Download GFP is the way to glow: bioimaging of the plant endomembrane

Journal of Microscopy, Vol. 214, Pt 2 May 2004, pp. 138–158
Received 22 October 2003; accepted 22 December 2003
INVITED REVIEW
Blackwell Publishing, Ltd.
GFP is the way to glow: bioimaging of the plant
endomembrane system
F. B R A N D I Z Z I *, S . L . I R O N S , J. J O H A N S E N , A . K O T Z E R &
U. N E U M A N N
Research School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy Lane,
Oxford OX3 0BP, U.K.
*Department of Biology, University of Saskatchewan, Saskatoon, Canada
Summary
It is less than a decade that the green fluorescent protein (GFP)
and its spectral variants have changed the approach to studying
the dynamics of the plant secretory pathway. GFP technology
has in fact shed new light on secretory events by allowing
bioimaging in vivo right to the heart of a plant cell. This review
highlights exciting discoveries and the most recent developments in the understanding of morphology and dynamics of
the plant secretory pathway achieved with the application of
fluorescent proteins.
Received 22 October 2003; accepted 22 December 2003
1. The plant secretory pathway
The endoplasmic reticulum (ER), the Golgi apparatus, the
vacuole and the plasma membrane constitute the major components of the plant secretory system (Fig. 1). In general, the
ER appears as the central factory dedicated to the synthesis of
proteins, lipids and glycans. ER proteins that are not retained
in the lumen of the ER or in the ER membrane are processed
and folded prior to their transport to the Golgi apparatus
(reviewed in Vitale & Denecke, 1999). In the Golgi, proteins
are further processed (reviewed in Dupree & Sherrier, 1998)
and then, if not resident in the Golgi, are sorted either to the
storage (for example in pea cotyledons via dense vesicles; Hohl
et al., 1996) or to the lytic vacuole via prevacuolar compartments (PVCs; Matsuoka & Neuhaus, 1999; Vitale & Raikhel,
1999) or multivesicular bodies (MVBs; Robinson et al., 1998).
Alternatively, proteins can be secreted at the plasma membrane
via vesicular transport (reviewed in Satiat-Jeunemaitre &
Hawes, 1993; Battey et al., 1999).
Recycling of membranes via an endocytic pathway contributes to the maintenance of the overall lipid and protein distribution between plasma membrane and secretory organelles
(reviewed in Hawes et al., 1995; in Battey et al., 1999; Crooks
Correspondence to: Dr Federica Brandizzi. Fax: +1 306 966 4461; e-mail:
[email protected]
et al., 1999). This would result in a movement of vesicles from
the plasma membrane to the partially coated reticulum (PCR),
and from this to either the Golgi or the MVBs and then to the
vacuole (Fowke et al., 1991; see also Hawes et al., 1999).
Our understanding of the dynamics of the secretory pathway in plant cells is often impaired by the difficulty of working
with such cells. The dynamics of the secretory pathway have
been exponentially clarified in recent years, thanks to new
tools becoming available, such as green fluorescent protein
(GFP) technology, that allow the study of dynamic cellular
processes in vivo. GFP is a self-assembling fluorescent protein
with a molecular mass of approximately 27 kDa. It emits green
fluorescence upon excitation with UV or blue light. As specific
secretory proteins or signals can be fused to GFP, usually
without altering their targeting, it is a useful alternative to
conventional dyes previously used to investigate endomembrane compartments in vivo. Moreover, GFP itself is not able
to cross most membranes, with the exception of the nuclear
membrane through the nuclear pores (Grebenok et al., 1997).
These are some of the most valuable features of GFP and its
spectral derivatives, especially considering that many of the
vital dyes available for studies in animal cells cannot be used
for investigating the biology of plant cells. In this respect,
GFP has played a central role in uncovering dynamic endomembrane events in vivo (for a review see also Brandizzi et al.,
2002b).
1.1. Introduction of GFP into the plant endomembrane studies
GFP was first identified in the jellyfish Aequoria victoria
(Shimomura et al., 1962) and purified and crystallized
later by Morise et al. (1974). About 20 years later, Prasher et al.
(1992) cloned the gfp gene. In 1994, it was first demonstrated
that the expression of the gene in heterologous organisms
generates fluorescence upon UV or blue light excitation
(Chalfie et al., 1994; Inouye & Tsuji, 1994).
In plants, the first reports of successful expression of unmodified wild-type gfp using cytoplasmic RNA viruses such
© 2004 The Royal Microscopical Society
G F P I S T H E WAY TO G L OW
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closer to the plant consensus and removal of the cryptic plant
intron (Haseloff et al., 1997) allowed a wider exploitation of
GFP as a fluorescent reporter for plant cell bioimaging.
2. Plant endomembranes highlighted by GFP
The introduction of GFP into the plant secretory pathway
mirrors a natural pathway of a secretory protein. GFP has in
fact first illuminated the ER and nuclear envelope (Boevink
et al., 1996) and then it has made its way to organelles downstream of the secretory pathway. In this section we highlight
the way that GFP has revealed the secretory pathway and
we focus on recent discoveries linked to GFP targeting and
expression in different secretory organelles.
2.1. The nuclear envelope
Fig. 1. Major organelles of the plant endomembrane system. (a)
Schematic diagram of the endomembrane organelles of the plant
secretory pathway. Material exiting the ER can be transported to the
protein storage vacuole (1) or passed to the Golgi. At the Golgi, the main
transport pathways are believed to lead to three different destinations: the
protein storage vacuole (PSV) via so-called dense vesicles (2), the plasma
membrane (PM)/apoplast via secretory vesicles (3) or the lytic vacuole
via clathrin-coated vesicles (4). On route to the lytic vacuole or the PM,
material passes through the prevacuolar compartment (PVC), which
may also be the sorting station for material entering from the PM via
endocytosis (5). (b) Transmission electron micrograph of Arabidopsis
suspension cultured cells prepared by high-pressure freezing and freeze
substitution, showing different organelles of the plant secretory pathway.
CW, cell wall (apoplast); ER, endoplasmic reticulum; G, Golgi apparatus;
LV, lytic vacuole; NE, nuclear envelope; PM, plasma membrane; T,
tonoplast. Scale bar = 500 nm.
as potato virus X and tobacco mosaic virus date back to 1995
(Baulcombe et al., 1995; Heinlein et al., 1995).
Owing to the presence of a cryptic intron recognizable by
plant mRNA splicing machinery, GFP technology had a difficult
birth in plant cell studies. Alteration of the codon usage to be
© 2004 The Royal Microscopical Society, Journal of Microscopy, 214, 138–158
The dynamics of the plant nuclear envelope (NE) are relatively
uncharacterized in comparison with many other membranes
of the plant endomembrane system. This is mostly due to a
lack of in vivo markers specific for the plant NE (Meier, 2001;
Irons et al., 2003). In the search for an NE-specific marker
that works in plants, the N-terminal domain of the human
lamin B receptor (LBR; Ellenberg et al., 1997) was fused to
GFP5 (Haseloff et al., 1997). Stable expression of the LBR-GFP5
fusion protein in tobacco plants and BY-2 suspension cells
gives specific labelling of the NE (Irons et al., 2003; Fig. 2a,b).
Immobile fluorescent punctate structures were observed in
interphase (Fig. 2a) and dividing cells (arrowhead, Fig. 2c green
image) and are likely to be membrane stacks arising as a result
of protein over-expression. Similar membrane stacks have
been observed in yeast cells expressing LBR (Smith & Blobel,
1994). During mitosis, the LBR-GFP5 fluorescence locates to
tubular membrane structures, co-localizing with a lumenal
ER marker constructed with the yellow fluorescent protein
(YFP) (Irons et al., 2003; Fig. 2c), showing that the protein
moves to the ER membranes during division as in animal cells.
The continuity of the NE and ER ensures that a secretory
protein, which labels the ER, labels the NE as well. This is illustrated by GFP targeted to the ER [signal peptide–GFP (sp-GFP);
Boevink et al., 1996], and of fluorescent proteins targeted
to and retained in the ER (e.g. sp-GFP-KDEL, Boevink et al.,
1996; sp-YFP-HDEL, calnexin-GFP, Irons et al., 2003). In the
case of soluble proteins, this is due to the continuity of the
NE and ER lumen. For membrane proteins, the nuclear pores
allow lateral diffusion of small proteins, with labelling of the
inner NE as well as the outer NE and ER. It is therefore interesting that in interphase the fluorescence of LBR-GFP5 localizes
exclusively to the NE, suggesting that specific retention signals
retain the protein in the NE.
A GFP fusion of the plant protein, matrix attachment region
binding filament-like protein (MFP1) associated factor 1 (MAF1;
Gindullis et al., 1999) has been located to the NE area in plant
cells and shows association with the nuclear matrix and
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F. B R A N D I Z Z I E T A L .
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G F P I S T H E WAY TO G L OW
nuclear periphery as well as structures further from the NE. A
GFP fusion of RanGAP, a protein mediating nuclear import via
activation of Ran GTPases, labels speckle-like structures at the
NE and the cytoplasm (Pay et al., 2002). Early papers using an
MFP1-GFP fusion showed labelling at the nuclear periphery
and through the cytoplasm (Gindullis & Meier, 1999). The
structures observed at the nuclear periphery in tobacco
suspension cells expressing MFP1-GFP have recently been
shown to be nucleoids of proplastids close to the NE ( Jeong
et al., 2003). This finding illustrates the need for high-resolution
microscopy beyond the light microscopy level for determination of protein location.
A plant protein that exclusively labels the NE remains elusive.
2.2. ER morphology and dynamics
The first published GFP fusion labelling of the plant ER was
achieved using the potato virus X expression system (Boevink
et al., 1996). In tobacco leaf cells GFP was targeted to the
lumen of the ER by fusing the coding sequence of the signal
peptide of the potato storage protein, patatin, to the N-terminus
of wild-type GFP. Substitution of the patatin signal peptide
with the sporamin signal peptide (PVX.sp-GFP) resulted in
brighter fluorescence, presumably reflecting a higher level of
translocation of GFP into the ER (Boevink et al., 1999).
It is known that soluble proteins remain in the ER due to a
C-terminal tetrapeptide (K/HDEL) retrieval signal encoded
in their primary amino acid sequence (Denecke et al., 1992).
Proteins escaping the ER and carrying the K/HDEL signal are
retrieved by a receptor that recognizes the signal (Semenza
et al., 1990; Lee et al., 1993). The construct PVX.sp-GFP without
a K/HDEL retrieval signal was secreted and accumulated in
the apoplast in older infections. When the KDEL tetrapeptide
was spliced to the carboxyl terminus of GFP, GFP was retained
in the ER (Boevink et al., 1996, 1999). With this marker, ER
appears as a relatively immobile but locally remodelling polygonal tubular network with variously shaped cisternae at the
cell cortex, with other more mobile tubules streaming through
the cytoplasm.
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A similar ER pattern has been identified in studies using
GFP spliced to ER native proteins and targeting motifs. These
include the soluble proteins such as sp-GFP-HDEL (Fig. 2d,f )
and colour variants (sp-YFP-HDEL; Irons et al., 2003), domains
of calreticulin (Brandizzi et al., 2003), BiP (Lee et al., 2002)
and the membrane protein calnexin (Irons et al., 2003), the dilysine motif GFP-tm-KKXX (Benghezal et al., 2000), and a
calmodulin-regulated Ca2+-ATPase (ACA2p; Hong et al., 1999).
ER structures highlighted by GFP are similar to those seen with
ER probes such as DiOC6 and hexyl rhodamine B, confirming
that GFP is correctly targeted to the ER (Quader, 1990).
sp-GFP-HDEL stably expressed in Arabidopsis highlights
a characteristic cortical tubular network in leaf cells
(Matsushima et al., 2002, 2003). In root cells, the ER showed
mobile ‘ER bodies’ as well as a fluorescent tubular network
(Hawes et al., 2001). The ER bodies are 0.5 µm wide and 5–
10 µm in length and are brightly fluorescent. Their presence
has also been demonstrated in wild-type plants by immunofluorescence, hence demonstrating that their presence is
not likely to be an artefact of a GFP fusion expression. A form of
β-glucosidase with an ER retention signal appears to be
the main constituent of the ER bodies (Matsushima et al.,
2003). The formation of ER bodies in leaves was observed
upon wounding (Matsushima et al., 2003), suggesting that
ER bodies may have a role in wounding response. For instance,
β-glucosidases may hydrolyse β-linked oligosaccharides (e.g.
cellobiose) involved in plant defence, hormone activation and
cell wall breakdown.
The movement of ER bodies within the ER lumen raises
questions about how proteins move within the ER. This opens
a new area of plant endomembrane study in which fluorescent
protein technology will be a key tool.
As well as providing a wealth of information on the dynamics
of the endomembrane system in ‘normal’ living plant cells,
fluorescent probes can visualize morphological changes occurring
in plant membranes during a pathogen attack. For example, the
effect of tobacco mosaic virus infection on the ER morphology
was followed with an ER-targeted GFP (Reichel & Beachy,
1998; Gillespie et al., 2002) and Takemoto et al. (2003) have
Fig. 2. Organelles of the early endomembrane system highlighted by fluorescent fusion proteins. (a) Confocal laser scanning (CLS) micrograph of an
interphase tobacco BY-2 cell expressing LBR-GFP5 (Irons et al., 2003). The fusion protein locates to the nuclear envelope. (b) Low-magnification CLS
micrograph of tobacco epidermal leaf cells stably expressing LBR-GFP5. Nuclear envelopes are highlighted by the fusion protein (green image,
arrowheads); nuclear contents are labelled with ethidium bromide (merged image, arrows). (c) CLS micrograph of BY-2 cells stably expressing LBR-GFP5
(green image) and a soluble ER-marker, spYFP-HDEL (red image; Irons et al., 2003), at different stages of division. The upper two cells are at the end of
mitosis, with the NE and phragmoplast fully formed. The lower cell is in anaphase, with the membranes of the mitotic apparatus forming tubular
structures (green image, arrows) as the chromosomes move towards the cell poles. Apart from the punctate structures, the GFP and YFP fusion proteins
label the same structures in the dividing cell (merged image), suggesting that the LBR-GFP5 protein locates to the ER during mitosis. (d) CLS micrograph of
a tobacco leaf protoplast transiently expressing sp-GFP-HDEL (Brandizzi et al., 2003). The cortical ER highlighted by this construct appears as a loose
network of tubules. (e) CLS micrograph of Golgi stacks in a BY-2 cell labelled with GFP-tagged mannosidase I (GmMan1-GFP; Nebenführ et al., 1999). The
image was created by merging 15 deconvolved images that were taken 0.5 µm apart at the centre of a BY-2 cell near the top of the nucleus. Micrograph
courtesy of A. Nebenführ. (f ) CLS micrograph of the cortical cytoplasm of a leaf epidermal cell transiently expressing both spGFP-HDEL (green image;
Batoko et al., 2000) and the Golgi marker ST-YFP (red image; Brandizzi et al., 2002a). Golgi stacks are in close proximity to the tubules of the ER network
(merged image). N, nucleus. Scale bars: a,d–f = 10 µm; b,c = 20 µm.
© 2004 The Royal Microscopical Society, Journal of Microscopy, 214, 138–158
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F. B R A N D I Z Z I E T A L .
investigated the effect of oomycete pathogen attack in
Arabidopsis on ER and Golgi membranes as well as microtubules
and microfilaments labelled with fluorescent proteins.
2.3. Golgi imaging in vivo
Studies on the dynamics of the plant Golgi are relatively young
in comparison with their mammalian counterparts, due to
the absence of genuine plant Golgi enzymes to be fused to GFP
and used as in vivo markers.
The first report on the visualization of the Golgi apparatus
in living plant cells with GFP dates back to 1998 (Boevink
et al., 1998), whereas the mammalian counterpart dates back
to 2 years earlier (Cole et al., 1996).
The Golgi apparatus in living plant cells has been first visualized with two different fluorescent constructs (Boevink et al.,
1998). GFP was fused (1) to the putative Arabidopsis K/HDEL
receptor, ERD2, homologous to a yeast and mammalian protein
that recycles soluble proteins with a K/HDEL tetrapeptide
back to the ER, and (2) to the transmembrane domain (TMD)
of a rat sialyl-transferase, a mammalian Golgi glycosylation
enzyme (ST-GFP). Both GFP-chimeras were found to localize
to fluorescent punctuate mobile structures, revealed to be
Golgi stacks by immunogold electron microscopy with a GFPantibody (Boevink et al., 1998). As native sialyl-transferases
have not been reported in plants, the targeting of the ST-GFP
to the plant Golgi suggests a common mechanism in plants
and mammals for targeting and retention of transferases in
Golgi membranes.
Subsequently, identification of novel probes has produced
more reports on the dynamics of the Golgi apparatus. For
example, Nebenführ et al. (1999) reported on Golgi targeting
in BY-2 cells of a soybean α-1,2 mannosidase I, the first enzyme
involved in the N-linked oligosaccharide pathway, fused to
GFP (Fig. 2e). The N-terminal TMD, including the flanking
amino acids from a plant N-acetylglucosaminyltransferase
I, has also been fused to GFP and was found to be targeted
to the Golgi in Nicotiana benthamiana plants with a tobacco
mosaic virus-mediated expression (Essl et al., 1999). The same
group reported later on Golgi targeting by an Arabidopsis β1,2-xylosyltransferase, a glycosyltransferase that is unique
to plants and some invertebrates (Dirnberger et al., 2002).
An N-glycan GFP-tagged xylosyltransferase has been found
associated with Golgi stacks of BY-2 cells preferentially located
in medial cisternae (Follet-Gueye et al., 2003). Dupree’s group
reported on the expression of a GONST1-YFP fusion that
localizes to small punctate structures in onion cells, interpreted
as being the Golgi apparatus (Baldwin et al., 2001).
The organization of the plant Golgi apparatus, at light microscope resolution, appears rather different from the mammalian Golgi. The plant Golgi is scattered in the cell as small
stacks in the cortical cytoplasm and within trans-vacuolar
strands of cytoplasm (Boevink et al., 1998; Wee et al., 1998;
Essl et al., 1999; Nebenführ et al., 1999).
In leaves, individual stacks of the Golgi apparatus appear
closely associated with the cortical ER network (Boevink et al.,
1998; Saint-Jore et al., 2002; Fig. 2f ). This is particularly
evident when imaging the ERD2-GFP fusion. In mammalian
cells, the homologue of the ERD2 protein has been identified
on the intermediate compartment between the ER and the
Golgi and on the Golgi itself (Griffiths et al., 1994). In plant
cells, the location of the receptor was, until recently, unknown.
Confocal microscopy of ERD2-GFP in Nicotiana leaves showed
the chimeric protein to be located to Golgi bodies and the
ER network. This close association of ER and Golgi has led to
the hypothesis, suggested by a few reports based on electron
microscopy, that the two organelles may have connections
and function as a secretory unit ( Juniper et al., 1982; Harris
& Oparka, 1983; Brandizzi et al., 2002c). However, the
nature and persistence of these connections have yet to be
established.
Movement of the Golgi over the ER is actin-dependent.
Rhodamine–phalloidin staining of tobacco leaf epidermal cells
expressing ERD2-GFP has revealed a close juxtaposition of the
ER and the Golgi to the actin cytoskeleton (Boevink et al.,
1998). More recently, the dynamic association of the Golgi
with actin has been shown in tobacco epidermal cells expressing fluorescent constructs for Golgi and actin cytoskeleton,
the latter being highlighted with an actin-binding region of a
mouse talin fused to YFP (Brandizzi et al., 2002c). Depolymerization of the actin network with drugs results in an inhibition
of Golgi movement and clustering of the fluorescent bodies
on small islands of lamellar ER within the cortical tubular
network (Boevink et al., 1998; Brandizzi et al., 2002c).
The dynamics of the plant Golgi over the ER network opens
up questions on the modality of ER-to-Golgi protein and
membrane transport and on the distribution of ER export sites.
It was postulated that ER-to-Golgi protein transport might
be linked to the discontinuity of Golgi movement (Nebenführ
et al., 1999). As Golgi stacks move, arrest and regain movement
after time intervals, cargo collection would occur during
Golgi arrest, possibly after transient detachment from actin
(‘stop-and-go model’). Alternatively, the Golgi movement in
plant cells would allow the Golgi continually to collect vesicles
budding from the ER (‘vacuum cleaner model’; Boevink et al.,
1998).
These models, which are as yet unsupported by any experimental evidence, do not exclude the possibility that the ER
and Golgi may behave as one dynamic system, either through
direct membrane continuities or through continuous vesicle
or tubule formation/fusion reactions (Brandizzi et al., 2002b;
reviewed in Neumann et al., 2003). In this view, ER-to-Golgi
transport may occur during Golgi movement.
2.4. Imaging the vacuole with GFP
The plant vacuole is a multifunctional organelle, which is
essential for growth and development. Unlike yeast vacuoles
© 2004 The Royal Microscopical Society, Journal of Microscopy, 214, 138–158
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or mammalian lysosomes, the plant vacuole often serves as
both storage and lytic compartment. In addition, the plant
vacuole acts as a pool for metabolic intermediates such
as organic acids and is involved in the regulation of turgor
pressure, detoxification of the cytosol and the maintenance
of cytosolic pH.
Both lytic and storage vacuoles may coexist in the same cell
(Paris et al., 1997). Lytic and protein storage vacuoles can be
distinguished by the presence of different specific aquaporins,
termed tonoplast intrinsic proteins (TIPs). The location
of several TIPs has been established through immunocytochemistry. α-TIP has been mainly assigned to protein storage
vacuoles ( Johnson et al., 1989). γ-TIP has been found in lytic
or degradative vacuoles (Paris et al., 1996; Reisen et al., 2003),
whereas δ-TIP has been found in pigment-containing vacuoles
( Jauh et al., 1998). The discovery of GFP and the formation of
TIP-GFP fusion proteins has allowed in vivo study of vacuolar
functions and dynamics.
In a random GFP::cDNA fusion screen, a δ-TIP fused to
GFP was demonstrated to be a vacuolar membrane protein in
Arabidopsis hypocotyl epidermal cells (Cutler et al., 2000). The
identity of this vacuolar membrane is at present unknown.
Reisen et al. (2003) recently reported on another aquaporin
isolated from cauliflower, BobTIP26-1, that has previously
been demonstrated to be an active aquaporin in Xenopus leavis
oocytes. BobTIP26-1 is a protein located in the tonoplast
(Barrieu et al., 1998) and is a specific marker for acidic, lytic
vacuoles. Reisen et al. (2003) fused the GFP sequence downstream of the BobTIP26-1 coding region and observed a
complex tonoplast labelling in Nicotiana tabacum cv. Wisconsin
38 suspension cells. Fluorescent patches were detected on the
tonoplast, which might suggest that the aquaporins are not
evenly distributed within the vacuolar membrane. In tobacco
leaf epidermal cells, BobTIP26-1-GFP evenly labels the
tonoplast (Fig. 3a). When BobTIP26-1-GFP was transiently
expressed in protoplasts isolated from tobacco suspension
cultures, the fusion protein was not detected on the tonoplast,
but in a fluorescent network resembling the ER. Reisen et al.
(2003) suggest that this might be due to BobTIP26-1-GFP en
route to the vacuolar membrane.
A study using γ-TIP-GFP to investigate the dynamics of the
vacuole reported that the tonoplast protein was targeted to
the vacuolar membranes in young Arabidopsis cotyledon cells
(Saito et al., 2002). In addition, it was found that the marker
protein also labelled some spherical structures (bulbs) that
were often observed within the lumen of vacuoles. These bulbs
were connected with the vacuolar membrane and were
moving around within or along the outline of the membrane.
In addition, larger bulbs were also shown to become elongated
and form tube-like structures. The intensity of the fluorescence
emitted from the γ-TIP-GFP-labelled bulbs was reported to be
several-fold higher than the adjacent vacuolar membrane.
This could be explained by the observation using transmission
electron microscopy that the bulbs consist of a double
© 2004 The Royal Microscopical Society, Journal of Microscopy, 214, 138–158
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Fig. 3. Fluorescent protein fusions highlights vacuole and plasma
membrane. (a) Low-magnification CLS micrograph of tobacco leaf
epidermal cells transiently expressing a GFP fusion to the tonoplast
intrinsic protein BobTIP26-1 (Reisen et al., 2003). The fusion protein
locates to the tonoplast of central lytic vacuole, thereby visualizing
the transvacuolar strands (arrowheads). The insert shows cytoplasmic
strands leading to the centre of a cell where the nucleus is located in a
cytoplasmic pocket in the middle of the vacuole. BobTIP26-1-GFP
construct courtesy of N. Leborgne-Castel. (b) CLS micrograph of a tobacco
leaf epidermal cell transiently expressing NtAQP1-YFP (A. Kotzer and
C. Hawes, unpublished data). This YFP fusion to the plasma membrane
intrinsic protein NtAQP1 exclusively labels the plasma membrane,
thereby outlining the cell shape. Note that in comparison with (a), no
cytoplasmic strands are highlighted. NtAQP1 construct (Siefritz et al.,
2001) courtesy of F. Siefritz. (c,d) Projection of CLS optical sections
through the roots of Arabidopsis cv. Wassylevskaja plantlets (Flückiger
et al., 2003), at an early stage of root hair formation, stably expressing
two different vacuolar GFP markers. (c) GFP-Chi (Di Sansebastiano et al.,
1998, 2001), a marker of the protein storage vacuole, accumulates
in small compartments. (d) Aleu-GFP (Di Sansebastiano et al., 2001), a
marker of the lytic vacuole, accumulates in the large central vacuole.
Micrographs courtesy of G. P. Di Sansebastiano. N, nucleus. Scale bars:
a–d = 25 µm; insert in a = 10 µm.
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F. B R A N D I Z Z I E T A L .
membrane. In the same study, another vacuolar membrane
protein, AtRab75c, was fused to GFP and expressed in Arabidopsis. The construct gave good vacuolar localization but,
interestingly, did not label any structures in the lumen of the
vacuole as seen for the γ-TIP-GFP (Saito et al., 2002). However,
although not fluorescent, bulbs are present in cotyledon
epidermal cells in GFP-AtRab75c-transformed Arabidopsis
as confirmed by electron microscopy. γ-TIP is an integral
membrane protein with six TMDs and probably forms a dimer
or tetramer like other aquaporins, whereas Rab75c belongs to
the Rab GTPase family and is attached to the membrane via
a C-terminal prenylation. Saito et al. (2002) proposed that the
difference in membrane attachment might be the reason for
their segregation into different structures. Alternatively, an
unknown mechanism might exclude GFP-AtRab75c from
the bulb region (Saito et al., 2002).
Uemura et al. (2002) used a vacuolar syntaxin-related
molecule, AtVam3/SYP22, fused to GFP, to study the vacuolar
dynamics in Arabidopsis. This protein has previously been
demonstrated by immunoelectron microscopy to locate to
the vacuolar membrane (Sato et al., 1997). The GFP construct
revealed complicated vacuolar architectures in various cell
types with internal membranous structures inside the central
vacuole in root epidermal cells. This was also observed in
mesophyll cell protoplasts. By three-dimensional modelling
and reconstruction from confocal images, the internal
membrane structures were organized as sheets and cylindrical structures. These structures were also highly dynamic and
were constantly remodelled. Interestingly, the fluorescence
intensity of this GFP construct, GFP-Vam3, on the cylindrical
structures was twice as strong as the outer-vacuolar membrane
(Saito et al., 2002). Uemura et al. (2002) concluded that this
intravacuolar membrane has a double membrane structure.
The streaming motion could be blocked with an actindepolymerizing agent, cytochalasin D, whereas microtubuledisrupting drugs had no effect on the movement of the sheet-like
structures. These data suggest that the dynamic movement
of the internal vacuolar membrane is actin-dependent, but
independent of microtubules. Uemura et al. (2002) concluded
that these sheet-like structures are possible transvacuolar
strands involved in cytoplasmic streaming.
They postulated two models for the formation of intravacuolar structures. In the ‘autophagic’ model, a small vacuole is
engulfed by a large vacuole, and remains enclosed in the
vacuolar lumen. The second model suggests that the vacuolar
membrane could be invaginated into the vacuolar lumen to
form sheet-like structures, which then become rounded.
A potassium channel protein (AtKCO1) fused to GFP locates
to the vacuolar membrane in transgenic tobacco BY-2 cells
(Czempinski et al., 2002). Confocal images showed that the
signal of the AtKCO1-GFP was present as a single fluorescent
line surrounding the large central vacuole. In addition,
individual cells contained multiple small vacuoles labelled
by GFP fluorescence. In addition, in some cells containing a single
large vacuole, a network of connecting strands most similar to
transvacuolar strands exhibited GFP fluorescence. Likewise,
an iron-regulated ABC transporter, IDI7, from barley roots fused
to GFP localized to the vacuolar membrane when transiently
expressed in suspension-cultured tobacco cells (Yamaguchi
et al., 2002). IDI7 is believed to be a tonoplast ABC transporter
and is thought to be involved in the export of certain substrates from the cytosol to the vacuoles. Finally, Thomine et al.
(2003) expressed a metal transporter of the NRAMP family,
AtNRAMP3, as a GFP fusion in onion cells and in Arabidopsis
protoplasts located to the vacuolar membrane.
The existence of more than one type of vacuole in some cells
implies that, in these cases, different mechanisms for tonoplast protein sorting coexist. It has been established for several
vacuolar membrane proteins that both the TMD and the
cytosolic tail are important for sorting to the tonoplast (Jiang
& Rogers, 1998). A C-terminal portion of α-TIP composed of
the sixth and last TMDs and the 17-amino-acid cytosolic tail
contains sufficient information for sorting to the tonoplast
(Hofte & Chrispeels, 1992).
Unlike the increasing amount of data collected using GFP
as a tag for soluble vacuolar proteins, little has been done to
investigate the actual transport pathway of vacuolar membrane proteins using GFP. Mitsuhashi et al. (2000) designed αTIP-GFP and γ-TIP-GFP constructs and both were localized to
a vacuolar membrane in BY-2 cells. Mitsuhashi et al. (2000)
concluded that both TIP-GFP fusions are targeted to the same
vacuole in BY-2 cells. This is in contrast to what Okita & Rogers
(1996) and Paris et al. (1996) reported, in which the two TIPs
localize to different vacuoles in the same cell in barley roots
and in maturing pea cotyledons. It will be interesting to reinforce these findings on the dynamics of coexisting vacuoles
with experiments based on co-expression of different TIP
chimeras with available GFP spectral variants. In the experiments of Mitsuhashi et al. (2000), GFP fluorescence was also
observed in the ER network, suggesting that both α-TIP-GFP
and γ-TIP-GFP are transported through the ER and then to the
vacuolar membranes.
2.5. Plasma membrane illuminated by GFP
The plasma membrane (PM) encloses the cell and is the prime
barrier between the cytosol and the extracellular environment.
The PM is a dynamic, fluid structure constantly changing
in composition and organization. Fusions of various plasma
membrane proteins to GFP have been used to label the PM, but
to date GFP has shed little light on the underlying mechanics
and dynamics of the PM.
GFP fusion proteins targeted to the PM have been constructed including artificial markers BP22-GFP and TM23GFP (Brandizzi et al., 2002a). BP22-GFP was designed by
adding three hydrophobic residues (LAL) to the TMD of the
pea vacuolar sorting receptor BP80 (Paris et al., 1997). This
addition resulted in fluorescence accumulation on the plasma
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G F P I S T H E WAY TO G L OW
membrane and in small punctate structures (Brandizzi et al.,
2002a). Like BP22-GFP, TM23-GFP, a fusion of the TMD of a
human lysosomal protein (LAMP1) to GFP, was targeted to the
PM (Brandizzi et al., 2002a).
In addition, the location of several endogenous/native
plasma membrane proteins has been demonstrated by the
use of GFP. PM proteins fused to GFP include the Rac-type
small GTPase ZeRAC2 (Nakanomyo et al., 2002), the tobacco
PM aquaporin NtAQP1 (Siefritz et al., 2001; A. Kotzer and
C. Hawes, unpublished observations; Fig. 3b), the ammonium
transporter LjAMT2;1 (Simon-Rosin et al., 2003) and the acylCoA binding protein ACBP1 (Li & Chye, 2003).
2.6. GFP and the apoplast
A GFP construct with the ER signal peptide but lacking the
retrieval signal (sp-GFP) has proven to be a useful marker for
following secretion in vivo (Boevink et al., 1999). Tobacco leaves
infected with this construct show very little fluorescence in
the ER, probably due to continuous secretion. Treating the
same leaves with cold shock or brefeldin A (BFA) caused an
inhibition of the GFP secretion, resulting in the build-up of
fluorescence in the ER (Boevink et al., 1999). These results were
similar to those reported for mammalian cell systems (Presley
et al., 1997). As will be discussed in detail below, transport of a
secreted form of GFP to the apoplast can also be inhibited by
co-expression of dominant-negative mutants of proteins
regulating secretion.
145
3. GFP sheds light on protein retention and targeting to the
endoplasmic reticulum
Mechanisms by which the cell exports secretory proteins but
retains resident ER proteins are the subject of intense investigation. Brandizzi et al. (2002a) investigated the influence of
the TMD length on the destination of membrane proteins
along the secretory pathway. The length of the TMD has been
found to be important for protein progression along the secretory pathway. In this respect, a GFP fusion to a TMD stretch
of 17 amino acids (aa) has been located to the NE and ER in
tobacco. Extension of this stretch by three further aa resulted
in the location to the Golgi, and a further elongation of the
TMD by another three aa resulted in the GFP fusion incorporation into the PM. This study highlights an analogous influence
of the TMD length on the destination of membrane proteins in
plants as found in yeast (Munro, 1995).
Irons et al. (2003) reported a TMD and cytosolic tail GFP
fusion of an Arabidopsis calnexin, which contains a dilysine
motif. The chimera locates to the NE and ER and it is highly
mobile in these membranes as revealed by fluorescence
recovery after photobleaching (FRAP) technique (Fig. 4a).
This construct may be retained in the ER by virtue of specific
signals present in the cytosolic tail of calnexin. Benghezal et al.
(2000) have shown that the C-terminal dilysine motif confers
ER localization to type I membrane proteins in plants. Cf-9 (a
resistance gene, conferring resistance against the fungal pathogen Cladosporium fulvum) is a type I membrane protein that
Fig. 4. Protein movement into organelles of the endomembrane system as shown by FRAP. (a) Photobleaching of a nuclear envelope (arrow) of a Nicotiana
tabacum leaf epidermal cell transformed with a calnexin-GFP construct (Irons et al., 2003). The images are prebleach (0.0 s), bleach event (7.4 s), partial
recovery (13.3 s) and full recovery (34.9 s). The frame indicates the area of photobleaching. (b) Photobleaching of a Golgi body in an N. tabacum leaf
epidermal cell expressing ERD2-GFP and treated with cytochalasin D as described in Brandizzi et al. (2002c). The images are prebleach (0.0 s), bleach
event (7.1 s), partial recovery (141.5 s) and full recovery (300.8 s). The frame indicates the area of photobleaching. Note that the recovery of fluorescence
in the nuclear envelope is much faster than the in the Golgi apparatus. Scale bars: a = 10 µm; b = 5 µm.
© 2004 The Royal Microscopical Society, Journal of Microscopy, 214, 138–158
146
F. B R A N D I Z Z I E T A L .
has a large extracytosolic leucine repeat domain, a single TMD
and a cytosolic tail carrying a C-terminal dilysine KKXX motif.
To study the ER retrieval mechanism motif of Cf-9, fusions of
GFP to the TMD and cytosolic tail of Cf-9, creating GFP-KKXX,
were generated (Benghezal et al., 2000). The location of GFPKKXX in the ER was verified by the co-localization of BiP and
GFP using double immunofluorescent labelling. Amino acid
substitution of KKXX to NNKK caused the secretion of most
of the fusion protein from the ER, proteolytic release of GFP
from its membrane anchor and loss of fluorescent properties.
The extent to which the GFP-NNKK fusion was retained in
the ER differed between different systems. In epidermal cells,
the GFP-NNKK still showed an ER pattern but the intensity of
fluorescence was much weaker than GFP-KKXX-expressing
cells. In BY-2 cells, this was not the case as the GFP-NNKK
showed no ER fluorescence.
Soluble reticuloplasmins are targeted to the ER by virtue of a
signal peptide. Signal peptides, like those of sporamin, chitinase
and patatin, were proven sufficient for targeting GFP to the
ER lumen (Boevink et al., 1996; Batoko et al., 2000; Brandizzi
et al., 2003).
The ER retention of soluble reticuloplasmins in plants
depends on the carboxyl-terminal signal K/HDEL (Denecke
et al., 1992). As mentioned above, ER-targeted GFP fused to K/
HDEL is retained in the ER (Boevink et al., 1996; Brandizzi et al.,
2003). However, when overexpressed, ER-targeted GFP-HDEL
(sp-GFP-HDEL) may reach the vacuole (Brandizzi et al., 2003).
This is consistent with the retrieval mechanism of reticuloplasmins being under the control of a saturable pathway. A
transmembrane receptor ERD2 has been identified in Arabidopsis
(Lee et al., 1993) and is likely to function in the same way as
the yeast and mammalian homologues in recognizing the ER
retention motif and to trigger retrograde transport of soluble
ER-resident proteins that have escaped. The localization of ERD2GFP on the Golgi and ER reflects the fact that the receptor is
likely to shuttle between the two organelles. However, the activity of the ERD2-GFP fusion has not yet been tested in plants.
Malfolded secretory proteins are retained in the ER until
they reach a final low-energy state conformation. How retention is accomplished is still a matter of debate. It is tempting
to suggest that retention may be operated by lumenal chaperones or alternatively that malfolding may prevent proteins
from being recognized by the ER-export machinery. If proteins
do not reach a proper conformation, they are generally
degraded. In plants, little is known about the mechanisms and
proteolytic systems that mediate protein quality control in the
secretory system. It is known that malfolded lumenal proteins
can be targeted to the vacuole via a Golgi-mediated pathway
(Pueyo et al., 1995; Coleman et al., 1996). Other malfolded
proteins are degraded via a BFA and heat-shock-independent
pathway in an unidentified cellular compartment (Pedrazzini
et al., 1997). The existence of a protein retrograde transport
and degradation pathway in plant cells has been suggested
by the analysis of the location of the ricin catalytic A subunit
in tobacco protoplasts (Frigerio et al., 1998). Brandizzi et al.
(2003) have identified a GFP fusion that is detected in the
cytosol and the nucleoplasm of tobacco cells in spite of the
presence of an N-terminal secretory signal peptide. In contrast
to secreted GFP, the fusion protein is retained in the cells where
it is degraded slowly, at a higher rate than the ER retained
GFP-HDEL. The fusion protein could not be stabilized by inhibitors of transport or the cytosolic proteasome. Brandizzi et al.
(2003) suggested that the fusion protein is disposed of from
the ER via a retrograde translocation back to the cytosol.
Moreover, accumulation in the nucleoplasm was shown to be
microtubule-dependent, unlike the well-documented diffusion
into the nucleoplasm of cytosolically expressed GFP. The apparent active transport of the GFP fusion into the nucleoplasm
may indicate a yet undiscovered feature of the ER-associated
protein degradation pathway and may explain the insensitivity
of degradation to proteasome inhibitors (Brandizzi et al., 2003).
4. GFP highlights protein movement towards cell
compartments
4.1. GFP shows that ER-to-Golgi transport does not require
cytoskeleton
GFP is an important tool for investigating ER-to-Golgi protein
transport in vivo. Saint-Jore et al. (2002) used BFA to establish
the involvement of the cytoskeleton in the transport to and from
the Golgi. In mammalian cells, BFA induces redistribution
of Golgi membrane proteins to the ER (Lippincott-Schwartz
et al., 1990; Robineau et al., 2000). After treatment with BFA,
fluorescent Golgi markers expressed in tobacco epidermal leaf
cells and in BY-2 cells are redistributed to the ER (Boevink et al.,
1998; Ritzenthaler et al., 2002; Saint-Jore et al., 2002). The
BFA effect is reversible upon drug washout (Satiat-Jeunemaitre
& Hawes, 1993; Satiat-Jeunemaitre et al., 1996; Saint-Jore
et al., 2002). Saint-Jore et al. (2002) proved that in the absence
of an intact actin and microtubule cytoskeleton, the BFA effect
and recovery were still taking place, indicating that movement
of proteins to and from the Golgi is cytoskeleton-independent.
These data were confirmed by Brandizzi et al. (2002c), who
used FRAP (Fig. 4b) experiments to investigate the requirements of an intact cytoskeleton for fluorescence recovery
into Golgi. FRAP experiments were performed on leaf tissues
expressing either ERD2-GFP or ST-GFP, and treated with cytoskeleton inhibitors. When the FRAP protocol was applied
to cells treated with actin depolymerizing agents, in order to
stop Golgi movement, the recovery of fluorescence in bleached
Golgi stacks occurred within 5 min of the bleaching event
(Fig. 4b). This shows that actin is not required for ER-to-Golgi
trafficking even though it is required for Golgi movement. As
Golgi still moved in the absence of microtubules, the concomitant use of an actin depolymerizing agent was required to
perform FRAP on immobile Golgi. Again, Golgi bodies re-gained
fluorescence within 5 min after the bleaching, indicating that
© 2004 The Royal Microscopical Society, Journal of Microscopy, 214, 138–158
G F P I S T H E WAY TO G L OW
neither actin filaments nor microtubules are necessary for
ER-to-Golgi protein transport. These results highlight a major
difference between the plant and mammalian Golgi, as it
appears that in plants, the cytoskeleton is not essential for
protein transport to the Golgi. Moreover, although the Golgi
marker ERD2-GFP was found distributed uniformly over the
Golgi and ST-GFP towards the trans-most cisternae (Boevink
et al., 1998), no substantial differences in the recovery times
upon photobleaching were recorded. In addition, the recovery
time of these two proteins was similar to the recovery time of a
glycosylated form of ST-GFP (Batoko et al., 2000).
4.1.1. Out of the Golgi: transport to the vacuole. A considerable
effort has been made over the last few years to identify and
characterize differentiated and specialized vacuoles in higher
plants. Recently, GFP has become a popular tool to unravel the
mechanisms of transport of soluble and membrane proteins
to the different vacuoles and to investigate the dynamics of the
plant vacuolar system. However, the acidic nature of certain
types of vacuoles has made it difficult to explore the accumulation of certain soluble markers, such as aleurain-GFP, for the
rapid degradation/quenching of GFP (Di Sansebastiano et al.,
1998). The low pH may be a determinant feature of vacuoles
with a primarily lytic function, and storage vacuoles need not
be acidic. Recently, using Arabidopsis cv. Columbia seedlings,
it has been shown that light-dependent hydrolytic enzymes
influence the stability of GFP and account for the reduction
of GFP fluorescence in lytic vacuoles (Tamura et al., 2003).
However, Di Sansebastiano et al. (2004) have not shown a
similar light-dependent effect on fluorescence levels of GFP
in lytic vacuoles when using another cultivar (Arabidopsis cv.
Wassylevskaja), suggesting that different ecotypes within the
same species may account for differences in the visibility of
GFP in the vacuolar system (Di Sansebastiano et al., 2004).
Soluble proteins reach the vacuole via information stored
in their C-terminus, N-terminus or in an internal fragment of
their propeptides. In the absence of such signalling peptides,
vacuolar proteins are secreted. Cleavable vacuolar sorting
signals include the N-terminal propeptide (NTPP) present in
sweet potato sporamin and the C-terminal propeptide (CTPP)
present in barley lectin (reviewed in Matsuoka & Neuhaus,
1999; Vitale & Raikhel, 1999). The N-terminal NTPP signals
contain an NPIR consensus amino acid motif that is necessary
for targeting sporamin to the vacuole (Matsuoka et al., 1995).
A consensus sequence has not yet been identified in the
C-terminus, but rather a common structural motif seems to
serve as a sorting signal in the CTPPs (reviewed in Matsuoka
& Neuhaus, 1999). CTPP- and NTPP-dependent pathways
are likely to be distinct.
Soluble proteins destined for the lytic vacuoles, such as
barley aleurain, are transported through the Golgi complex.
From the Golgi apparatus, the proteins are transported to a
prevacuolar compartment (PVC) in clathrin-coated vesicles
(CCVs; Paris et al., 1996; Vitale & Raikhel, 1999). A type I trans© 2004 The Royal Microscopical Society, Journal of Microscopy, 214, 138–158
147
membrane glycoprotein, BP80, probably acts as a receptor for
protein delivery to the lytic vacuole (Matsuoka et al., 1997;
Paris et al., 1997).
Lumenal vacuolar proteins might reach the protein storage
vacuole in three different ways. The first route is through
autophagy of ER-released storage protein aggregates (Levanony
et al., 1992). In the second route, precursor-accumulating
(PAC) vesicles may be released from the ER and mediate
transport of storage proteins directly to the storage vacuole as
reported in pumpkin and castor bean seeds (Hara-Nishimura
et al., 1998). The third route includes transport via the Golgi
complex where storage vacuole proteins are sorted in dense
vesicles from other proteins in the secretory pathway as
demonstrated for barley lectin (Hohl et al., 1996). Each transport pathway implies a first sorting event in the ER where
some proteins are transported to the Golgi complex whereas
others would aggregate and be released in the cytosol or be
packed into PAC vesicles for transport to the storage vacuole.
Di Sansebastiano et al. (2001) described protein trafficking
to two different types of vacuoles in protoplasts by the fusion
of GFP to two vacuolar sorting determinants (VSD). One GFP,
mGFP5, was targeted to a pH-neutral vacuole by the C-terminal
vacuolar sorting signal (VSS) of tobacco chitinase A (see also
Fig. 3c). The construct was demonstrated to accumulate in an
organelle different from the organelle accumulating the stain
neutral red, which on proteonation is trapped within acidic
compartments. It was concluded that the fusion to the Cterminus VSS of chitinase A is sufficient to send a secreted GFP
to the storage vacuole. A construct made by fusing the NTPP
of barley aleurain to mGFP5 was localized to compartments
smaller than 2 µm and no fluorescence was detectable in the
main lytic vacuole. The authors suggested that the lack of
fluorescence in the main lytic vacuole might be due to the
rapid degradation of GFP in acidic environments and that
the observed fluorescent smaller compartments might be
intermediates in the transport pathway of aleurain (see also
Fig. 3d). Fusion of the pro-peptide of aleurain to a brighter and
more stable form of GFP, mGFP6, targeted the construct to the
small intermediate compartments in addition to the central
vacuole. Neutral red was demonstrated to accumulate in
the same compartment as mGFP6-aleurain, indicating a lytic
vacuolar location for aleurain. This also clearly indicates the
sensitivity of mGFP5 to an acidic environment.
Recently, Flückiger et al. (2003) made stable Arabidopsis
transformants with soluble vacuolar GFP constructs. It was
evident that the distribution of the fluorescence of GFP-Chi
and GFP-Aleu was different in elongated root cells. The GFPChi was located in the ER, small vacuoles and occasionally in
the large central vacuole, whereas GFP-Aleu accumulated in
the large lytic vacuole (Fig. 3c,d). In addition, the organization
of the vacuolar system was investigated in different tissues of
Arabidopsis cv. Wassylevskaja plantlets. In contrast to the
location of GFP-Chi in elongated root cells, GFP-Chi was found
in root apex cells to accumulate in the ER and in different
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F. B R A N D I Z Z I E T A L .
smaller vacuolar compartments. These results highlight the
differences in protein targeting due to tissue specificity and
cellular differentiation (Flückiger et al., 2003).
The location of another soluble vacuolar protein, 2S
albumin, a major storage protein, was investigated using GFP
(Mitsuhashi et al., 2000). A construct, consisting of a signal
peptide (sp) fused to GFP followed by the C-terminal 18-aa
peptide (2SC), sp-GFP-2SC, was found to be able to bind to
PV72, a putative sorting receptor for the storage vacuole.
Fluorescence was observed in a large compartment as well as
in the ER network and in the nuclear envelope. The large compartment was also stainable with BCECF, which is fluorescent
in vegetative vacuoles in BY-2 cells. Some small particles were
also labelled by sp-GFP-2SC. Mitsuhashi et al. (2000) postulated that because these particles show a similar size as the
Golgi complex, the C-terminal peptide of 2S albumin functions
as a targeting signal for vacuoles via the Golgi complex.
Mitsuhashi et al. (2000) also designed a GFP fusion of sp-GFP
and the C-terminal region of pumpkin PV72 that included
the TMD and the cytosolic tail. This construct located to small
particles in 3-day-old BY-2 cells. The authors concluded that
these particles were consistent with fluorescent Golgi complexes and that the C-terminal part of PV72 is responsible for
the presence of the receptor in the Golgi complex. An observation on these experiments is that no co-localization with a
Golgi marker was performed, and that these ‘small particles’
might also correspond to PAC vesicles that Hara-Nishimura
et al. (1998) previously demonstrated PV72 to locate to. In
10-day-old callus cells, the fluorescence of sp-GFP-PV72 was
observed within the vacuoles and the construct was demonstrated to be proteolytically cleaved by immunoblot analysis.
Mitsuhashi et al. (2000) suggest that like pea BP-80, the
vacuolar sorting receptor for the lytic vacuole, PV72, might
cycle between the Golgi complex and a prevacuolar compartment of the storage vacuole.
5. GFP technology helps to locate regulatory small GTPases
and to shed light on their function
In eukaryotic cells, the model that transport vesicles mediate
protein transport between the various compartments of the
endomembrane system is generally accepted. The formation,
transport and fusion with a target membrane of these vesicular
shuttles are orchestrated by a plethora of proteins. Among
these regulatory proteins, Ras-related small GTPases play
an important role (Vernoud et al., 2003). Whereas Arf and
Sar GTPases are predominantly involved in the formation of
vesicles and cargo packaging (Chavrier & Goud, 1999), Rab
proteins are mainly thought to act as key regulators of the
fusion of vesicles with their appropriate target membrane
(Zerial & McBride, 2001).
GFP technology has increased our understanding of the
function of small GTPases in trafficking events between
compartments of the plant endomembrane system (Figs 5–8).
Fig. 5. Effect of wild-type and mutant forms of Sar1p on the transport
of Golgi and vacuolar fluorescent markers. (a–c) CLS micrographs of
Arabidopsis protoplasts expressing ERD2-GFP, a Golgi/ER marker, alone
(a) and alongside AtSar1 (b) or AtSar1 H74L (5c). When expressed alone
(a) or with wild-type AtSar1 (b), GFP fluorescence is mainly in the form of
punctate structures corresponding to individual Golgi stacks, whereas
co-expression of AtSar1 H74L leads to fluorescence accumulation of
AtErd2-GFP in an ER-like pattern (c). (d–f ) CLS micrographs of Arabidopsis
protoplasts expressing a GFP fusion to the vacuolar soluble protein
sporamin alone (d) and alongside AtSar1 (e) or AtSar1 H74L (f). When
expressed alone (d) or with wild-type AtSar1 (e), sporamin-GFP locates to
the lumen of the central vacuole and to additional punctate structures,
whereas when co-expressed with AtSar1 H74L, sporamin-GFP fluorescence
accumulates in the ER (f ). Scale bars = 10 µm. (a) to (f ) are from Takeuchi
et al. (2000) (figs 3a,c,e, 7a,c,e). Copyright Blackwell Publishing, Oxford,
U.K., and reprinted with permission.
Two major GFP-based approaches have been adopted. The
first consists of expressing GFP fusions of small GTPases in
order to identify their subcellular distribution, which can be
indicative of the organelles involved in the transport step(s)
regulated by that particular gene product (Ueda et al., 2001;
Cheung et al., 2002; Inaba et al., 2002; Bolte et al., 2004). The
second approach is based on different GFP markers either of
one specific endomembrane organelle or of a whole transport
pathway (Andreeva et al., 2000; Batoko et al., 2000; Saint-Jore
et al., 2002; Sohn et al., 2003). In co-expression experiments,
the influence of small GTPases on the intracellular distribution/transport of a given GFP marker can then be studied with
the confocal laser scanning microscope. In this type of study,
defined point mutations predicted to disrupt GTP binding and
hydrolysis activity of small GTPases (resulting in constitutively
active or inactive mutant forms) are often introduced in their
coding sequence. The influence of the wild-type and the different mutant forms on the intracellular distribution/transport
of GFP markers can then be compared. Finally, in a combination of both approaches, dual colour imaging experiments are
performed in which fluorescent protein-tagged small GTPases
(i.e. GFP) are co-expressed with fluorescent markers based on
a spectral derivative [i.e. YFP, and the cyan fluorescent protein
© 2004 The Royal Microscopical Society, Journal of Microscopy, 214, 138–158
G F P I S T H E WAY TO G L OW
Fig. 6. Effect of wild-type and mutant forms of Arf1 and Arf3 on the
transport of Golgi and plasma membrane GFP markers. GFP fluorescence
in green, chlorophyll autofluorescence in red. (a–c) CLS micrographs of
Arabidopsis protoplasts transiently expressing the Golgi marker ST-GFP
alongside Arf1 (a) or Arf1[T31N] (b) or Arf3[T31N] (c). When expressed
with wild-type Arf1 (a) or Arf3[T31N] (c), GFP fluorescence is in the form
of punctate structures corresponding to individual Golgi stacks, whereas
co-expression of Arf1[T31N] leads to a diffuse GFP pattern (b). (d–g) CLS
micrographs of Arabidopsis protoplasts transiently expressing the plasma
membrane marker H+-ATPase-GFP alone (d) and alongside Arf1 (e) or
Arf1[T31N] (f,g). When expressed alone (d) or with wild-type Arf1 (e),
H+-ATPase-GFP is exclusively located at the plasma membrane (arrows).
Co-expression with Arf1[T31N] leads to fluorescence accumulation of
the plasma membrane marker in punctate structures and aggregates (f,g,
arrowheads). CH, chloroplasts. Scale bars: d–e = 20 µm. (a)–(c) are from
Lee et al. (2002) (fig. 2A,a,b,c). (d) to (g) are from Lee et al. (2002)
(fig. 6A,a,b,c-3,c-2). Copyright the American Society of Plant Biologists
and reprinted with permission.
(CFP)]. This last method offers the possibility to correlate
unequivocally, in a cell population, the phenotype of individual
cells to the effect of GTPases.
5.1. Regulatory proteins of ER-Golgi/Golgi-ER transport steps
5.1.1. Sar1p. In mammalian and yeast cells, protein transport
from the ER to the Golgi is mediated by COPII-vesicles. Cargo
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149
packaging and vesicle formation at this level requires initial
binding to the ER membrane of the Sar1p GTPase (reviewed by
Barlowe, 2002). Two tobacco and Arabidopsis Sar1p homologues were shown to be able to complement the lethal yeast
∆sar1 mutant (Takeuchi et al., 1998). Co-expression of one of
two mutant forms of the tobacco Sar1p impaired in guanine
nucleotide interactions (T34N and H74L) with either a
secreted soluble or a Golgi membrane GFP marker led to an
accumulation of fluorescence in the ER of Nicotiana clevelandii
leaf epidermal cells (Andreeva et al., 2000). Fluorescence also
accumulated in the ER, though to a lesser extent, when the
GFP markers were co-expressed with a third mutant form of
tobacco Sar1p (N129I) and with an entire wild-type ORF in
anti-sense orientation (Andreeva et al., 2000). Similar results
were obtained by Takeuchi et al. (2000), in which one of two
Golgi GFP markers and a wild-type or mutant form (H74L) of
Arabidopsis Sar1p (AtSar1) were encoded in the same T-DNA.
The Golgi markers relocated from the Golgi to the ER in tobacco
BY-2 cells as well as in Arabidopsis suspension protoplasts
when simultaneously expressed with AtSar1 H74L (Takeuchi
et al., 2000; Fig. 5a–c). In addition, AtSar1 H74L, in contrast to
the wild-type protein, had a drastic effect on the intracellular
distribution of a soluble GFP marker targeted to the lytic vacuole
(sporamin-GFP). In Arabidopsis suspension protoplasts, fluorescence distribution of sporamin-GFP changed from a predominantly vacuolar location to an ER-like network pattern
(Takeuchi et al., 2000; Fig. 5d–f ). A biochemical approach
confirmed that Sar1p is necessary for transport of proteins to
the Golgi in tobacco leaf protoplasts (Phillipson et al., 2001).
5.1.2. Arf1, Arf3. Arf1 GTPases are involved in vesicle budding steps by recruiting COPI coatomer components as well
as clathrin coats. In mammalian cells, COPI vesicles mediate
retrograde protein transport between the Golgi cisternae and
from ER-Golgi-intermediate compartments back to the ER;
their involvement in anterograde protein transport is still
debated (Spang, 2002). In plant cells, Arf1 has been located
to the Golgi by immunocytochemical means both by light
microscopy in BY-2 cells (Ritzenthaler et al., 2002) and in
maize root tip cells (Couchy et al., 2003), as well as by electron
microscopy in maize and Arabidopsis root tip cells (Pimpl et al.,
2000). In a GFP-based study, Arabidopsis Arf1-GFP (AtArf1-GFP)
was shown to accumulate in BFA-sensitive, Golgi-reminiscent
punctate fluorescent structures in both Arabidopsis protoplasts
and BY-2 cells (Takeuchi et al., 2002). The effect of different
forms of AtArf1 on various fluorescent markers (three Golgi
markers, one vacuolar marker) was evaluated principally
in tobacco BY-2 cells using an approach similar to the one
described above for Sar1p in which the marker protein and the
untagged small GTPase are expressed from the same T-DNA
(Takeuchi et al., 2000). Expression of the GTP-locked AtArf1
Q71L or the GDP-locked AtArf1 T31N resulted in the relocation of ERD2-GFP fluorescence from the Golgi to the ER, whereas
two other fluorescent Golgi markers were not affected or at
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F. B R A N D I Z Z I E T A L .
least did not relocate to the ER. In addition, the transport
of sporamin-GFP to the lytic vacuole was inhibited with the
marker accumulating in the ER when AtArf1 Q71L was
present. The authors suggested that Arf1 is primarily required
for retrograde Golgi-to-ER trafficking and that disruption of
Arf1 activity leads indirectly to perturbation of ER-to-Golgi
trafficking (Takeuchi et al., 2002).
Functional differences between the two Arabidopsis isoforms
Arf1 and Arf3 were described by Lee et al. (2002) in experiments
performed in Arabidopsis leaf protoplasts in which either the
wild-type or GDP-binding dominant-negative mutants of Arf1
and Arf3 (Arf1[T31N] and Arf3[T31N]) were co-transfected
alongside one or two cellular markers tagged by either GFP or
the red fluorescent protein (RFP). In the presence of Arf1[T31N]
but not of Arf1, Arf3 and Arf3[T31N], the Golgi marker
ST-GFP relocated to the ER, which was found to display
profound morphological changes (Fig. 6a–c). Arf1[T31N] and
Arf3[T31N] also had a different effect on the trafficking
of H+-ATPase-GFP. Although the presence of Arf1 and Arf3
wild-type as well as of Arf3[T31N] did not affect the targeting
of H+-ATPase:GFP to the plasma membrane (Fig. 6d,e), H+ATPase:GFP fluorescence was in the form of punctate stains
or aggregates and not at the plasma membrane or in the ER
when co-expressed with Arf1[T31N] (Fig. 6f,g). Interestingly,
BFA caused a similar change in distribution of H+-ATPase:GFP,
suggesting that Arf1 may act via a BFA-sensitive factor as in
yeast and mammalian cells (Lee et al., 2002).
These reports clearly show that Arf1 mutants do not always
lead to a relocation into the ER of markers of different cellular
compartments (Golgi, vacuole, plasma membrane) although
all of these markers are transported from the ER to the Golgi
(AtArf1 Q71L even had different effects on markers of the same
organelle, the Golgi). This indicates that inhibition of transport
by Arf1 mutants cannot be easily explained either by a general
blockage of anterograde ER-to-Golgi transport or by a blockage
of retrograde Golgi-to-ER transport. In this respect it is interesting to note that biochemical data indicate that Arf1 might
not only influence transport steps of soluble cargo to the plasma
membrane but also have an influence on the BP80-mediated
transport of soluble cargo to the vacuole (Pimpl et al., 2003).
5.1.3. Rab1 and Rab2 homologues. Whereas Sar1p and Arf1
linked to vesicle formation and recruitment of vesicle coat
proteins, other small GTPases act predominantly at the level of
vesicle fusion with the target membrane. One example for a
small GTPase apparently involved at this particular transport
level of anterograde ER-to-Golgi protein trafficking is Rab1,
controlling tethering and fusion events at the Golgi level
(Lupashin & Waters, 1997; Moyer et al., 2001). Batoko et al.
(2000) used both biochemical and GFP techniques to investigate the function of an Arabidopsis Rab1 (AtRab1) homologue
in tobacco. Co-expression of the dominant inhibitory mutant
AtRab1b(N121I) alongside a secreted form of GFP (secGFP)
resulted in GFP fluorescence accumulating in tobacco leaf
epidermal cells in an ER-reminiscent dynamic reticulate
pattern, whereas the fluorescence pattern of secGFP was unaltered when co-expressed with wild-type AtRab1b (Fig. 7a–e).
AtRab1b(N121I), when co-expressed alongside the Golgi
marker ST-GFP, was shown to have no effect on the BFAinduced redistribution of the Golgi marker back into the ER
(Saint-Jore et al., 2002). However, recovery of Golgi fluorescence upon BFA removal was seriously reduced in the presence
of AtRab1b(N121I) (Fig. 7f,g) whereas the BFA recovery
phenotype could be rescued when the wild-type protein was
co-expressed alongside AtRab1b(N121I). These data strongly
suggest that AtRab1b regulates anterograde rather than
retrograde transport between the ER and the Golgi. However,
although providing strong evidence for the involvement of
Rab1 in ER-to-Golgi trafficking events in plant cells, neither of
the two studies (Batoko et al., 2000; Saint-Jore et al., 2002)
could distinguish between a blockage of protein export out
of the ER and blockage of protein import into the Golgi.
Tobacco Rab2 (NtRab2), a homologue of the mammalian
Rab2 GTPase, has recently been investigated in tobacco pollen
tubes as well as in leaf epidermal cells (Cheung et al., 2002). A
GFP fusion of NtRab2 in pollen tubes was shown to locate
Fig. 7. Effect of wild-type and mutant forms of plant Rab1 and Rab2 proteins on ER-to-Golgi transport as illustrated by the use of various GFP markers.
(a – c) Low-magnification CLS micrographs of tobacco leaf epidermal cells transformed with a secreted GFP marker, secGFP, alone (a) and alongside AtRab1b
(b) or AtRab1b(N121I) (c). Epidermal cells of tobacco leaf areas infiltrated with secGFP alone (a) or secGFP and AtRab1b (b) show little or no intracellular
fluorescence. Intracellular secGFP fluorescence is clearly visible when leaf areas are co-transformed with AtRab1b(N121I) (c). (d,e) High-magnification
CLS micrographs showing that, when co-expressed with AtRab1b(N121I), the pattern of intracellular secGFP fluorescence (d) resembles the fluorescence
pattern caused by expression of GFP-HDEL, a soluble ER marker (e). (f,g) CLS micrographs of tobacco leaf epidermal cells expressing the Golgi marker STGFP and after 8 h recovery in water from BFA treatment in the absence (f ) or in the presence of AtRab1b(N121I) (g). In comparison with the control cell (f),
BFA recovery is inhibited by the presence of AtRab1b(N121I), as indicated by the ER-like pattern of ST-GFP fluorescence (g). (h–j) Fluorescence micrographs of tobacco pollen tubes expressing the Golgi-marker ERD2-GFP alongside wild-type and mutant forms of NtRab2. When co-expressed with wildtype NtRab2, ERD2-GFP locates to Golgi stacks (h), whereas co-expression with NtRab2(S20N) (i) or NtRab2(N119I) ( j) reduces the Golgi location of
ERD2-GFP. Micrograph (i) courtesy of A. Cheung. (k,l) Fluorescence micrographs of tobacco pollen tubes expressing the plasma membrane marker Aha1GFP alongside wild-type NtRab2 (k) or NtRab2(S20N) (l). When co-expressed with NtRab2, Aha-GFP labels the plasma membrane (k), whereas co-expression
with NtRab2(S20N) leads to intracellular accumulation of Aha1-GFP. Scale bars: a–c,f,g = 25 µm; d–e,k = 10 µm; h,j,l = 20 µm. (a) to (e) are from Batoko
et al. (2000) (fig. 4A,B). (h), ( j) and (l) from Cheung et al. (2002) (figs 4C,4E, 5D,E). Copyright the American Society of Plant Biologists and reprinted with
permission. (f) to (g) are from Saint-Jore et al. (2002) (fig. 8g,h). Copyright Blackwell Publishing, Oxford, U.K., and reprinted with permission.
© 2004 The Royal Microscopical Society, Journal of Microscopy, 214, 138–158
G F P I S T H E WAY TO G L OW
© 2004 The Royal Microscopical Society, Journal of Microscopy, 214, 138–158
151
152
F. B R A N D I Z Z I E T A L .
Fig. 8. Location of wild-type and mutant forms of Rab proteins involved in post-Golgi transport. (a) CLS micrograph of tobacco BY-2 cells simultaneously
expressing GFP-Pra2 (green image) and RFP-Pra3 (red image) (Inaba et al., 2002). The merged image shows that the two closely related Ypt3/Rab11
homologues locate to different organelles of the endomembrane system. Micrograph courtesy of Y. Nagano. (b) CLS micrograph of Arabidopsis protoplasts
expressing Ara6-GFP, a homologue to mammalian Rab5, and labelled with the putative endocytic marker FM4-64. Mobile punctate structures and
spherical organelles labelled with GFP (green image, arrowheads) were also labelled with FM4-64 (red image, arrowheads), as shown by the merged
image (arrowheads). (c) CLS micrograph of Arabidopsis protoplasts expressing Ara6Q93L-GFP and labelled with the putative endocytic marker FM4-64.
Ara6Q93L-GFP predominantly locates to the tonoplast and to spherical structures larger than those labelled by wild-type Ara6-GFP (green image; compare
with b). These spherical structures were also labelled by FM4-64 (red and merged images). N, nucleus. Scale bars: b–c = 10 µm. (b) and (c) are reprinted,
with permission, from Ueda et al. (2001) (figs 6D,E,F and 7M).
to BFA-sensitive punctate structures, identified as individual
Golgi stacks by immunogold TEM with polyclonal anti-GFP
serum. GFP fusions of two dominant-negative inhibitory
mutants, GFP-NtRab2(S20N) and GFP-NtRab2(N119I),
located to the pollen tube cytosol. Untagged versions of the
two mutant proteins inhibited the transport of Golgi-resident,
plasma membrane, and secreted GFP marker proteins to their
usual destination in pollen tubes (Fig. 7h–l). For instance,
the ERD2-GFP was shown to change from a predominantly
Golgi location to the ER when co-expressed with the
dominant-negative inhibitory mutants of NtRab2 (Fig. 7h–j).
NtRab2(S20N) and NtRab2(N119I) also had an inhibitory
effect on pollen tube growth. Thus, Cheung et al. (2002)
proposed a role for NtRab2 in ER-to-Golgi traffic. Interestingly,
in contrast to pollen tubes, the GFP fusion of the wild-type
NtRab2 did not target Golgi stacks in tobacco leaf protoplasts
© 2004 The Royal Microscopical Society, Journal of Microscopy, 214, 138–158
G F P I S T H E WAY TO G L OW
and epidermal cells. However, a GFP fusion of an Arabidopsis
Rab2 homologue, AtRab2a (now called AtRabB1b), successfully
targeted the fusion to Golgi stacks in tobacco leaf epidermal
cells (Neumann et al., 2003). This may suggest a role of
AtRab2a in a traffic event between the Golgi and a downstream
compartment (U. Neumann, I. Moore, C. Hawes and H. Batoko,
unpublished observations).
5.2. Regulatory proteins of post-Golgi transport steps
5.2.1. Rab11 (and Rab25) homologues. GFP-based studies on
plant homologues of the mammalian Rab11 subclass suggest
that closely related Rab proteins locate to different compartments and might fulfil different functions (Inaba et al., 2002;
Fig. 8a). GFP fusions of Pra2 and Pra3, two Pisum sativum
Rab11/Ypt3 homologues, have been located to punctate
structures in stably transformed tobacco BY-2 cells. Whereas
GFP-Pra3 fluorescence was not affected by BFA treatment, the
punctate fluorescent GFP-Pra2 structures relocated to ERreminiscent membranes after BFA treatment, suggesting that
GFP-Pra2 is targeted to Golgi stacks. However, GFP-Pra2 also
labelled BFA-insensitive structures thought to be endosomallike compartments. Co-localization experiments of GFP-Pra3
with a fusion between AtVTI11 – a marker of the ‘trans-Golgi
network’ (TGN) and the PVC – and RFP resulted in partial
overlap of the two fluorochromes, suggesting that GFP-Pra3
locates to the TGN and/or the PVC.
5.2.2. Rab5 homologues. GFP-based technology has also highlighted functional diversification among plant homologues
of mammalian Rab5 GTPases (Ueda et al., 2001; Sohn et al.,
2003; Bolte et al., 2004). The location of GFP-tagged versions
of two Rab5 homologues, Ara6 and Ara7, was investigated in
Arabidopsis protoplasts (Ueda et al., 2001). Ara6 is a member
of a novel, plant-unique type of Rab GTPases that lacks the
C-terminal region, essential for attachment to membranes
and subcellular localization. Instead, these unique Rabs
are modified at the N-terminus for N-myristoylation and
palmitoylation (Ueda et al., 2001). Ara6-GFP was shown to label
both punctate and spherical structures (in addition to some
PM and ER labelling in some cells), whereas GFP-Ara7, the
conventional Rab5 homologue, was predominantly detected
on punctate structures and less often on spherical organelles.
Dual-colour imaging showed that Ara6-GFP- or GFP-Ara7positive structures were also labelled with the styryl dye
FM4-64, a putative marker of the endocytic pathway (Fig. 8b).
GFP fusions of the GTPase-deficient mutants, Ara6Q93L-GFP
and GFP-Ara7Q69L, both located to aggregates of large spherical
structures (FM4-64 positive; Fig. 8c), to the tonoplast and small
punctate structures. Whereas Ara6Q93L-GFP was occasionally
detected on the PM, GFP-Ara7Q69L never was. Ueda et al. (2001)
conclude that both Ara6 and Ara7 locate to endosomal-like
organelles and regulate membrane fusion in the early endocytic
pathway. However, based on the slightly different location of their
© 2004 The Royal Microscopical Society, Journal of Microscopy, 214, 138–158
153
GTPase-deficient mutants as well as on differences in expression
pattern and in the amino acid sequence of the effector domain,
Ara6 and Ara7 might regulate fusion of different endosomal
populations in Arabidopsis protoplasts (Ueda et al., 2001).
Another member of the plant-specific Rab GTPases lacking
C-terminal isoprenylation has also been isolated from a salinitytolerant plant, Mesembryanthemum crystallinum (Bolte et al.,
2000). Termed m-Rabmc, it is closely related to the Arabidopsis homologue Ara6 and correspondingly undergoes N-myristoylation
(Bolte et al., 2004). In contrast to results shown by Ueda and
co-workers on Ara6, m-Rabmc appears to be involved in the
vacuolar trafficking pathway (Bolte et al., 2004). m-Rabmc has
been co-located with markers for the prevacuolar compartment of the lytic vacuole (BP80, Pep12). These localization
studies were carried out by immunolabelling or by a combination
of expression of m-Rabmc-CFP constructs and immunolabelling.
Both approaches resulted in a co-localization of m-Rabmc on
more than 80% of prevacuole. It has also been shown that mRabmc co-located partially on the Golgi apparatus. Importantly,
it has been demonstrated that m-Rabmc was implicated in the
transport of the soluble marker protein aleurain-GFP, which is
targeted to the acidic vacuole (Bolte et al., 2004).
A similar role in trafficking of soluble cargo from the PVC to
the central vacuole was also suggested for the third Arabidopsis
isoform closely related to mammalian Rab5, Rha1 (Sohn
et al., 2003). In Arabidopsis protoplasts, the dominant-negative
inhibitory mutant Rha1[S24N] (either tagged with the small
epitope haemagglutinin, HA, or fused to RFP) was shown to
inhibit transport of GFP fused to the soluble vacuolar markers
sporamin (spo-GFP) and Arabidopsis aleurain-like protein
(AALP-GFP) to the central vacuole, presumably at the level of
PVCs. Interestingly, RFP-Ara7[S24N] but not Ara6[S47N]HA caused the same altered distribution of spo-GFP. Biochemical
methods confirmed that vacuolar trafficking of spo-GFP and
AALP-GFP is inhibited, but also revealed that spo-GFP and
AALP-GFP are secreted into the culture medium under the
influence of HA-Rha1[S24N] and RFP-Rha1[S24N], respectively (Sohn et al., 2003).
Thus, questions regarding the location and function of the
plant Rab5 homologues have yet to be conclusively answered.
Concluding remarks
Within a short time, fluorescent proteins have become invaluable tools for in vivo investigations of the plant secretory pathway dynamics and regulation. However, as with any powerful
technique, GFP technology has to be used wisely to avoid
artefacts and data misinterpretations. For example, the nature
of expression systems, whether by virus, agrobacterium or
other transformation methods, may involve protein overexpression. As such, it is important to bear in mind the possible
effect that protein over-expression may have on the plant
endomembrane system. Therefore, the value of controls
on transformation levels and ensuring that the labelling with
154
F. B R A N D I Z Z I E T A L .
Fig. 9. Comparison of the location of two different Golgi markers in tobacco BY-2 cells with the CLSM (a,c) and the TEM (d). Although both GFP fusion
proteins, ST-GFP (Batoko et al., 2000; Saint-Jore et al., 2002) and GFP-XylT36 (Follet-Gueye et al., 2003) locate to punctate structures when seen with the
CLSM (a,b, respectively), their location within the Golgi stack, as seen by immunogold labelling using polyclonal anti-GFP antibodies, is different. Whereas
ST-GFP locates towards the trans-half of the Golgi stack (b), GFP-XylT36 is restricted to medial cisternae (d). Scale bars: a = 50 µm; b = 20 µm;
c,d = 100 nm. (c) and (d) are reproduced, with permission, from Follet-Gueye et al. (2003) (fig. 6A,B).
GFP fusions is due to correct targeting should not be underestimated. Over-expression of protein may lead to ‘over-spill’ to
other cellular compartments including the Golgi, vacuole and
tonoplast, or even distension of the ER tubules as seen in cells
over-expressing non-GFP-labelled protein (Crofts et al., 1999).
In addition, GFP tagging of proteins may result in loss of a
specific function or, worse, acquisition of new function.
Another possible limit of GFP technology could arise from
the production of such artefacts in reporter systems involving
malfolding of proteins and malfunction. Furthermore, as
for any other fluorescent probe, the sensitivity of GFP and its
spectral variants to pH and proteolysis should be considered
when investigating different organelles. One example is the
vacuole, where GFP may accumulate but its fluorescence may
be easily quenched by the acidic pH and specific experimental
procedures may need to be adopted in order to visualize GFP
fluorescence (Tamura et al., 2003). Finally, yet importantly,
the expression and fluorescence of a GFP reporter may vary
among different reporter systems (Di Sansebastiano et al.,
2004).
It should also be noted that organelles highlighted by GFP
that may have similar appearance to others described in the
literature (e.g. a dot or a ring) might not necessarily be the
same. Therefore, attempts to establish the subcellular location of novel protein markers fused to GFP should always be
accompanied by co-localization studies with other fluorescent markers that label specific compartments. Alternatively,
immunogold with GFP antiserum applied to TEM should be
used to investigate the nature of the GFP labelling at high
resolution (see Fig. 9).
As for any other light-microscopy-based technique, GFP
technology also has its limits with regard to suborganelle
© 2004 The Royal Microscopical Society, Journal of Microscopy, 214, 138–158
G F P I S T H E WAY TO G L OW
resolution. Figure 9 illustrates that two GFP fusion proteins
locating to the Golgi bodies in tobacco BY-2 cells show a different
location within the stack with the aid of electron microscopy.
Modifications of GFP resulting in variations in both emission
and excitation wavelengths (Haseloff et al., 1999), coupled
with the development of other fluorescent proteins such as
the recently described red fluorescent protein (DsRed, Matz
et al., 1999), will permit two- or even three-colour detection
of organelles in vivo (reviewed in Brandizzi et al., 2002b). The
advent of new photoactivatable proteins, such as the photoactivatable GFP (Patterson & Lippincott-Schwartz, 2002)
and the kindling RFP (Chudakov et al., 2003), coupled with
improvements in imaging and analysis techniques, will offer
an impressive armoury for the investigation and understanding
of the dynamic nature of living plant cells.
Acknowledgements
We thank all those who have kindly provided micrographs:
H. Batoko, A. Cheung, G. P. Di Sansebastiano, M. L. Follet-Gueye,
I. Hwang, Y. Nagano, A. Nakano, A. Nebenführ, C. Saint-Jore,
M. Takeuchi and T. Ueda. We also thank F. Siefritz and
N. Leborgne-Castel for the generous gift of the NtAQP1 cDNA
and of the BobTIP26-1::GFP construct, respectively. We
acknowledge the Biotechnology and Biological Sciences
Research Council for supporting the work undertaken in
our laboratory. Finally, we would like to thank Chris Hawes for
having been a very good friend and an excellent tutor during
our residency at Oxford Brookes.
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