scholarly journals Phase separation induced by cohesin SMC protein complexes

2020 ◽  
Author(s):  
Je-Kyung Ryu ◽  
Celine Bouchoux ◽  
Hon Wing Liu ◽  
Eugene Kim ◽  
Masashi Minamino ◽  
...  
Keyword(s):  
Phase Separation ◽  
Genome Organization ◽  
Weak Interactions ◽  
Protein Complexes ◽  
Yeast Cells ◽  
Liquid Droplets ◽  
Independent Manner ◽  
Reversible Dissociation

AbstractCohesin is a key protein complex that organizes the spatial structure of chromosomes during interphase. Here, we show that yeast cohesin shows pronounced clustering on DNA in an ATP-independent manner, exhibiting all the hallmarks of phase separation. In vitro visualization of cohesin on DNA shows DNA-cohesin clusters that exhibit liquid-like behavior. This includes mutual fusion and reversible dissociation upon depleting the cohesin concentration, increasing the ionic strength, or adding 1,6-hexanediol, conditions that disrupt weak interactions. We discuss how bridging-induced phase separation can explain the DNA-cohesin clustering through DNA-cohesin-DNA bridges. We confirm that, in vivo, a fraction of cohesin associates with chromatin in yeast cells in a manner consistent with phase separation. Our findings establish that SMC proteins can exhibit phase separation, which has potential to clarify previously unexplained aspects of in vivo SMC behavior and constitute an additional principle by which SMC complexes impact genome organization.One sentence summaryYeast cohesin complex is observed to phase separate with DNA into liquid droplets, which it accomplishes by ATP-independent DNA bridging.

scholarly journals Bridging-induced phase separation induced by cohesin SMC protein complexes

2021 ◽  
Vol 7 (7) ◽  
pp. eabe5905
Author(s):  
Je-Kyung Ryu ◽  
Céline Bouchoux ◽  
Hon Wing Liu ◽  
Eugene Kim ◽  
Masashi Minamino ◽  
...  
Keyword(s):  
Phase Separation ◽  
Genome Organization ◽  
Fluorescence Recovery After Photobleaching ◽  
Protein Complexes ◽  
Yeast Cells ◽  
Base Pairs ◽  
Fundamental Building Block ◽  
Structural Maintenance Of Chromosome

Structural maintenance of chromosome (SMC) protein complexes are able to extrude DNA loops. While loop extrusion constitutes a fundamental building block of chromosomes, other factors may be equally important. Here, we show that yeast cohesin exhibits pronounced clustering on DNA, with all the hallmarks of biomolecular condensation. DNA-cohesin clusters exhibit liquid-like behavior, showing fusion of clusters, rapid fluorescence recovery after photobleaching and exchange of cohesin with the environment. Strikingly, the in vitro clustering is DNA length dependent, as cohesin forms clusters only on DNA exceeding 3 kilo–base pairs. We discuss how bridging-induced phase separation, a previously unobserved type of biological condensation, can explain the DNA-cohesin clustering through DNA-cohesin-DNA bridges. We confirm that, in yeast cells in vivo, a fraction of cohesin associates with chromatin in a manner consistent with bridging-induced phase separation. Biomolecular condensation by SMC proteins constitutes a new basic principle by which SMC complexes direct genome organization.

scholarly journals Pat1 promotes processing body assembly by enhancing the phase separation of the DEAD-box ATPase Dhh1 and RNA

2018 ◽  
Author(s):  
Ruchika Sachdev ◽  
Maria Hondele ◽  
Miriam Linsenmeier ◽  
Pascal Vallotton ◽  
Christopher F. Mugler ◽  
...  
Keyword(s):  
Phase Separation ◽  
Direct Interaction ◽  
Liquid Droplets ◽  
Processing Bodies ◽  
Dead Box ◽  
The Dead ◽  
Processing Body ◽  
Liquid Liquid Phase Separation

AbstractProcessing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid-liquid phase separation and are implicated in the decay or storage of mRNAs. How PB assembly is regulated in cells remains unclear. We recently identified the ATPase activity of the DEAD-box protein Dhh1 as a key regulator of PB dynamics and demonstrated that Not1, an activator of the Dhh1 ATPase and member of the CCR4-NOT deadenylase complex inhibits PB assembly in vivo [Mugler et al., 2016]. Here, we show that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1. Intriguingly, in vivo PB dynamics can be recapitulated in vitro, since Pat1 enhances the phase separation of Dhh1 and RNA into liquid droplets, whereas Not1 reverses Pat1-Dhh1-RNA condensation. Overall, our results uncover a function of Pat1 in promoting the multimerization of Dhh1 on mRNA, thereby aiding the assembly of large multivalent mRNP granules that are PBs.

scholarly journals Paraspeckles: Paragons of functional aggregation

2015 ◽  
Vol 210 (4) ◽  
pp. 527-528 ◽  
Author(s):  
Edward Courchaine ◽  
Karla M. Neugebauer
Keyword(s):  
Gene Expression ◽  
Phase Separation ◽  
Low Complexity ◽  
Nuclear Body ◽  
Liquid Droplets ◽  
Cell Biol

Low-complexity proteins undergo phase separation in vitro, forming hydrogels or liquid droplets. Whether these form in vivo, and under what conditions, is still unclear. In this issue, Hennig et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201504117) show that formation of the paraspeckle, a nuclear body that regulates gene expression, requires low-complexity prion-like domains (PLDs) within paraspeckle proteins. The same proteins were shown to form hydrogels, shedding light on the role of “functional aggregation” in nuclear substructure.

scholarly journals Pat1 promotes processing body assembly by enhancing the phase separation of the DEAD-box ATPase Dhh1 and RNA

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Ruchika Sachdev ◽  
Maria Hondele ◽  
Miriam Linsenmeier ◽  
Pascal Vallotton ◽  
Christopher F Mugler ◽  
...  
Keyword(s):  
Phase Separation ◽  
Direct Interaction ◽  
Liquid Droplets ◽  
Processing Bodies ◽  
Dead Box ◽  
The Dead ◽  
Processing Body ◽  
Liquid Liquid Phase Separation

Processing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid–liquid phase separation and are implicated in the decay or storage of mRNAs. How PB assembly is regulated in cells remains unclear. Previously, we identified the ATPase activity of the DEAD-box protein Dhh1 as a key regulator of PB dynamics and demonstrated that Not1, an activator of the Dhh1 ATPase and member of the CCR4-NOT deadenylase complex inhibits PB assembly in vivo (Mugler et al., 2016). Here, we show that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1. Intriguingly, in vivo PB dynamics can be recapitulated in vitro, since Pat1 enhances the phase separation of Dhh1 and RNA into liquid droplets, whereas Not1 reverses Pat1-Dhh1-RNA condensation. Overall, our results uncover a function of Pat1 in promoting the multimerization of Dhh1 on mRNA, thereby aiding the assembly of large multivalent mRNP granules that are PBs.

From the common molecular basis of the AAA protein to various energy-dependent and -independent activities of AAA proteins

2008 ◽  
Vol 36 (1) ◽  
pp. 68-71 ◽  
Author(s):  
Teru Ogura ◽  
Yuka Matsushita-Ishiodori ◽  
Ai Johjima ◽  
Masayo Nishizono ◽  
Shingo Nishikori ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Facilitated Diffusion ◽  
Independent Manner ◽  
Aromatic Residue ◽  
Aaa Atpase ◽  
Central Pore ◽  
Aaa Protein

AAA (ATPase associated with various cellular activities) proteins remodel substrate proteins and protein complexes upon ATP hydrolysis. Substrate remodelling is diverse, e.g. proteolysis, unfolding, disaggregation and disassembly. In the oligomeric ring of the AAA protein, there is a conserved aromatic residue which lines the central pore. Functional analysis indicates that this conserved residue in AAA proteases is involved in threading unfolded polypeptides. Katanin and spastin have microtubule-severing activity. These AAA proteins also possess a conserved aromatic residue at the central pore, suggesting its importance in their biological activity. We have constructed pore mutants of these AAA proteins and have obtained in vivo and in vitro results indicating the functional importance of the pore motif. Degradation of casein by the Escherichia coli AAA protease, FtsH, strictly requires ATP hydrolysis. We have constructed several chimaeric proteases by exchanging domains of FtsH and its homologues from Caenorhabditis elegans mitochondria, and examined their ATPase and protease activities in vitro. Interestingly, it has been found that some chimaeras are able to degrade casein in an ATP-independent manner. The proteolysis is supported by either ATP[S] (adenosine 5′-[γ-thio]triphosphate) or ADP, as well as ATP. It is most likely that substrate translocation in these chimaeras occurs by facilitated diffusion. We have also investigated the roles of C. elegans p97 homologues in aggregation/disaggregation of polyglutamine repeats, and have found that p97 prevents filament formation of polyglutamine proteins in an ATP-independent fashion.

scholarly journals Binding to m6A RNA promotes YTHDF2-mediated phase separation

2019 ◽  
Author(s):  
Jiahua Wang ◽  
Liyong Wang ◽  
Jianbo Diao ◽  
Yujiang Geno Shi ◽  
Yang Shi ◽  
...  
Keyword(s):  
Phase Separation ◽  
Low Complexity ◽  
Rna Degradation ◽  
Biological Processes ◽  
Liquid Droplets ◽  
P Granules ◽  
Defective Mutant ◽  
In Cells

AbstractAs the most abundant modification on mRNA in mammal, N6-Methyladenosine (m6A) has been demonstrated to play important roles in various biological processes including mRNA splicing, translation and degradation. m6A reader proteins have been shown to play central roles in these processes. One of the m6A readers, YTHDF2 is localized to the P granules, which are liquid-like droplets where RNA degradation occurs. How YTHDF2 is localized to P granules is unknown. Here we provide evidence that YTHDF2 forms liquid droplets and phase separate, mediated by its low complexity (LC) domains. Interestingly, the ability to phase separate is robustly stimulated by m6A RNAs in vitro. In vivo, YTHDF2 phase separation may in fact be dependent on m6A RNA and YTHDF2 binding to m6A RNA, since a YTHDF2 m6A-binding defective mutant or a wildtype YTHDF2 assayed in cells lacking m6A RNAs, both fail to phase separate. The ability of phase separate is not limited to YTHDF2; we find other members of the YTH-domain m6A readers can also undergo phase separation. Our findings suggest that m6A RNA induced phase separation of m6A readers may play an important role in their distributions to different phase-separated compartments in cells.

scholarly journals Phase separation of Mer2 organizes the meiotic loop-axis structure of chromatin during meiosis I

2020 ◽  
Author(s):  
Bin Tsai ◽  
Wei Liu ◽  
Dashan Dong ◽  
Kebin Shi ◽  
Liangyi Chen ◽  
...  
Keyword(s):  
Phase Separation ◽  
Binding Motif ◽  
Liquid Droplets ◽  
Meiotic Cell ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Intrinsically Disordered ◽  
Meiosis I

Sexually reproducing organisms acquire genetic diversity through meiotic recombination during meiosis I, which is initiated via programmed DNA double-strand breaks (DSBs) induced by Spo11-containing machinery in each meiotic cell. The combination of programmed DSB sites in each meiotic cell must be diverse, which requires a certain degree of randomness in the distribution of DSBs. The formation of programmed DSBs requires a preestablished loop-axis structure of chromatin. Here, we demonstrate that the axial element protein Mer2 undergoes liquid-liquid phase separation in vitro and in vivo through its intrinsically disordered C-terminal domain. A DNA binding motif within its central domain is responsible for bringing DNA into Mer2 liquid droplets and Mer2-DNA complex could assemble into filamentous structures extending from the droplets. These results suggest that phase separation of Mer2 drives the formation of a droplet-loop structure of meiotic chromatin to facilitate and to diversify programmed DSB formation.

Photoinhibition in vivo and in vitro Involves Weakly Coupled Chlorophyll–Protein Complexes‡¶

2002 ◽  
Vol 75 (6) ◽  
pp. 613 ◽  
Author(s):  
Stefano Santabarbara ◽  
Ilaria Cazzalini ◽  
Andrea Rivadossi ◽  
Flavio M. Garlaschi ◽  
Giuseppe Zucchelli ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Weakly Coupled ◽  
Chlorophyll Protein Complexes ◽  
Chlorophyll Protein

scholarly journals FORMULATION AND IN VITRO, IN VIVO EVALUATION OF PRONIOSOMAL GEL OF NEOMYCIN SULPHATE

2019 ◽  
pp. 156-163
Author(s):  
AMOL SHETE ◽  
PRIYANKA THORAT ◽  
RAJENDRA DOIJAD ◽  
SACHIN SAJANE
Keyword(s):  
Phase Separation ◽  
Skin Irritation ◽  
Entrapment Efficiency ◽  
Separation Method ◽  
Gelling Agent ◽  
In Vivo Evaluation ◽  
Skin Delivery ◽  
Span 60

Objective: The objectives of present investigation were to prepare and evaluate proniosomes of neomycin sulphate (NS) by coacervation phase separation method by using sorbitan monostearate (span 60) and lecithin as a surfactant to increase the penetration through the skin and study the effect of concentration of the same. Methods: Proniosomes of neomycin sulphate (NS) were prepared by coacervation phase separation method by using span 60 and lecithin. The effect of concentration of span 60 and lecithin was studied by factorial design. The prepared proniosomes were converted to gel by using carbopol as a gelling agent. The prepared formulations were evaluated for entrapment efficiency, in vitro drug diffusion, in vitro antibacterial activity and in vivo skin irritation test etc. Results: All Formulation showed the percentage entrapment efficiency in the range 38.31±0.05% to 77.96±0.06%, good homogeneity and gel was easily spreadable with minimal of shear. Optimized formulation showed enhanced rate of diffusion in vitro, increase in zone of inhibition against staphylococcus aureus, no skin irritation and showed good stability. Conclusion: The results of present study indicates that proniosomal gel formulated by using combination of span 60, Lecithin, cholesterol can be used to enhance skin delivery of NS because of excellent permeation of drug. Developed proniosomal gel formulation was promising carrier for NS

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