Differential dynamic microscopy of weakly scattering and polydisperse protein-rich clusters

Safari, Mohammad S.; Vorontsova, Maria A.; Poling-Skutvik, Ryan; Vekilov, Peter G.; Conrad, Jacinta C.

doi:10.1103/physreve.92.042712

Cited by 40 publications

(59 citation statements)

References 55 publications

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“…] with µ 2 and µ 3 being the second and third order polydispersity indices [19,24]. Alternatively, we found that a stretched exponential also fit our data well: [25,26].…”

Section: Differential-dynamic Microscopysupporting

confidence: 52%

“…It allows one to extract, from a series of timeresolved images, the temporal decay rate of density fluctuations over a range of spatial frequencies which provides the ensemble transport dynamics within the field-of-view. DDM has been shown to work on images where single particles or molecules are clearly resolved [18] as well as on images where lower signal-to-background ratios prohibit single particle localization [19].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Light-sheet microscopy with digital Fourier analysis measures transport properties over large field-of-view

Wulstein¹,

Regan²,

Robertson‐Anderson³

et al. 2016

Opt. Express

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Using light-sheet microscopy combined with digital Fourier methods we probe the dynamics of colloidal samples and DNA molecules. This combination, referred to as selective-plane illumination differential dynamic microscopy (SPIDDM), has the benefit of optical sectioning to study, with minimal photobleaching, thick samples allowing us to measure the diffusivity of colloidal particles at high volume fractions. Further, SPIDDM exploits the inherent spatially-varying thickness of Gaussian light-sheets. Where the excitation sheet is most focused, we capture high spatial frequency dynamics as the signal-to-background is high. In thicker regions, we capture the slower dynamics as diffusion out of the sheet takes longer.

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“…] with µ 2 and µ 3 being the second and third order polydispersity indices [19,24]. Alternatively, we found that a stretched exponential also fit our data well: [25,26].…”

Section: Differential-dynamic Microscopysupporting

confidence: 52%

Section: Introductionmentioning

confidence: 99%

Light-sheet microscopy with digital Fourier analysis measures transport properties over large field-of-view

Wulstein¹,

Regan²,

Robertson‐Anderson³

et al. 2016

Opt. Express

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“…Three behaviors of the p53 R248Q aggregates cohere with previous observations of mesoscopic protein-rich clusters of both globular proteins and wild type p53. 38,43,49,50 The average cluster radius R is, first, steady and, second, independent of the protein concentration C0. The third characteristic cluster behavior exhibited by the p53 R248 aggregates is the near-exponential increase of N and φ2 with C0 (Figure 2 h,j).…”

Section: Mesoscopic Protein-rich Clusters In Solutions Of P53 R248qmentioning

confidence: 97%

Mesoscopic Liquid Clusters Represent a Distinct Condensate of Mutant p53

Yang

Saeedi

Davtyan

et al. 2020

Preprint

Self Cite

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The oncogenic properties of mutant p53 have been ascribed to destabilization of the p53 conformation, followed by aggregation into insoluble fibrils. Here we combine immunofluorescent 3D confocal microscopy of breast cancer cells expressing the p53 mutant Arg248Gln (R248Q) with light scattering from solutions of the purified protein and molecular simulations to probe the mechanisms that govern phase behaviors of the mutant across multiple length scales, from cellular to molecular. We establish that p53 R248Q forms mesoscopic proteinrich clusters, an anomalous liquid phase with several unique properties. We demonstrate that the clusters host and facilitate the nucleation of amyloid fibrils. The distinct characteristics of the clusters of R248Q and wild-type p53 and theoretical models indicate that p53 condensation into clusters is driven by the structural destabilization of the core domain and not by interactions of its extensive disordered region. Two-step nucleation of mutant p53 amyloids suggests means to IntroductionThe wild type p53 protein is a potent cancer suppressor, which is inactivated in almost every tumor, either through mutations in the TP53 gene (in 50% or more of human cancers) or deregulation of its associated pathways. 1-6 By contrast, p53 mutants emerge as potent cancer promoters because they exert a dominant-negative (DN) effect on the wild-type variant and also display oncogenic gain-of-function (GOF) properties by inhibiting other cancer suppressors. 1 Several mechanisms of cancer promotion by mutant p53 have been discussed. [6][7][8][9][10][11][12] It was recently suggested that the structural destabilization of the p53 mutants and the associated aggregation into insoluble, degradation resistant structures, designated fibrils, may play a decisive role in their oncogenicity. 2,[13][14][15] Concurrently, fibril suppression, for instance, by stabilization of the mutant p53 conformation, has been identified as a general way to fight cancer. 2,16 A parallel development identified a new form of biological organization, liquid-liquid phase separation, which constitutes several common membraneless organelles such as nucleoli, Cajal bodies, and P granules. [17][18][19][20][21] Many proteins forming dense liquid condensates in live cells incorporate significant disordered regions and condensation at the low concentrations of these proteins in the nucleoplasm and cytoplasm has been attributed to stronger intermolecular attraction due to the unstructured segments. 19,20 Furthermore, it is well-appreciated that protein dense liquids can serve as precursors and facilitators of higher order organization such as ordering into crystals 22-25 and the formation of polymers of sickle hemoglobin. 26,27 Theoretical analyses and recent experiments suggest that a similar precursor mechanism would apply to the nucleation of amyloid fibrils. [28][29][30][31][32][33]

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“…of size or velocity). For moderately polydisperse samples, a cumulant analysis such as the one used in DLS has been proven to be applicable 42,45 , and there are no barriers to employing more complex inversion procedures, as we will discuss further below. It is also worth mentioning that VPT may pose also some constraints on the imaging methods and/or on the samples.…”

Section: Established Points Of Strenghts and Limiting Factorsmentioning

confidence: 99%

Perspective: Differential dynamic microscopy extracts multi-scale activity in complex fluids and biological systems

Cerbino

Cicuta

2017

The Journal of Chemical Physics

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Differential dynamic microscopy (DDM) is a technique that exploits optical microscopy to obtain local, multi-scale quantitative information about dynamic samples, in most cases without user intervention. It is proving extremely useful in understanding dynamics in liquid suspensions, soft materials, cells and tissues. In DDM, image sequences are analyzed via a combination of image differences and spatial Fourier transforms to obtain information equivalent to that obtained by means of light scattering techniques. Compared to light scattering, DDM offers obvious advantages, principally (a) simplicity of setup; (b) possibility of removing static contributions along the optical path; (c) power of simultaneous different microscopy contrast mechanisms; (d) flexibility of choosing an analysis region, analogous to a scattering volume. For many questions, DDM has also advantages compared to segmentation/tracking approaches and to correlation techniques like Particle Image Velocimetry. The very straightforward DDM approach, originally demonstrated with bright field microscopy of aqueous colloids, has lately been used to probe a variety of other complex fluids and biological systems with many different imaging methods, including dark-field, differential interference contrast, wide-field, light-sheet, and confocal microscopy. The number of adopting groups is rapidly increasing and so are the applications. Here, we briefly recall the working principles of DDM, we highlight its advantages and limitations, we outline recent experimental breakthroughs, and we provide a perspective on future challenges and directions. DDM can become a standard primary tool in every laboratory equipped with a microscope, at the very least as a first bias-free automated evaluation of the dynamics in a system.

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Differential dynamic microscopy of weakly scattering and polydisperse protein-rich clusters

Cited by 40 publications

References 55 publications

Light-sheet microscopy with digital Fourier analysis measures transport properties over large field-of-view

Light-sheet microscopy with digital Fourier analysis measures transport properties over large field-of-view

Mesoscopic Liquid Clusters Represent a Distinct Condensate of Mutant p53

Perspective: Differential dynamic microscopy extracts multi-scale activity in complex fluids and biological systems

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