REVIEW

Lipid rafts signaling detected by FRET-based molecularbiosensors

Jihye Seong

Received: 24 April 2015 / Accepted: 30 April 2015 / Published online: 12 May 2015

� The Korean Society for Applied Biological Chemistry 2015

Abstract Cells recognize and process various extracel-

lular signals via transmembrane receptors and membrane-

associated signaling molecules. Therefore, the precise

regulation of these signaling events at the plasma mem-

brane, which occur spatiotemporally, is necessary for

proper cellular functions such as cell proliferation, migra-

tion, and survival. The plasma membrane may contain

dynamic microdomains called lipid rafts, which are sug-

gested to be crucial for the regulation of efficient and

specific signaling pathways. However, because of the

limitation in methodologies, the specific molecular

mechanisms underlying dynamic signaling events at lipid

rafts are largely unknown. This review discusses the tra-

ditional biochemical methods for the visualization of lipid

rafts and the related signaling events at these mi-

crodomains. In addition, the review reports on fluorescence

resonance energy transfer (FRET)-based molecular

biosensors with lipid rafts targeting sequences as a pow-

erful tool for live-cell imaging of spatiotemporal signaling

events at lipid rafts. In particular, examples of dynamic

lipid rafts signaling mechanisms visualized by FRET-based

biosensors in live cells are covered in the last section.

Keywords Acylation � Biosensor � Fluorescenceresonance energy transfer � Lipid rafts � Live-cell imaging �Prenylation � Signaling

Introduction

Lipid rafts at plasma membrane

According to the fluid mosaic model proposed by Singer

and Nicolson in 1972, the plasma membrane was thought

to be a 2D phospholipid bilayer containing peripheral and

integral proteins floating freely in this bilayer (Singer and

Nicolson 1972). Since then, however, studies in a model

lipid bilayer system and in living cells have reported that

the lipid bilayer is not a static homogenous fluid. These

studies indicate that the plasma membrane contains deter-

gent-resistant microdomains of more ordered states

(Thompson and Tillack 1985; Brown and Rose 1992; Sil-

vius et al. 1996; Schroeder et al. 1998). After two decades,

Simons and colleagues postulated the ‘‘lipid rafts hy-

pothesis’’, defining that these membrane microdomains

may float around the surrounding lipid fluid like a raft

(Simons and Ikonen 1997). Lipid rafts are enriched with

sphingolipids and cholesterol, and they may be more

tightly packed because of these lipid compositions. These

distinct physicochemical properties may allow the segre-

gated distribution of membrane-associated proteins in and

outside the lipid rafts. For example, glycosylphos-

phatidylinositol (GPI)-anchored proteins and saturated long

fatty acids are shown to be strongly attached to lipid rafts

microdomains (Brown and London 1998; Simons and

Toomre 2000). The selective segregation of specific sig-

naling proteins is involved in the tight regulation of signal

transduction on the plasma membrane. For example, in T

cell antigen receptor signaling (Horejsi 2003), neurotrans-

mitter signaling (Allen et al. 2007), and membrane traf-

ficking (Hanzal-Bayer and Hancock 2007), extracellular

signals have been suggested to be sorted and transferred

inside the cells more efficiently through the clustering of

J. Seong (&)

Center for Neuro-Medicine, Brain Science Institute, Korea

Institute of Science and Technology (KIST), Hwarangno 14-gil

5, Seongbuk-gu, Seoul 136-791, Republic of Korea

e-mail: jseong@kist.re.kr

123

J Korean Soc Appl Biol Chem (2015) 58(5):629–636 Online ISSN 2234-344X

DOI 10.1007/s13765-015-0082-2 Print ISSN 1738-2203

lipid rafts. Moreover, lipid rafts are believed to be of var-

ious sizes (from 10 nm to submicrometer range) and highly

dynamic in their assembly/disassembly in response to ex-

tracellular signals. Considering that the resolution of con-

ventional microscopy is several hundred micrometers, it

has been a major challenge to clearly understand the

mechanisms underlying the involvement of these small,

heterogeneous, and highly dynamic microdomains in the

regulation of signal transduction pathways in live cells.

Visualization of lipid rafts

To understand the highly dynamic features of membrane

microdomains, numerous studies have been tried to visu-

alize lipid rafts using fluorescent lipid/protein analogs or

lipid-binding toxins/antibodies (Ishitsuka et al. 2005).

For example, fluorescein–polyethylene glycol–cholesterol,

Cy5-dimyristoyl-sn-glycero-phosphatidylethanolamine, and

GPI linked to green fluorescent protein (GFP) have been

used as fluorescent analogs of lipid rafts components:

cholesterol, saturated long fatty acids, and GPI-associated

proteins, respectively (Schutz et al. 2000; Sato et al. 2004;

Sharma et al. 2004). In addition, non-toxic recombinant

derivatives of pore-forming toxins, such as lysenin, cholera

toxin subunit B, and aerolysin, have been used for the lipid

raft markers because they specifically bind to sphin-

gomyelin, glycosphingolipid GM1, and GPI-associated

proteins, respectively (Kenworthy et al. 2000; Shakor et al.

2003; Skocaj et al. 2013).

These fluorescent analogs and probes are useful tools to

visualize lipid rafts microdomains, although the size of the

individual raft seems to be beyond the resolution of the

current conventional microscopy. Super-resolution imaging

techniques have recently begun to be applied to imaging lipid

rafts, and they include stimulated emission depletion

(STED), photoactivated localization microscopy (PALM),

and stochastic optical reconstruction microscopy (STORM)

(Simons and Gerl 2010; Owen et al. 2012; Owen and Gaus

2013). Various cutting-edge optical methods have also been

applied such as fluorescence resonance energy transfer

(FRET), fluorescence lifetime imaging (FLIM), fluorescence

recovery after photobleaching (FRAP), and fluorescence

correlation spectroscopy (FCS) (Lagerholm et al. 2005; Rao

and Mayor 2005; de Almeida et al. 2009). These optical

techniques are expected to provide more accurate informa-

tion of lipid rafts dynamics and the spatiotemporal signaling

events at these microdomains.

Biochemical methods to study lipid rafts signaling

The functions of lipid rafts in signal transduction have been

studied using several biochemical methods. First, using the

detergent-resistant property of lipid rafts, these membrane

fractions can be extracted with cold non-ionic detergent

(e.g., Triton X-100 at 4 �C), and then separated by sucrose

gradient ultracentrifugation (Simons and Ikonen 1997;

Hooper 1999). In fact, lipid rafts are also called detergent-

resistant membranes (DRMs). The distribution of signaling

proteins in separated DRMs can be measured by im-

munoblotting. However, this method has been controver-

sial because the results are largely affected by conditions of

the extraction (e.g., the types and concentrations of deter-

gents) (Hooper 1999). The proteins weakly attached to

lipid rafts may not be successfully isolated by the detergent

extraction method, and it is also difficult to capture the

dynamic distribution of signaling proteins, which occurs

during signal transduction in live cells.

Another typical method to study lipid rafts signaling is

to deplete cholesterol with methyl-b-cyclodextrin (MbCD)to measure the effect of rafts disruption (Simons and

Toomre 2000). Other approaches to disrupt the integrity of

lipid rafts have also been applied such as the inhibition of

cholesterol biosynthesis by Lovastatin, the addition of ex-

ogenous cholesterol, and the sequestration of cholesterol by

filipin or saponin (Simons and Toomre 2000). However,

these methods may promote some nonspecific artifacts, as

cholesterol is not simply the major component of lipid rafts

but is also involved in many cellular functions. In addition,

MbCD may work nonspecifically on other hydrophobic

molecules (Zidovetzki and Levitan 2007). These methods

are also limited to detecting dynamic signaling events

spatiotemporally at lipid rafts in live cells.

FRET-based molecular biosensor in lipid rafts signaling

FRET-based molecular biosensor

Fluorescent lipid/protein analogs or probes are useful tools

to investigate the distribution of proteins of interest on

membrane microdomains. However, simple fluorescent

tagging is not enough to visualize real activity of target

proteins on lipid rafts. Traditional biochemical assays also

have limitations for the detection of dynamic subcellular

protein activities in live cells. To visualize real-time activity

of signaling proteins in live cells, genetically encoded

molecular biosensors have been developed based on FRET

(Zhang et al. 2002; Aoki et al. 2013). FRET is a physical

phenomenon observed between donor and acceptor FPs at a

proximal distance within 10 nm. In addition to the proximal

distance, the emission spectrum of the donor FP should

overlap with the excitation spectrum of the effector FP to

achieve successful FRET. For example, the emission spec-

trum of cyan FP (CFP) largely overlap with the excitation

spectrum of yellow FP (YFP), thus they are widely used as a

FRET pair (Zhang et al. 2002; Aoki et al. 2013).

630 J Korean Soc Appl Biol Chem (2015) 58(5):629–636

123

Various FRET-based biosensors can be designed to vi-

sualize different signaling events (Zhang et al. 2002; Gaits

and Hahn 2003; Aoki et al. 2013). For example, molecular

biosensors detecting posttranslational modifications, such

as phosphorylation and methylation, can be designed to

include a specific substrate and its sensory domain between

a FRET pair. Some ligand-sensing biosensors contain only

a sensory domain between a FRET pair, and upon ligand

binding to the sensory domain, its conformation is altered

to cause FRET changes. Protease biosensors have a specific

substrate site for cleavage between a FRET pair. All of

these FRET biosensors are designed to induce huge con-

formational changes after specifically sensing the target

signaling events, resulting in significant changes in the

FRET level. Thus, by detecting FRET level changes, we

can monitor the dynamic signaling events in live cells. In

addition to these intra-molecular FRET biosensors, mole-

cular biosensors based on inter-molecular FRET have also

been developed to visualize the interactions/proximity be-

tween two target proteins, with each fused to either the

donor or acceptor FPs. Therefore, we can now monitor

real-time molecular interactions and dynamic signaling

events in live cells by measuring the changes in the FRET

level of these genetically encoded molecular biosensors.

Lipid rafts targeting signals

Genetically encoded FRET biosensors can be further tar-

geted to the subcellular compartments of the cell to detect

local activities of signaling molecules in live cells. Thus,

the integration of the lipid rafts targeting motif with FRET

biosensors allows the visualization of real-time signaling

events at lipid rafts. Zacharias et al. have shown that acy-

lated, but not prenylated, monomeric FPs can be parti-

tioned into membrane microdomains (Melkonian et al.

1999; Zacharias et al. 2002). When monomeric CFP or

YFP are genetically fused to the acylation sequences for

myristoylation and palmitoylation, which are from the Src

family kinase member Lyn kinase, efficient clustering be-

tween CFP and YFP is detected by FRET and this can be

completely inhibited by MbCD. In contrast, clustering of

monomeric CFP and YFP with the prenylation sequence

(i.e., the geranylgeranylation motif derived from Rho

GTPase) does not respond to MbCD treatment. In addition,

the prenylated FP is not clustered with the acylated FP,

suggesting that different lipid modifications can be used as

targeting signals to different membrane microdomains.

These results also indicate that the acylation, but not the

prenylation, motif is sufficient to target a protein at lipid

raft microdomains. Therefore, the addition of lipid

modification sequences to FRET biosensors can be applied

to the visualization of dynamic signaling events at mem-

brane microdomains in living cells (Fig. 1).

FRET-based visualization of signaling events at lipid

rafts

Src kinase

Src kinase plays important roles in many cellular functions

such as cell migration, proliferation, and survival, and is

often found to be hyper-activated in many cancers (Thomas

and Brugge 1997; Martin 2001; Mitra and Schlaepfer

2006). Src kinases interact with various downstream sig-

naling molecules, including p130CAS, paxillin, focal ad-

hesion kinase, Ras/Mitogen-activated protein kinase

(MAPK), and phosphatidylinositol 3-kinase (PI3 K)/Akt.

Therefore, the tight regulation of spatiotemporal Src ac-

tivity is required to control these multiple signaling events

accurately and more efficiently. In response to various

extracellular signals such as growth factors, Src has been

suggested to be activated near the plasma membrane

(Sandilands et al. 2004). However, whether Src resides in

lipid rafts remains controversial, most likely because of the

limitation of previous tools to investigate signaling events

in these dynamic subcellular regions.

To visualize Src activity in and outside lipid rafts, Seong

et al. (2009) constructed FRET-based Src biosensors that

target these regions (Wang et al. 2005; Seong et al. 2009).

The Src biosensor is composed of a Src substrate peptide

derived from p130CAS, a flexible linker, and the SH2

domain between enhanced CFP (ECFP) and a YFP variant

(YPet). When active Src kinase phosphorylates the sub-

strate, the phosphorylated tyrosine in the substrate binds to

the nearby SH2 domain in the biosensor. This intra-mole-

cular interaction would cause a huge conformational

change of the biosensor, and the strong FRET between

ECFP and YPet would be significantly decreased. There-

fore, by measuring changes in the FRET level, real-time

Src activation in live cells can be visualized. This biosensor

is further targeted to lipid rafts or non-raft regions with

different lipid modifications (i.e., acylation or prenylation

sequences) as discussed in the previous section (Zacharias

et al. 2002).

Surprisingly, the results utilizing these subcellular-tar-

geted FRET-based Src biosensors showed the faster and

stronger Src activation outside lipid rafts in response to

growth factors and chemical stimulations (Seong et al.

2009). The quantitative analysis of Src activation by cal-

culating kinetic factors confirmed different kinetics of Src

activations in and outside lipid rafts (Seong et al. 2009).

Further experiments have revealed that relatively slower

Src activation on lipid rafts is dependent on actin-mediated

transportation, while fast Src activation in non-raft regions

is independent of the actin cytoskeleton (Seong et al.

2009). These results suggest the existence of two distinct

Src populations that are differentially regulated by the

J Korean Soc Appl Biol Chem (2015) 58(5):629–636 631

123

cytoskeleton. These two populations may be crucial in

mediating different signaling pathways more efficiently.

For example, Src on lipid rafts regions has been shown to

be involved in PI3 K/Akt signaling, while Src at non-raft

regions regulates MAPK/extracellular signal-related kinase

(ERK) pathways (de Diesbach et al. 2008).

Focal adhesion kinase (FAK)

Cells connect to the extracellular matrix through trans-

membrane receptor integrins and many associated signal-

ing/structural molecules at focal adhesions, which are

specialized membrane structures (Huttenlocher and Hor-

witz 2011). FAK, which is a direct downstream molecule

of the integrin signaling pathway at focal adhesions, plays

key roles in cell adhesion and migration (Mitra et al. 2005;

Mitra and Schlaepfer 2006). However, whether lipid rafts

are involved in FAK functions remains unclear. It has been

of particular interests whether physicochemically defined

lipid rafts microdomains are related to functionally spe-

cialized membrane structures (i.e., focal adhesions).

To compare FAK activity in and outside lipid rafts of

plasma membrane in live cells, Seong et al. (2011) created

a FRET-based FAK biosensor with targeting signals to

lipid rafts or non-raft regions (Seong et al. 2011). The FAK

biosensor is designed to include a specific substrate con-

taining its autophosphorylation site Y397, a linker, and the

Src SH2 domain between ECFP and YPet. Activated FAK

phosphorylates the substrate, and the intra-molecular in-

teraction between the phosphorylated substrate and the

SH2 domain in the biosensor causes conformational

changes, resulting in modifications in the FRET level. For

targeting signals, the same strategies with Src biosensor

were applied (i.e., acylation sequences for lipid rafts teth-

ering and prenylation site for non-rafts targeting).

In response to growth factors and integrin signaling, the

FAK biosensor at lipid rafts shows the strong FRET re-

sponse, while at non-raft regions, a very minimal response

is displayed. These results indicate that FAK functions

mainly at lipid rafts. Thus, it is an interesting observation

given that its close partner, the Src kinase, functions both in

and outside lipid rafts and furthermore, the stronger Src

activity has been seen at non-raft regions (Seong et al.

2009). Therefore, the mode of molecular interactions may

be different at subcellular compartments. The mechanisms

underlying the interaction between FAK and Src in re-

sponse to extracellular stimulations have also been inves-

tigated utilizing lipid rafts-targeted FAK and Src

biosensors in live cells. Results have demonstrated that in

response to growth factor stimulation, Src kinase activity is

required for FAK activation, whereas FAK regulates Src

activation in the integrin signaling pathway (Seong et al.

2011). These findings suggest that the mode of molecular

interactions at subcellular regions can be further altered in

response to different physiological stimulations.

PI3 K/Akt signaling

The PI3 K/Akt signaling pathway is crucial in cell survival,

motility, and proliferation, and is also involved in the de-

velopment of cancer (Brazil and Hemmings 2001; Man-

ning and Cantley 2007; Wong et al. 2010). After growth

factor stimulation, activated PI3 K produces phos-

phatidylinositol (3,4,5) triphosphate (PIP3), which is

Fig. 1 Targeting strategies of

FRET-based biosensor to

membrane microdomains.

FRET-based FAK biosensor is

targeted to lipid rafts or non-raft

regions with different lipid

modification sequences. The

lipid rafts-targeted FAK

biosensor reports higher ECFP/

FRET emission ratios indicating

stronger FAK activity at lipid

rafts in live cells

632 J Korean Soc Appl Biol Chem (2015) 58(5):629–636

123

negatively regulated by phosphatase and tensin homolog

deleted on chromosome 10 (PTEN). PIP3 then recruits Akt

to the plasma membrane, which is then phosphorylated by

phosphoinositide-dependent kinase 1 (PDK1), and is sub-

sequently fully activated by multiple proteins including

mammalian target of rapamycin complex (mTORC). Lipid

rafts have been suggested to be involved in PI3 K/Akt

signaling, but the regulatory mechanisms of dynamic Akt

activation at membrane microdomains are unclear.

Gao and Zhang (2008) constructed a FRET-based Akt

biosensor consisting of a substrate from FOXO1 and its

binding domain forkhead-associated domain (FHA1) be-

tween the FRET pair Cerulean and cpV E172 (Gao and

Zhang 2008). This Akt biosensor has been further targeted

in or outside lipid rafts by different lipid modifications. In

response to platelet-derived growth factors (PDGF) or in-

sulin-like growth factor-1 (IGF-1), these Akt biosensors

have reported the activation of Akt, both in and outside

lipid rafts. However, Akt activation in lipid rafts has been

shown to be faster and stronger than that of outside lipid

rafts.

In addition, when MbCD, which disrupts lipid rafts in-

tegrity, was treated, the PDGF-stimulated Akt activity at

lipid rafts, but not at non-raft regions, was decreased,

suggesting two independent Akt populations at membrane

microdomains. For IGF-1 stimulation, however, MbCDinhibits Akt activation both in and outside lipid rafts, and

interestingly the Akt activation at non-raft regions is

completely blocked. These findings suggest that IGF-1-

induced Akt activation at non-raft regions may be depen-

dent on the one at lipid rafts. Thus, stimulation by different

growth factors may result in different mechanisms of Akt

activation at membrane microdomains.

Gao et al. (2011) further investigated the regulatory

mechanisms of the compartmentalized activities of PI3 K/

Akt at membrane microdomains (Gao et al. 2011). This

study investigated at which membrane microdomains the

Akt-related signaling molecules are localized to regulate

the spatiotemporal activation of Akt. Therefore, they con-

structed a FRET-based biosensor to detect the activity of

PDK1, an upstream signaling molecule for Akt activation.

The PDK1 biosensor is composed of the full-length PDK1

molecule flanked by a FRET pair, ECFP and Citrine, and is

designed to directly detect the conformational change of

PDK1 by FRET measurement. The biosensor is targeted in

or outside lipid rafts with lipid modification sequences.

Results from these FRET-based PDK1 biosensors have

indicated that the activation of PDK1 is exclusively ob-

served in lipid rafts, which may explain the higher Akt

activity at this region. In contrast, PTEN, a negative

regulator for Akt activity, has been shown to be mainly

located at non-raft regions, and the altered distribution of

PTEN inhibits proper Akt activation. These also support

the regulatory mechanism of the higher Akt activity at lipid

rafts. Therefore, the precise spatiotemporal activities of

related signaling molecules are crucial for the proper sig-

naling of PI3 K/Akt.

Cyclic adenosine monophosphate (cAMP)/protein

kinase A (PKA)

Cyclic adenosine monophosphate is a second messenger

converted from adenosine triphosphate by adenylyl cyclase

(AC), which is activated by the G-protein-coupled receptor

(GPCR) on the plasma membrane (Chin et al. 2002).

Protein kinase A is a traditional target of the second mes-

senger cAMP and is involved in many cellular functions

such as gene expression and proliferation (Tasken and

Aandahl 2004). A-kinase anchoring protein (AKAP) (e.g.,

Ezrin) is known to localize PKA to appropriate subcellular

regions, which is crucial for PKA functions. However, how

PKA activity is regulated in and outside lipid rafts of the

plasma membrane is unclear.

To visualize PKA activity, Depry et al. (2011) devel-

oped the FRET-based PKA biosensor, which is composed

of the PKA substrate and forkhead-associated domain

(FHA1) between Cerulean and cpVenus (Depry et al.

2011). This FRET biosensor has also been further targeted

to membrane microdomains by lipid modifications. Results

from this study show that the basal PKA activity at resting

state is higher at lipid rafts than non-raft regions, and this

activity is dependent on PKA localization and lipid rafts

integrity. Interestingly, the disruption of lipid rafts by

MbCD induces a stronger and prolonged PKA response,

and this finding suggests that lipid rafts may be involved in

the proper desensitization of the b-adrenergic receptor

(bAR)-induced cAMP response (Depry et al. 2011). In fact,

bAR is shown to be localized at lipid rafts (Depry et al.

2011). It is possible that the disruption of lipid rafts may

cause the slower desensitization of this GPCR, because

lipid rafts are also suggested to be involved in endocytosis

and membrane trafficking (Hanzal-Bayer and Hancock

2007).

cAMP/exchange proteins directly activated by cAMP

(Epac)

The production of cAMP also regulates other signaling

molecules such as Epac (Bos 2003). Epac is a recently

found guanine exchange factor (GEF) that works on the

small GTPase, Rap1. It plays important roles in insulin

secretion, vascular permeability, and cardiac contraction.

The activation of Epac results from its conformational

changes upon cAMP binding. Therefore, spatiotemporal

distribution of cAMP is likely to control the cAMP-induced

Epac signaling pathway at subcellular levels.

J Korean Soc Appl Biol Chem (2015) 58(5):629–636 633

123

To visualize the distribution of cAMP at membrane

microdomains in live cells, DiPilato et al. (2004) designed

a FRET-based cAMP biosensor, which is composed of full-

length Epac1 between ECFP and citrine (DiPilato et al.

2004). Findings from this biosensor system have shown

that increased production of cAMP directly binds to Epac1

in the biosensor, causing the conformational change of the

biosensor and the subsequent changes in the FRET level.

This biosensor has been further targeted to lipid rafts by

acylation sequences (DiPilato and Zhang 2009). Upon ac-

tivation of the GPCR, bAR, this biosensor shows local andtransient increases in cAMP production in live cells. In-

terestingly, this cAMP activity is enhanced by the treat-

ment of MbCD, suggesting that lipid rafts may be involved

in the desensitization of bAR-induced cAMP signaling.

The prostaglandin E receptor is another GPCR located in

non-raft regions, and with the treatment of MbCD has been

shown not to enhance the prostaglandin E receptor-induced

cAMP response (DiPilato and Zhang 2009). Therefore, the

negative role of lipid rafts in cAMP signaling may be de-

pendent on the type and/or distribution of GPCR.

Agarwal et al. (2014) also constructed a FRET-based

cAMP biosensor. This biosensor is composed of Epac2

between EYFP and ECFP, and is targeted to lipid rafts or

non-raft regions of the plasma membrane by lipid modifi-

cation sequences (Agarwal et al. 2014). Results from this

group have revealed that basal cAMP activity is sig-

nificantly higher in non-raft regions at plasma membrane,

because the biosensors tethered at non-raft regions show

more sensitive FRET response to the inhibition of AC, a

positive regulator of cAMP. The FRET response of the

biosensors at non-raft regions also show a less sensitive

response to the inhibition of phosphodiesterase (PDE),

which is involved in the cAMP metabolism. Therefore,

different levels of cAMP production in and outside lipid

rafts may be due to the compartmentalized regulation of

the positive/negative regulatory molecules such as AC and

PDE.

Rac1

Rac1, a member of Rho GTPases, regulates cell adhesion

and motility through cytoskeletal reorganization (Jaffe and

Hall 2005). GDP-bound Rac1 is inactive and associated

with GDP-dissociation inhibitor (GDI) in the cytosol. Rac1

gets activated when GTP exchange factors (GEFs) replaces

GDP to GTP, and the dissociation of GDI exposes its

membrane targeting signals, leading Rac1 to plasma

membrane. Rac1-bound GTP can be hydrolyzed to GDP by

GTPase-activating protein (GAP), inactivating Rac1. The

precise location of Rac1 is required for the correct inter-

action with downstream signaling molecules. A previous

study has suggested that Rac1 may function in lipid raft

microdomains (del Pozo et al. 2004).

To investigate the distribution of Rac1 at membrane

microdomains of live cells, Moissoglu et al. (2014) de-

signed a FRET-based system consisting of GFP-tagged

Rac1 and mCherry fluorescence proteins fused to acyla-

tion/prenylation sequences (Moissoglu et al. 2014). In this

system, the acylated or prenylated mCherry FP is readily

located in or outside lipid rafts. When the activated Rac1

translocates to each microdomain of the plasma mem-

brane, FRET would be observed between Rac1-attached

GFP and mCherry that is tethered in or outside lipid rafts.

The FRET results have shown that Rac1 is located both in

lipid rafts and non-raft regions on the plasma membrane.

Further studies with a Rac GEF (Tiam) and a Rac GAP

(b2-chimaerin) have indicated that GEF activates both

Fig. 2 Summary of compartmentalized signaling pathways detected by FRET

634 J Korean Soc Appl Biol Chem (2015) 58(5):629–636

123

Rac1 populations in and outside lipid rafts, while GAP

preferentially inactivates the Rac1 population in non-raft

regions. Thus, the selective function of the negative

regulator in the membrane compartments may contribute

to the preferential Rac1 activity that occurs at lipid rafts.

These FRET imaging system findings provide an insight

into the molecular mechanism of the differential Rac1

activity at membrane microdomains in live cells.

Conclusion and perspectives

In this review, several lipid rafts signaling pathways visu-

alized by FRET biosensors were discussed (Summarized in

Fig. 2). FRET-based molecular biosensors have been used

as powerful tools to visualize real-time signaling events in

live cells with high spatiotemporal resolutions. Therefore,

the integration of these biosensors with subcellular target-

ing signals (i.e., acylation/prenylation sequences) success-

fully provides a unique strategy to precisely monitor

dynamic signaling events and to study the molecular

regulatory mechanisms at these membrane microdomains.

FRET biosensors with lipid modification sequences pro-

vide a much deeper understanding of the distribution,

functional activities, and regulatory mechanisms of crucial

signaling molecules (e.g., Src, FAK, PI3 K/Akt, cAMP/

PKA, and Rac1) at membrane microdomains. These sig-

naling pathways are also related to diseases such as can-

cers. Therefore, subcellular-targeted FRET biosensors can

contribute to the development of new diagnostic tools, drug

screening methods, and therapeutic strategies. Recent ad-

vances in super-resolution optical techniques such as STED

microscopy, PALM, and STORM are promising to visu-

alize nanometer-sized individual lipid raft and the local

signaling pathways more accurately. The combination of

these new optical techniques with FRET-based biosensors

will provide a more in-depth understanding of lipid rafts

signaling pathways with super spatiotemporal resolutions.

Acknowledgments This work is supported by the Korea Institute of

Science and Technology (KIST-2E25240, 2E25473).

Conflict Interests The author declares no competing interests.

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