scholarly journals Heat shock chaperone HSPB1 regulates cytoplasmic TDP-43 phase separation and liquid-to-gel transition

2021 ◽  
Author(s):  
Shan Lu ◽  
Jiaojiao Hu ◽  
Bankhole Aladesuyi ◽  
Alexander Goginashvili ◽  
Sonia Vazquez-Sanchez ◽  
...  
Keyword(s):  
Phase Separation ◽  
Heat Shock ◽  
Motor Neurons ◽  
Rna Binding ◽  
Isotope Labeling ◽  
Small Heat Shock Protein ◽  
Proteasome Inhibition ◽  
Low Complexity ◽  
Spinal Motor Neurons ◽  
Hsp70 Family

Abstract While the RNA binding protein TDP-43 reversibly phase separates within nuclei into complex droplets (anisosomes) with TDP-43-containing liquid outer shells and liquid centers of HSP70 family chaperones, cytoplasmic aggregates of TDP-43 are hallmarks of multiple neurodegenerative diseases, including ALS. Here we show that transient oxidative stress, proteasome inhibition, or inhibition of HSP70’s ATP-dependent chaperone activity provokes reversible cytoplasmic TDP-43 de-mixing and transition from liquid to gel/solid, independent of RNA binding or stress granules. Isotope labeling mass spectrometry is used to identify that phase separated cytoplasmic TDP-43 is primarily bound by the small heat shock protein HSPB1. Binding is direct, mediated through TDP-43’s RNA binding and low complexity domains. HSPB1 partitions into TDP-43 droplets, inhibits TDP-43 assembly into fibrils, and is essential for disassembly of stress-induced, TDP-43 droplets. Decrease of HSPB1 promotes cytoplasmic TDP-43 de-mixing and mislocalization. HSPB1 depletion is identified within ALS-patient spinal motor neurons containing aggregated TDP-43. These findings identify HSPB1 to be a regulator of cytoplasmic TDP-43 phase separation and aggregation.

scholarly journals Heat shock chaperone HSPB1 regulates cytoplasmic TDP-43 phase separation and liquid-to-gel transition

2021 ◽  
Author(s):  
Shan Lu ◽  
Jiaojiao Hu ◽  
Olubankole Aladesuyi Arogundade ◽  
Alexander Goginashvili ◽  
Sonia Vazquez-Sanchez ◽  
...  
Keyword(s):  
Phase Separation ◽  
Heat Shock ◽  
Motor Neurons ◽  
Rna Binding ◽  
Isotope Labeling ◽  
Small Heat Shock Protein ◽  
Proteasome Inhibition ◽  
Low Complexity ◽  
Spinal Motor Neurons ◽  
Hsp70 Family

While the RNA binding protein TDP-43 reversibly phase separates within nuclei into complex droplets (anisosomes) with TDP-43-containing liquid outer shells and liquid centers of HSP70 family chaperones, cytoplasmic aggregates of TDP-43 are hallmarks of multiple neurodegenerative diseases, including ALS. Here we show that transient oxidative stress, proteasome inhibition, or inhibition of HSP70's ATP-dependent chaperone activity provokes reversible cytoplasmic TDP-43 de-mixing and transition from liquid to gel/solid, independent of RNA binding or stress granules. Isotope labeling mass spectrometry is used to identify that phase separated cytoplasmic TDP-43 is primarily bound by the small heat shock protein HSPB1. Binding is direct, mediated through TDP-43's RNA binding and low complexity domains. HSPB1 partitions into TDP-43 droplets, inhibits TDP-43 assembly into fibrils, and is essential for disassembly of stress-induced, TDP-43 droplets. Decrease of HSPB1 promotes cytoplasmic TDP-43 de-mixing and mislocalization. HSPB1 depletion is identified within ALS-patient spinal motor neurons containing aggregated TDP-43. These findings identify HSPB1 to be a regulator of cytoplasmic TDP-43 phase separation and aggregation.

scholarly journals TDP-43 and HSP70 phase separate into anisotropic, intranuclear liquid spherical annuli

2020 ◽  
Author(s):  
Haiyang Yu ◽  
Shan Lu ◽  
Kelsey Gasior ◽  
Digvijay Singh ◽  
Olga Tapia ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Proteasome Inhibition ◽  
Chaperone Activity ◽  
Cell Nuclei ◽  
The Core ◽  
Hsp70 Family ◽  
Age Related ◽  
Annulus Formation

AbstractThe RNA binding protein TDP-43 naturally phase separates within cell nuclei and forms cytoplasmic aggregates in age-related neurodegenerative diseases. Here we show that acetylation-mediated inhibition of TDP-43 binding to RNA produces co-de-mixing of acetylated and unmodified TDP-43 into symmetrical, intranuclear spherical annuli whose shells and cores have liquid properties. Shells are anisotropic, like liquid crystals. Consistent with our modelling predictions that annulus formation is driven by components with strong self-interactions but weak interaction with TDP-43, the major components of annuli cores are identified to be HSP70 family proteins, whose chaperone activity is required to maintain liquidity of the core. Proteasome inhibition, mimicking reduction in proteasome activity during aging, induces TDP-43-containing annuli in neurons in rodents. Thus, we identify that TDP-43 phase separation is regulated by acetylation, proteolysis, and ATPase-dependent chaperone activity of HSP70.One Sentence SummaryAcetylation of TDP-43 drives its phase separation into spherical annuli that form a liquid-inside-a-liquid-inside-a-liquid.

scholarly journals FUS-ALS mutants alter FMRP phase separation equilibrium and impair protein translation

2021 ◽  
Vol 7 (30) ◽  
pp. eabf8660
Author(s):  
Nicol Birsa ◽  
Agnieszka M. Ule ◽  
Maria Giovanna Garone ◽  
Brian Tsang ◽  
Francesca Mattedi ◽  
...  
Keyword(s):  
Phase Separation ◽  
Motor Neurons ◽  
Rna Binding ◽  
Fragile X ◽  
Protein Translation ◽  
Fused In Sarcoma ◽  
Mental Retardation Protein ◽  
Lateral Sclerosis

FUsed in Sarcoma (FUS) is a multifunctional RNA binding protein (RBP). FUS mutations lead to its cytoplasmic mislocalization and cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Here, we use mouse and human models with endogenous ALS-associated mutations to study the early consequences of increased cytoplasmic FUS. We show that in axons, mutant FUS condensates sequester and promote the phase separation of fragile X mental retardation protein (FMRP), another RBP associated with neurodegeneration. This leads to repression of translation in mouse and human FUS-ALS motor neurons and is corroborated in vitro, where FUS and FMRP copartition and repress translation. Last, we show that translation of FMRP-bound RNAs is reduced in vivo in FUS-ALS motor neurons. Our results unravel new pathomechanisms of FUS-ALS and identify a novel paradigm by which mutations in one RBP favor the formation of condensates sequestering other RBPs, affecting crucial biological functions, such as protein translation.

scholarly journals HSP70 chaperones RNA-free TDP-43 into anisotropic intranuclear liquid spherical shells

Science ◽  
2020 ◽  
Vol 371 (6529) ◽  
pp. eabb4309 ◽  
Author(s):  
Haiyang Yu ◽  
Shan Lu ◽  
Kelsey Gasior ◽  
Digvijay Singh ◽  
Sonia Vazquez-Sanchez ◽  
...  
Keyword(s):  
Rna Binding ◽  
Solid Phase ◽  
Liquid Core ◽  
Proteasome Inhibition ◽  
Crystal Formation ◽  
Spherical Shells ◽  
Hsp70 Family ◽  
Age Related ◽  
Recognition Motifs

The RNA binding protein TDP-43 forms intranuclear or cytoplasmic aggregates in age-related neurodegenerative diseases. In this study, we found that RNA binding–deficient TDP-43 (produced by neurodegeneration-causing mutations or posttranslational acetylation in its RNA recognition motifs) drove TDP-43 demixing into intranuclear liquid spherical shells with liquid cores. These droplets, which we named “anisosomes”, have shells that exhibit birefringence, thus indicating liquid crystal formation. Guided by mathematical modeling, we identified the primary components of the liquid core to be HSP70 family chaperones, whose adenosine triphosphate (ATP)–dependent activity maintained the liquidity of shells and cores. In vivo proteasome inhibition within neurons, to mimic aging-related reduction of proteasome activity, induced TDP-43–containing anisosomes. These structures converted to aggregates when ATP levels were reduced. Thus, acetylation, HSP70, and proteasome activities regulate TDP-43 phase separation and conversion into a gel or solid phase.

scholarly journals Illuminating ALS Motor Neurons With Optogenetics in Zebrafish

2021 ◽  
Vol 9 ◽  
Author(s):  
Kazuhide Asakawa ◽  
Hiroshi Handa ◽  
Koichi Kawakami
Keyword(s):  
Spinal Cord ◽  
Motor Neurons ◽  
Rna Binding ◽  
The Body ◽  
Optical Methods ◽  
Light Illumination ◽  
Whole Cells ◽  
Spinal Motor Neurons ◽  
Spinal Motor

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by progressive degeneration of motor neurons in the brain and spinal cord. Spinal motor neurons align along the spinal cord length within the vertebral column, and extend long axons to connect with skeletal muscles covering the body surface. Due to this anatomy, spinal motor neurons are among the most difficult cells to observe in vivo. Larval zebrafish have transparent bodies that allow non-invasive visualization of whole cells of single spinal motor neurons, from somas to the neuromuscular synapses. This unique feature, combined with its amenability to genome editing, pharmacology, and optogenetics, enables functional analyses of ALS-associated proteins in the spinal motor neurons in vivo with subcellular resolution. Here, we review the zebrafish skeletal neuromuscular system and the optical methods used to study it. We then introduce a recently developed optogenetic zebrafish ALS model that uses light illumination to control oligomerization, phase transition and aggregation of the ALS-associated DNA/RNA-binding protein called TDP-43. Finally, we will discuss how this disease-in-a-fish ALS model can help solve key questions about ALS pathogenesis and lead to new ALS therapeutics.

scholarly journals The low-complexity domain of the FUS RNA binding protein self-assembles via the mutually exclusive use of two distinct cross-β cores

2021 ◽  
Vol 118 (42) ◽  
pp. e2114412118
Author(s):  
Masato Kato ◽  
Steven L. McKnight
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Low Complexity ◽  
Terminal Region ◽  
Aqueous Environment ◽  
Fused In Sarcoma ◽  
Biochemical Assays ◽  
Biologic Function

The low-complexity (LC) domain of the fused in sarcoma (FUS) RNA binding protein self-associates in a manner causing phase separation from an aqueous environment. Incubation of the FUS LC domain under physiologically normal conditions of salt and pH leads to rapid formation of liquid-like droplets that mature into a gel-like state. Both examples of phase separation have enabled reductionist biochemical assays allowing discovery of an N-terminal region of 57 residues that assembles into a labile, cross-β structure. Here we provide evidence of a nonoverlapping, C-terminal region of the FUS LC domain that also forms specific cross-β interactions. We propose that biologic function of the FUS LC domain may operate via the mutually exclusive use of these N- and C-terminal cross-β cores. Neurodegenerative disease–causing mutations in the FUS LC domain are shown to imbalance the two cross-β cores, offering an unanticipated concept of LC domain function and dysfunction.

scholarly journals Interplay Between Short-range Attraction and Long-range Repulsion Controls Reentrant Liquid Condensation of Ribonucleoprotein-RNA Complexes

2019 ◽  
Author(s):  
Ibraheem Alshareedah ◽  
Taranpreet Kaur ◽  
Jason Ngo ◽  
Hannah Seppala ◽  
Liz-Audrey Djomnang Kounatse ◽  
...  
Keyword(s):  
Phase Separation ◽  
Long Range ◽  
Short Range ◽  
Rna Binding ◽  
Driving Forces ◽  
Low Complexity ◽  
Model Systems ◽  
Supramolecular Organization ◽  
Cluster Phase ◽  
Rna Complexes

AbstractIn eukaryotic cells, ribonucleoproteins (RNPs) form mesoscale condensates by liquid-liquid phase separation that play essential roles in subcellular dynamic compartmentalization. The formation and dissolution of many RNP condensates are finely dependent on the RNA-to-RNP ratio, giving rise to a window-like phase separation behavior. This is commonly referred to as reentrant liquid condensation (RLC). Here, using RNP-inspired polypeptides with low-complexity RNA-binding sequences as well as the C-terminal disordered domain of the ribonucleoprotein FUS as model systems, we investigate the molecular driving forces underlying this non-monotonous phase transition. We show that an interplay between short-range cation-π attractions and long-range electrostatic forces governs the heterotypic RLC of RNP-RNA complexes. Short-range attractions, which can be encoded by both polypeptide chain primary sequence and nucleic acid base sequence, are activated by RNP-RNA condensate formation. After activation, the short-range forces regulate material properties of polypeptide-RNA condensates and subsequently oppose their reentrant dissolution. In the presence of excess RNA, a competition between short-range attraction and long-range electrostatic repulsion drives the formation of a colloid-like cluster phase. With increasing short-range attraction, the fluid dynamics of the cluster phase is arrested, leading to the formation of a colloidal gel. Our results reveal that phase behavior, supramolecular organization, and material states of RNP-RNA assemblies are controlled by a dynamic interplay between molecular interactions at different length scales.

scholarly journals Distinct roles of hnRNPH1 low-complexity domains in splicing and transcription

2021 ◽  
Vol 118 (50) ◽  
pp. e2109668118
Author(s):  
Ga Hye Kim ◽  
Ilmin Kwon
Keyword(s):  
Phase Separation ◽  
Transcriptional Activation ◽  
Rna Splicing ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Lymphoblastic Leukemia ◽  
Large Family ◽  
Low Complexity ◽  
Reversible Polymers

Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins that control key events in RNA biogenesis under both normal and diseased cellular conditions. The low-complexity (LC) domain of hnRNPs can become liquid-like droplets or reversible amyloid-like polymers by phase separation. Yet, whether phase separation of the LC domains contributes to physiological functions of hnRNPs remains unclear. hnRNPH1 contains two LC domains, LC1 and LC2. Here, we show that reversible phase separation of the LC1 domain is critical for both interaction with different kinds of RNA-binding proteins and control of the alternative-splicing activity of hnRNPH1. Interestingly, although not required for phase separation, the LC2 domain contributes to the robust transcriptional activation of hnRNPH1 when fused to the DNA-binding domain, as found recently in acute lymphoblastic leukemia. Our data suggest that the ability of the LC1 domain to phase-separate into reversible polymers or liquid-like droplets is essential for function of hnRNPH1 as an alternative RNA-splicing regulator, whereas the LC2 domain may contribute to the aberrant transcriptional activity responsible for cancer transformation.

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