Ratiometric Imaging Using a Single Dye Enables Simultaneous Visualization of Rac1 and Cdc42 Activation

MacNevin, Christopher J.; Toutchkine, Alexei; Marston, Daniel J.; Hsu, Chia Wen; Tsygankov, Denis; Li, Li; Liu, Bei; Qi, Timothy; Nguyen, Dan Vinh; Hahn, Klaus M.

doi:10.1021/jacs.5b09764

Cited by 24 publications

(32 citation statements)

References 36 publications

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“…RapR-Src-cerulean-myc (19), GFP-FRB (8), and RapR-p38α (8) have already been described. Rac1-FLARE and the control plasmids expressing Y-PET and Turq (29) were gifts from Klaus Hahn, University of North Carolina, Chapel Hill, NC. HA-Akt1 construct was a gift from John O'Bryan, University of Illinois-Chicago, Chicago.…”

Section: Methodsmentioning

confidence: 99%

“…One of the key pathways for the stimulation of membrane protrusions is signaling through small GTPase Rac1 (25,26). To assess changes in Rac1 activity mediated by regulation of RapR-Src-as2, we used a previously described biosensor for Rac1 (27)(28)(29). Our studies reveal that activation of RapR-Src-as2 leads to robust stimulation of Rac1 at the cell periphery (SI Appendix, Fig.…”

Section: Dissecting Src-mediated Signaling Pathways Using Engineeredmentioning

confidence: 99%

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Mimicking transient activation of protein kinases in living cells

Klomp

Huyot

Ray

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Physiological stimuli activate protein kinases for finite periods of time, which is critical for specific biological outcomes. Mimicking this transient biological activity of kinases is challenging due to the limitations of existing methods. Here, we report a strategy enabling transient kinase activation in living cells. Using two proteinengineering approaches, we achieve independent control of kinase activation and inactivation. We show successful regulation of tyrosine kinase c-Src (Src) and Ser/Thr kinase p38α (p38), demonstrating broad applicability of the method. By activating Src for finite periods of time, we reveal how the duration of kinase activation affects secondary morphological changes that follow transient Src activation. This approach highlights distinct roles for sequential Src-Rac1-and Src-PI3K-signaling pathways at different stages during transient Src activation. Finally, we demonstrate that this method enables transient activation of Src and p38 in a specific signaling complex, providing a tool for targeted regulation of individual signaling pathways.any physiological and pathological processes are regulated by protein kinases. Therefore, dissection of kinase functions helps to uncover molecular mechanisms and guides the development of new therapeutic strategies (1). However, progress in our understanding of kinase-mediated signaling is often hampered by the limitations of available tools (2). Pharmacological inhibitors of kinases have been valuable for the study of kinase functions, but they often do not provide the desired specificity and they are not available for the majority of kinases (1, 3). Also, the application of inhibitors is limited to the identification of processes affected by the inactivation of the kinase. Activation of a kinase is often achieved by the treatment of cells with growth factors or other extracellular stimuli. However, this approach triggers a multitude of parallel signaling pathways, which significantly complicates the analysis of individual kinase functions (4). Specific activation or inactivation of a kinase can be achieved by genetic modifications. However, this method is susceptible to compensatory artifacts and does not allow us to control the level and timing of kinase activation. Thus, development of novel tools that combine high specificity and tight temporal control of kinase activity remains a necessity. Under physiological conditions, kinases are activated for a finite period, and the duration of this activation is critical for eliciting specific biological outcomes (5, 6). Therefore, to mimic the biological activity of a kinase we need to use methods that allow for transient activation of a kinase with precise temporal control. An optogenetic approach has been successfully used for transient regulation of Raf kinase by dimerization (7). However, this method relies on the fact that Raf dimerization is required for its activation and thus has limited applicability to other kinases. Therefore, development of broadly applicable methods for tightly contro...

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Section: Methodsmentioning

confidence: 99%

Section: Dissecting Src-mediated Signaling Pathways Using Engineeredmentioning

confidence: 99%

Mimicking transient activation of protein kinases in living cells

Klomp

Huyot

Ray

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…However, quantification of FRET requires calibration of the fluorophore concentrations and undesired signals from donor emission bleed-through, moreover, direct acceptor excitation have to be considered. In a FRET independent approach, the CRIB domain of the effector WASP labeled with an environment-sensitive dye displayed an increase in fluorescence intensity upon binding to the activated Cdc42 GTPase (21,22).…”

mentioning

confidence: 99%

Spatiotemporal imaging of small GTPases activity in live cells

Voss

Krüger

Koch

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Ras-like small GTPases function as molecular switches and regulate diverse cellular events. To examine the dynamics of signaling requires spatiotemporal visualization of their activity in the cell. Current small GTPase sensors rely on specific effector domains that are available for only a small number of GTPases and compete for endogenous regulator/effector binding. Here, we describe versatile conformational sensors for GTPase activity (COSGAs) based on the conserved GTPase fold. Conformational changes upon GDP/GTP exchange were directly observed in solution, on beads, and in live cells by Förster resonance energy transfer (FRET). The COSGAs allow for monitoring of Rab1 and K-Ras activity in live cells using fluorescence lifetime imaging microscopy. We found that Rab1 is largely active in the cytoplasm and inactive at the Golgi, suggesting that the Golgi serves as the terminal of the Rab1 functional cycle. K-Ras displays polarized activity at the plasma membrane, with less activity at the edge of the cell and membrane ruffles.

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“…In addition, ratiometric probes are preferred, because they are less influenced by extraneous factors, such as a photo‐bleaching or changes of local concentration of the probe. A few ratiometric fluorophores detecting solvent polarity have been reported, including MCY‐BF 2 , mero199 and MBQ, and few ratiometric fluorescent L‐amino acids such as 3HCaa and M3HFaa, which have been used to study biomolecular interactions. However, they have very low quantum yields in water: <0.001, 0.05, 0.08, 0.019 and 0.078 respectively.…”

Section: Figurementioning

confidence: 99%

A Polarity‐Sensitive Fluorescent Amino Acid and its Incorporation into Peptides for the Ratiometric Detection of Biomolecular Interactions

et al. 2019

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A fluorescent amino acid containing our recently developed 1,5‐naphthyridin‐2(1H)‐one based‐ fluorophore, which is a structural component of the fluorescent natural compounds amarastelline A and nigakinone, has been developed. It has useful functions, that is, solvent‐polarity‐dependent change of fluorescence ratio at 370/480 nm and strong fluorescence in both aqueous solution and less polar organic solvents. To demonstrate its utility, it was incorporated into a C5 peptide with the aim of detecting the interaction of this peptide with calmodulin. As expected, the fluorescence ratio at 370/480 nm of the peptide was changed by the calmodulin only in the presence of Ca2+, thus indicating that the fluorescent peptide could sense the conformational change of calmodulin induced by Ca2+, followed by its interaction. These results also suggest that this fluorescent amino acid as well as its precursor, a succinimidyl ester, could be applicable for detecting various biomolecular interactions.

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Ratiometric Imaging Using a Single Dye Enables Simultaneous Visualization of Rac1 and Cdc42 Activation

Cited by 24 publications

References 36 publications

Mimicking transient activation of protein kinases in living cells

Mimicking transient activation of protein kinases in living cells

Spatiotemporal imaging of small GTPases activity in live cells

A Polarity‐Sensitive Fluorescent Amino Acid and its Incorporation into Peptides for the Ratiometric Detection of Biomolecular Interactions

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