Discovery of a phase-separating small molecule that selectively sequesters tubulin in cells

Ado, Genyir; Noda, Naotaka; Vũ, Huệ Thị; Perron, Amélie; Mahapatra, Amarjyoti Das; Arista, Karla Pineda; Yoshimura, Hidetoshi; Packwood, Daniel M.; Ishidate, Fumiyoshi; Sato, Shin; Ozawa, Takeaki; Uesugi, Motonari

doi:10.1039/d1sc07151c

Cited by 10 publications

(4 citation statements)

References 44 publications

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“…We then wondered if the internal compartments were indeed liquid droplets. We performed fluorescence recovery after photobleaching (FRAP) experiments to determine the fluidity and diffusion characteristics of the droplets, using Nile red as a probe molecule (Figure d–f). , Upon photobleaching (λ ex = 515 nm, 100% intensity), the fluorescence of droplets recovered up to 70–90% of its initial value in 20 s for all of the time points (Figures S10 and S11). The diffusion coefficients of the dye inside the droplet were determined from the FRAP results (Figure f and Figures S10 and S11), revealing a slight decreasing trend in diffusion coefficient as the assembly time increases.…”

Section: Resultsmentioning

confidence: 99%

Elastic Network of Droplets for Underwater Adhesives

Jo,

Sim

2023

J. Am. Chem. Soc.

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Section: Resultsmentioning

confidence: 99%

Elastic Network of Droplets for Underwater Adhesives

Jo,

Sim

2023

J. Am. Chem. Soc.

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“…Liquid-liquid phase separation (LLPS) is a process in which a homogenous solution separates into two or more liquid phases. 1 This phenomenon is observed in various molecular solutions including small molecules, 2 polymers, 3 peptides, 4 and proteins. 5 LLPS of proteins is commonly observed in living systems and plays an important role in transcription, DNA repair, chromatin organization, and disease formation.…”

Section: Introductionmentioning

confidence: 99%

Revealing nanoscale structure and interfaces of protein and polymer condensates via cryo-electron microscopy

RIZVI,

Favetta,

Jaber

et al. 2024

Preprint

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Liquid-liquid phase separation (LLPS) is a ubiquitous demixing phenomenon observed in various molecular solutions, including in polymer and protein solutions. Demixing of solutions results in condensed, phase separated droplets which exhibit a range of liquid-like properties driven by transient intermolecular interactions. Understanding the organization within these condensates is crucial for deciphering their material properties and functions. This study introduces a streamlined methodology for imaging three different classes of condensates, proteins, complex coacervates, and non-ionic block copolymer coacervates, using cryo-electron microscopy (cryo-EM). The method involves initiating condensate formation on electron microscopy grids to control droplet size and stage in the phase separation process. Results from cryo-EM reveal distinct nanoscale networks and interfaces in the condensate samples. We further investigate these structures using cryo-electron tomography which provides 3D reconstructions, uncovering porous internal structures, unique core-shell morphologies, and inhomogeneities within the nanoscale organization of protein condensates. Comparison with dry-state transmission electron microscopy emphasizes the importance of preserving the hydrated structure for accurate structural analysis. We correlate the internal structure of condensates with their material properties by performing viscosity measurements that support that more viscous condensates exhibit denser internal assemblies. Our findings contribute to a comprehensive understanding of condensate structure and this streamlined methodology provides a versatile tool for exploring various phase-separated systems and their nanoscale structures.

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“…The only related examples in the literature are some small molecules that could act as a biphasic modulator of the protein LLPS, [21][22][23][24][25] and the small molecule huezole that can form phase-separated droplets. [26] Tracing the formation and interaction of phase-separating molecules often relies on optical microscopes. For macromolecules, fluorescent labeling, either chemical tagging of a small fluorescent molecule or genetic fusion with fluorescent proteins, can render the phase-separated coacervates fluorescent without changing their phase-separating property.…”

Section: Introductionmentioning

confidence: 99%

“…Notwithstanding, nonpeptide‐like low‐molecular‐weight compounds that spontaneously phase separate in solutions are relatively scarce. The only related examples in the literature are some small molecules that could act as a biphasic modulator of the protein LLPS, [21–25] and the small molecule huezole that can form phase‐separated droplets [26] …”

Section: Introductionmentioning

confidence: 99%

Photo‐Responsive Phase‐Separating Fluorescent Molecules for Intracellular Protein Delivery

Bao,

Chen,

et al. 2023

Angew Chem Int Ed

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Cellular membranes, including the plasma and endosome membranes, are barriers to outside proteins. Various vehicles have been devised to deliver proteins across the plasma membrane, but in many cases, the payload gets trapped in the endosome. Here we designed a photo‐responsive phase‐separating fluorescent molecule (PPFM) with a molecular weight of 666.8 daltons. The PPFM compound condensates as fluorescent droplets in the aqueous solution by liquid‐liquid phase separation (LLPS), which disintegrate upon photoirradiation with a 405 nm LED lamp within 20 min or a 405 nm laser within 3 min. The PPFM coacervates recruit a wide range of peptides and proteins and deliver them into mammalian cells. Photolysis disperses the payload from condensates into the cytosolic space. Altogether, a type of small molecules that are photo‐responsive and phase separating are discovered; their coacervates can serve as transmembrane vehicles for intracellular delivery of proteins, whereas photo illumination triggers the cytosolic distribution of the payload.

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Discovery of a phase-separating small molecule that selectively sequesters tubulin in cells

Abstract: Phase-separated membraneless organelles or biomolecular condensates play diverse functions in cells, however recapturing their characteristics using small organic molecules has been a challenge. In the present study, cell-lysate-based screening of...

Cited by 10 publications

References 44 publications

Elastic Network of Droplets for Underwater Adhesives

Elastic Network of Droplets for Underwater Adhesives

Revealing nanoscale structure and interfaces of protein and polymer condensates via cryo-electron microscopy

Photo‐Responsive Phase‐Separating Fluorescent Molecules for Intracellular Protein Delivery

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