scholarly journals Liquid-liquid phase separation promotes animal desiccation tolerance

2020 ◽  
Vol 117 (44) ◽  
pp. 27676-27684
Author(s):  
Clinton Belott ◽  
Brett Janis ◽  
Michael A. Menze
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Stress Responses ◽  
Desiccation Tolerance ◽  
Molecular Mechanisms ◽  
Insect Cells ◽  
Regulation Of Gene Expression ◽  
Ectopic Expression ◽  
Liquid Phase Separation ◽  
Liquid Liquid Phase Separation

Proteinaceous liquid-liquid phase separation (LLPS) occurs when a polypeptide coalesces into a dense phase to form a liquid droplet (i.e., condensate) in aqueous solution. In vivo, functional protein-based condensates are often referred to as membraneless organelles (MLOs), which have roles in cellular processes ranging from stress responses to regulation of gene expression. Late embryogenesis abundant (LEA) proteins containing seed maturation protein domains (SMP; PF04927) have been linked to storage tolerance of orthodox seeds. The mechanism by which anhydrobiotic longevity is improved is unknown. Interestingly, the brine shrimpArtemia franciscanais the only animal known to express such a protein (AfrLEA6) in its anhydrobiotic embryos. Ectopic expression ofAfrLEA6 (AWM11684) in insect cells improves their desiccation tolerance and a fraction of the protein is sequestered into MLOs, while aqueousAfrLEA6 raises the viscosity of the cytoplasm. LLPS ofAfrLEA6 is driven by the SMP domain, while the size of formed MLOs is regulated by a domain predicted to engage in protein binding.AfrLEA6 condensates formed in vitro selectively incorporate target proteins based on their surface charge, while cytoplasmic MLOs formed inAfrLEA6-transfected insect cells behave like stress granules. We suggest thatAfrLEA6 promotes desiccation tolerance by engaging in two distinct molecular mechanisms: by raising cytoplasmic viscosity at even modest levels of water loss to promote cell integrity during drying and by forming condensates that may act as protective compartments for desiccation-sensitive proteins. Identifying and understanding the molecular mechanisms that govern anhydrobiosis will lead to significant advancements in preserving biological samples.

scholarly journals Poly-glutamine-dependent self-association as a potential mechanism for regulation of androgen receptor activity

2021 ◽  
Author(s):  
Josep Rizo ◽  
Carlos M. Roggero ◽  
Victoria Esser ◽  
Lingling Duan ◽  
Allyson M. Rice ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Phase Separation ◽  
Androgen Receptor ◽  
Liquid Phase ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Catalytic Domain ◽  
Low Complexity ◽  
Liquid Phase Separation ◽  
Liquid Liquid Phase Separation

The androgen receptor (AR) plays a central role in prostate cancer. Development of castration resistant prostate cancer (CRPC) requires androgen-independent activation of AR, which involves its large N-terminal domain (NTD) and entails dramatic epigenetic changes depending in part on histone lysine demethylases (KDMs) that interact with AR. The AR-NTD is rich in low-complexity sequences, including a polyQ repeat. Longer polyQ sequences were reported to decrease transcriptional activity and to protect against prostate cancer. However, the molecular mechanisms underlying these observations are unclear. Using NMR spectroscopy, here we identify weak interactions between the AR-NTD and the KDM4A catalytic domain, and between the AR ligand-binding domain and a central KDM4A region that also contains low-complexity sequences. We also show that the AR-NTD can undergo liquid-liquid phase separation in vitro, with longer polyQ sequences phase separating more readily. Moreover, longer polyQ sequences hinder nuclear localization in the absence of hormone and increase the propensity for formation of AR-containing puncta in the nucleus of cells treated with dihydrotestosterone. These results lead us to hypothesize that polyQ-dependent liquid-liquid phase separation may provide a mechanism to decrease the transcriptional activity of AR, potentially opening new opportunities to design effective therapies against CRPC.

scholarly journals A generic approach to study the kinetics of liquid–liquid phase separation under near-native conditions

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Joris Van Lindt ◽  
Anna Bratek-Skicki ◽  
Phuong N. Nguyen ◽  
Donya Pakravan ◽  
Luis F. Durán-Armenta ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Cell Biology ◽  
Molecular Mechanisms ◽  
Stress Protein ◽  
Low Complexity ◽  
Liquid Phase Separation ◽  
Phase Separation Behavior ◽  
Liquid Liquid Phase Separation

AbstractUnderstanding the kinetics, thermodynamics, and molecular mechanisms of liquid–liquid phase separation (LLPS) is of paramount importance in cell biology, requiring reproducible methods for studying often severely aggregation-prone proteins. Frequently applied approaches for inducing LLPS, such as dilution of the protein from an urea-containing solution or cleavage of its fused solubility tag, often lead to very different kinetic behaviors. Here we demonstrate that at carefully selected pH values proteins such as the low-complexity domain of hnRNPA2, TDP-43, and NUP98, or the stress protein ERD14, can be kept in solution and their LLPS can then be induced by a jump to native pH. This approach represents a generic method for studying the full kinetic trajectory of LLPS under near native conditions that can be easily controlled, providing a platform for the characterization of physiologically relevant phase-separation behavior of diverse proteins.

scholarly journals Tau N-Terminal Inserts Regulate Tau Liquid-Liquid Phase Separation and Condensates Maturation in a Neuronal Cell Model

2021 ◽  
Vol 22 (18) ◽  
pp. 9728
Author(s):  
Chengchen Wu ◽  
Junyi Zhao ◽  
Qiuping Wu ◽  
Qiulong Tan ◽  
Qiong Liu ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Molecular Mechanisms ◽  
Cell Model ◽  
Neuronal Cell ◽  
Liquid Phase Separation ◽  
Assembly Mechanism ◽  
Tau Isoforms ◽  
Liquid Liquid Phase Separation

The microtubule-associated protein tau can undergo liquid–liquid phase separation (LLPS) to form membraneless condensates in neurons, yet the underlying molecular mechanisms and functions of tau LLPS and tau droplets remain to be elucidated. The human brain contains mainly 6 tau isoforms with different numbers of microtubule-binding repeats (3R, 4R) and N-terminal inserts (0N, 1N, 2N). However, little is known about the role of N-terminal inserts. Here we observed the dynamics of three tau isoforms with different N-terminal inserts in live neuronal cell line HT22. We validated tau LLPS in cytoplasm and found that 2N-tau forms liquid-like, hollow-shell droplets. Tau condensates became smaller in 1N-tau comparing with 2N-tau, while no obvious tau accumulated dots were shown in 0N-tau. The absence of N-terminal inserts significantly affected condensate colocalization of tau and p62. The results reveal insights into the tau LLPS assembly mechanism and functional effects of N-terminal inserts in tau.

scholarly journals Poly-glutamine-dependent self-association as a potential mechanism for regulation of androgen receptor activity

PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0258876
Author(s):  
Carlos M. Roggero ◽  
Victoria Esser ◽  
Lingling Duan ◽  
Allyson M. Rice ◽  
Shihong Ma ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Phase Separation ◽  
Androgen Receptor ◽  
Liquid Phase ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Muscular Atrophy ◽  
Low Complexity ◽  
Liquid Phase Separation ◽  
Liquid Liquid Phase Separation

The androgen receptor (AR) plays a central role in prostate cancer. Development of castration resistant prostate cancer (CRPC) requires androgen-independent activation of AR, which involves its large N-terminal domain (NTD) and entails extensive epigenetic changes depending in part on histone lysine demethylases (KDMs) that interact with AR. The AR-NTD is rich in low-complexity sequences, including a polyQ repeat. Longer polyQ sequences were reported to decrease transcriptional activity and to protect against prostate cancer, although they can lead to muscular atrophy. However, the molecular mechanisms underlying these observations are unclear. Using NMR spectroscopy, here we identify weak interactions between the AR-NTD and the KDM4A catalytic domain, and between the AR ligand-binding domain and a central KDM4A region that also contains low-complexity sequences. We also show that the AR-NTD can undergo liquid-liquid phase separation in vitro, with longer polyQ sequences phase separating more readily. Moreover, longer polyQ sequences hinder nuclear localization in the absence of hormone and increase the propensity for formation of AR-containing puncta in the nucleus of cells treated with dihydrotestosterone. These results lead us to hypothesize that polyQ-dependent liquid-liquid phase separation may provide a mechanism to decrease the transcriptional activity of AR, potentially opening new opportunities to design effective therapies against CRPC and muscular atrophy.

scholarly journals Dengue and Zika virus capsid proteins bind to membranes and self-assemble into liquid droplets with nucleic acids

2021 ◽  
Author(s):  
Ernesto E. Ambroggio ◽  
Guadalupe Soledad Costa Navarro ◽  
Luis Benito Pérez Socas ◽  
Luis A. Bagatolli ◽  
Andrea V. Gamarnik
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Nucleic Acids ◽  
Molecular Mechanisms ◽  
Liquid Phase Separation ◽  
Capsid Proteins ◽  
Particle Assembly ◽  
Virus Capsid ◽  
Liquid Liquid Phase Separation ◽  
Er Membrane

Liquid-liquid phase separation is prone to occur when positively charged proteins interact with nucleic acids. Here, we studied biophysical properties of Dengue (DENV) and Zika (ZIKV) virus capsid proteins to understand the process of RNA genome encapsidation. In this route, the capsid proteins efficiently recruit the viral RNA at the ER membrane to yield nascent viral particles. However, little is known either about the molecular mechanisms by which multiple copies of capsid proteins assemble into nucleocapsids or how the nucleocapsid is recruited and wrapped by the ER membrane during particle morphogenesis. Here, we measured relevant interactions concerning the viral process using purified DENV and ZIKV capsids proteins, membranes mimicking the ER lipid composition and nucleic acids at in vitro conditions. We found that both ZIKV and DENV capsid proteins bound to liposomes at liquid-disordered phase regions and docked exogenous membranes and RNA molecules. When the proteins bound nucleic acids, droplet liquid-liquid phase separation was observed. We characterized these liquid condensates by measuring nucleic acid partition constant and the extent of water dipolar relaxation observing a cooperative process for the formation of the new phase that involves a distinct water organization. Our data supports a new model in which capsid-RNA complexes directly bind the ER membrane, seeding the process of RNA recruitment for viral particle assembly. These results contribute to understand the viral nucleocapsid formation as a stable liquid-liquid phase transition, which could be relevant for Dengue and Zika gemmation, opening new avenues for antiviral intervention.

scholarly journals Liquid-Liquid Phase Separation of Tau Driven by Hydrophobic Interaction Facilitates Fibrillization of Tau

2021 ◽  
Vol 433 (2) ◽  
pp. 166731
Author(s):  
Yanxian Lin ◽  
Yann Fichou ◽  
Andrew P. Longhini ◽  
Luana C. Llanes ◽  
Pengyi Yin ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Hydrophobic Interaction ◽  
Liquid Phase Separation ◽  
Liquid Liquid Phase Separation

scholarly journals Amyloid Aggregation under the Lens of Liquid–Liquid Phase Separation

2020 ◽  
pp. 368-378
Author(s):  
Yanting Xing ◽  
Aparna Nandakumar ◽  
Aleksandr Kakinen ◽  
Yunxiang Sun ◽  
Thomas P. Davis ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Amyloid Aggregation ◽  
Liquid Liquid Phase Separation

scholarly journals Observation of liquid-liquid phase separation of ataxin-3 and quantitative evaluation of its concentration in a single droplet using Raman microscopy

2021 ◽  
Author(s):  
Kazuki Murakami ◽  
Shinji Kajimoto ◽  
Daiki Shibata ◽  
Kunisato Kuroi ◽  
Fumihiko Fujii ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Neurodegenerative Diseases ◽  
Protein Aggregation ◽  
Quantitative Evaluation ◽  
Raman Microscopy ◽  
Liquid Phase Separation ◽  
Biological Processes ◽  
Single Droplet ◽  
Liquid Liquid Phase Separation

Liquid–liquid phase separation (LLPS) plays an important role in a variety of biological processes and is also associated with protein aggregation in neurodegenerative diseases. Quantification of LLPS is necessary to...

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