Considering Discrete Protein Pools when Measuring the Dynamics of Nuclear Membrane Proteins

Zuleger, Nikolaj; Kelly, David A.; Schirmer, Eric C.

doi:10.1007/978-1-62703-526-2_20

Cited by 3 publications

(3 citation statements)

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“…2012; Hardy, 2012; Zuleger et al . 2013). The simplicity of the fundamental idea of FRAP combined with less stringent technical requirements, comprehensible data analysis and the ability to cover long time scales offer a good explanation, although this is difficult to prove.…”

Section: Applications Of Frapmentioning

confidence: 99%

Fluorescence recovery after photobleaching in material and life sciences: putting theory into practice

Lorén¹,

Hagman²,

Jonasson

et al. 2015

Quart. Rev. Biophys.

131

121

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Fluorescence recovery after photobleaching (FRAP) is a versatile tool for determining diffusion and interaction/binding properties in biological and material sciences. An understanding of the mechanisms controlling the diffusion requires a deep understanding of structure-interaction-diffusion relationships. In cell biology, for instance, this applies to the movement of proteins and lipids in the plasma membrane, cytoplasm and nucleus. In industrial applications related to pharmaceutics, foods, textiles, hygiene products and cosmetics, the diffusion of solutes and solvent molecules contributes strongly to the properties and functionality of the final product. All these systems are heterogeneous, and accurate quantification of the mass transport processes at the local level is therefore essential to the understanding of the properties of soft (bio)materials. FRAP is a commonly used fluorescence microscopy-based technique to determine local molecular transport at the micrometer scale. A brief high-intensity laser pulse is locally applied to the sample, causing substantial photobleaching of the fluorescent molecules within the illuminated area. This causes a local concentration gradient of fluorescent molecules, leading to diffusional influx of intact fluorophores from the local surroundings into the bleached area. Quantitative information on the molecular transport can be extracted from the time evolution of the fluorescence recovery in the bleached area using a suitable model. A multitude of FRAP models has been developed over the years, each based on specific assumptions. This makes it challenging for the non-specialist to decide which model is best suited for a particular application. Furthermore, there are many subtleties in performing accurate FRAP experiments. For these reasons, this review aims to provide an extensive tutorial covering the essential theoretical and practical aspects so as to enable accurate quantitative FRAP experiments for molecular transport measurements in soft (bio)materials.

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Section: Applications Of Frapmentioning

confidence: 99%

Fluorescence recovery after photobleaching in material and life sciences: putting theory into practice

Lorén¹,

Hagman²,

Jonasson

et al. 2015

Quart. Rev. Biophys.

131

121

View full text Add to dashboard Cite

show abstract

“…FRAP, is the most widely used technique by experimental biologists because of the relative ease of performing confocal FRAP in the majority of the biological domains. This technique is also very sensitive towards the bound or immobile fractions of the molecules under study (Costantini & Snapp, 2013;González-González et al, 2012;Hardy, 2012;Zuleger et al, 2013). However, there are major limitation associated with the FRAP technique.…”

mentioning

confidence: 99%

Line-FRAP, A Versatile Method to Measure Diffusion Rates In Vitro and In Vivo

Dey

Marciano

Nunes-Alves

et al. 2021

Journal of Molecular Biology

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Section: Da = Kbt/6phramentioning

confidence: 99%

Line-FRAP, a versatile method based on fluorescence recovery after photobleaching to measure diffusion ratesin vitroandin vivo

Dey

Marciano

Schreiber

2020

Preprint

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AbstractA cell is a densely packed conglomerate of macromolecules, where diffusion is essential for their function. The crowded conditions may affect diffusion both through hard (occluded space) and soft (weak, non-specific) interactions. Multiple-methods have been developed to measure diffusion rates at physiological protein concentrations within cells, however, each of them has its limitations. Here, we introduce Line-FRAP, a method based on measuring recovery of photobleaching under a confocal microscope that allows diffusion rate measurements in versatile environments using standard equipment. Implementation of Line mode to the classical FRAP technique improves the time resolution in data acquisition. We also introduce an updated method for data analysis. Together, these contribute towards better estimation of Diffusion coefficients in a wide range of environments. We evaluated the method using different proteins either chemically labelled or by fusion to YFP. The calculated diffusion rates were comparable to literature data as measured in vitro, using artificial crowders, in HeLa cells and in E.coli. Diffusion coefficients in HeLa was ~2.5-fold slower and in E.coli 15-fold slower than measured in buffer. Moreover, we show that increasing the osmotic pressure on E.coli further decreases diffusion by 2-3 fold. The method presented here is easy to apply on a standard confocal microscope, fits a large range of molecules with different sizes and provides robust results in any conceivable environment and protein concentration.

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Considering Discrete Protein Pools when Measuring the Dynamics of Nuclear Membrane Proteins

Cited by 3 publications

References 24 publications

Fluorescence recovery after photobleaching in material and life sciences: putting theory into practice

Fluorescence recovery after photobleaching in material and life sciences: putting theory into practice

Line-FRAP, A Versatile Method to Measure Diffusion Rates In Vitro and In Vivo

Line-FRAP, a versatile method based on fluorescence recovery after photobleaching to measure diffusion ratesin vitroandin vivo

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