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Abstract

  1. Top of page
  2. Abstract
  3. FRET and FRET-based probes
  4. FRET probes for phosphorylation
  5. Small GTPases
  6. Activity changes of the Rho-family of GTPases during cell division
  7. Membrane ruffling
  8. Future directions
  9. Acknowledgments
  10. References

A hallmark of cancer cells is their uncontrolled activation of growth signal transduction cascades comprised of oncogene products. Overexpression and activating mutations of the growth factor receptors Ras and Raf are frequently observed in human cancer cells. Several research groups, including our own, have been developing probes based on the principle of fluorescence (Förster) resonance energy transfer (FRET) to visualize how signaling molecules, including oncogene products, are regulated in normal and cancerous cells in the living state. In this review, we will briefly introduce the principle of FRET-based probes, present an overview of the probes reported to date, and discuss the perspectives of these probes and fluorescent imaging systems in cancer biology. (Cancer Sci 2006; 97: 8–15)

Cancer is a genetic disease caused by alterations in genes that regulate various aspects of cellular functions.(1) Among these, genes that encode proteins with important roles in the diverse arrays of growth signaling cascades frequently suffer from amplification and mutations in human cancers. Of note, many components of these growth signaling cascades were originally identified as oncogene products, and it is therefore not surprising that hyperactivation of these signaling cascades leads to abnormal cellular proliferation. For the last two decades, scientists have been enthusiastically deciphering the complex networks of signaling molecules, primarily by isolating novel binding partners using molecular and biochemical approaches. An illustration of one of the best-characterized growth signaling cascades is shown in Fig. 1. Upon activation by extracellular stimuli, such as epidermal growth factor (EGF), receptors at the plasma membrane become phosphorylated and recruit adaptor molecules, followed by activation of the small G protein Ras, thereby leading to the activation of many downstream signaling networks, including the ERK MAP kinase cascade. Although we now possess a plethora of information about the participating molecules and their networks, we still know little about the spatiotemporal regulation of these signaling molecules, such as how they are regulated over short and long time courses, or where in the cells they are activated and inactivated. The lack of such spatiotemporal information can mainly be ascribed to the inherent properties of the biochemical techniques used. Thus, the development of new techniques has been awaited for visualization of the signal transduction cascades in living cells in both physiological and cancerous contexts.

image

Figure 1. Schematic view of a signal transduction event. Upon growth factor stimuli, receptors on the plasma membrane become tyrosine-phosphorylated and recruit adaptor proteins. The adaptor proteins directly or indirectly bring their binding partner, a guanine nucleotide exchange factor (GEF), which promotes guanine nucleotide exchange of GDP for GTP and activates G proteins. The GTP-bound active G proteins bind to effectors and transduce signals for biological outputs, such as cytoskeletal rearrangement or proliferation. GTPase-activating proteins (GAP) negatively regulate the G protein activity.

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The discovery of green fluorescent protein (GFP) has facilitated a huge amount of progress toward investigating the localization of molecules of interest. Based on GFP technology, we and others have been trying to establish probes to visualize the biochemical interactions among signaling molecules in order to understand their spatiotemporal regulation. Here, we summarize the probes developed recently by ourselves and other researchers, and the findings obtained using these probes. As the detailed experimental procedure can be found in our previous review article(2) and is also available in our website (http://www.biken.osaka-u.ac.jp/biken/shuyouvirus/e-phogemon/index.htm), we only present brief details regarding the basis of fluorescence (Förster) resonance energy transfer (FRET) and the experimental procedure in this article.

FRET and FRET-based probes

  1. Top of page
  2. Abstract
  3. FRET and FRET-based probes
  4. FRET probes for phosphorylation
  5. Small GTPases
  6. Activity changes of the Rho-family of GTPases during cell division
  7. Membrane ruffling
  8. Future directions
  9. Acknowledgments
  10. References

FRET is a process by which a fluorophore (donor) in an excited state non-radiatively transfers its energy to a neighboring fluorophore (acceptor),(3) thereby causing the acceptor to emit fluorescence at its characteristic wavelength. FRET depends on a proper spectral overlap between the donor and the acceptor, the distance between the two fluorophores and their relative orientation. In general, FRET probes are classified into two types: intramolecular (or unimolecular) and intermolecular (or bimolecular).(4) Both types of probe have their merits and demerits. For instance, intermolecular probes are powerful tools for investigating the presence of a specified domain in cells(5–7) but are not usually appropriate for visualizing signaling events. Thus, we will concentrate on presenting an overview of the intramolecular FRET probes in this review and Table 1.

Table 1. Signal transduction probes based on green fluorescent protein and fluorescence (Förster) resonance energy transfer
TargetStructureNameReference
Receptors/tyrosine kinases
 EGFRCFP-SH2(Shc)-substrate-YFP 15
 Abl/EGF-RYFP-CrkII-CFPPicchu11
 AblCFP-CrkII-YFP 15
 Insulin RCFP-substrate(PI3K)-SH2-YFPPhocus16
 SrcTM(Cbp)-YFP-Cbp(substrate)-SH2 (Csk)-CFPChimera20
 SrcCFP-SH2(Src)-substrate-YFP 15,19
Serine/threonine kinases
 RafYFP-Raf-CFPPrin23
 PKAGFP-KID-BFP 45
 PKACFP-substrate(14.3.3)-YFPAKAR24
 PKARGFP-substrate(Kemptide)-BGFPART25
 AKT/PKBCFP-14.3.3-substrate-YFPAktus46
 AKT/PKBGFP-AKT-red-shifted YFP 47
 PKCCFP-FHA2(Rad53P)-substrate-YFPCKAR26
Small GTPases
 RasYFP-Ras-RBD(Raf)-CFPRaichu-Ras9
 Rap1YFP-Ras-RBD(Raf)-CFPRaichu-Rap19
 RalAYFP-RalA-RalBP1-RBD-CFPRaichu-RalA34
 RhoAYFP-RhoA-RBD(PKN)-CFPRaichu-RhoA29
 RhoAYFP-RBD-CFP 29
 RacYFP-CRIB(PAK)-Rac-CFPRaichu-Rac128
 Cdc42YFP-CRIB(PAK)-Cdc42-CFPRaichu-Cdc4228
 Rac, Cdc42YFP-CRIB(PAK)-CFPRaichu-CRIB28
 Cdc42YFP-GBD-ACV (N-WASP)-CFP 37
 Cdc42YFP-GBD-CFP 37
 Cdc42Cdc42-YFP-GBD-ACV-CFPGEF sensor37
 N-WASP/Cdc42CFP-N-WASP-YFPN-WASP-BS39

The first intramolecular GFP-based FRET indicator, Cameleon, was reported in 1997 (Fig. 2A), and monitors the intracellular calcium concentration by using calmodulin.(8) However, the difficulties associated with the development of intramolecular FRET probes subsequently hampered further success until 2001, when we reported a FRET probe for the Ras oncogene product(9) (Fig. 2B). Intramolecular FRET probes are comprised of four modules: the donor, the acceptor and a pair of molecules, which are expected to interact with each other. A cyan-emitting mutant of GFP (CFP) and a yellow-emitting mutant of GFP (YFP) are now used as the donor and acceptor molecules, respectively, rather than the original pair of a blue-emitting mutant of GFP (BFP) and GFP.(10) The difficulties associated with the development of intramolecular FRET probes mainly involve the design of the probes themselves. Although the amount of available structural data for proteins continues to increase very rapidly, data for protein–protein complexes are comparatively scarce. Thus, in many instances, there is little rationale for the design drawings of the probe. Here, we present an overview of the probes developed to date in order to outline both their design and usage.

image

Figure 2. Structures of the Raichu/Picchu FRET probes. Two conformations, namely off (upper) and on (lower), are shown schematically for each probe. (A) Cameleon: Binding of calcium (green ball) to calmodulin (CaM) induces a conformational change in Cameleon, thereby bringing the donor (BFP) into close proximity with the acceptor (GFP) and evoking sensitized FRET. (B) Raichu-Ras: GTP-bound Ras in the probe binds intramolecularly with the Ras-binding domain of Raf, which results in increased FRET from CFP and YFP. (C) Picchu: CrkII, which is composed of Src homology (SH) 2 and SH3 domains, is sandwiched between YFP and CFP. Upon activation of Abl kinase or epidermal growth factor receptor (EGFR), tyrosine 221 (denoted as Y221) is phosphorylated (indicated as P, and circled in red). Intramolecular binding of the SH2 domain to Y221 increases the level of FRET. (D) Src monitor: The SH2 domain of Src and a substrate peptide sequence (subpep) are sandwiched between YFP and CFP. Upon activation of Src, the tyrosine in the substrate peptide is phosphorylated (indicated as P, and circled in red) and then recognized by the SH2 domain of the probe. (E) Prin-c-Raf: Raf kinase is sandwiched between YFP and CFP. Raf adopts two conformations, namely the closed inactive and open active forms. FRET is high and low in the former and latter conformations, respectively. Upon activation, the Ras-binding domain (RBD) and kinase region of the probe bind to Ras and MEK, respectively, concomitant with a decrease in FRET. (F) AKAR and ART: In AKAR, the phosphoserine-binding domain of 14–3-3τ and a substrate peptide sequence (subpep) are sandwiched between YFP and CFP. Protein kinase A (PKA) phosphorylates the serine residue in the substrate region (indicated as P, and circled in red), which is then recognized by the phosphoserine-binding domain of 14–3-3τ. In ART, red- and blue-shifted GFP (RGFP and BGFP) are fused to the substrate-peptide sequence of Kemptide. The conformational change of Kemptide induced by serine phosphorylation (indicated by the circled P) is monitored by FRET. (G) CKAR: The FHA2 phosphothreonine-binding domain from Rad51 (denoted as FHA2) and a substrate peptide sequence (subpep) are sandwiched between CFP and YFP. A conformational change of the probe is induced by protein kinase C (PKC)-dependent phosphorylation (indicated by the circled P).

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FRET probes for phosphorylation

  1. Top of page
  2. Abstract
  3. FRET and FRET-based probes
  4. FRET probes for phosphorylation
  5. Small GTPases
  6. Activity changes of the Rho-family of GTPases during cell division
  7. Membrane ruffling
  8. Future directions
  9. Acknowledgments
  10. References

EGF receptor: New insights into lateral diffusion and signaling at endosomes

To investigate the origin of the phosphorylation wave in response to growth factor stimulation in living cells, we developed a FRET probe designated phosphorylation indicator of CrkII chimeric unit (Picchu)(11) (Fig. 2C). The backbone of Picchu is the adaptor protein CrkII, and this is sandwiched between CFP and YFP. CrkII belongs to a family of adaptor proteins containing Src homology (SH) 2 and SH3 domains. EGF stimulation phosphorylates tyrosine 221 (Tyr221) of CrkII, and the phosphorylated Tyr221 is recognized intramolecularly by the SH2 domain of CrkII. This intramolecular binding of the SH2 domain to phosphorylated Tyr221 brings CFP into close proximity with YFP, thereby increasing the level of FRET from CFP to YFP. Therefore, Picchu reflects the level of EGF receptor (EGFR) activation. The wave of EGF-initiated phosphotyrosine has been observed to move from the periphery of the cell to the center.(11)

The lateral propagation of EGFR phosphorylation has also been monitored using this FRET probe. When cells were stimulated locally with EGF, tyrosine phosphorylation and Ras activation were found to be restricted to the site of EGF application.(12) However, in cells overexpressing EGFR or preincubated with endocytosis inhibitors, tyrosine phosphorylation and Ras activation were spread over the entire cell. These observations indicate that activated EGFR is rapidly downregulated by endocytosis, and that this negative regulatory mechanism can be saturated by overexpression of EGFR. As EGFR overexpression has been reported in many types of human cancer, these findings suggest that non-physiological lateral propagation of EGFR activation could explain the growth factor independence of many cancer cells.

Epidermal growth factor receptor activation by its ligand triggers rapid endocytosis of EGF–EGFR complexes. However, it has been proposed that this endocytosis not only delivers EGFR to the degradation machineries but also provides a signaling platform within cells.(13) To monitor the activity of EGFR, a modified Picchu probe, designated Picchu-Z, was developed. In Picchu-Z, a pair of synthetic amphipathic helices, WinZipA2 and WinZipB1, was used to bind Picchu non-covalently to the C-terminus of EGFR.(14) Using this probe, it has been shown that a high level of tyrosine phosphorylation is retained after endocytosis, up to the point when EGFR has translocated to the perinuclear region.

Other probes similar to Picchu have also been reported for monitoring tyrosine phosphorylation. For example, another probe for EGFR involving the SH2 domain of Shc and a phosphorylation substrate peptide has been reported.(15) Furthermore, activation of the insulin receptor has been monitored by a probe named phocus.(16)

Src and Abl: Tyrosine kinases meet membrane ruffles

v-Src, the first oncogene product to be discovered, was also the first protein to be identified as a tyrosine kinase.(17) Its cellular homolog, c-Src, is known to regulate integrin–cytoskeleton interactions, which are essential for the transduction of mechanical stimuli. A FRET probe for Src was established originally using the SH2 domain of Src and its substrate peptide EIYGEF(18) (Fig. 2C), and has been improved recently by using the substrate region of p130Cas.(19) Using this FRET probe and laser-tweezer traction on fibronectin-coated beads, rapid distal Src activation and a slower directional wave propagation of Src activation along the plasma membrane have been observed. This force-induced directional and long-range activation of Src requires intact actin filaments and microtubules.

Src activation by EGF has also been monitored using a different FRET indicator named Chimera, which is composed of YFP at the N-terminus, followed by the substrate domain of Cbp, the SH2 domain of Csk and finally CFP.(20) The function of Src is negatively regulated by phosphorylation of the C-terminal regulatory site mediated by Csk, which belongs to the cytoplasmic tyrosine kinases, while Cbp phosphorylated by Src is recognized by the SH2 domain of Csk. For membrane targeting of this probe, the transmembrane domain of Cbp was fused at the N-terminus. EGF stimulation was found to upregulate the FRET of this indicator at membrane ruffles in COS1 cells.(20)

In general, these reporters are composed of CFP, a phosphotyrosine-binding domain, a consensus substrate for Src and Abl, and YFP.(15) Because Crk is phosphorylated by Abl, Picchu is utilized as an Abl monitor. A monitor similar to Picchu has also been constructed and used as an Abl activation indicator in cells stimulated with EGF and platelet-derived growth factor (PDGF).(15)

Raf: Translocation, conformational change and ignition

The three Raf genes, namely A-RAF, B-RAF and C-RAF, encode cytoplasmic serine/threonine kinases that are situated downstream of Ras and upstream of mitogen-activated protein kinase (MEK).(21) B-RAF somatic missense mutations are found in 66% of malignant melanomas and at lower frequencies in a wide range of other human cancers.(22) Although there is no doubt that Raf is regulated by Ras, a few questions about their molecular mechanisms still remain to be answered. For example, there is a dispute regarding whether or not the role of Ras to Raf is limited to membrane translocation. In other words, it remains unclear whether or not the Ras-induced conformational change of Raf has any role in signal transduction from Raf to MEK. To assess the conformational change of Raf, a unimolecular FRET indicator, designated Photomeric Raf indicator (Prin)-c-Raf, was constructed(23) (Fig. 2C). In agreement with previous models, c-Raf was found to adopt two conformations: open active and closed inactive forms. Ras binding transforms c-Raf from the closed to the open conformation, resulting in c-Raf binding to MEK. It was found that, in the presence of a cytoplasmic RasV12 mutant, the c-Raf–Ras complex bound to, but failed to phosphorylate, MEK. In contrast, the cytoplasmic RasV12 mutant significantly enhanced MEK phosphorylation by a membrane-targeted c-Raf. Thus, that study clearly demonstrated that Ras activates Raf in two ways, namely induction of the conformational change of Raf essential for binding to its substrate, MEK, and recruitment of Raf to the plasma membrane where it becomes catalytically activated.

Protein kinase A and protein kinase C: A-kinase activity reporter and C-kinase activity reporter

The FRET probe developed to monitor protein kinase A (PKA) activation, designated A-kinase activity reporter (AKAR), comprises CFP, a phosphoamino acid-binding domain (14–3-3τ), a consensus substrate peptide for PKA, and YFP.(24) Another probe for monitoring PKA activity, named ART (cAMP-responsive tracer), was developed by sandwiching Kemptide (a substrate for PKA) between red- and blue-shifted GFP (RGFP and BGFP, respectively).(25) These probes have been shown to be able to monitor the level of cAMP upon ligand stimulation.

C-kinase activity reporter (CKAR), which is similar to AKAR, was designed to monitor the level of protein kinase C (PKC) activation.(26) By simultaneously monitoring FRET and PKC translocation to the plasma membrane, the membrane targeting and activation of PKC were dissected for the first time. It has also been revealed that PKC activation and translocation are coupled with an increased level of calcium, but not phosopholipase C (PLC) activation, which yields diacylglycerol, a potent activator of PKC.

Small GTPases

  1. Top of page
  2. Abstract
  3. FRET and FRET-based probes
  4. FRET probes for phosphorylation
  5. Small GTPases
  6. Activity changes of the Rho-family of GTPases during cell division
  7. Membrane ruffling
  8. Future directions
  9. Acknowledgments
  10. References

The Ras superfamily of GTPases, including Ras, Rho, Rab, Arf and Ran, are molecular switches that perceive changes in the extracellular or intracellular environment, and transduce these changes into downstream effectors that regulate a wide spectrum of cellular functions, such as remodeling of the actin cytoskeleton, membrane trafficking, transcriptional activation and cell cycle progression. Among these, Ras is very well known for its high mutation rate in human cancers. However, accumulating evidence is beginning to reveal that other member of the Ras superfamily of GTPases are also involved in many aspects of oncogenesis. For instance, overexpression of Rho superfamily members, including Rho, Rac and Cdc42, has been reported in various human cancers.(27) GTPases cycle between GDP-bound inactive and GTP-bound active forms (Fig. 1). Upon stimulation, guanine-nucleotide exchange factors (GEF) convert the GDP-bound inactive form into the GTP-bound active form. The activity of GEF is counter-balanced by GTPase-activating proteins (GAP), which promote GTP hydrolysis. We previously developed GFP-based FRET probes for GTPases, which are collectively designated ‘Ras and interacting protein chimeric unit’ (Raichu) probes.(9) Using a similar strategy, we also established indicators for the Rho family.(28,29)

Ras and Rap: Plasma membrane or not plasma membrane, localization matters

The 36 members of the human Ras family can be further divided into Ras, Ral and Rap subfamilies according to their amino acid sequence similarities.(30) Rap1, also known as Krev1 and Smg21A, was originally isolated as an antagonist of Ras.(31) Even though Ras and Rap1 share a high sequence homology, and therefore bind to a common set of effectors, it has remained elusive why these two proteins induce such different cellular responses. The Raichu probes for Ras and Rap1 revealed that EGF activates Ras and Rap1 at distinct intracellular compartments.(9) Specifically, EGF activated Ras and Rap1 at the peripheral plasma membrane and the intracellular perinuclear region of COS-1 cells, respectively (Fig. 3). Activation of Rap1 at the perinuclear region was also observed in cAMP-stimulated cells expressing Epac, a cAMP-responsive Rap1 GEF. Similar to EGF stimulation, cAMP activated Ras at the peripheral plasma membrane in cells expressing e-GRF, a recombinant cAMP-responsive Ras GEF. As the distributions of Epac and e-GRF were very similar to each other, this does not appear to account for the discrepancy between the activation maps of Ras and Rap1. Importantly, probes with reduced sensitivity to GAP showed uniform activation in the EGF-stimulated cells. These data suggest that GAP primarily dictate the spatial regulation of the Ras family of G proteins, whereas GEF primarily determine the timing of Ras activation.(32)

image

Figure 3. Examples of Ras and Rap imaging. (A) COS-7 cells expressing Raichu-Ras were stimulated with 50 ng/mL of epidermal growth factor. FRET and differential interference contrast images were recorded at the indicated time points. (B) HeLa cells expressing Raichu-Rap1 and Epac were stimulated with 50 µM and 100 µM of forskolin and the phosphodiesterase inhibitor IBMX, respectively, to elevate intracellular cAMP. FRET images were recorded at the indicated time points.

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RalA: Convergence of signals at lamellipodia

Ral GEF and RalA play critical roles in the Ras-mediated transformation and tumorigenic growth of human cells.(33) Upon EGF stimulation, activated Ras recruits Ral GEF, thereby activating RalA. By using FRET-based probes, EGF-induced RalA activation was found to occur locally at EGF-induced nascent lamellipodia, whereas both Ras activation and Ras-dependent translocation of Ral GEF occurred more diffusely at the plasma membrane under similar conditions.(34) These observations demonstrate that the spatial regulation of RalA is conducted by a mechanism distinct from the temporal regulation conducted by Ras-dependent plasma membrane recruitment of Ral GEF. This EGF-induced RalA activation was not observed under conditions where lamellipodial protrusion was severely impaired. Conversely, EGF-induced lamellipodial protrusion was inhibited by microinjection of inhibitory mutants of RalA effectors. Thus, there is a positive feedback loop between membrane ruffling and RalA activation. Such positive feedback loops involving reorganization of the cytoskeleton can only be analyzed using probes that can retrieve information for both the activity and the morphology. Analyses with RalA have shown that FRET probes are ideal for this purpose.

RhoA, Rac and Cdc42: Regulators of cell shape

A major function of the Rho-family of GTPases is to regulate the organization of the actin cytoskeleton, and filopodia, lamellipodia and stress fibers are regarded as typical phenotypes of activated Cdc42, Rac and Rho, respectively.(35) There are several probes available for monitoring the activities of Rho GTPases in living cells (Table 1).(36,37)

Activity changes of the Rho-family of GTPases during cell division

  1. Top of page
  2. Abstract
  3. FRET and FRET-based probes
  4. FRET probes for phosphorylation
  5. Small GTPases
  6. Activity changes of the Rho-family of GTPases during cell division
  7. Membrane ruffling
  8. Future directions
  9. Acknowledgments
  10. References

The Raichu probes have been used to image the activities of the Rho-family of GTPases during cell division of HeLa cells. The activities of RhoA, Rac1 and Cdc42 were high at the plasma membrane during interphase, and then decreased rapidly upon entry into M phase. After anaphase, the RhoA activity increased at the plasma membrane, including the cleavage furrow. Rac1 activity was suppressed at the spindle midzone and only increased at the plasma membrane of the polar sides after telophase. Cdc42 activity was suppressed at the plasma membrane and high at the intracellular membrane compartments during cytokinesis.(29) These observations revealed for the first time that the activities of the Rho-family of GTPases are differentially regulated both spatially and temporally during cytokinesis.

Membrane ruffling

  1. Top of page
  2. Abstract
  3. FRET and FRET-based probes
  4. FRET probes for phosphorylation
  5. Small GTPases
  6. Activity changes of the Rho-family of GTPases during cell division
  7. Membrane ruffling
  8. Future directions
  9. Acknowledgments
  10. References

Membrane ruffling, which is observed at the front of invasive cells, is regarded as one of the hallmarks of Rac activation. However, recent data obtained with FRET probes has shown that membrane ruffling is a collaborative effort by not only Rac but also Rho and Cdc42. First, it was reported that Rac1 and Cdc42 appear to synergistically induce lamellipodia and membrane ruffles in EGF-stimulated Cos1 cells and A431 cells.(38) In these cells, both Rac1 and Cdc42 are transiently activated at the diffuse area of the plasma membrane, followed by lamellipodial protrusion and membrane ruffling. Although Rac1 activity subsides rapidly, Cdc42 activity is sustained at the lamellipodia. In agreement with this finding, a critical role of Cdc42 in these EGF-induced morphological changes has been demonstrated.(38) The critical role of Cdc42 at membrane ruffles was further supported by the finding that its effector, Neural Wiskott–Aldrich syndrome protein (N-WASP), is activated at membrane ruffles, as evaluated using an N-WASP biosensor (N-WASP-BS).(39)

RhoA has also been found to cooperate with Rac1 to induce membrane ruffles. The first clue was obtained by FRET imaging of migrating cells.(40) In contrast to previous reports, RhoA was activated not only in the contractile tail but also at the leading edge of migrating cells (Fig. 4). In agreement of the sustained RhoA activity at membrane ruffles, a dominant-negative mutant of RhoA was found to inhibit the induction of membrane ruffles. Through these studies, it has become clear that lamellipodial protrusion of the plasma membrane is the primary effect of Rac activation, whereas membrane ruffling, a backward movement of the plasma membrane, is caused by the cooperation of not only Rac but also Cdc42 and RhoA.

image

Figure 4. Rho is activated both at the trailing and leading edges of migrating cells. HeLa cells expressing Raichu-RhoA were replated onto glass-bottom dishes coated with collagen to observe their stochastic migration. FRET and differential interference contrast (DIC) images were recorded at the indicated time points. The white scale bars represent 10 µm.

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Future directions

  1. Top of page
  2. Abstract
  3. FRET and FRET-based probes
  4. FRET probes for phosphorylation
  5. Small GTPases
  6. Activity changes of the Rho-family of GTPases during cell division
  7. Membrane ruffling
  8. Future directions
  9. Acknowledgments
  10. References

To date, FRET probes have revealed abnormal lateral propagation of EGFR in cancer cells, unexpected roles of RhoA and Cdc42 at membrane ruffles, the importance of GAP for spatial regulation of Ras, and so on. The increasing number of available FRET probes encourages us to apply this FRET imaging technique in various fields of cancer research. At the same time, however, it is also necessary for us to spend more effort in optimizing both the probes and the imaging systems in order to increase the amount of information we can retrieve from living cells.

FRET probes are also applicable to the screening of regulators. For example, a rapid and simple assay for the analysis of putative GEF and GAP for small GTPases has been established with Raichu probes.(41) Recent success in FRET detection using a commercially available fluorescent activated cell sorter will further widen the applications of FRET probes.(42,43) It should be emphasized that the use of fluorescence-activated cell sorter (FACS) can significantly increase the signal-to-noise ratio at the expense of spatial information.

Another potential and promising usage of FRET probes is their application to three-dimensional cultures ex vivo, or even in vivo. For thick specimens, such as reconstituted glands or cysts in three-dimensional cultures, two-photon excitation fluorescence microscopy can be used for FRET detection. Furthermore, microscopes with needle-type lenses or connected with glass-fibers, which are now available commercially, will enable us to visualize FRET probes in living animals. However, the preparation of transgenic animals expressing FRET probes is facing some difficulties. First, the expression level of the FRET probe in animals is usually very low. This may be ascribable to the toxicity of FRET probes when expressed for a long period. Second, it is preferable to express the FRET probe in a small fraction of the cells of interest. For example, if all cells in an organ of interest expressed the probe, we could not quantitate the level of FRET in a cell due to the stray light. Thus, a method to introduce the FRET probe only in the cells of interest is required. By injecting the plasmid pRaichu-RhoA, a successful image of RhoA activity was obtained from a zebra fish embryo.(44) Thus, although there still remain many obstacles to be overcome, it will not be long before we achieve our long-standing dream of observing the activity of oncogenes in living animals.

Acknowledgments

  1. Top of page
  2. Abstract
  3. FRET and FRET-based probes
  4. FRET probes for phosphorylation
  5. Small GTPases
  6. Activity changes of the Rho-family of GTPases during cell division
  7. Membrane ruffling
  8. Future directions
  9. Acknowledgments
  10. References

This work was supported by a grant from the Ministry of Education, Science, Sports and Culture of Japan, and a grant from the Health Science Foundation, Japan. We thank members of the Matsuda laboratory for technical advice and helpful input.

References

  1. Top of page
  2. Abstract
  3. FRET and FRET-based probes
  4. FRET probes for phosphorylation
  5. Small GTPases
  6. Activity changes of the Rho-family of GTPases during cell division
  7. Membrane ruffling
  8. Future directions
  9. Acknowledgments
  10. References
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