Ubiquitin‐Modulated Phase Separation of Shuttle Proteins: Does Condensate Formation Promote Protein Degradation?

AbstractThe active zone of a presynaptic nerve terminal defines sites for neurotransmitter release. Its protein machinery may be organized through liquid–liquid phase separation, a mechanism for the formation of membrane-less subcellular compartments. Here, we show that the active zone protein Liprin-α3 rapidly and reversibly undergoes phase separation in transfected HEK293T cells. Condensate formation is triggered by Liprin-α3 PKC-phosphorylation at serine-760, and RIM and Munc13 are co-recruited into membrane-attached condensates. Phospho-specific antibodies establish phosphorylation of Liprin-α3 serine-760 in transfected cells and mouse brain tissue. In primary hippocampal neurons of newly generated Liprin-α2/α3 double knockout mice, synaptic levels of RIM and Munc13 are reduced and the pool of releasable vesicles is decreased. Re-expression of Liprin-α3 restored these presynaptic defects, while mutating the Liprin-α3 phosphorylation site to abolish phase condensation prevented this rescue. Finally, PKC activation in these neurons acutely increased RIM, Munc13 and neurotransmitter release, which depended on the presence of phosphorylatable Liprin-α3. Our findings indicate that PKC-mediated phosphorylation of Liprin-α3 triggers its phase separation and modulates active zone structure and function.

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Membrane-associated phase separation: organization and function emerge from a two-dimensional milieu

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjab010 ◽

2021 ◽

Author(s):

Jonathon A Ditlev

Keyword(s):

Phase Separation ◽

Cell Biology ◽

Cellular Environment ◽

Condensate Formation ◽

Associated Proteins ◽

Available Technology ◽

And Function ◽

Surrounding Membrane

Abstract Liquid‒liquid phase separation (LLPS) of biomolecules has emerged as an important mechanism that contributes to cellular organization. Phase separated biomolecular condensates, or membrane-less organelles, are compartments composed of specific biomolecules without a surrounding membrane in the nucleus and cytoplasm. LLPS also occurs at membranes, where both lipids and membrane-associated proteins can de-mix to form phase separated compartments. Investigation of these membrane-associated condensates using in vitro biochemical reconstitution and cell biology has provided key insights into the role of phase separation in membrane domain formation and function. However, these studies have generally been limited by available technology to study LLPS on model membranes and the complex cellular environment that regulates condensate formation, composition, and function. Here, I briefly review our current understanding of membrane-associated condensates, establish why LLPS can be advantageous for certain membrane-associated condensates, and offer a perspective for how these condensates may be studied in the future.

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Nuclear condensates of the Polycomb protein chromobox 2 (CBX2) assemble through phase separation

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.006620 ◽

2018 ◽

Vol 294 (5) ◽

pp. 1451-1463 ◽

Cited By ~ 92

Author(s):

Roubina Tatavosian ◽

Samantha Kent ◽

Kyle Brown ◽

Tingting Yao ◽

Huy Nguyen Duc ◽

...

Keyword(s):

Phase Separation ◽

Polycomb Group ◽

Site Directed Mutagenesis ◽

Developmental Defects ◽

Heterochromatin Formation ◽

Intrinsically Disordered ◽

Polycomb Protein ◽

Condensate Formation

Polycomb group (PcG) proteins repress master regulators of development and differentiation through organization of chromatin structure. Mutation and dysregulation of PcG genes cause developmental defects and cancer. PcG proteins form condensates in the cell nucleus, and these condensates are the physical sites of PcG-targeted gene silencing via formation of facultative heterochromatin. However, the physiochemical principles underlying the formation of PcG condensates remain unknown, and their determination could shed light on how these condensates compact chromatin. Using fluorescence live-cell imaging, we observed that the Polycomb repressive complex 1 (PRC1) protein chromobox 2 (CBX2), a member of the CBX protein family, undergoes phase separation to form condensates and that the CBX2 condensates exhibit liquid-like properties. Using site-directed mutagenesis, we demonstrated that the conserved residues of CBX2 within the intrinsically disordered region (IDR), which is the region for compaction of chromatin in vitro, promote the condensate formation both in vitro and in vivo. We showed that the CBX2 condensates concentrate DNA and nucleosomes. Using genetic engineering, we report that trimethylation of Lys-27 at histone H3 (H3K27me3), a marker of heterochromatin formation produced by PRC2, had minimal effects on the CBX2 condensate formation. We further demonstrated that the CBX2 condensate formation does not require CBX2–PRC1 subunits; however, the condensate formation of CBX2–PRC1 subunits depends on CBX2, suggesting a mechanism underlying the assembly of CBX2–PRC1 condensates. In summary, our results reveal that PcG condensates assemble through liquid–liquid phase separation (LLPS) and suggest that phase-separated condensates can organize PcG-bound chromatin.

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NBR1 directly promotes the formation of p62 – ubiquitin condensates via its PB1 and UBA domains

10.1101/2020.09.18.303552 ◽

2020 ◽

Author(s):

Adriana Savova ◽

Julia Romanov ◽

Sascha Martens

Keyword(s):

Phase Separation ◽

Wild Type ◽

Selective Autophagy ◽

Ubiquitinated Proteins ◽

Cargo Receptor ◽

Condensate Formation ◽

Uba Domain ◽

Reconstituted System ◽

Pb1 Domain

SummarySelective autophagy removes harmful intracellular structures such as ubiquitinated, aggregated proteins ensuring cellular homeostasis. This is achieved by the encapsulation of this cargo material within autophagosomes. The cargo receptor p62/SQSTM1 mediates the phase separation of ubiquitinated proteins into condensates, which subsequently become targets for the autophagy machinery. NBR1, another cargo receptor, is a crucial regulator of condensate formation. The mechanisms of the interplay between p62 and NBR1 are not well understood. Employing a fully reconstituted system we show that two domains of NBR1, the PB1 domain which binds to p62 and the UBA domain which binds to ubiquitin, are required to promote p62-ubiquitin condensate formation. In cells, acute depletion of endogenous NBR1 reduces formation of p62 condensates, a phenotype that can be rescued by re-expression of wild-type NBR1, but not PB1 or UBA domain mutants. Our results provide mechanistic insights into the role of NBR1 in selective autophagy.

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Natural and designed proteins inspired by extremotolerant organisms can form condensates and attenuate apoptosis in human cells

10.1101/2021.10.01.462432 ◽

2021 ◽

Author(s):

Mike T. Veling ◽

Dan T. Nguyen ◽

Nicole N. Thadani ◽

Michela E. Oster ◽

Nathan J. Rollins ◽

...

Keyword(s):

Phase Separation ◽

Intrinsically Disordered Proteins ◽

Human Cells ◽

Disordered Proteins ◽

Cellular Protection ◽

Intrinsically Disordered ◽

Apoe Protein ◽

Condensate Formation ◽

Associated Proteins ◽

Induced Apoptosis

ABSTRACTMany organisms can survive extreme conditions and successfully recover to normal life. This extremotolerant behavior has been attributed in part to repetitive, amphipathic, and intrinsically disordered proteins that are upregulated in the protected state. Here, we assemble a library of approximately 300 naturally-occurring and designed extremotolerance-associated proteins to assess their ability to protect human cells from chemically-induced apoptosis. We show that proteins from tardigrades, nematodes, and the Chinese giant salamander are apoptosis protective. Notably, we identify a region of the human ApoE protein with similarity to extremotolerance-associated proteins that also protects against apoptosis. This region mirrors the phase separation behavior seen with such proteins, like the tardigrade protein CAHS2. Moreover, we identify a synthetic protein, DHR81, that shares this combination of elevated phase separation propensity and apoptosis protection. Finally, we demonstrate that driving protective proteins into the condensate state increases apoptosis protection, and highlight the ability for DHR81 condensates to sequester caspase-7. Taken together, this work draws a link between extremotolerance-associated proteins, condensate formation, and human cellular protection.

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Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1820683116 ◽

2019 ◽

Vol 116 (33) ◽

pp. 16326-16331 ◽

Cited By ~ 21

Author(s):

Florian Heinkel ◽

Libin Abraham ◽

Mary Ko ◽

Joseph Chao ◽

Horacio Bach ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Phase Separation ◽

Lipid Bilayers ◽

Abc Transporter ◽

Single Molecule ◽

Intracellular Signaling ◽

Intrinsically Disordered ◽

Regulatory Module ◽

Condensate Formation ◽

Associated Proteins

Phase separation drives numerous cellular processes, ranging from the formation of membrane-less organelles to the cooperative assembly of signaling proteins. Features such as multivalency and intrinsic disorder that enable condensate formation are found not only in cytosolic and nuclear proteins, but also in membrane-associated proteins. The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected hosts, has a cytoplasmic regulatory module consisting of 2 phosphothreonine-binding Forkhead-associated domains joined by an intrinsically disordered linker with multiple phospho-acceptor threonines. Here we demonstrate that the regulatory modules of Rv1747 and its homolog in Mycobacterium smegmatis form liquid-like condensates as a function of concentration and phosphorylation. The serine/threonine kinases and sole phosphatase of Mtb tune phosphorylation-enhanced phase separation and differentially colocalize with the resulting condensates. The Rv1747 regulatory module also phase-separates on supported lipid bilayers and forms dynamic foci when expressed heterologously in live yeast and M. smegmatis cells. Consistent with these observations, single-molecule localization microscopy reveals that the endogenous Mtb transporter forms higher-order clusters within the Mycobacterium membrane. Collectively, these data suggest a key role for phase separation in the function of these mycobacterial ABC transporters and their regulation via intracellular signaling.

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Concentration and dosage sensitivity of proteins driving liquid-liquid phase separation

10.1101/2021.02.19.430946 ◽

2021 ◽

Author(s):

Nazanin Farahi ◽

Tamas Lazar ◽

Shoshana J. Wodak ◽

Peter Tompa ◽

Rita Pancsa

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Liquid Phase Separation ◽

Condensate Formation ◽

Associated Proteins ◽

Liquid Liquid Phase Separation ◽

In Cells ◽

Dosage Sensitivity

AbstractLiquid-liquid phase separation (LLPS) is a molecular process that leads to the formation of membraneless organelles (MLOs), i.e. functionally specialized liquid-like cellular condensates formed by proteins and nucleic acids. Integration of data on LLPS-associated proteins from dedicated databases revealed only modest overlap between them and resulted in a confident set of 89 human LLPS driver proteins. Since LLPS is highly concentration-sensitive, the underlying experiments are often criticized for applying higher-than-physiological protein concentrations. To clarify this issue, we performed a naive comparison of in vitro applied and quantitative proteomics-derived protein concentrations and discuss a number of considerations that rationalize the choice of apparently high in vitro concentrations in most LLPS studies. The validity of in vitro LLPS experiments is further supported by in vivo phase-separation experiments and by the observation that the corresponding genes show a strong propensity for dosage sensitivity. This observation implies that the availability of the respective proteins is tightly regulated in cells to avoid erroneous condensate formation. In all, we propose that although local protein concentrations are practically impossible to determine in cells, proteomics-derived cellular concentrations should rather be considered as lower limits of protein concentrations, than strict upper bounds, to be respected by in vitro experiments.

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Polycomb Cbx2 Condensates Assemble through Phase Separation

10.1101/468926 ◽

2018 ◽

Cited By ~ 1

Author(s):

Roubina Tatavosian ◽

Samantha Kent ◽

Kyle Brown ◽

Tingting Yao ◽

Huy Nguyen Duc ◽

...

Keyword(s):

Phase Separation ◽

Polycomb Group ◽

Developmental Defects ◽

Intrinsically Disordered ◽

Condensate Formation ◽

Development And Differentiation ◽

Polycomb Repressive Complex 1 ◽

Liquid Liquid Phase Separation

AbstractPolycomb group (PcG) proteins are master regulators of development and differentiation. Mutation and dysregulation of PcG genes cause developmental defects and cancer. PcG proteins form condensates in the nucleus of cells and these condensates are the physical sites of PcG-targeted gene silencing. However, the physiochemical principles underlying the PcG condensate formation remain unknown. Here we show that Polycomb repressive complex 1 (PRC1) protein Cbx2, one member of the Cbx family proteins, contains a long stretch of intrinsically disordered region (IDR). Cbx2 undergoes phase separation to form condensates. Cbx2 condensates exhibit liquid-like properties and can concentrate DNA and nucleosomes. We demonstrate that the conserved residues within the IDR promote the condensate formation in vitro and in vivo. We further indicate that H3K27me3 has minimal effects on the Cbx2 condensate formation while depletion of core PRC1 subunits facilitates the condensate formation. Thus, our results reveal that PcG condensates assemble through liquid-liquid phase separation (LLPS) and suggest that PcG-bound chromatin is in part organized through phase-separated condensates.

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m6A-binding YTHDF proteins promote stress granule formation by modulating phase separation of stress granule proteins

10.1101/694455 ◽

2019 ◽

Cited By ~ 6

Author(s):

Ye Fu ◽

Xiaowei Zhuang

Keyword(s):

Phase Separation ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Super Resolution ◽

Stress Granule ◽

Granule Formation ◽

Intrinsically Disordered ◽

Condensate Formation ◽

Resolution Imaging

AbstractDiverse RNAs and RNA-binding proteins form phase-separated, membraneless granules in cells under stress conditions. However, the role of the prevalent mRNA methylation, m6A, and its binding proteins in stress granule (SG) assembly remain unclear. Here, we show that m6A-modified mRNAs are enriched in SGs, and that m6A-binding YTHDF proteins are critical for SG formation. Depletion of YTHDF1/3 inhibits SG formation and recruitment of m6A-modified mRNAs to SGs. Both the N-terminal intrinsically disordered region and the C-terminal m6A-binding YTH domain of YTHDF proteins are crucial for SG formation. Super-resolution imaging further reveals that YTHDF proteins are in a super-saturated state, forming clusters that reside in the periphery of and at the junctions between SG core clusters, and promote SG phase separation by reducing the activation energy barrier and critical size for condensate formation. Our results reveal a new function and mechanistic insights of the m6A-binding YTHDF proteins in regulating phase separation.

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Phase separation of RNA-binding protein promotes polymerase engagement and transcription

10.1101/2021.03.26.436939 ◽

2021 ◽

Author(s):

Wen Shao ◽

Xianju Bi ◽

Boyang Gao ◽

Jun Wu ◽

Yixuan Pan ◽

...

Keyword(s):

Phase Separation ◽

Rna Binding ◽

Rna Binding Proteins ◽

Embryonic Stem ◽

Transcription Control ◽

Pol Ii ◽

Genome Wide ◽

Condensate Formation ◽

Active Transcription ◽

Biochemical Analyses

An RNA-involved phase-separation model has been proposed for transcription control. Yet, the molecular links that connect RNA binding to the transcription machinery remain missing. Here we find RNA-binding proteins (RBPs) constitute half of the chromatin proteome in embryonic stem cells (ESCs), and some are colocalized with RNA polymerase (Pol) II at promoters and enhancers. Biochemical analyses of representative RBPs--such as PSPC1 and PTBP1--show that the paraspeckle protein PSPC1 not only prevents the RNA-induced premature release of Pol II, and also makes use of RNA as multivalent molecules to promote Pol II engagement and activity, by enhancing the phase separation and subsequent phosphorylation and release of polymerase condensates. In ESCs, auxin-induced acute degradation of PSPC1 leads to genome-wide defects in Pol II phosphorylation and chromatin-binding and nascent transcription. We propose that the synergistic interplay of RBPs and RNA aids in the rate-limiting step of polymerase condensate formation to promote active transcription.

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