Visualizing the Signal Transduction Pathways in Living Cells with GFP-Based FRET Probes

Kurokawa, Kenji; Takaya, Akiyuki; Terai, Kenta; Fujioka, Aki

doi:10.1267/ahc.37.347

Cited by 17 publications

(20 citation statements)

References 43 publications

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“…Data obtained with RaichuRhoA fused to the carboxy-terminus of RhoA was available as supplementary data (Supplementary Figure 1 and Supplementary Video SupFig1HeLa.mov). The detailed comparison of the probes has been described elsewhere (Kurokawa et al, 2004b).…”

Section: Rhoa Is Activated At the Leading Edge Of Migrating Cellsmentioning

confidence: 99%

Localized RhoA Activation as a Requirement for the Induction of Membrane Ruffling

Kurokawa

Matsuda

2005

MBoC

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172

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We examined the spatio-temporal activity of RhoA in migrating cells and growth factor-stimulated cells by using probes based on the principle of fluorescence resonance energy transfer. In HeLa cells migrating at a low cell density, RhoA was activated both at the contractile tail and at the leading edge. However, RhoA was activated only at the leading edge in MDCK cells migrating as a monolayer sheet. In growth factor-stimulated Cos1 and NIH3T3 cells, the activity of RhoA was greatly decreased at the plasma membrane, but remained high at the membrane ruffles in nascent lamellipodia. These observations are in agreement with the proposed role played by RhoA in stress fiber formation, but they also implicated RhoA in the regulation of membrane ruffling, the induction of which is a typical phenotype of activated Rac. In agreement with this view, dominant negative RhoA was found to inhibit membrane ruffling induced by active Rac. Furthermore, we found that Cdc42 activity was also required for high RhoA activity in membrane ruffles. Finally, we found that mDia1, but not ROCK, was stably associated with membrane ruffles. In conclusion, these results suggested that RhoA cooperates with Rac1 and Cdc42 to induce membrane ruffles via the recruitment of mDia. INTRODUCTIONCell migration is an essential process in embryonic development, wound repair, immune surveillance, and tumor cell metastasis, and such migration is known to be regulated by growth factors, the extracellular matrix, and other extracellular components. Such extracellular cues are known to elicit various intracellular responses in the organization of both the actin and the microtubule cytoskeletons, as well as that of vesicular transport pathway and gene transcription (Mitchison and Cramer, 1996;Lauffenburger and Horwitz, 1996;Michaelson et al., 2001). It is generally accepted that the driving force for short-term cell movement is provided by the dynamic reorganization of the actin cytoskeleton, which directs the protrusion of the cell membrane at the front of the cell, as well as retraction at the rear. On the other hand, efficient and long-term migration requires the stabilization of cell polarity, which is achieved through the reorganization of the microtubule cytoskeleton (Lauffenburger and Horwitz, 1996;Gundersen and Cook, 1999;Etienne-Manneville and Hall, 2002;Raftopoulou and Hall, 2004). Therefore, the spatial regulation of the actin as well as the microtubule cytoskeleton is a critical component in the regulation of cell migration.Rho-family GTPases function as critical molecular switches in transducing extracellular signals to both the actin and microtubule cytoskeleton (Ridley, 2001; EtienneManneville and Hall, 2002). For example, RhoA triggers actin stress fiber formation; Rac1 induces lamellipodia and membrane ruffles; and Cdc42 elicits the formation of filopodia, i.e., protrusive actin (Mitchison and Cramer, 1996;Hall, 1998). At the leading edge of motile cells, filopodia, lamellipodia, and membrane ruffles are often recognized, suggesting the loc...

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Section: Rhoa Is Activated At the Leading Edge Of Migrating Cellsmentioning

confidence: 99%

Localized RhoA Activation as a Requirement for the Induction of Membrane Ruffling

Kurokawa

Matsuda

2005

MBoC

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172

170

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“…Thus, we developed specific anti-R-Ras sera and a probe for R-Ras activity based on the principle of fluorescence resonance energy transfer (FRET), a technique that has been shown to be extremely useful for the spatiotemporal analysis of small GTPases (Kurokawa et al, 2004b). Using these tools, we found that endogenous R-Ras is enriched and activated at endosomes and that these R-Ras proteins promote exocytosis by activating RalA.…”

Section: Introductionmentioning

confidence: 99%

R-Ras Regulates Exocytosis by Rgl2/Rlf-mediated Activation of RalA on Endosomes

Takaya

Kamio

Masuda

et al. 2007

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R-Ras is a Ras-family small GTPase that regulates various cellular functions such as apoptosis and cell adhesion. Here, we demonstrate a role of R-Ras in exocytosis. By the use of specific anti-R-Ras antibody, we found that R-Ras was enriched on both early and recycling endosomes in a wide range of cell lines. Using a fluorescence resonance energy transfer-based probe for R-Ras activity, R-Ras activity was found to be higher on endosomes than on the plasma membrane. This high R-Ras activity on the endosomes correlated with the accumulation of an R-Ras effector, the Rgl2/Rlf guanine nucleotide exchange factor for RalA, and also with high RalA activity. The essential role played by R-Ras in inducing high levels of RalA activity on the endosomes was evidenced by the short hairpin RNA (shRNA)-mediated suppression of R-Ras and by the expression of R-Ras GAP. In agreement with the reported role of RalA in exocytosis, the shRNA of either R-Ras or RalA was found to suppress calcium-triggered exocytosis in PC12 pheochromocytoma cells. These data revealed that R-Ras activates RalA on endosomes and that it thereby positively regulates exocytosis. INTRODUCTIONR-Ras is a Ras-family GTPase and its amino acid sequence is 55% identical to those of the classical types of Ras (H-, K-, N-Ras, collectively referred to hereafter as "Ras") (Lowe et al., 1987). As is the case with the other Ras-family GTPases, R-Ras is regulated primarily by two classes of protein, guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP). Reflecting the high sequence similarity among Ras-family GTPases, many GEFs and GAPs for R-Ras catalyze other Ras-family GTPases as well (Ohba et al., 2000). Furthermore, R-Ras is known to interact with many effectors of Ras, such as Raf-1, Ral GEFs, and the p110␣ subunit of phosphoinositide 3-kinase (PI3K) (Rey et al., 1994;Marte et al., 1997). Despite this redundancy between R-Ras and Ras, R-Ras exhibits various properties that are distinct from those of Ras. For example, R-Ras preferentially activates Ral GEFs and PI3K, but it does not activate Raf (Huff et al., 1997;Rodriguez-Viciana et al., 2004). The transforming activity of constitutively active R-Ras is substantially less potent than that of the constitutively active Ras (Cox et al., 1994), although it should be noted that a recent report has suggested the involvement of R-Ras in human gastric cancer (Nishigaki et al., 2005). Meanwhile, R-Ras is known to regulate cell adhesion, cell spreading, and phagocytosis through the activation of integrin (Zhang et al., 1996;Keely et al., 1999; Berrier et al., 2000;Self et al., 2001). R-Ras-null mice have recently been shown to exhibit excessive vascular responses, in spite of the fact that they are otherwise normal (Komatsu and Ruoslahti, 2005). This phenotype seems to reflect higher levels of expression of R-Ras in smooth muscle cells, including blood vessel cells. The results obtained with R-Ras-null mice have also demonstrated that an R-Ras defect can be almost entirely compensated for by other ...

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“…In general, green fluorescent protein (GFP)-based FRET sensors are classified into two types: bimolecular and unimolecular sensors (Miyawaki, 2003;Kurokawa et al, 2004). For bimolecular sensors, donor (CFP) and acceptor (YFP) are fused to protein A (e.g., the sensor domain) and protein B (e.g., the detector domain), respectively (Fig.…”

Section: Advantages Of Unimolecular Fret Sensorsmentioning

confidence: 99%

“…This is because with a unimolecular sensor protein A and protein B are placed in close proximity, and thus, protein B can easily find protein A. This will increase the percentage of real FRET signals versus undesired signals arising from donor emission bleedthrough and direct acceptor excitation (Hailey et al, 2002;Kurokawa et al, 2004). Furthermore, perturbation of endogenous signaling is reduced when using a unimolecular sensor instead of a bimolecular sensor (Miyawaki, 2003).…”

Section: Wwwintechopencommentioning

confidence: 99%