How to make FRET biosensors for Rab family GTPases

Ishido, Nanako; Kobayashi, Hotaka; Sako, Yasushi; Arai, Takao; Fukuda, Mistunori; Nakamura, Takeshi

doi:10.5772/16984

Cited by 3 publications

(2 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Among those, Raichu (which stands for Ras and interacting protein chimeric unit) FRET sensors have been developed for Ras and Rap1 ( Figure 4c; Mochizuki et al, 2001), RhoA (Pertz, Hodgson, Klemke, & Hahn, 2006), and Rab5 (Kitano, Nakaya, Nakamura, Nagata, & Matsuda, 2008). The Raichu FRET sensors consist of four key functional components: a donor, an acceptor, a GTPase and a GTPase‐binding domain from the binding partner of the GTPase (Ishido et al, 2011). Like other fluorescent reporters (Bivona et al, 2004) of GTPase activity, Raichu‐Rap reflects the GTP‐binding state of Rap1, with Ras‐binding domain of Raf binding to Ras‐GTP, drawing the donor and acceptor close to each other.…”

Section: Signaling Eventsmentioning

confidence: 99%

Bioengineering tools for probing intracellular events in T lymphocytes

Zhang

Mariano

Ando

et al. 2020

WIREs Mechanisms of Disease

View full text Add to dashboard Cite

T lymphocytes are the central coordinator and executor of many immune functions. The activation and function of T lymphocytes are mediated through the engagement of cell surface receptors and regulated by a myriad of intracellular signaling network. Bioengineering tools, including imaging modalities and fluorescent probes, have been developed and employed to elucidate the cellular events throughout the functional lifespan of T cells. A better understanding of these events can broaden our knowledge in the immune systems biology, as well as accelerate the development of effective diagnostics and immunotherapies. Here we review the commonly used and recently developed techniques and probes for monitoring T lymphocyte intracellular events, following the order of intracellular events in T cells from activation, signaling, metabolism to apoptosis. The techniques introduced here can be broadly applied to other immune cells and cell systems.

show abstract

Section: Signaling Eventsmentioning

confidence: 99%

Bioengineering tools for probing intracellular events in T lymphocytes

Zhang

Mariano

Ando

et al. 2020

WIREs Mechanisms of Disease

View full text Add to dashboard Cite

show abstract

“…In a biosensor, the donor and acceptor fluorophore are bound to the extremities of proteins of interest, forming unimolecular FRET sensors (Ishido et al, 2011). A conformational change of the fluorescent pair is induced through stimulation, resulting in FRET (Ai et al, 2008;Rizzo et al, 2004).…”

Section: Techniquementioning

confidence: 99%

Multifocal multiphoton microscopy with adaptive optical correction

et al. 2013

View full text Add to dashboard Cite

Multiphoton microscopy (MPM) is a remarkably versatile technique in biologicalimaging. MPM provides increased depth over confocal imaging and can be combined with other imaging techniques such as fluorescence lifetime imaging microscopy (FLIM), adding functional information. FLIM read--out is relatively straightforward using time--correlated single photon counting (TCSPC). Fluorescence lifetime detection enhances the power of multiphoton imaging to allow three dimensional, concentration independent, measurements of environmental parameters such as pH, Oxygen tension and Ca 2+ in addtion to the interaction or conformational modification of proteins by Förster resonant energy transfer (FRET); the latter is a particular focus of the Dimbleby research groups at King's CollegeLondon. However, there are significant limitations in both FLIM and MM.Limitations of TCSPC--FLIM include prolonged acquisition times along with signal and resolution degradation as a function of depth. This thesis demonstrates advancements multiphoton fluorescence lifetime imaging through improvements in two principal areas: speed and resolution at depth.In order to improve acquistion rates a multifocal multiphoton microscope (MMM) capable of rapid, parallelized TCSPC--FLIM was developed --MegaFLI. Acquisitions demonstrate rapid 3--dimensional, high temporal resolution FLIM in vivo Zebrafish.Performed by massively parallel excitation/detection the speed is signficantly improved by a factor of 64.In parallel to the MegaFLI project, a second microscope employing adaptive optical correction has been developed. The introduction of Adaptive Optics (AO) serves to improve imaging quality by counteracting the refractive index heterogeneities introduced by the sample, limiting the imaging depth. Incorporated with a single beam scanning FLIM system, a pupil--segmentation AO--TCSPC--FLIM demonstrates improved signal--to--noise ratio (SNR) and resolution, permiting a more accurate determination of fluorescent lifetime in turbid media. 3 DECLARATION OF AUTHORSHIPThe work present is my own work with the exception of TRI2, which was developed The introduction (Chapter 1) concentrates on optical methods developed in the context of multiphoton microscopy (MPM) or more specifically two--photon microscopy (TPM) (Denk et al., 1990). TPM enables long--term imaging of in vivo biological specimens, image generation at increased tissue depth, and higher signal--to--noise images compared to wide--field and confocal approaches (Helmchen and In comparison to confocal microscopy, two--photon microscopy (TPM) offers considerable benefits since excitation is confined to a femtolitre volume in the vicinity of the focal plane, reducing overall photobleaching and phototoxicity of thick samples (Centonze and White, 1998;Denk et al., 1990;Helmchen and Denk, 2005;Williams et al., 2001). Out--of--focus excitation is avoided due to the localization of the excitation making the confocal pinhole obsolete (Amos and Where is the numerical aperture of the obj...

show abstract

Rab10 inactivation promotes AMPAR insertion and spine enlargement during long-term potentiation

Wang

Nishiyama

Parra-Bueno

et al. 2022

Preprint

View full text Add to dashboard Cite

Rab-dependent membrane trafficking is critical for changing the structure and function of dendritic spines during synaptic plasticity. Here, we developed highly sensitive sensors to monitor Rab protein activity in single dendritic spines undergoing structural long-term potentiation (sLTP) in rodent organotypic hippocampal slices. During sLTP, Rab10 was persistently inactivated (>30 min) in the stimulated spines, whereas Rab4 was transiently activated over ~5 min. Inhibiting or deleting Rab10 enhanced sLTP, electrophysiological LTP and AMPA receptor (AMPAR) insertion during sLTP. In contrast, disrupting Rab4 impaired sLTP only in the first few minutes, and decreased AMPAR insertion during sLTP. Thus, our results suggest that Rab10 and Rab4 oppositely regulate AMPAR trafficking during sLTP, and disinhibition of Rab10 signaling gates the induction of LTP and associated spine structural plasticity.

show abstract

How to make FRET biosensors for Rab family GTPases

Cited by 3 publications

References 56 publications

Bioengineering tools for probing intracellular events in T lymphocytes

Bioengineering tools for probing intracellular events in T lymphocytes

Multifocal multiphoton microscopy with adaptive optical correction

Rab10 inactivation promotes AMPAR insertion and spine enlargement during long-term potentiation

Contact Info

Product

Resources

About