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Tanja Mittag - Instruct Biennial Structural Biology Conference 2024 - "New Directions for Structural Biology"

• 0:01 Introduction: Tanja Mittag discusses protein condensates, focusing on the protein HNRNPA1, which contains intrinsically disordered regions (IDRs) or low complexity domains.

• 0:58 IDRs and Phase Separation: These IDRs, which don't use the full amino acid alphabet, are crucial for mediating phase separation, a process where proteins form distinct liquid-like droplets within cells.

• 1:33 Phase Separation in Biology: Phase separation occurs under physiological conditions and is driven by multivalent interactions, forming a 3D network. Examples include stress granules and other cellular bodies.

• 3:49 Importance of Biomolecular Condensates: Dozens of bodies, including nucleoli and stress granules, are not membrane bound, but concentrate specific components. Phase separation offers answers to how this is achieved.

• 5:09 Condensate Properties: Cellular bodies' properties are dynamic, and their components enter and leave, so their properties help in understanding their biology.

• 5:20 Phase Separation Beyond Bodies: Phase separation mediates other cellular processes, such as heterochromatin formation, RNA metabolism, membrane receptor clustering, autophagy, endocytosis, and nuclear pore complex selectivity.

• 6:10 "Cell Biology 2.0": Phase separation's role in compartmentalization is changing how we view cell biology.

• 6:22 Multivalent Interactions and Phase Diagrams: The driving force for phase separation depends on the valency (number of interaction sites) of proteins. Phase diagrams illustrate the concentrations required for phase separation.

• 7:35 IDR Phase Separation Mechanism: The low complexity domain of HNRNPA1 undergoes phase separation, driven primarily by aromatic residues ("stickers") within its sequence.

• 9:03 Investigating Self-Association: NMR spectroscopy and small-angle X-ray scattering (SAXS) revealed that aromatic residues mediate self-interactions and compaction, crucial for phase separation.

• 10:37 Stickers and Spacers Model: A "stickers and spacers" model was developed, where aromatic residues ("stickers") promote phase separation, while intervening "spacer" residues modulate interactions and solubility.

• 11:10 Coarse-Grained Simulations: Simulations using this model, developed in collaboration with the Pappu Lab, accurately predicted phase behavior, confirming the importance of sticker interactions.

• 12:38 Modifying Interaction Strength: Different aromatic residues (tyrosine, phenylalanine) have varying interaction strengths, influencing the driving force for phase separation.

• 13:10 Spacer Residue Effects: Spacer residues like glycine and serine also contribute; bulkier residues (serine) can suppress phase separation due to their larger effective solvation volume.

• 16:07 Model Validation: The sticker-spacer model accurately describes phase behavior and has been extended to other protein types, including folded proteins with associative patches.

• 17:01 Unequal Ion Partitioning: The dilute and dense phases in condensates can have different ion concentrations, leading to electric potentials between the phases.

• 18:03 Measuring Ion Concentrations: Techniques like inductively coupled plasma mass spectrometry (ICP-MS) and ion-selective electrodes measure ion concentrations in the different phases.

• 20:31 Implications of Electric Potentials: These electric potentials, influenced by molecular charge, ion type, and salt concentration, may have functional implications, potentially affecting cellular processes.

• 21:41 Condensates as Capacitors: The electric potential indicates that condensates can store charge and potentially do work, influencing cellular functions like transcription.

• 23:09 Viscoelastic Properties: Condensates are "network fluids" with viscoelastic properties, influenced by the strength and lifetime of the interactions within them.

• 24:14 Microrheology Measurements: Passive microrheology with optical tweezers measures the viscoelastic properties (storage and loss modulus) of condensates.

• 25:42 Modulating Viscoelasticity: Changing the "sticker" strength (e.g., using phenylalanine, tyrosine, or tryptophan) alters the viscoelastic properties of condensates.

• 26:45 Condensate Aging: Over time, condensates can become less dynamic, a process linked to neurodegenerative diseases.

• 29:13 Manipulating Aging: Manipulating the sequence (e.g., replacing glycine with serine) can alter the timescale of condensate aging.

• 29:45 Creep Test: A "creep test" using optical tweezers showed that aged condensates can transition into viscoelastic solids.

• 31:20 Fibril Formation: Condensate interfaces can enhance the nucleation of fibrils, while the interior can suppress fibril formation, potentially acting as a protective mechanism in cells.

• 33:35 Overlapping Grammars: Condensate and fibril formations have distinct, but overlapping, molecular grammars. Condensate formation is driven by sticker-sticker interactions, while fibrils rely on "zipper-zipper" interactions in beta sheets.

• 33:53 Summary: Condensates are dynamic network fluids with properties that can influence cellular functions, enhance or suppress reactions, and promote reactions through electric potentials at their interfaces.