Single-molecule and ensemble methods to probe RNP nucleation and condensate properties

Rhine, Kevin; Skanchy, Sophie; Myong, Sua

doi:10.1016/j.ymeth.2021.02.012

Cited by 11 publications

(6 citation statements)

References 31 publications

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“…FRAP is a well-known and effective fluorescence microscopy approach for getting information on the dynamics of mobile fluorescent molecules at the micrometer scale. FRAP may also be utilized to obtain quantitative information on the diffusion process, transient binding events, and guided transport on a micrometer scale . FRAP, a method used for measuring the molecular diffusion coefficient in soft materials, has been applied to a variety of media, ranging from thin films to live cells. − Diffusion may occur across a wide variety of periods, from seconds to years, as shown by FRAP experiments.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence Recovery after Photobleaching in Colloidal Science: Introduction and Application

Moud

2022

ACS Biomater. Sci. Eng.

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FRAP (fluorescence recovery after photo bleaching) is a method for determining diffusion in material science. In industrial applications such as medications, foods, Medtech, hygiene, and textiles, the diffusion process has a substantial influence on the overall qualities of goods. All these complex and heterogeneous systems have diffusion-based processes at the local level. FRAP is a fluorescence-based approach for detecting diffusion; in this method, a high-intensity laser is made for a brief period and then applied to the samples, bleaching the fluorescent chemical inside the region, which is subsequently filled up by natural diffusion. This brief Review will focus on the existing research on employing FRAP to measure colloidal system heterogeneity and explore diffusion into complicated structures. This description of FRAP will be followed by a discussion of how FRAP is intended to be used in colloidal science. When constructing the current Review, the most recent publications were reviewed for this assessment. Because of the large number of FRAP articles in colloidal research, there is currently a dearth of knowledge regarding the growth of FRAP’s significance to colloidal science. Colloids make up only 2% of FRAP papers, according to ISI Web of Knowledge.

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Section: Introductionmentioning

confidence: 99%

Fluorescence Recovery after Photobleaching in Colloidal Science: Introduction and Application

Moud

2022

ACS Biomater. Sci. Eng.

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“…We next sought to test the hypothesis that N1ICD forms nuclear phase condensates by characterizing the biophysical properties of phase-separated nuclear N1ICD::GFP foci using Fluorescence Recovery After Photobleaching (FRAP). 26,[55][56][57] Using this approach, we observed that following complete photobleaching of individual nuclear foci, there is a 46%(+/-14) mobile fraction of the total N1ICD::GFP signal within a single focus over 300 seconds. We also determined that N1ICD::GFP exhibited a time-to-half recovery of 24(+/-5) seconds within individual nuclear foci (Figure 3C/D, M.2).…”

Section: Biophysical Characterization Of N1icd Nuclear Focimentioning

confidence: 99%

Notch1 forms nuclear transcriptional condensates that drive target gene expression

Foran

Hallam

Megaly

et al. 2023

Preprint

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The Notch receptor is a titratable, context-specific counter of intercellular interactions that translates productive interactions with ligands on neighbouring cells into corresponding changes in gene expression via the nuclear localization of the Notch intracellular Domain (NICD). Using an Optogenetic Notch1 construct in combination with a live imaging transcriptional reporter and super-resolution imaging, we show that the N1ICD activates gene expression through spontaneous self-assembly into transcriptional condensates whose phase separation is driven by C-terminal Intrinsically Disordered Regions (IDR) of the N1ICD. N1ICD condensates recruit and encapsulate a broad set of core transcriptional proteins, thereby facilitating gene expression and promoting super enhancer-looping. We produced a model of Notch1 activity, whereby discrete changes in nuclear NICD abundance is translated into precise changes in target gene expression through the assembly of phase separated N1ICD molecular crucibles that catalyze gene expression in a concentration-dependent manner by enriching essential transcriptional machineries at target genomic loci.

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“…In this approach, individual condensates are optically captured from a solution in two different laser beams and are brought into close proximity to initiate coalescence. The kinetics of droplet fusion and relaxation can be quantitatively determined from fusion videos ( 33 , 34 ). The major experimental techniques for studying physical properties of condensates are described in ( 34 ).…”

Section: Phases Versus Structurementioning

confidence: 99%

A sePARate phase? Poly(ADP-ribose) versus RNA in the organization of biomolecular condensates

Alemasova

Lavrik

2022

Nucleic Acids Research

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Condensates are biomolecular assemblies that concentrate biomolecules without the help of membranes. They are morphologically highly versatile and may emerge via distinct mechanisms. Nucleic acids–DNA, RNA and poly(ADP-ribose) (PAR) play special roles in the process of condensate organization. These polymeric scaffolds provide multiple specific and nonspecific interactions during nucleation and ‘development’ of macromolecular assemblages. In this review, we focus on condensates formed with PAR. We discuss to what extent the literature supports the phase separation origin of these structures. Special attention is paid to similarities and differences between PAR and RNA in the process of dynamic restructuring of condensates during their functioning.

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Single-molecule and ensemble methods to probe RNP nucleation and condensate properties

Cited by 11 publications

References 31 publications

Fluorescence Recovery after Photobleaching in Colloidal Science: Introduction and Application

Fluorescence Recovery after Photobleaching in Colloidal Science: Introduction and Application

Notch1 forms nuclear transcriptional condensates that drive target gene expression

A sePARate phase? Poly(ADP-ribose) versus RNA in the organization of biomolecular condensates

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