Analysis of a RanGTP-regulated gradient in mitotic somatic cells

Kaláb, Petr; Pralle, Arnd; Isacoff, Ehud Y.; Heald, Rebecca; Weis, Karsten

doi:10.1038/nature04589

Cited by 352 publications

(363 citation statements)

References 18 publications

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“…Remarkable studies have enabled the discovery of intracellular gradients of RanGTP, stimulated by RCC1 and emanating from chromosomes, which can be described as a reaction-diffusion process that supports the spindle machinery in cell division 27,29,[35][36][37] . Interestingly, by manipulating RanQ69L-GTP and RCC1 signalling grafted to magnetic nanoparticles, we have demonstrated experimentally how cascading reaction-diffusion signals may directly influence the position of asymmetric microtubule arrays.…”

Section: Discussionmentioning

confidence: 99%

“…The expression of plasmids and purification of recombinant proteins were realized as described previously 29,43,44 . The plasmids for Escherichia coli expression of RanQ69L-GTP (pQE32-Ran, 6His Tag) and RCC1 (pQE60-Rcc1, 6His Tag) were provided by I. Mattaj (EMBL).…”

Section: Methodsmentioning

confidence: 99%

“…To test the ability of Ran-NPs to interact with importins, we used the fluorescence resonance energy transfer (FRET) biosensor Rango (Ran-regulated importin-b cargo) (Fig. 2c,d) 29 . This FRET sensor has two conformations.…”

Section: Magnetic Nanoparticles Controlling Microtubule Assemblymentioning

confidence: 99%

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Spatiotemporal control of microtubule nucleation and assembly using magnetic nanoparticles

et al. 2013

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Decisions on the fate of cells and their functions are dictated by the spatiotemporal dynamics of molecular signalling networks. However, techniques to examine the dynamics of these intracellular processes remain limited. Here, we show that magnetic nanoparticles conjugated with key regulatory proteins can artificially control, in time and space, the Ran/RCC1 signalling pathway that regulates the cell cytoskeleton. In the presence of a magnetic field, RanGTP proteins conjugated to superparamagnetic nanoparticles can induce microtubule fibres to assemble into asymmetric arrays of polarized fibres in Xenopus laevis egg extracts. The orientation of the fibres is dictated by the direction of the magnetic force. When we locally concentrated nanoparticles conjugated with the upstream guanine nucleotide exchange factor RCC1, the assembly of microtubule fibres could be induced over a greater range of distances than RanGTP particles. The method shows how bioactive nanoparticles can be used to engineer signalling networks and spatial self-organization inside a cell environment.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Spatiotemporal control of microtubule nucleation and assembly using magnetic nanoparticles

et al. 2013

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“…A cell extract droplet has been used as a model system for investigating microtubule nucleation and assembly by magnetic control. Gradient of GTPase RanGTP, driven by the GTP exchange factor RCC1, has been shown to stabilize and promote microtubule assembly in extracts (53). Magnetic nanoparticles conjugated to Ran can be added to the extract and encapsulated in single emulsion droplets.…”

Section: Magnetic Control Of Intracellular Signalingmentioning

confidence: 99%

Biophysical Tools for Cellular and Subcellular Mechanical Actuation of Cell Signaling

Liu

2016

Biophysical Journal

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The ability to spatially control cell signaling can help resolve fundamental biological questions. Optogenetic and chemical dimerization techniques along with fluorescent biosensors to report cell signaling activities have enabled researchers to both visualize and perturb biochemistry in living cells. A number of approaches based on mechanical actuation using forcefield gradients have emerged as complementary technologies to manipulate cell signaling in real time. This review covers several technologies, including optical, magnetic, and acoustic control of cell signaling and behavior and highlights some studies that have led to novel insights. I will also discuss some future direction on repurposing mechanosensitive channel for mechanical actuation of spatial cell signaling.

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“…The specially designed molecules are known as FRET biosensors and can be classified into two categories, intermolecular and intramolecular (single-molecule) FRET biosensors (4). Because of their high signal/noise ratio and wider application in live imaging, and because their use does not require calculation of the corrective FRET efficiency, many laboratories have preferentially created single-molecule FRET biosensors that monitor specific molecules such as kinases (6)(7)(8)(9)(10), proteases (11)(12)(13), GTPases (14)(15)(16)(17) or lipids (18). Recently, a breakthrough was reported in the generation of transgenic mice expressing single-molecular FRET biosensors, in which the activities of numerous kinases, proteases, or GTPases are visualized in living tissues and organs (19)(20)(21).…”

mentioning

confidence: 99%

Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy

Hirata

Kiyokawa

2016

Biophysical Journal

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Fö rster (or fluorescence) resonance energy transfer (FRET) is a nonradiative energy transfer process between two fluorophores located in close proximity to each other. To date, a variety of biosensors based on the principle of FRET have been developed to monitor the activity of kinases, proteases, GTPases or lipid concentration in living cells. In addition, generation of biosensors that can monitor physical stresses such as mechanical power, heat, or electric/magnetic fields is also expected based on recent discoveries on the effects of these stressors on cell behavior. These biosensors can now be stably expressed in cells and mice by transposon technologies. In addition, two-photon excitation microscopy can be used to detect the activities or concentrations of bioactive molecules in vivo. In the future, more sophisticated techniques for image acquisition and quantitative analysis will be needed to obtain more precise FRET signals in spatiotemporal dimensions. Improvement of tissue/organ position fixation methods for mouse imaging is the first step toward effective image acquisition. Progress in the development of fluorescent proteins that can be excited with longer wavelength should be applied to FRET biosensors to obtain deeper structures. The development of computational programs that can separately quantify signals from single cells embedded in complicated three-dimensional environments is also expected. Along with the progress in these methodologies, two-photon excitation intravital FRET microscopy will be a powerful and valuable tool for the comprehensive understanding of biomedical phenomena.Since the discovery of green fluorescent protein (GFP) (1), scientists have been widely employing this gene-encoded fluorescent protein (FP) to investigate the spatiotemporal dynamics of molecules with subcellular resolution. One of the applications of FP engineering is the development of biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) (2). FRET is a nonradiative energy transfer process between two fluorophores located in close proximity to each other, and its efficiency is strongly dependent on the distance between the fluorophores. More precisely, the energy transfer rate (k t ) from a donor to an acceptor fluorophore is calculated in the following Förster's equation;where J is the overlap of donor emission and acceptor excitation spectra, k 2 is an orientation factor of transition moment (a factor determined by the relative orientation of the two fluorophores), n is a refractive index, R is the distance between the donor and acceptor fluorophores, and k f is the donor fluorescence emission speed. Therefore, FRET efficiency is determined by the distance and orientation of the two fluorophores. To measure FRET efficiency, there are roughly two methods; ratiometric analysis and lifetime analysis. Ratiometric analysis utilizes fluorescence intensity of an acceptor (e.g., yellow FP (YFP)) and a donor (e.g., cyan FP (CFP)) upon the donor excitation. Because accept...

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Analysis of a RanGTP-regulated gradient in mitotic somatic cells

Cited by 352 publications

References 18 publications

Spatiotemporal control of microtubule nucleation and assembly using magnetic nanoparticles

Spatiotemporal control of microtubule nucleation and assembly using magnetic nanoparticles

Biophysical Tools for Cellular and Subcellular Mechanical Actuation of Cell Signaling

Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy

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