirus is a model system – major cause of acute gastroenteritis in infants and young children worldwide Viral capsids are found in the viral factory (black dots in the dark grey structure). Viral capsids “bud” from the viral factory Novoa et al. (2005) Biol of the Cell, 97, 147 Geiger et al. (2021) EMBO J, e107711  Liquid-like viral factories can be “dissolved” 1,6-hexanediol (1,6-HD) is a chemical probe to differentiate between liquid-like and gel-like states of membraneless organelles Early infection viral factories are completely dissolved by 1,6-HD. When 1,6-HD is removed they re-appear. At 12 hours post infection, only some smaller structures were dissolved, suggesting that later factories are more gel-like. In the presence of molecules that dissolve viral factories, the virus replicates slower Geiger et al. (2021) EMBO J, e107711  RSV replication is blocked by a condensate hardening drug Respiratory syncytial virus (RSV), a common respiratory virus that usually causes mild, cold-like symptoms. RSV nucleoprotein (N), phosphoprotein (P) are key to form viral factories. Treatment with small molecules reduces fluidity of viral factories and decreases viral replication CPM Condensates CAN be targeted to inhibit virus FRAP replication in vivo analysis of RSV factories Drug treatment A3E in cells reduces virus replication Risso-Ballester et al. (2021) Nature, 595, 596  Summary Membraneless organelles are key organisers of the cell. Act as storage compartments and reaction crucibles. Liquid-liquid phase separation can be used to explain their formation. Membraneless organelles comprise RNA, RNA binding proteins. Liquid-to-solid transitions are associated with disease Stress granules are membraneless organelles that store non-translating mRNA. Many viral proteins undergo phase separation in cells to form factories that promote virus replication/assembly. Tools (FRAP, 1,6-HD) can probe membraneless organelles in cells.  Learning Outcomes Understand the behavior of membraneless organelles. Understand the mechanism of liquid-liquid phase separation that is used to explain the formation of membranless organelles. Knowledge of the key drivers of phase separation. Understand how phase separation can be modulated in cells. Understand that liquid-to-solid transitions are associated with disease. Understand the function, assembly and regulation of stress granule formation. Understand how phase separation contributes to virus infection.