scholarly journals Spider silk self-assembly via modular liquid-liquid phase separation and nanofibrillation

2020 ◽  
Vol 6 (45) ◽  
pp. eabb6030
Author(s):  
Ali D. Malay ◽  
Takehiro Suzuki ◽  
Takuya Katashima ◽  
Nobuaki Kono ◽  
Kazuharu Arakawa ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Self Assembly ◽  
Spider Silk ◽  
Solid Phase ◽  
Silk Gland ◽  
Liquid Phase Separation ◽  
Hierarchical Organization ◽  
Amino Terminal ◽  
Liquid Liquid Phase Separation

Spider silk fiber rapidly assembles from spidroin protein in soluble state via an incompletely understood mechanism. Here, we present an integrated model for silk formation that incorporates the effects of multiple chemical and physical gradients on the different spidroin functional domains. Central to the process is liquid-liquid phase separation (LLPS) that occurs in response to multivalent anions such as phosphate, mediated by the carboxyl-terminal and repetitive domains. Acidification coupled with LLPS triggers the swift self-assembly of nanofibril networks, facilitated by dimerization of the amino-terminal domain, and leads to a liquid-to-solid phase transition. Mechanical stress applied to the fibril structures yields macroscopic fibers with hierarchical organization and enriched for β-sheet conformations. Studies using native silk gland material corroborate our findings on spidroin phase separation. Our results suggest an intriguing parallel between silk assembly and other LLPS-mediated mechanisms, such as found in intracellular membraneless organelles and protein aggregation disorders.

scholarly journals Prion-Like Proteins in Phase Separation and Their Link to Disease

Biomolecules ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1014
Author(s):  
Macy L. Sprunger ◽  
Meredith E. Jackrel
Keyword(s):  
Protein Folding ◽  
Phase Transitions ◽  
Phase Separation ◽  
Liquid Phase ◽  
Protein Misfolding ◽  
Solid Phase ◽  
Liquid Phase Separation ◽  
Therapeutic Approaches ◽  
Important Physiological Process ◽  
Liquid Liquid Phase Separation

Aberrant protein folding underpins many neurodegenerative diseases as well as certain myopathies and cancers. Protein misfolding can be driven by the presence of distinctive prion and prion-like regions within certain proteins. These prion and prion-like regions have also been found to drive liquid-liquid phase separation. Liquid-liquid phase separation is thought to be an important physiological process, but one that is prone to malfunction. Thus, aberrant liquid-to-solid phase transitions may drive protein aggregation and fibrillization, which could give rise to pathological inclusions. Here, we review prions and prion-like proteins, their roles in phase separation and disease, as well as potential therapeutic approaches to counter aberrant phase transitions.

scholarly journals Pericentromeric heterochromatin is hierarchically organized and spatially contacts H3K9me2 islands in euchromatin

2019 ◽  
Author(s):  
Yuh Chwen G. Lee ◽  
Yuki Ogiyama ◽  
Nuno M. C. Martins ◽  
Brian J. Beliveau ◽  
David Acevedo ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Genome Stability ◽  
Repetitive Sequences ◽  
Liquid Phase Separation ◽  
Hierarchical Organization ◽  
Pericentromeric Heterochromatin ◽  
Chromosome Dynamics ◽  
Technical Challenges ◽  
Liquid Liquid Phase Separation

AbstractMembraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, our current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. We investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, we reveal a hierarchical organization of the PCH domains into distinct “territories.” Strikingly, H3K9me2/3-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2/3 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. Our findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin.Author summaryThe three dimensional (3D) organization of genomes in cell nuclei can influence a wide variety of genome functions. However, most of our understanding of this critical architecture has been limited to the gene-rich euchromatin, and largely ignores the gene-poor and repeat-rich pericentromeric heterochromatin, or PCH. PCH comprises large part of most eukaryotic genomes, forms 3D PCH domains in nuclei, and plays vital role in chromosome dynamics and genome stability. In this study, we developed a new method that overcomes the technical challenges imposed by the highly repetitive PCH DNA, and generated a comprehensive picture of its 3D organization. Combined with image analyses, we revealed a hierarchical organization of the PCH domains. Surprisingly, we showed that distant euchromatic regions enriched for repressive epigenetic marks also dynamically interact with the main PCH domains. These 3D interactions are mediated by liquid-liquid phase separation mechanisms, similar to how oil and vinegar separate in salad dressing, and can influence the fitness of individuals. Our discoveries have strong implications for how seemingly “junk” DNA could impact functions in the gene-rich euchromatin.

scholarly journals Liquid-liquid phase separation of full-length prion protein initiates conformational conversion in vitro

2020 ◽  
Author(s):  
Hiroya Tange ◽  
Daisuke Ishibashi ◽  
Takehiro Nakagaki ◽  
Yuzuru Taguchi ◽  
Yuji O. Kamatari ◽  
...  
Keyword(s):  
Phase Transition ◽  
Phase Separation ◽  
Liquid Phase ◽  
Prion Protein ◽  
Solid Phase ◽  
Terminal Region ◽  
Liquid Phase Separation ◽  
Solid Phase Transition ◽  
Liquid Liquid Phase Separation

AbstractPrion diseases are characterized by accumulation of amyloid fibrils. The causative agent is an infectious amyloid that is comprised solely of misfolded prion protein (PrPSc). Prions can convert PrPC to proteinase-resistant PrP (PrP-res) in vitro; however, the intermediate steps involved in the spontaneous conversion remain unknown. We investigated whether recombinant prion protein (rPrP) can directly convert into PrP-res via liquid-liquid phase separation in the absence of PrPSc. We found that rPrP underwent liquid-liquid phase separation at the interface of the aqueous two-phase system (ATPS) of polyethylene glycol (PEG) and dextran, whereas single-phase conditions were not inducible. Fluorescence recovery assay after photobleaching revealed that the liquid-solid phase transition occurred within a short time. The aged rPrP-gel acquired proteinase-resistant amyloid accompanied by β-sheet conversion, as confirmed by western blotting, Fourier transform infrared spectroscopy, and Congo red staining. The reactions required both the N-terminal region of rPrP (amino acids 23-89) and kosmotropic salts, suggesting that the kosmotropic anions may interact with the N-terminal region of rPrP to promote liquid-liquid phase separation. Thus, structural conversion via liquid–liquid phase separation and liquid–solid phase transition are intermediate steps in the conversion of prions.

scholarly journals Capillary flow experiments for thermodynamic and kinetic characterization of protein liquid-liquid phase separation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Emil G. P. Stender ◽  
Soumik Ray ◽  
Rasmus K. Norrild ◽  
Jacob Aunstrup Larsen ◽  
Daniel Petersen ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Amyloid Fibrils ◽  
Capillary Flow ◽  
Solid Phase ◽  
Liquid Phase Separation ◽  
Dead Box ◽  
Phase Concentration ◽  
Liquid Liquid Phase Separation

AbstractLiquid-liquid phase separation or LLPS of proteins is a field of mounting importance and the value of quantitative kinetic and thermodynamic characterization of LLPS is increasingly recognized. We present a method, Capflex, which allows rapid and accurate quantification of key parameters for LLPS: Dilute phase concentration, relative droplet size distributions, and the kinetics of droplet formation and maturation into amyloid fibrils. The binding affinity between the polypeptide undergoing LLPS and LLPS-modulating compounds can also be determined. We apply Capflex to characterize the LLPS of Human DEAD-box helicase-4 and the coacervate system ssDNA/RP3. Furthermore, we study LLPS and the aberrant liquid-to-solid phase transition of α-synuclein. We quantitatively measure the decrease in dilute phase concentration as the LLPS of α-synuclein is followed by the formation of Thioflavin-T positive amyloid aggregates. The high information content, throughput and the versatility of Capflex makes it a valuable tool for characterizing biomolecular LLPS.

scholarly journals Tau conformers in FTLD-MAPT undergo liquid-liquid phase separation and perturb the nuclear envelope

2020 ◽  
Author(s):  
Sang-Gyun Kang ◽  
Zhuang Zhuang Han ◽  
Nathalie Daude ◽  
Emily McNamara ◽  
Serene Wohlgemuth ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Nuclear Envelope ◽  
Solid Phase ◽  
Critical Mass ◽  
Liquid Phase Separation ◽  
Liquid Droplets ◽  
Cytoplasmic Transport ◽  
End Stage ◽  
Liquid Liquid Phase Separation

AbstractRecent studies show that a single MAPT gene mutation can promote alternative tau misfolding pathways engendering divergent forms of frontotemporal dementia and that under conditions of molecular crowding, the repertoire of tau forms can include liquid-liquid phase separation (LLPS). We show here that following pathogenic seeding, tau condenses on the nuclear envelope (NE) and disrupts nuclear-cytoplasmic transport (NCT). Interestingly, NE fluorescent tau signals and small fluorescent inclusions behaved as demixed liquid droplets in living cells. Thioflavin S-positive intracellular aggregates were prevalent in tau-derived inclusions with a size bigger than 3 μm2, indicating that a threshold of critical mass in the liquid state condensation may drive liquid-solid phase transitions. Our findings indicate that tau undergoing LLPS is more toxic amongst a spectrum of alternative conformers; LLPS droplets on the NE that disrupt NCT serve to trigger cell death and can act as nurseries for fibrillar structures abundantly detected in end-stage disease.

Polymerization-Induced Self-Assembly Promoted by Liquid–Liquid Phase Separation

2019 ◽  
Vol 8 (8) ◽  
pp. 943-946 ◽  
Author(s):  
Yi Ding ◽  
Qingqing Zhao ◽  
Lei Wang ◽  
Leilei Huang ◽  
Qizhou Liu ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Self Assembly ◽  
Liquid Phase Separation ◽  
Liquid Liquid Phase Separation
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