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Endocytic and Secretory Traffic in Arabidopsis Merge in theTrans-Golgi Network/Early Endosome, an Independent andHighly Dynamic Organelle W
Corrado Viotti,a,1 Julia Bubeck,b,1 York-Dieter Stierhof,c Melanie Krebs,b Markus Langhans,a Willy van den Berg,d
Walter van Dongen,d Sandra Richter,e Niko Geldner,f Junpei Takano,g Gerd Jurgens,e Sacco C. de Vries,d
David G. Robinson,a and Karin Schumacherb,2
a Department of Cell Biology, Heidelberg Institute for Plant Sciences, University of Heidelberg, 69120 Heidelberg, Germanyb Department of Developmental Biology, Heidelberg Institute for Plant Sciences, University of Heidelberg, 69120 Heidelberg,
GermanycMicroscopy Unit, Center for Plant Molecular Biology, University of Tubingen, 72076 Tubingen, Germanyd Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, The Netherlandse Developmental Genetics, Center for Plant Molecular Biology, University of Tubingen, 72076 Tubingen, Germanyf Department of Plant Molecular Biology, University of Lausanne, 1015 Lausanne, Switzerlandg Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
Plants constantly adjust their repertoire of plasma membrane proteins that mediates transduction of environmental and
developmental signals as well as transport of ions, nutrients, and hormones. The importance of regulated secretory and
endocytic trafficking is becoming increasingly clear; however, our knowledge of the compartments and molecular ma-
chinery involved is still fragmentary. We used immunogold electron microscopy and confocal laser scanning microscopy to
trace the route of cargo molecules, including the BRASSINOSTEROID INSENSITIVE1 receptor and the REQUIRES HIGH
BORON1 boron exporter, throughout the plant endomembrane system. Our results provide evidence that both endocytic
and secretory cargo pass through the trans-Golgi network/early endosome (TGN/EE) and demonstrate that cargo in late
endosomes/multivesicular bodies is destined for vacuolar degradation. Moreover, using spinning disc microscopy, we
show that TGN/EEs move independently and are only transiently associated with an individual Golgi stack.
INTRODUCTION
As a consequence of their sessile lifestyle, plants have to be able
to rapidly adapt their functional responses to environmental
cues. In this regard, data have been accumulating that indicate
that the repertoire of plasma membrane (PM) proteins is highly
dynamic and is constantly being adjusted to suit the plant’s
needs. These include receptors mediating the transduction of
environmental and developmental signals aswell as transporters
for ions, nutrients, and hormones. By regulating the density of
these proteins at the PM through the secretory and endocytic
pathways, the plant can effectively adapt to new environmen-
tal conditions. Although our knowledge of the compartments
through which endocytic cargo passes is still rudimentary
(Robinson et al., 2008), one striking example of a regulatory
switch between the recycling and degradative pathways of endo-
cytosis is the boron (B) exporter REQUIRES HIGH BORON1
(BOR1; Takano et al., 2002). Under steady state conditions in the
presence of low B, BOR1 is found principally at the PM, with a
fraction undergoing constitutive cycling. To avoid B toxicity,
BOR1 is rapidly internalized and targeted for vacuolar degrada-
tion after sensing high external B concentrations (Takano et al.,
2005). Unlike BOR1, the steady state distribution of the brassi-
nosteroid receptor BRASSINOSTEROID INSENSITIVE1 (BRI1;
Li and Chory, 1997; Friedrichsen et al., 2000) at the PM does
not change after application of the ligand (Geldner et al., 2007).
However, BRI1 has been shown to cycle between the PM and
brefeldin A (BFA)-sensitive endosomal compartments, suggest-
ing that BRI1 is also subject to constitutive endocytic recycling
(Geldner et al., 2007). Increasing endosomal concentrations of
BRI1 lead to enhanced BR signaling, indicating that plants, like
animals, use endosomes as signaling platforms (Russinova et al.,
2004; Geldner et al., 2007). Nevertheless, a fraction of endocy-
tosed BRI1 molecules is also targeted to the vacuole (Geldner
et al., 2007), so it remains to be determined from which endo-
somal compartments BRI1 can recycle to the PMand fromwhich
point on it becomes destined for degradation. Thus, BRI1 and
BOR1 provide evidence that different modes of endocytosis
coexist in plant cells, although the respective trafficking path-
ways have not been precisely defined (Geldner and Jurgens,
2006).
1 These authors contributed equally to this work.2 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Karin Schumacher([email protected]).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.109.072637
The Plant Cell, Vol. 22: 1344–1357, April 2010, www.plantcell.org ã 2010 American Society of Plant Biologists
Based on rapid staining with the endocytic tracer FM4-64
and the colocalization of TGN markers and endocytosed PM
proteins in the core of BFA compartments, there is now com-
pelling evidence that the trans-Golgi network (TGN), or a
subdomain of it, acts as an early endosome (EE) (Dettmer
et al., 2006; Lam et al., 2007; Chow et al., 2008). However,
evidence for the passage of endocytosed PM proteins through
the TGN/EE is limited to the cytokinesis-specific syntaxin
KNOLLE, which is removed from the cell plate during M phase
(Reichardt et al., 2007).
The TGN was originally defined as a clathrin-coated, tubular
network contained within the matrix of a Golgi stack (Staehelin
and Moore, 1995). However, TGN-like structures have also been
observed more distant from Golgi stacks, and this variation in
distance has been ascribed to amaturation process that involves
sloughing off at the trans-mostGolgi cisterna (Hillmer et al., 1988;
Toyooka et al., 2009). More recently, electron tomography has
provided evidence for a structural differentiation of the TGN into
so-called early and late subcompartments (Staehelin and Kang,
2008), although kinetic studies are needed to confirm this clas-
sification. The extent to which the TGN/EE participates in secre-
tory traffic to the PM is also controversial. The study of Toyooka
et al. (2009) suggests that part of the TGN separates from the rest
to form a cluster of secretory vesicles, which eventually fuse with
the PM. On the other hand, it has recently been suggested that
cellulose synthase complexesmight not pass through the TGN/EE
Figure 1. The TGN Is an Independent Organelle That Undergoes Reversible Homotypic Association.
Spinning disc confocal microscopy of hypocotyl cells expressing VHA-a1-RFP and ST-GFP. Numbers = seconds; bars = 5 mM.
(A) The TGN is a highly mobile and independent organelle (white arrows, time point from 0 to 11.8 s) that temporarily pauses and becomes closely
associated with a Golgi stack (blue arrowheads, 1.2 to 15.4 s). Homotypic association of individual TGNs was also observed (red arrows, 13.0 to 15.4 s),
and sometimes a thin protrusion between the interacting partners was visible (yellow arrows, 10.6 s). Not all TGNs are competent for this interaction
(magenta arrow, 8.3 and 9.5 s).
(B) A dissociation event for an elongated TGN shared by two Golgi stacks (white arrows, 0 to 9.5 s).
Dynamics of the Trans-Golgi Network/Early Endosome 1345
when they move to the PM, thus leading the authors to question
the role of the TGN in secretion (Crowell et al., 2009).
Although the ARF GTPase exchange factor GNOM (Geldner
et al., 2003) identifies a recycling endosome, a morphological
characterization of this compartment at the EM level is lacking.
GNOM-bearingmembranes and TGN/EEmarkers accumulate in
BFA compartments (Dettmer et al., 2006; Lam et al., 2007; Chow
et al., 2008), but it remains to be seen whether the recycling
endosome represents an independent compartment, a subdo-
main of the TGN/EE, or perhaps a compartment that matures out
of the latter. Recycling to the PM has also been suggested to
occur from a compartment characterized by the presence of
SORTING NEXIN1 (SNX1) in Arabidopsis thaliana (Jaillais et al.,
2006, 2008). SNX1 is part of the vacuolar sorting receptor-
recycling protein complex (retromer) that was previously re-
ported to be localized to the multivesicular body/prevacuolar
compartment (MVB/PVC), the equivalent of the late endosome in
plants (Oliviusson et al., 2006). However, more recently, SNX1
was shown to localize to the TGN/EE (Niemes et al., 2010a),
showing that the plant late endosome has an equivalent function
as its counterpart from mammals, from which receptor recycling
also does not occur (Braulke and Bonifacino, 2009).
Clearly, the routes of cargo molecules and the compartments
of the plant endosomal system need to be better characterized.
In particular, it remains to be demonstrated through which
domains/compartments receptors and transporters pass on
their way to the PM after synthesis and then subsequently during
endocytosis: Are they the same or are the synthetic and
endocytic populations kept separate? To reconcile the differen-
tiation of the TGN into early and late subcompartments in terms
of secretion (Staehelin andKang, 2008) with its function as an EE,
we performed spinning disc confocal microscopy on an Arabi-
dopsis line expressing Golgi and TGN markers. This revealed
that TGN/EEs frequently display independent movement and are
only transiently associated with individual Golgi stacks. To trace
the fate of internalized PM proteins and to distinguish between
endocytic and secretory cargo in the TGN, we used immunogold
electron microscopy (IEM) and confocal laser scanning micros-
copy (CLSM) to determine the route(s) of BRI1 and BOR1
throughout the plant endomembrane system. Our results con-
firm that both endocytic and secretory cargo pass through the
TGN/EE and demonstrate that PM proteins found in late endo-
somes/MVBs are indeed destined for vacuolar degradation.
Moreover, CLSM analysis after inducible expression of secretory
green fluorescent protein (secGFP) and BRI1-yellow fluorescent
protein (YFP) as well as immunogold-EM of a xyloglucan epitope
confirmed that at least some secretory cargo molecules pass
through the TGN en route to the PM.
Figure 2. The Identity and Independence of the TGN Is Affected by ConcA.
Immunogold labeling of VHA-a1 and SYP61-CFP in ConcA-treated cells. (A) to (D), silver-enhanced cryosections; (E) to (H), HM20 resin sections (see
Methods). Bars = 250 nm.
(A) and (E) VHA-a1 and SYP61 are highly specific markers for the plant TGN/EE.
(B) to (D) and (F) to (H) In the presence of ConcA, VHA-a1 and SYP61-CFP were additionally found on Golgi stacks, and a clear spatial separation
between the Golgi apparatus and the TGN was no longer recognizable. Furthermore, ConcA induces the proliferation of the Golgi cisternae,
accompanied by a loss of identity of the two compartments.
1346 The Plant Cell
RESULTS
Independent Movement and Dynamic Association of the
TGN/EE with Golgi Stacks
To better characterize the temporal and spatial relationship
between Golgi stacks and TGN/EE, we performed spinning
disc confocal microscopy on seedlings expressing both the
TGN/EE marker VHA-a1-red fluorescent protein (RFP) (Dettmer
et al., 2006) and the trans-Golgi marker N-ST-YFP (Grebe et al.,
2003). Hypocotyl cells of etiolated seedlings were chosen since
their relatively thin peripheral layer of cytoplasm allowed us to
follow the dynamics of the organelles in an almost two-dimensional
plane. We found that the TGN behaved as a highly mobile and
independent organelle (Figure 1A, white arrow; see Supplemen-
tal Movie 1 online) that temporarily paused and became closely
associated with a Golgi stack (Figure 1A, arrowhead; see Sup-
plemental Movie 1 online). Furthermore, association of individual
TGNs was observed (Figure 1A, red arrow; see Supplemental
Movie 1 online). Sometimes, a thin protrusion between the
partners was visible before the actual merging occurred (Figure
1A, yellow arrow). Evidently, not all TGNswere competent for this
interaction (Figure 1A, magenta arrow). Association seemed to
be balanced by dissociation events as shown in Figure 1B and
Supplemental Movie 2 online for an elongated TGN shared by
two Golgi stacks.
Although the TGN can move as an independent compart-
ment, we previously showed that genetic and pharmacological
inhibition of V-ATPase activity not only affects the TGN/EE but
also causes a loss of recognizable Golgi morphology (Dettmer
et al., 2005,2006; Brux et al., 2008). To test if V-ATPase activity
is required for the identity of the TGN/EE, we analyzed the
distribution of VHA-a1 and SYP61, two highly specific markers
for the plant TGN/EE (Figures 2A and 2E; Dettmer et al., 2006;
Robert et al., 2008) in meristematic root cells after a mild
treatment with the V-ATPase inhibitor concanamycin (ConcA).
In the presence of 0.5mMConcA, the number of Golgi cisternae
increased from 5 6 1 to 8 6 1 (Table 1), and a clear spatial
separation between the Golgi stack and the TGN was no longer
recognizable (Figures 2B to 2D and 2F to 2H). Furthermore,
VHA-a1 and SYP61 were no longer restricted to the TGN but
were additionally found on Golgi cisternae, indicating that
V-ATPase activity is required for the identity of the TGN/EE
(Figures 2B to 2D and 2F to 2H, Table 1).
Endocytic Cargo Passes through the TGN
To define the endosomal compartments through which BRI1 and
BOR1 pass after their internalization from the PM, we traced these
cargoes by IEM. Seedlings expressing BRI1-GFP (Friedrichsen
et al., 2000) or BOR1-GFP (Takano et al., 2010) were high-pressure
frozen, freeze-substituted, and embedded in Lowicryl HM20 resin.
Embedded samples in which the GFP signal, despite the thickness
of the resin block, was still detectable were selected (see Supple-
mental Figure 1 online). Affinity-purified YFP antibodies were used
for the IEM. In meristematic root cells, BRI1-GFP was only detect-
able in the region of the TGN and in MVBs (Figure 3A, Table 1). The
tubular-vesicular TGN/EE was positively labeled even when not
obviously associated with a Golgi stack (Figure 3B). BFA treatment
causes the aggregation of endosomal compartments, including the
TGN/EE (Grebe et al., 2003; Dettmer et al., 2006; Robinson et al.,
2008). Accordingly, the core of the BFA compartment was posi-
tively labeled with BRI1-GFP (Figure 3C). Nevertheless, punctae
surrounding the core were also visible (Figure 3C, arrows). IEM
analysis confirmed the presence of BRI1-GFP both in the core of
the BFA compartment and inMVBs (Figures 3D and 3E). Treatment
with the protein synthesis inhibitor cycloheximide (CHX; see Sup-
plemental Figures 2Aand 2B online) did not prevent the accumu-
lation of BRI1-GFP in the core of the BFA compartment (Figures 3F
and 3H), and BRI1 was still detected at the TGN/EE (Figure 3G),
indicating that the signal was not only due to secretory cargo.
Endocytic Cargo in MVBs Is Destined for Degradation
The PI3-kinase inhibitor wortmannin (Wm) (Emans et al., 2002)
causes swelling of MVBs, in part due to homotypic fusion events
Table 1. Quantitative Analysis of IEM
Protein
Golgi TGN MVB
Treatment No. Labeled No. Gold/mm2 No. Cisternae Labeled (%) No. Gold/mm2 Labeled (%) No. Gold/mm2 Inner Gold
BRI1-GFP control 9% 1.1 6 0.4 5 6 1 66% 22.7 6 2.1 66% 20.4 6 2.8 93% 6 5%
BRI1-GFP +CHX 11% 1.3 6 0.5 5 6 1 59% 18.6 6 2.5 58% 18,6 6 3.5 88% 6 7%
BOR1-GFP �B 11% 1.1 6 0.3 5 6 1 25% 5.2 6 1.0 19% 3.3 6 0.9* n.a.
BOR1-GFP +B 11% 1.3 6 0.4 5 6 1 66% 29.1 6 2.3 68% 21.8 6 2.2 85% 6 4%
BOR1-GFP +CHX, +B 10% 1.5 6 0.4 5 6 1 59% 20.5 6 1.7 60% 21.3 6 3.3 87% 6 5%
BP80 Control 12% 1.3 6 0.5 5 6 1 81% 42.2 6 3.5 76% 39.4 6 3.9 30% 6 5%
hs:BRI1-YFP Control 48% 8.3 6 1.0 5 61 50% 18.1 6 2.1 19% 3.1 6 0.8* n.a.
hs:BRI1-YFP +Wm 54% 9.5 6 1.4 5 6 1 50% 17.2 6 2.5 18% 3.8 6 0.8* n.a.
SYP61-CFP Control 12% 1.6 6 0.6 5 6 1 85% 47.4 6 3.9 10% 2.4 6 1.4* n.a.
SYP61-CFP +ConcA 63% 9.2 6 1.1 8 6 1 81% 31.9 6 2.7 9% 2.7 6 1.5* n.a.
Labeling density is expressed as the number of gold particles/mm2 6 SE, number of cisternae 6 SD, and percentage of inner gold particles 6 SD.
Asterisk indicates that labeling density (#5 gold/mm2) comparable to background labeling (see Supplemental Table 1 online). n.a., not available; gold
particles were too few to allow analysis. For further details (total number of compartments analyzed and total number of gold particles), see
Supplemental Table 1 online.
Dynamics of the Trans-Golgi Network/Early Endosome 1347
(Wang et al., 2009). As a result, fluorescently tagged PVC
markers like the vacuolar sorting receptor BP80 or the Arabi-
dopsis Rab5-like proteins ARA6 and ARA7 show a ring-like
appearance after Wm treatment (Tse et al., 2004; Lam et al.,
2007; Reichardt et al., 2007). However, in no cases did BRI1-
labeled endosomes show a ring-like appearance in response to
Wm. We considered this to be a consequence of the fact that
BRI1 was always localized on the inner vesicles of the MVBs
(Figures 3A, 3E, and 4A, Table 1), rather than preferentially on the
boundarymembrane, as is the case for BP80 (Figure 4B, Table 1;
see Supplemental Figure 3 online). Also, when roots were treated
with both CHX and Wm, IEM showed that BRI1-GFP is present
exclusively on the inner vesicles (Figure 4A), althoughMVBswere
swollen. A similar distribution pattern was detected for the
Figure 3. Endocytosed BRI1-GFP Is Found at the TGN and in MVBs.
Immunogold labeling and CLSM analyses of root tips from BRI1-GFP Arabidopsis plants. m, multivesicular body; t, TGN; g, Golgi; er, endoplasmic
reticulum; b, BFA compartment. The symbol “/” means that the second inhibitor was added with the first still present. IEM bars = 200 nm; CLSM
bars = 5 mm.
(A) BRI1-GFP localizes to both the TGN and MVB (arrowheads).
(B) The TGN is positively labeled (arrowheads) also when it is not closely associated with the Golgi stacks.
(C) The core of the BFA compartment is labeled, and a surrounding punctate pattern is also visible (arrows).
(D) and (E) IEM confirmed the localization of BRI1-GFP in the core of the BFA compartment and on the MVB (arrowheads).
(F) The protein synthesis inhibitor CHX does not prevent the accumulation of BRI1-GFP in the core of the BFA compartment (arrowheads).
(G) In the presence of CHX, BRI1 is still detected at the TGN (arrowheads).
(H) In the presence of CHX and BFA, BRI1 is detected in the BFA compartment (arrowheads).
1348 The Plant Cell
PM-based boron exporter BOR1. Under low boron conditions,
BOR1 was not detectable in any endosomal compartment.
However, after 30 min of exposure to boron, BOR1-GFP was
localized both to the TGN and to the MVB (Figure 4C). The TGN
signal was confirmed also in the presence of CHX (Figure 4D).
Moreover, BOR1 also was specifically localized to the inner
vesicles of the MVB (Figures 4E and 4F, Table 1), pointing to its
subsequent degradation in the vacuole, since after 70min of high
boron treatment the GFP signal had almost disappeared (see
Supplemental Figure 4 online). These data indicate that at least
themajority of PMproteins found inMVBswill not recycle back to
the PM, implying that recycling is more likely to take place at the
TGN, through a TGN subdomain or a completely independent
recycling endosome derived from it.
Secretory Traffic Passes through the TGN
To determine if secretory cargo passes the TGN on its way to
the PM, we used two transgenic Arabidopsis lines expres-
sing either secGFP, a secreted version of GFP (Zheng et al.,
2004), or BRI1-YFP (Geldner et al., 2007), both under the con-
trol of a heat shock–inducible promoter. After heat shock,
secGFP was weakly detected only in the apoplastic space
(Figure 5A), whereas seedlings treated with ConcA showed a
GFP signal inside the cells that partially overlapped with the
endocytic tracer FM4-64 (Figure 5B). Similarly, BRI-YFP, which
localized to the PM after heat shock (Figure 5D), accumulated
intracellularly and was colocalized with FM4-64 after treatment
with ConcA (Figure 5E). In the presence of BFA, secGFP was
hard to detect but present in the core of the BFA compartment
(Figure 5C), whereas inducible expression of BRI1-YFP led to
much stronger labeling (Figure 5F). All inhibitor treatments were
reversible within 2 h after washout and had no adverse effect on
cell viability (see Supplemental Figure 5 online). Immunogold
labeling on ultrathin HM20 resin sections showed that
BRI1-YFP was already detectable in both the Golgi apparatus
and the TGN 1 h after the induction (Figure 6A, Table 1), a time
point at which, by CLSM, no BRI1-YFP signal was detectable at
thePM (seeSupplemental Figure 2Conline).UponWmtreatment,
Golgi stacksandTGNwere specifically labeled,whereas theMVB
was not (Figure 6B, Table 1). Finally, in the additional presence of
BFA (Figure 6C) and ConcA (Figure 6D), the Golgi stacks and the
respective TGN-derived inhibitor-induced compartments were
specifically labeled.
Xyloglucans are processed in the Golgi apparatus (Keegstra
and Raikhel, 2001), and CCRC-M1, an antibody directed
against fucose-containing xyloglucans (Puhlmann et al.,
1994), labels not only the Golgi stack but also a TGN-like/
post-Golgi compartment (Moore and Staehelin, 1988; Moore
et al., 1991). We used this antibody to detect fucosylated
xyloglucans in Arabidopsis root tips via IEM analysis. Immuno-
gold labeling of ultrathin thawed cryosections of high-pressure
frozen, freeze-substituted, and rehydrated samples showed
that the TGN was labeled by CCRC-M1 (Figures 7A and 7B).
Moreover, after treatment with ConcA or BFA, the ConcA-
induced aggregates (Figures 7C and 7D) or the BFA compart-
ments (Figures 7E to 7G), respectively, were intensely labeled,
confirming that both inhibitors affect secretory trafficking and
Figure 4. BRI1-GFP and BOR1-GFP, but Not BP80, Are Found on the Inner Vesicles of MVBs.
Immunogold labeling of BP80, BRI1-GFP, or BOR1-GFP in Arabidopsis root tips. m, multivesicular body; t, TGN; v, vacuole; g, Golgi. Bars = 200 nm.
(A) BRI1-GFP is found on the inner vesicles of MVBs (highlighted by circles).
(B) By contrast, BP80 localizes on the boundary membrane of MVBs (arrowheads).
(C) BOR1-GFP localizes both to the TGN and to the MVB after 30 min of exposure to boron (arrowheads).
(D) BOR1-GFP localizes to the TGN (arrow) after 30 min of exposure to boron also in the presence of CHX.
(E) and (F) BOR1-GFP always localizes to the inner vesicles of MVBs, after either 30 or 70 min of exposure to boron.
Dynamics of the Trans-Golgi Network/Early Endosome 1349
that the secretory cargo that passes through the TGN also
accumulates in the core of BFA compartments.
DISCUSSION
The TGN/EE: A Golgi-Derived Independent Organelle
The TGN is considered to be part of the plant Golgi apparatus
and is localized within a ribosome-excluding Golgi matrix (Moore
and Staehelin, 1988). It only leaves the matrix as a consequence
of maturation and sloughing off (Mollenhauer, 1971; Mollenhauer
andMorre, 1991; Toyooka et al., 2009). A differentiation into early
and late subcompartments of the TGN, based upon electron
tomography (Staehelin and Kang, 2008), would therefore ap-
pear to support this model for Golgi function. However, live-cell
imaging using spinning disc confocal microscopy has revealed
novel and unexpected information about the TGN/EE, which
causes us to reconsider the nature of this compartment. The
TGN/EE was seen to leave Golgi stacks and become associated
with others, often bypassing the most immediate Golgi. These
observations are not in accordancewith a subdivision of the TGN
based on degrees of maturation of an indivisible Golgi-TGN/EE
unit. The TGN/EE should therefore now be recognized as being a
dynamic and independent compartment that only temporarily
moves together with an individual Golgi stack.
Surprisingly, we observed that distinct TGN/EE units could
associate with one another (Figure 8). This homotypic interaction
was preceded by the formation of a thin tube-like protrusion that
connected the TGN/EEs. It would appear that the association
process is reversible, since dissociation events were also de-
tected. In in vitro assays, the yeast Tlg SNARE complex has been
shown to be required for homotypic fusion (Brickner et al., 2001),
and the early endosomal antigen 1 is involved in homotypic
fusion ofmammalian early endosomal vesicles (Mills et al., 1998).
The dynamic behavior of the plant TGN/EE may also involve
membrane fusion and fission, but proof of this awaits the
availability of suitable protocols for purification and content
mixing of TGN/EE vesicles.
Although BFA-sensitive guanine-nucleotide exchange factors
for the ADP-ribosylation factor GTPases (ARF-GEFs) are the
known molecular targets of BFA, and BFA treatment of plants
lacking the BFA-resistant Golgi-localized ARF-GEF GNOM-
LIKE1 prevents COPI vesicle formation (Richter et al., 2007), it
is not at all clear how the prevention of coat protein attachment
by BFA eventually leads to the formation of BFA compartments.
Perhaps a BFA-sensitive ARF-GEF interfering with fission would
explain the huge aggregates of TGN/EEs observed in root cells
after BFA treatment.
The TGN and Endocytic Recycling
Based on studies with the endocytic tracer FM4-64, two com-
partments have been identified in Arabidopsis and in general in
plants: the tubular-vesicular TGN and the MVB (Dettmer et al.,
2006; Lam et al., 2007; Otegui and Spitzer, 2008; Robinson et al.,
2008). If the TGN, the main sorting hub for the secretory and
vacuolar traffic is also an early endosomal compartment, PM
proteins detected in this compartment could therefore be either
newly synthesized or internalized. In this investigation, we have
been able to differentiate between secretory and endocytic
cargo in the TGN, and, as a result, have confirmed the dual
function for the TGN/EE.
The PM-localized brassinosteroid receptor BRI1 and the
boron exporter BOR1 are both internalized via endocytosis
(Russinova et al., 2004; Takano et al., 2005), but while most of
BRI1 seems to recycle back to the PM (Geldner et al., 2007),
BOR1 is rapidly degraded when high amounts of boron are
supplied (Takano et al., 2005). We localized both BRI1 and BOR1
to the TGN and the MVB. Combined with the kinetics of FM4-64
endocytosis (Dettmer et al., 2006), this strongly suggests that the
Figure 5. The Effects of ConcA and BFA on hs:secGFP and hs:BRI1-
YFP.
CLSM images of cells in the root elongation zone of hs:secGFP ([A] to
[C]) or hs:BRI1-YFP ([D] to [F]) seedlings. Bars = 10 mM.
(A) and (D) Untreated cells expressing secGFP (A) or BRI1-YFP (D) 5 h
after heat shock.
(B) and (E) FM4-64 was added 5 min before ConcA. Thirty minutes after
addition of ConcA, the heat shock started. After 5 h of expression, a
strong intracellular signal of both secGFP (B) or BRI1-YFP (E) was
detectable.
(C) and (F) BFA was added 30min before the heat shock. Five hours after
the heat shock, root cells were stained with FM4-64 for 30 min. Both
secGFP (C) and BRI1-YFP (F) were detected in the core of the BFA
compartment.
1350 The Plant Cell
TGN is indeed the first compartment to be reached by endocy-
tosed PM proteins, and it is reasonable to hypothesize that
recycling back to the PM (for BRI1) starts from the TGN. In
Arabidopsis, a recycling endosome (RE) has been characterized
by the presence of the ARF GTPase exchange factor GNOM
(Geldner et al., 2003), but its identity at the EM level is lacking.
However, the GNOM compartment is BFA sensitive and, like the
TGN, is also found in the core of the BFA compartment. Never-
theless, differences in the BFA sensitivities of the two compart-
ments have been reported (Geldner et al., 2009), suggesting that
the TGN and RE may be separate entities. Thus, it remains
unclear whether the RE is physically separate and independent
of the TGN or is merely a subdomain of the TGN. The situation
becomes even more complicated when one looks at the endo-
somal marker GTPases Rab A2 and A3. During interphase, they
partially overlap with the TGN marker VHA-a1 but, unlike VHA-
a1, become incorporated into the cell plate during cytokinesis
(Chow et al., 2008).
In this investigation, we confirmed the presence of rapidly
internalized BRI1 and BOR1 in the TGN. By inhibiting protein
synthesis with cycloheximide, we distinguished between secre-
tory and endocytic populations of these two PM proteins in the
TGN. Under these conditions, positive immunogold signals were
still obtained in the TGN. This demonstrates that, at any one time,
at least part of the signals for BRI1 and BOR1 found in the TGN
are endocytic in origin.
The TGN and Secretory Traffic to the PM
Classically, the TGN is regarded as the final sorting station for
secretory cargo to the PM or the vacuole (Staehelin and Moore,
1995). Some secretory cargo molecules, including certain pec-
tins and the cellulose synthase complex, seem to exit the Golgi
stack before reaching the TGN (Moore et al., 1991; Zhang and
Staehelin, 1992; Crowell et al., 2009). Surprisingly, direct evi-
dence of secretory cargo passing through the TGN is limited, and
we have therefore made use of inducible cargo molecules. Both
secGFP and BRI1-YFP accumulated intracellularly after ConcA
treatment and based on costaining with FM4-64 both molecules
were trapped in TGN-derived membranes. In Arabidopsis root
cells, BFA does not block secretion (Grebe et al., 2003; Zheng
et al., 2004), but we found that, after prolonged incubation with
BFA, secGFP was weakly detectable in the core of the BFA com-
partment, indicating that it does pass through the TGN. Accumu-
lation of BRI1-YFP in BFA compartments could be due to both
secretory and endocytic trafficking. Nevertheless, BRI1-YFP was
detectable in the TGN 1 h after induction, which argues that it
passes through the TGN on its way to the PM. Currently available
means to interfere with endocytosis are not sufficient to fully
exclude recycling. Improved tools for in vivo imaging are needed to
better differentiate between endocytic and secretory cargo.
On the other hand, the presence of fucosylated xyloglucans
indicates that secretory cargo is found in the TGN and the BFA
compartment, since there is no evidence for endocytic recycling
Figure 6. Newly Synthesized BRI1-YFP Passes through Both the Golgi and the TGN.
Immunogold labeling of hs:BRI1-YFP in Arabidopsis root tips in the presence of different combinations of inhibitors. g, Golgi; t, TGN; m, MVB; b, BFA
compartment. Bars = 200 nm.
(A) After a 1-h heat shock treatment, seedlings were immediately high-pressure frozen and freeze substituted. hs:BRI1-YFP localizes both to the Golgi
apparatus and the TGN (arrows).
(B) Treatment with the endocytosis inhibitor Wm started 30 min before the heat shock. hs:BRI1-YFP again localized to both the Golgi and the TGN
(arrows), whereas the MVB was not labeled.
(C) Treatment with Wm and BFA started 30 min before the heat shock. hs:BRI1-YFP localized both to the Golgi apparatus and the core of the BFA
compartment (arrows).
(D) Treatment with Wm and ConcA started 30 min before the heat shock. hs:BRI1-YFP localized to the TGN (arrows).
Dynamics of the Trans-Golgi Network/Early Endosome 1351
of xyloglucans. This is not the casewith pectins, whose presence
in BFA compartments has been interpreted to represent endo-
cytic cargo (Baluska et al., 2002, 2005; Samaj et al., 2005). This
conclusion is largely based on the specificity of a rhamnoga-
lacturonan antibody used for identifying cross-linked pectins.
However, based on immunoblotswith purified epitopes, this anti-
body binds non-cross-linked pectins with the same affinity as
cell wall–derived cross-linked pectins (Matoh et al., 1998).
Moreover, there is no evidence that BFA treatment prevents
continued synthesis of pectins in the Golgi apparatus. Thus, it
cannot be claimed with certainty that the presence of pectins in
the BFA compartment is due to endocytosis. We therefore
conclude that although not all secretory cargo may have to
pass through the TGN/EE, it should still be considered as a
secretory compartment.
DoMVBs Function in Recycling?
Even though BRI1 is mostly recycled back to the PM, its
estimated half-life is 5 h (Geldner et al., 2007), implying that at
any time a significant proportion should be found in the pathway
leading to vacuolar degradation. We therefore interpret the
immunogold labeling of the inner vesicles of the MVB for both
BRI1 and BOR1 as indicating that these vesicles are destined for
degradation after fusion of the MVB with the vacuole. The
presence of BRI1 on the inner vesicles of the MVB is also in
accordance with the assumption that signal transduction can
only be switched off when the cytosolic C-terminal kinase
domain of BRI1 no longer faces the cytosol. Therefore, we
consider it unlikely that recycling to the PM takes place from
MVBs. Labeling of the inner vesicles also rules out the possibility
that recyclingmight occur through the direct fusion of MVBswith
the PM, as thePMproteinswould be released into the apoplast in
an exosome-like fashion (An et al., 2007).
Based on the presence of vacuolar sorting receptors (VSRs;
Tse et al., 2004) and retromer, a protein complex believed to
recycle VSRs (Oliviusson et al., 2006), the MVB/PVC has been
regarded as a recycling compartment (Foresti and Denecke,
2008; Jaillais et al., 2008; Otegui and Spitzer, 2008). In mamma-
lian cells, which have separate TGN and EE compartments,
recycling of the lysosomal acid hydrolase receptor mannosyl
6-phosphate to the TGN occurs mainly from the EE via retromer
(Bonifacino and Hurley, 2008). Although some additional recy-
cling from late endosomal compartments cannot be excluded
(Ghosh et al., 2003; Braulke and Bonifacino, 2009), the coat
protein for facilitating this event is unclear since the previously
favored candidate, TIP47, has now been shown to have other
functions (Bulankina et al., 2009). Plant retromer interacts with
VSRs (Oliviusson et al., 2006), but the recycling of VSRs from the
plant MVB/PVC has recently been challenged by Niemes et al.
Figure 7. Xyloglucans Accumulate after ConcA Treatment and in BFA Compartments.
Immunogold labeling of a fucosylated xyloglucan epitope with CCRC-M1 antibodies. b, BFA compartment; cw, cell wall; g, Golgi stack; t, TGN. Bars =
250 nm in (A) to (D), (F), and (G) and 2 mm in (E).
(A) Labeling of the rims of Golgi stacks and TGN with silver-enhanced Nanogold-F(ab)2.
(B) Labeling of the rims of Golgi stacks (arrowheads) and TGN (arrows) with 12-nm gold particles coupled to IgG. Clustered marker molecules represent
secretory vesicles.
(C) and (D) Labeling of aggregated large Golgi-derived secretory vesicles after treatment with ConcA.
(C) Vesicles close to a Golgi stack.
(E) to (G) Labeling of BFA compartments after treatment with BFA.
(E) Overview showing two vesicles aggregates.
(F) Enlarged BFA compartment.
(G) BFA compartment with attached Golgi stack.
1352 The Plant Cell
(2010a) who have shown that both the small (sorting nexins) and
large subunit of retromer locate to the TGN. Nevertheless, the
presence of VSRs at the MVB/PVC raises the question of
whether they are functionally relevant in this compartment.
In addressing this problem, it must first be stated that VSRs (or
reporters like GFP-BP80) do not always accumulate in the MVB/
PVC. Indeed, the predominant location of VSRs in Arabidopsis
roots is the TGN (Reichardt et al., 2007; this article). Second,
there is now evidence that VSRs may already interact with their
soluble vacuolar cargo ligands in the endoplasmic reticulum
(Niemes et al., 2010b). Third, Niemes et al. (2010a) have shown
that the steady state distribution of VSRs in tobacco (Nicotiana
tabacum) protoplasts can be shifted upstream from the MVB/
PVC to the TGN without affecting vacuolar cargo transport.
Together, these results cast doubt on the functional relevance of
VSRs in the MVB/PVC, suggesting that their presence in this
compartment is a consequence of a gradual accumulation of
inactive receptors destined for degradation (Niemes et al.,
2010a). However, in contrast with BRI1 and BOR1, the VSR
BP80 (80 kD binding protein) is mainly localized to the boundary
membrane of the MVB. In mammalian cells, ubiquitinylation
plays a decisive role in targeting molecules for rapid ESCRT
(endosomal sorting complex required for transport)-mediated
internalization (Raiborg and Stenmark, 2009), and inArabidopsis,
the ESCRT-related CHMP1A and CHMP1B (CHARGED MULTI-
VESICULAR BODY PROTEIN 1A/1B) proteins mediate the sort-
ing of auxin carriers into the internal vesicles of the MVB (Spitzer
et al., 2009). We might therefore assume that for the purpose of
rapid degradation, BOR1, and a portion of BRI1, become
ubiquitinylated and gain access to the interior of the MVB with
the help of the ESCRT complex (Hurley and Emr, 2006). How-
ever, the ubiquitinylation of the cytosolic tail of an intracellular
receptor would effectively curtail its role in recycling, and this
may be the reason that VSRs are not detectable at high levels
inside the MVB. It is also in agreement with the fact that under
conditions of high overexpression, BP80 has been detected at
the tonoplast (da Silva et al., 2005; Fung et al., 2005), which
would indicate that the limiting membrane of the MVB has been
incorporated into the tonoplast. Despite the foregoing argu-
ments, we cannot rule out the possibility of a nonretromer-
mediated recycling of VSRs from theMVB/PVC. However, in this
regard, it should be kept in mind that the cycling of mannosyl
6-phosphate receptors between the TGN and the EE in mam-
malian cells is reflected at the ultrastructural level: large numbers
of budding profiles for CCV and other vesicles are seen at the
TGN and the EE shows extensive tubulation (due to the attach-
ment of the sorting nexins). By contrast, although the plant TGN
has lots of tubules together with vesicle-budding profiles, the
MVB/PVC, in both chemically and high-pressure frozen-fixed
specimens reveals neither tubular extensions nor vesicle bud-
ding profiles.
As a result of the observations presented here, a number of
additional questions concerning the TGN/EE as an independent
compartment now arise. We do not know how long a TGN/EE as
such survives, nor do we know anything about the mechanisms
of TGN-Golgi stack recognition and reattachment. However, the
major challenge will be to understand how the dynamic behavior
of the TGN/EE, with its complex tasks, is made compatible with
its role as the central hub for endocytic and secretory trafficking.
METHODS
Plant Materials and Growth Conditions
Plants expressing BRI1-GFP (Friedrichsen et al., 2000) were a gift
from Joanne Chory. Plants expressing hs:BRI1-YFP or BOR1:BOR1-GFP
were previously described (Geldner et al., 2007; Takano et al., 2010). All
plants used were Arabidopsis thaliana ecotype Columbia-0. Three- to
five-day-old BRI1-GFP, hs:secGFP, and hs:BRI1-YFP seedlings were
grown onMurashige and Skoog (MS) medium + 1% sucrose. The bor1-1/
Figure 8. Model Illustrating the Multiple Functions of the TGN in Plants.
The TGN can be found either separated or closely associated to the Golgi stack. TGNs can associate homotypically, and the process is reversible. BFA
causes the formation of large TGN-derived agglomerates (BFA compartments); we propose that this drug inhibits the process of dissociation between
TGNs.
Dynamics of the Trans-Golgi Network/Early Endosome 1353
BOR1:BOR1-GFP plants were grown in MGRL medium (Fujiwara et al.,
1992) containing 50 mM Fe-EDTA and 0.3 mM boric acid (2B) or 100 mM
boric (+B). All the seedlingswere grown at 228C,with cycles of 16 h of light
and 8 h of dark for 3 to 5 d.
Construction of hs:secGFP
SecGFP as an XhoI/HindIII fragment was amplified from the secGFP
construct of Batoko et al. (2000) using the following primers: (sense)
59-CTGATCAACTCGAGGGATCCAAGGAGATATAACAATGAA-39 and
(antisense) 59-CGTACGGTAAGCTTTTATTTGTATAGTTCATCCATG-
CCA-39, and then subcloned into pGII-HS-tNos (Geldner et al., 2007).
Inhibitor Treatments and FM4-64 Staining
Seedlings were incubated in 1 mL of liquid medium (half-strength MS
medium þ 0.5% sucrose, pH 5.8) containing 45 or 90 mM BFA, 0.5 mM or
2 mM ConcA, 20 mM Wm, 50 mM CHX, or combinations of these inhib-
itors. Endocytosis and colocalization studies were performed with 2 mM
FM4-64. The seedlings were incubated with inhibitors and dye at room
temperature or at 378C for the indicated times. The following stock
solutions were used: 50mMBFA in DMSO:ethanol (1:1), 10 mMConcA in
DMSO, 20mMWm in DMSO, 50mMCHX in DMSO, and 4mMFM4-64 in
DMSO.
Protein Extraction and Immunoblot Analysis
For total protein extracts, 4-d-old etiolated seedlings were ground on ice
and resuspended in extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 0.5% Triton X-100, and 13 Complete protease-inhibitor mix
[Roche]). Protein gel blots and immunodetection were performed as
previously described (Pimpl et al., 2006), using affinity-purified rabbit YFP
antibodies (0.62 mg/mL).
Affinity-Purified YFP Antibodies
YFP was produced in Escherichia coli BL21(DE3) as a fusion protein with
an intein-chitin binding domain (CBD) tag in plasmid vector pTYB11 as
described by Visser et al. (2002). The fusion protein was bound to a
column of chitin beads, and the tag was removed on-column by intein-
mediated splicing induced with 50 mM DTT. Anti-YFP antibodies were
raised in rabbits by four subcutaneous injections, each with 100 mg
purified YFP (Eurogentec). Anti-YFP IgG was purified from the rabbit
serum on a 7-mL column of chitin beads to which 15 mg of the CBD-
intein-YFP fusion protein was bound. Briefly, 15mL of serumwas cleared
by centrifugation and loaded onto the column in 20mMNaPi and 100mM
NaCl, pH 7.0. After extensive washing with the same buffer to remove
noninteracting proteins, anti-YFP IgG was eluted from the column by
decreasing the pH from 3.2 to 3.5 with a steep, 2-mL linear 0 to 20%
gradient of 0.1 M Na-citrate, pH 2.7, in 100 to 80% 20 mM NaPi and 100
mM NaCl, pH 7.0, followed by elution with three volumes of 20% 0.1 M
Na-citrate, pH 2.7, in 80% 20 mM NaPi and 100 mM NaCl, pH 7.0. One-
milliliter fractions of IgG eluting from the column (monitored by ab-
sorbance at 280 nm) were collected in tubes containing 0.1 mL 1 M
unbuffered KPiPi, pH;10, to bring the pH after elution immediately to pH
6.0 to 7.
Transmission Electron Microscopy
For Figures 2E to 2H, 3, 4, and 6, 3- to 5-d-old root tips from Arabidopsis
were high-pressure frozen as described by Bubeck et al. (2008). Freeze
substitution was performed with a Leica EM AFS2 freeze substitution unit
in dry acetone supplementedwith 0.2 to 0.4%uranyl acetate at2858C for
16 h before warming up to2508Cover a 5-h period. Roots were infiltrated
and embedded in Lowicryl HM20 (Polysciences) at 2508C and UV
polymerized for 3 d. Ultrathin sections were incubated with antibodies
against GFP and BP80 at a dilution of 1:1400 and 1:50, respectively.
Gold-conjugated secondary antibodies (BioCell GAR10) were used at a
dilution of 1:50 in PBS supplemented with 1% (w/v) BSA. Sections were
poststained with aqueous uranyl acetate/lead citrate and examined in a
JEM 1400 transmission electron microscope (JEOL) operating at 80 kV.
For Figures 2A to 2D and 7, 3- to 5-d-old seedlings (controls or treated
withConcA for 45min or 45mMBFA for 75min) were high-pressure frozen
as described by Ripper et al. (2008). Frozen samples were freeze-
substituted in acetone containing 0.075% OsO4, 0.5% glutaraldehyde,
and 0.25% uranyl acetate and 2% water (2908C, 72 h; 2608C, 8 h;
2308C, 8 h; Ripper et al., 2008). Samples were washed and rehydrated
with acetone and water containing 0.5% and 0.25% gibberellin, respec-
tively. After additional postfixation in water with 0.35% gibberellin at 08C
for 45 min, samples were washed with water and infiltrated in a mixture of
polyvinylpyrrolidone and sucrose (1.8 M sucrose/20% polyvinylpyrroli-
done; Tokuyasu, 1989) and mounted in a cryo-ultramicrotome (Leica) for
cryosectioning at 21158C. Ultrathin thawed cryosections were labeled
with mAb CCRC-M1 (1:5; Zhang and Staehelin, 1992; Carbosource
Services, University of Georgia), and goat anti-mouse F(ab9)2 coupled to
Nanogold (1:70; Nanoprobes) or goat anti-mouse IgG coupled to 12-nm
gold (1:30; Dianova). Nanogold was silver-enhanced with HQSilver (8.5
min, 228C; Nanoprobes). Labeled and silver-enhanced sections were
embedded in methyl cellulose containing 0.3 to 0.45% uranyl acetate.
Quantitative Analysis of IEM
The quantitative analysis was conducted on ultrathin sections previously
immunolabeled at the most convenient dilution of the respective anti-
bodies. The sections were analyzed and every endosomal compartment
encountered during the screening of cells that did not present folding,
scratches, or any source of unspecific labeling was taken into consider-
ation. The area of the single compartments was calculated using the
image processing program ImageJ (http://rsbweb.nih.gov/ij/). Standard
errors and standard deviations were calculated using Excel (Microsoft).
CLSM
Seedlings were mounted in half-strength MS liquid medium and were
observed under a Zeiss Axiovert LSM510 Meta CLSM using a
C-Apochromat 363/1.2 W corr water immersion objective. At the
Metadetector, the main beam splitters (HFT) 514 and 488 were used.
The following fluorophores (excited and emitted by frame switching in the
multitracking mode) were used: GFP (488 nm [40% laser power]/496 to
518 nm), YFP (514 nm [40% laser power]/518 to 539 nm], FM4-64 (488 nm
[10% laser power]/625 to 689 nm together with GFP or 514 nm [20% laser
power]/636 to 689 nm togetherwith YFP). Pinholeswere adjusted to 1 airy
unit for each wavelength. Postacquisition image processing was per-
formed using the Zeiss LSM 5 image browser and CorelDrawX4.
Spinning Disc Confocal Microscopy
Seedswere surface sterilized and stratified at 48C for 2 d before plating on
half-strength MS and 0.7% agar at pH 5.8. After 2 h of light exposure, the
plates were wrapped in aluminum foil and the seeds were grown for 3
to 4 d at 228C in the vertical position. Seedlings were mounted in half-
strength MS liquid medium. Imaging was performed on a Nikon Ti
inverted confocal microscope featuring a Perkin-Elmer Ultra-View spin-
ning disc confocal using a 360 water immersion objective. YFP was
excited at 488 nm, and RFP was excited at 568 nm. Excitation switching
and shutteringwere performed using theUltra VIEWdiscriminationmode,
and emission filtering was accomplished using the UltraVIEW emission
wheel 527 (W55) for YFP and 455 (W80), 615 (W70) for RFP. Images were
1354 The Plant Cell
acquired with a Hamamatsu EM-CCD high-sensitive (black and white)
camera, driven by Volocity software (Improvision, Volocity Version 5.1.0).
Typical exposures were 102 or 152 ms for ST-YFP and 717 or 849 ms for
VHA-a1-RFP.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: VHA-a1 (At2g28520), BRI1 (At4g38400), BOR1 (At2g47160),
SYP61 (At1g28490), and BP-80 (At3g52850).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. The BRI1-GFP Signal Is Detectable through
the Entire Thickness of the Lowicryl HM20 Resin Block.
Supplemental Figure 2. Efficiency of Cycloheximide Treatment and
Time Course of hs:BRI1-YFP Induction.
Supplemental Figure 3. Immunogold Labeling of Endogenous BP80
in Root Tips from Wild-Type Arabidopsis Plants.
Supplemental Figure 4. Upon High Boron Conditions, BOR1-GFP Is
Endocytosed and Undergoes Rapid Degradation.
Supplemental Figure 5. Effects of 5 h ConcA and BFA Treatment Are
Reversible.
Supplemental Table 1. Further Details on Quantitative Analysis of
Immunogold Electron Microscopy.
Supplemental Movie 1. Spinning Disc Confocal Microscopy Show-
ing an Association Event among TGNs.
Supplemental Movie 2. Spinning Disc Confocal Microscopy Show-
ing a Dissociation Event among TGNs.
Supplemental References.
Supplemental Movie Legends.
ACKNOWLEDGMENTS
We thank Natasha Raikhel for providing the SYP61-CFP line. We
acknowledge Stefan Hillmer for expert advice with IEM, Silke Niemes
for help with CLSM, and Stephanie Gold, Dagmar Ripper, and Barbara
Maier for technical assistance. We thank Ulrike Engel and Christian
Ackermann from the Nikon Imaging Center at BioQuant, University of
Heidelberg. This work was supported by the Deutsche Forschungsge-
meinshaft through SFB 446 (K.S. and Y.-D.S.), RO440/14-1 (D.G.R.),
and the ERA-PG project PRECIAR (C.V. and W.v.D.).
Received November 9, 2009; revised March 22, 2010; accepted April 9,
2010; published April 30, 2010.
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Dynamics of the Trans-Golgi Network/Early Endosome 1357
DOI 10.1105/tpc.109.072637; originally published online April 30, 2010; 2010;22;1344-1357Plant Cell
Vries, David G. Robinson and Karin SchumacherBerg, Walter van Dongen, Sandra Richter, Niko Geldner, Junpei Takano, Gerd Jürgens, Sacco C. de
Corrado Viotti, Julia Bubeck, York-Dieter Stierhof, Melanie Krebs, Markus Langhans, Willy van denEndosome, an Independent and Highly Dynamic Organelle
Merge in the Trans-Golgi Network/EarlyArabidopsisEndocytic and Secretory Traffic in
This information is current as of August 26, 2018
Supplemental Data /content/suppl/2010/04/12/tpc.109.072637.DC1.html
References /content/22/4/1344.full.html#ref-list-1
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