scholarly journals Liquid-liquid phase separation and liquid-to-solid transition mediate α-synuclein amyloid fibril containing hydrogel formation

2019 ◽  
Author(s):  
Soumik Ray ◽  
Nitu Singh ◽  
Satyaprakash Pandey ◽  
Rakesh Kumar ◽  
Laxmikant Gadhe ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Droplet ◽  
Liquid Phase Separation ◽  
Amyloid Formation ◽  
Liquid To Solid Transition ◽  
Phase Separation Behavior ◽  
Separation Behavior ◽  
Liquid Liquid Phase Separation

SUMMARYα-Synuclein (α-Syn) aggregation and amyloid formation is directly linked with Parkinson’s disease (PD) pathogenesis. However, the early events involved in this process remain unclear. Here, using in vitro reconstitution and cellular model, we show that liquid-liquid phase separation (LLPS) of α-Syn precedes its aggregation. In particular, in vitro generated α-Syn liquid-like droplets eventually undergo a liquid-to-solid transition and form amyloid-hydrogel containing oligomers and fibrillar species. Factors known to aggravate α-Syn aggregation such as low pH, phosphomimic substitution, and familial PD mutation also promote α-Syn LLPS and its subsequent maturation. We further demonstrate α-Syn liquid droplet formation in cells, under oxidative stress. These cellular α-Syn droplets eventually transform into perinuclear aggresomes, the process regulated by microtubules. The present work provides detailed insights into the phase separation behavior of natively unstructured α-Syn and its conversion to a disease-associated aggregated state, which is highly relevant in PD pathogenesis.

scholarly journals A generic approach for studying the kinetics of liquid-liquid phase separation under near-native conditions

2019 ◽  
Author(s):  
Joris van Lindt ◽  
Anna Bratek-Skicki ◽  
Donya Pakravan ◽  
Ludo Van Den Bosch ◽  
Dominique Maes ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Cell Biology ◽  
Low Complexity ◽  
Liquid Phase Separation ◽  
Ph Values ◽  
Phase Separation Behavior ◽  
Kinetics Of ◽  
Separation Behavior ◽  
Liquid Liquid Phase Separation

Understanding the kinetics and underlying physicochemical forces of liquid-liquid phase separation (LLPS) is of paramount importance in cell biology, requiring reproducible methods for the analysis of often severely aggregation-prone proteins. Frequently applied approaches, such as dilution of the protein from an urea-containing solution or cleavage of its fused solubility tag, however, often lead to very different kinetic behaviors. Here we suggest that at extreme pH values even proteins such as the low-complexity domain (LCD) of hnRNPA2, TDP-43, and NUP-98 can be kept in solution, and then their LLPS can be induced by a jump to native pH, resulting in a system that can be easily controlled. This approach represents a generic method for studying LLPS under near native conditions, providing a platform for studying the phase-separation behavior of diverse proteins.

scholarly journals Stress induces dynamic, cytotoxicity-antagonizing TDP-43 nuclear bodies via paraspeckle lncRNA NEAT1-mediated liquid-liquid phase separation

2019 ◽  
Author(s):  
Chen Wang ◽  
Yongjia Duan ◽  
Gang Duan ◽  
Qiangqiang Wang ◽  
Kai Zhang ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Mammalian Cells ◽  
Liquid Droplet ◽  
Super Resolution ◽  
Liquid Phase Separation ◽  
Nuclear Bodies ◽  
List Type ◽  
Liquid Liquid Phase Separation

Graphic AbstractHighlights(Up to four bullet points. The length of each highlight cannot exceed 85 characters, including spaces)Stress induces phase-separated TDP-43 NBs to alleviate cytotoxicityThe two RRMs interact with different RNAs and act distinctly in the assembly of TDP-43 NBsLncRNA NEAT1 promotes TDP-43 LLPS and is upregulated in stressed neuronsThe ALS-causing D169G mutation is NB-defective and forms pTDP-43 cytoplasmic fociSummaryDespite the prominent role of TDP-43 in neurodegeneration, its physiological and pathological functions are not fully understood. Here, we report an unexpected function of TDP-43 in the formation of dynamic, reversible, liquid droplet-like nuclear bodies (NBs) in response to stress. Formation of NBs alleviates TDP-43-mediated cytotoxicity in mammalian cells and fly neurons. Super-resolution microscopy reveals a “core-shell” organization of TDP-43 NBs, antagonistically maintained by the two RRMs. TDP-43 NBs are partially colocalized with nuclear paraspeckles, whose scaffolding lncRNA NEAT1 is dramatically upregulated in stressed neurons. Moreover, increase of NEAT1 promotes TDP-43 liquid-liquid phase separation (LLPS) in vitro. Finally, we uncover that the ALS-associated mutation D169G impairs the NEAT1-mediated TDP-43 LLPS and NB assembly, causing excessive cytoplasmic translocation of TDP-43 to form stress granules that become phosphorylated TDP-43 cytoplasmic foci upon prolonged stress. Together, our findings suggest a stress-mitigating role and mechanism of TDP-43 NBs, whose dysfunction may be involved in ALS pathogenesis.

scholarly journals Targeting NSD2-mediated SRC-3 liquid–liquid phase separation sensitizes bortezomib treatment in multiple myeloma

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jing Liu ◽  
Ying Xie ◽  
Jing Guo ◽  
Xin Li ◽  
Jingjing Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Acquired Drug Resistance ◽  
Bortezomib Treatment ◽  
Liquid Liquid Phase Separation

AbstractDevelopment of chemoresistance is the main reason for failure of clinical management of multiple myeloma (MM), but the genetic and epigenetic aberrations that interact to confer such chemoresistance remains unknown. In the present study, we find that high steroid receptor coactivator-3 (SRC-3) expression is correlated with relapse/refractory and poor outcomes in MM patients treated with bortezomib (BTZ)-based regimens. Furthermore, in immortalized cell lines, high SRC-3 enhances resistance to proteasome inhibitor (PI)-induced apoptosis. Overexpressed histone methyltransferase NSD2 in patients bearing a t(4;14) translocation or in BTZ-resistant MM cells coordinates elevated SRC-3 by enhancing its liquid–liquid phase separation to supranormally modify histone H3 lysine 36 dimethylation (H3K36me2) modifications on promoters of anti-apoptotic genes. Targeting SRC-3 or interference of its interactions with NSD2 using a newly developed inhibitor, SI-2, sensitizes BTZ treatment and overcomes drug resistance both in vitro and in vivo. Taken together, our findings elucidate a previously unrecognized orchestration of SRC-3 and NSD2 in acquired drug resistance of MM and suggest that SI-2 may be efficacious for overcoming drug resistance in MM patients.

scholarly journals G-quadruplex DNA inhibits unwinding activity but promotes liquid–liquid phase separation by the DEAD-box helicase Ded1p

2021 ◽  
Author(s):  
Jun Gao ◽  
Zhaofeng Gao ◽  
Andrea A. Putnam ◽  
Alicia K. Byrd ◽  
Sarah L. Venus ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Intrinsically Disordered ◽  
Dead Box ◽  
G4 Dna ◽  
G Quadruplex ◽  
The Dead ◽  
Liquid Liquid Phase Separation

G-quadruplex (G4) DNA inhibits RNA unwinding activity but promotes liquid–liquid phase separation of the DEAD-box helicase Ded1p in vitro and in cells. This highlights multifaceted effects of G4DNA on an enzyme with intrinsically disordered domains.

scholarly journals Liquid-liquid phase separation of the chromosomal passenger complex drives parallel bundling of midzone microtubules

2022 ◽  
Author(s):  
Ewa Niedzialkowska ◽  
Tan M Truong ◽  
Luke A Eldredge ◽  
Stefanie Redemann ◽  
Denis Chretien ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Chromosome Segregation ◽  
Dynamic Structure ◽  
Liquid Phase Separation ◽  
Chromosomal Passenger Complex ◽  
Chromosomal Passenger ◽  
Spindle Midzone ◽  
Liquid Liquid Phase Separation

The spindle midzone is a dynamic structure that forms during anaphase, mediates chromosome segregation, and provides a signaling platform to position the cleavage furrow. The spindle midzone comprises two antiparallel bundles of microtubules (MTs) but the process of their formation is poorly understood. Here, we show that the Chromosomal Passenger Complex (CPC) undergoes liquid-liquid phase separation (LLPS) to generate parallel MT bundles in vitro when incubated with free tubulin and GTP. MT bundles emerge from CPC droplets with protruding minus-ends that then grow into long, tapered MT structures. During this growth, the CPC in condensates apparently reorganize to coat and bundle the resulting MT structures. CPC mutants attenuated for LLPS or MT binding prevented the generation of parallel MT bundles in vitro and reduced the number of MTs present at spindle midzones in HeLa cells. Our data uncovers a kinase-independent function of the CPC and provides models for how cells generate parallel-bundled MT structures that are important for the assembly of the mitotic spindle.

scholarly journals Liquid–Liquid Phase Separation in Crowded Environments

2020 ◽  
Vol 21 (16) ◽  
pp. 5908 ◽  
Author(s):  
Alain A. M. André ◽  
Evan Spruijt
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Macromolecular Crowding ◽  
Liquid Phase Separation ◽  
The Impact ◽  
Crowded Environments ◽  
Liquid Liquid Phase Separation ◽  
In Cells

Biomolecular condensates play a key role in organizing cellular fluids such as the cytoplasm and nucleoplasm. Most of these non-membranous organelles show liquid-like properties both in cells and when studied in vitro through liquid–liquid phase separation (LLPS) of purified proteins. In general, LLPS of proteins is known to be sensitive to variations in pH, temperature and ionic strength, but the role of crowding remains underappreciated. Several decades of research have shown that macromolecular crowding can have profound effects on protein interactions, folding and aggregation, and it must, by extension, also impact LLPS. However, the precise role of crowding in LLPS is far from trivial, as most condensate components have a disordered nature and exhibit multiple weak attractive interactions. Here, we discuss which factors determine the scope of LLPS in crowded environments, and we review the evidence for the impact of macromolecular crowding on phase boundaries, partitioning behavior and condensate properties. Based on a comparison of both in vivo and in vitro LLPS studies, we propose that phase separation in cells does not solely rely on attractive interactions, but shows important similarities to segregative phase separation.

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