EasyFRAP-web: a web-based tool for the analysis of fluorescence recovery after photobleaching data

Koulouras, Grigorios; Παναγόπουλος, Ανδρέας; Rapsomaniki, Maria Anna; Giakoumakis, Nickolaos Nikiforos; Taraviras, Stavros; Lygerou, Zoi

doi:10.1093/nar/gky508

Cited by 143 publications

(120 citation statements)

References 22 publications

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“…FRAP experiments were performed using a 100X oil immersion lens in a Nikon Eclipse Ti based confocal system. The analysis was done using Fiji and easyfrap (https://easyfrap.vmnet.upatras.gr/) [34, 50].…”

Section: Methodsmentioning

confidence: 99%

A role of cellular translation regulation associated with toxic Huntingtin protein

Joag

Ghatpande

Sarkar

et al. 2019

Preprint

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Huntington's disease (HD) is a severe neurodegenerative disorder caused by poly Q repeat expansion in the Huntingtin (Htt) gene. While the Htt amyloid aggregates are known to affect many cellular processes, its role in translation is not addressed. Here we report pathogenic Htt expression causes protein synthesis deficit in cells. We find a functional prion-like protein, the translation regulator Orb2 to be sequestered by Htt aggregates. Coexpression of Orb2 can partially rescue the lethality associated with poly Q expanded Htt. These findings can be relevant for HD as human homologs of Orb2 also can be sequestered by pathogenic Htt aggregates. Our work suggests that translation dysfunction could be one of the contributors in the pathogenesis of HD and new therapies targeting protein synthesis pathways might help alleviate disease symptoms.

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Section: Methodsmentioning

confidence: 99%

A role of cellular translation regulation associated with toxic Huntingtin protein

Joag

Ghatpande

Sarkar

et al. 2019

Preprint

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“…We measured the fluorescence intensity in the bleach zone as well as the whole cell and background in order to correct for photobleaching and background signal. Immobile and mobile fractions were calculated manually and confirmed using the easyFRAP web tool 26 . Fitting of the average curve was performed with OriginPro 8 (Originlab).…”

Section: Methodsmentioning

confidence: 99%

Dynamic regulation of CD45 by tetraspanin CD53

Dunlock

Arp

Jansen

et al. 2019

Preprint

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T cells are central to the adaptive immune response, playing a role in both the direct and indirect killing of pathogens and transformed cells. The activation of T cells is the result of a complex signaling cascade, initiated at the T cell receptor (TCR), and ending with the induction of proliferation. CD45, a member of the protein tyrosine phosphatase family, is one of the most abundant membrane proteins on T cells and functions by regulating activation directly downstream of the TCR. As a result of alternative splicing, CD45 can be expressed in multiple isoforms, naive T cells express the CD45RA isoform, while activated T cells gain expression of CD45RO, which has been proposed to increase signaling. Though the importance of CD45 in TCR signaling, proliferation and cytokine production is well established, little is known about the regulation of CD45 activity. We discovered that the immune-specific tetraspanin CD53 directly affects the stability and function of CD45RO in T cells. We have identified CD53 as a T cell co-stimulatory molecule in primary human and murine cells. Furthermore, we have shown that the absence of CD53 leads to an altered CD45 isoform expression as a result of decreased CD45RO stability on the cell surface. This instability was accompanied by increased mobility as measured by FRAP. Together, this indicates that CD53 functions as a stabilizer of CD45RO, and therefore as a positive regulator of TCR signaling at the T cell surface. Our data provides novel insight into the role of tetraspanins in the regulation of immune signaling and may provide a new avenue for the regulation of T cell signaling.

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“…We have students fit their measurements of fluorescence recovery to a single exponential function to make the analysis and corresponding code as simple as possible. However, in future semesters we may turn to freely available software capable of using more sophisticated recovery models (81).…”

Section: Performing Fluorescence Recovery After Photobleaching Expmentioning

confidence: 99%

Liquid–Liquid Phase Separation: Undergraduate Labs on a New Paradigm for Intracellular Organization

Riedstra

McGorty

2020

The Biophysicist

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Recent work has shown that the intracellular environment is organized not only through membrane-bound organelles but also through fluid droplets that emerge through liquid–liquid phase separation (LLPS). Intracellular LLPS has attracted recent attention because these fluid droplets, termed biomolecular condensates or membraneless organelles, seem to play important roles in cells' responses to stress, gene regulation, and pathologies. Our understanding of intracellular LLPS has advanced through many quantitative biophysical techniques. Here, we describe a set of undergraduate lab activities that highlight these biophysical techniques. We use various optical microscopy methods and quantitative image analysis to characterize the physical properties of a model aqueous system that exhibits liquid–liquid phase separation. These lab activities can form a multiweek module that exposes students to this exciting new and interdisciplinary field that investigates how phase transitions organize the cell interior.

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EasyFRAP-web: a web-based tool for the analysis of fluorescence recovery after photobleaching data

Cited by 143 publications

References 22 publications

A role of cellular translation regulation associated with toxic Huntingtin protein

A role of cellular translation regulation associated with toxic Huntingtin protein

Dynamic regulation of CD45 by tetraspanin CD53

Liquid–Liquid Phase Separation: Undergraduate Labs on a New Paradigm for Intracellular Organization

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