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1.
Nature ; 599(7885): 503-506, 2021 11.
Article in English | MEDLINE | ID: mdl-34552246

ABSTRACT

All structures within living cells must form at the right time and place. This includes condensates such as the nucleolus, Cajal bodies and stress granules, which form via liquid-liquid phase separation of biomolecules, particularly proteins enriched in intrinsically disordered regions (IDRs)1,2. In non-living systems, the initial stages of nucleated phase separation arise when thermal fluctuations overcome an energy barrier due to surface tension. This phenomenon can be modelled by classical nucleation theory (CNT), which describes how the rate of droplet nucleation depends on the degree of supersaturation, whereas the location at which droplets appear is controlled by interfacial heterogeneities3,4. However, it remains unknown whether this framework applies in living cells, owing to the multicomponent and highly complex nature of the intracellular environment, including the presence of diverse IDRs, whose specificity of biomolecular interactions is unclear5-8. Here we show that despite this complexity, nucleation in living cells occurs through a physical process similar to that in inanimate materials, but the efficacy of nucleation sites can be tuned by their biomolecular features. By quantitatively characterizing the nucleation kinetics of endogenous and biomimetic condensates in living cells, we find that key features of condensate nucleation can be quantitatively understood through a CNT-like theoretical framework. Nucleation rates can be substantially enhanced by compatible biomolecular (IDR) seeds, and the kinetics of cellular processes can impact condensate nucleation rates and specificity of location. This quantitative framework sheds light on the intracellular nucleation landscape, and paves the way for engineering synthetic condensates precisely positioned in space and time.


Subject(s)
Biomolecular Condensates/chemistry , Cell Line, Tumor , Female , Humans , Kinetics , Thermodynamics
2.
Proc Natl Acad Sci U S A ; 116(51): 25440-25445, 2019 12 17.
Article in English | MEDLINE | ID: mdl-31772016

ABSTRACT

Lipid droplets are cytoplasmic microscale organelles involved in energy homeostasis and handling of cellular lipids and proteins. The core structure is mainly composed of two kinds of neutral lipids, triglycerides and cholesteryl esters, which are coated by a phospholipid monolayer and proteins. Despite the liquid crystalline nature of cholesteryl esters, the connection between the lipid composition and physical states is poorly understood. Here, we present a universal intracellular phase diagram of lipid droplets, semiquantitatively consistent with the in vitro phase diagram, and reveal that cholesterol esters cause the liquid-liquid crystal phase transition under near-physiological conditions. We moreover combine in vivo and in vitro studies, together with the theory of confined liquid crystals, to suggest that the radial molecular alignments in the liquid crystallized lipid droplets are caused by an anchoring force at the droplet surface. Our findings on the phase transition of lipid droplets and resulting molecular organization contribute to a better understanding of their biological functions and diseases.


Subject(s)
Lipid Droplets/chemistry , Animals , Cell Line , Cholesterol Esters/chemistry , Crystallization , Humans , Mice , Phase Transition
3.
J Biochem ; 175(2): 179-186, 2024 Feb 25.
Article in English | MEDLINE | ID: mdl-37993409

ABSTRACT

Liquid-liquid phase separation has been recognized as universal mechanisms in living cells for the formation of RNA-protein condensates and ordered lipid domains. These biomolecular condensates or domains nucleate, diffuse and interact with each other across physical dimensions to perform their biological functions. Here we summarize key features of biophysical principles underlying the multi-dimensional condensation of RNA-protein condensates and ordered lipid domains, which are related to nuclear transcription, and signaling on cell membranes. Uncovering physicochemical factors that govern the spatiotemporal coupling of those condensates presents a new avenue in their functions and associated human diseases.


Subject(s)
Phase Separation , RNA , Humans , Biophysics , Cell Membrane , Lipids
4.
J Chem Phys ; 138(17): 174907, 2013 May 07.
Article in English | MEDLINE | ID: mdl-23656159

ABSTRACT

By use of the single-molecule observation, we count the number of DNA double-strand breaks caused by γ-ray irradiation with genome-sized DNA molecules (166 kbp). We find that P1, the number of double-strand breaks (DSBs) per base pair per unit Gy, is nearly inversely proportional to the DNA concentration above a certain threshold DNA concentration. The inverse relationship implies that the total number of DSBs remains essentially constant. We give a theoretical interpretation of our experimental results in terms of attack of reactive species upon DNA molecules, indicating the significance of the characteristics of genome-sized giant DNA as semiflexible polymers for the efficiency of DSBs.


Subject(s)
DNA Breaks, Double-Stranded/radiation effects , DNA/genetics , Bacteriophage T4/genetics , Bacteriophage T4/radiation effects , DNA/chemistry , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Viral/chemistry , DNA, Viral/genetics , Dose-Response Relationship, Radiation , Escherichia coli/genetics , Escherichia coli/radiation effects , Gamma Rays , Humans , Models, Biological , Probability
5.
Nat Commun ; 12(1): 1561, 2021 03 10.
Article in English | MEDLINE | ID: mdl-33692345

ABSTRACT

Recent studies suggest that transcriptional activators and components of the pre-initiation complex (PIC) form higher order associations-clusters or condensates-at active loci. Considerably less is known about the distribution of repressor proteins responsible for gene silencing. Here, we develop an expression assay in living Ciona embryos that captures the liquid behavior of individual nucleoli undergoing dynamic fusion events. The assay is used to visualize puncta of Hes repressors, along with the Groucho/TLE corepressor. We observe that Hes.a/Gro puncta have the properties of viscous liquid droplets that undergo limited fusion events due to association with DNA. Hes.a mutants that are unable to bind DNA display hallmarks of liquid-liquid phase separation, including dynamic fusions of individual condensates to produce large droplets. We propose that the DNA template serves as a scaffold for the formation of Hes condensates, but limits the spread of transcriptional repressors to unwanted regions of the genome.


Subject(s)
Ciona/metabolism , Embryo, Nonmammalian/metabolism , Animals , Ciona/genetics , Gene Expression Regulation, Developmental/genetics , Gene Expression Regulation, Developmental/physiology , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic/genetics , Transcription, Genetic/physiology
6.
Nat Cell Biol ; 22(10): 1187-1196, 2020 10.
Article in English | MEDLINE | ID: mdl-32929202

ABSTRACT

Membraneless organelles or condensates form through liquid-liquid phase separation1-4, which is thought to underlie gene transcription through condensation of the large-scale nucleolus5-7 or in smaller assemblies known as transcriptional condensates8-11. Transcriptional condensates have been hypothesized to phase separate at particular genomic loci and locally promote the biomolecular interactions underlying gene expression. However, there have been few quantitative biophysical tests of this model in living cells, and phase separation has not yet been directly linked with dynamic transcriptional outputs12,13. Here, we apply an optogenetic approach to show that FET-family transcriptional regulators exhibit a strong tendency to phase separate within living cells, a process that can drive localized RNA transcription. We find that TAF15 has a unique charge distribution among the FET family members that enhances its interactions with the C-terminal domain of RNA polymerase II. Nascent C-terminal domain clusters at primed genomic loci lower the energetic barrier for nucleation of TAF15 condensates, which in turn further recruit RNA polymerase II to drive transcriptional output. These results suggest that positive feedback between interacting transcriptional components drives localized phase separation to amplify gene expression.


Subject(s)
Cell Nucleolus/metabolism , Gene Expression Regulation , Intrinsically Disordered Proteins/metabolism , Organelles/metabolism , RNA Polymerase II/metabolism , TATA-Binding Protein Associated Factors/metabolism , Animals , Cell Nucleolus/genetics , Cytoplasm/metabolism , Humans , Intrinsically Disordered Proteins/chemistry , Intrinsically Disordered Proteins/genetics , Mice , Organelles/genetics , Phase Transition , RNA Polymerase II/chemistry , RNA Polymerase II/genetics , TATA-Binding Protein Associated Factors/chemistry , TATA-Binding Protein Associated Factors/genetics
7.
Sci Rep ; 8(1): 17769, 2018 12 11.
Article in English | MEDLINE | ID: mdl-30538268

ABSTRACT

Inkjet printing is of growing interest due to the attractive technologies for surface patterning. During the printing process, the solutes are transported to the droplet periphery and form a ring-like deposit, which disturbs the fabrication of high-resolution patterns. Thus, controlling the uniformity of particle coating is crucial in the advanced and extensive applications. Here, we find that sweet coffee drops above a threshold sugar concentration leave uniform rather than the ring-like pattern. The evaporative deposit changes from a ring-like pattern to a uniform pattern with an increase in sugar concentration. We moreover observe the particle movements near the contact line during the evaporation, suggesting that the sugar is precipitated from the droplet edge because of the highest evaporation and it causes the depinning of the contact line. By analyzing the following dynamics of the depinning contact line and flow fields and observing the internal structure of the deposit with a FIB-SEM system, we conclude that the depinned contact line recedes due to the solidification of sugar solution without any slip motion while suppressing the capillary flow and homogeneously fixing suspended particles, leading to the uniform coating. Our findings show that suppressing the coffee-ring effect by adding sugar is a cost-effective, easy and nontoxic strategy for improving the pattern resolution.

8.
Article in English | MEDLINE | ID: mdl-26172746

ABSTRACT

The behavior of long DNA molecules in a cell-sized confined space was investigated. We prepared water-in-oil droplets covered by phospholipids, which mimic the inner space of a cell, following the encapsulation of DNA molecules with unfolded coil and folded globule conformations. Microscopic observation revealed that the adsorption of coiled DNA onto the membrane surface depended on the size of the vesicular space. Globular DNA showed a cell-size-dependent unfolding transition after adsorption on the membrane. Furthermore, when DNA interacted with a two-phase membrane surface, DNA selectively adsorbed on the membrane phase, such as an ordered or disordered phase, depending on its conformation. We discuss the mechanism of these trends by considering the free energy of DNA together with a polyamine in the solution. The free energy of our model was consistent with the present experimental data. The cooperative interaction of DNA and polyamines with a membrane surface leads to the size-dependent behavior of molecular systems in a small space. These findings may contribute to a better understanding of the physical mechanism of molecular events and reactions inside a cell.


Subject(s)
DNA, Viral/chemistry , DNA, Viral/metabolism , Lipid Bilayers/metabolism , Models, Molecular , Adsorption , Bacteriophage T4 , Cell Membrane/chemistry , Cell Membrane/metabolism , Lipid Bilayers/chemistry , Nucleic Acid Conformation , Spermidine/chemistry , Thermodynamics
9.
J Phys Chem B ; 118(36): 10688-94, 2014 Sep 11.
Article in English | MEDLINE | ID: mdl-25133311

ABSTRACT

Spontaneous generation of DNA-enclosing liposomes from a DNA-lipid blend film is investigated. The special properties of the lipid vesicles, namely, micrometer size, unilamellarity, and dense polymer encapsulation acquired by the dehydration-rehydration process, are physicochemically revealed. We found that the formation of giant unilamellar vesicles encapsulating DNAs are governed by micropatterns of the films, such as dots and network patterns. From the results, we proposed a plausible physical mechanism for the dehydration-rehydration process, making it possible to optimize the encapsulation of any agent.


Subject(s)
DNA/chemistry , Phosphatidylcholines/chemistry , Unilamellar Liposomes/chemistry , Animals , Bacteriophage T4 , Bacteriophage lambda , Desiccation , Microscopy, Confocal , Osmotic Pressure , Salmon , Viscoelastic Substances/chemistry , Water/chemistry
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