Download Nanoscale Architecture of Endoplasmic Reticulum Export Sites and

Update on Electron Tomography Analysis of Plant Golgi Stacks
Nanoscale Architecture of Endoplasmic Reticulum Export
Sites and of Golgi Membranes as Determined by
Electron Tomography1[W]
L. Andrew Staehelin and Byung-Ho Kang*
Molecular Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309 (L.A.S.);
and Microbiology and Cell Science Department and Integrated Center for Biotechnology Research,
University of Florida, Gainesville, Florida 32611 (B.-H.K.)
Modern architecture is guided by the axiom ‘‘form
follows function,’’ which emphasizes the need for the
shape of a building or an object to reflect its intended
function or purpose. Biologists tend to prefer the
phrase ‘‘form begets function,’’ because in living organisms, form not only reflects on function but also
defines many functional attributes. This relationship
between biological structure and function explains
why one of the central goals of 21st century cell
biologists is to provide a seamless link of structural
understanding between the macroscopic level of tissue
organization to the molecular and even atomic level
organization of the building blocks of cells and tissues.
In turn, this structural knowledge provides architectural constraints for developing testable hypotheses
for how macromolecular complexes, organelles, cells,
and organs operate on a functional level. The goal of
this Update is to summarize new information on the
three-dimensional (3D) architecture of the membrane
systems of the secretory pathway of plant cells produced by dual-axis electron tomography of cells preserved by high-pressure freezing/freeze-substitution
techniques. These studies have redefined our understanding of the nanoscale organization of the membranes of endoplasmic reticulum (ER) export sites,
Golgi stacks, trans-Golgi network (TGN) cisternae,
and associated vesicles and scaffolds, and by doing so
have led to many new insights into the functional
organization of these membrane systems. In addition,
these studies have created a bridge between live
cell imaging by confocal microscopy on one hand
and biochemical and molecular investigations on the
other.
1
This work was supported by the National Institutes of Health
(grant no. GM–61306 to L.A.S.).
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Byung-Ho Kang ([email protected]).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.108.120618
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HIGH-PRESSURE FREEZING AND
LOW-TEMPERATURE PROCESSING
METHODS ARE ESSENTIAL TOOLS FOR
ELECTRON TOMOGRAPHY STUDIES
OF CELLS
The quality of a final electron tomogram is determined primarily by the quality of the initial specimen
preservation. The membranous organelles of the secretory pathway are highly dynamic cellular structures
that undergo changes in organization in the time range
of seconds and even fractions of seconds. Thus, to
preserve these membrane systems in their natural state
for electron microscope (EM) analysis requires fixation
methods that can stabilize cellular structures in a fraction of a second and processing techniques that can
preserve the stabilized structures until they have been
immobilized in polymerized resin.
Chemical fixatives such as glutaraldehyde and osmium tetroxide preserve cellular structures by crosslinking cellular molecules, thereby preserving their
spatial organization. However, because these chemical
reactions are relatively slow and highly selective, immobilization of membranes typically takes minutes,
and even then not all molecules are fixed (Buckley, 1973;
Mersey and McCully, 1978). In practical terms, considering that plant Golgi stacks can travel at speeds of 3 to 4
mm/s along actin filaments (Boevink et al., 1998;
Nebenführ et al., 1999) and that chemical fixatives
cross-link and thereby immobilize different cellular
structures at different rates, it is easy to imagine how a
given Golgi stack that was close to an ER export site at
the onset of fixation can be separated from this site by
microns of distance by the time the fixation process is
completed. Because additional changes in membrane
architecture occur during chemical dehydration prior
to embedding, it is obvious that the analysis of chemically fixed samples by electron tomography will yield
information that is of limited value in terms of defining
the macromolecular architecture of dynamic organelles.
These limitations of chemical fixatives can be largely
overcome with the use of cryofixation techniques, among
which high-pressure freezing is the most widely used
(Kiss and Staehelin, 1995). Unlike chemical fixatives,
cryofixation methods immobilize all cellular molecules
Plant Physiology, August 2008, Vol. 147, pp. 1454–1468, www.plantphysiol.org Ó 2008 American Society of Plant Biologists
Electron Tomography Analysis of Plant Golgi Stacks
(proteins, lipids, carbohydrates, water, salts) simultaneously, and this physical stabilization occurs in approximately 1 ms, which is fast enough to capture most
transient membrane events of interest to cell biologists.
Samples preserved in this manner can then be freezesubstituted at 280°C to 290°C prior to being infiltrated
in resin and fixed in 3D space by resin polymerization
(Morphew, 2007). Due to the tight binding of water
molecules to plant cell wall polysaccharides, freezesubstitution of plant cells takes longer than that of
animal cells. Similarly, optimal preservation of freezesubstituted plant cells requires very slow rates of resin
infiltration (see Segui-Simarro et al., 2004; Austin et al.,
2005). For immunolabeling experiments, the best results are achieved when the freeze-substituted samples
are infiltrated with Lowicryl HM20 at 250°C to 260°C
and polymerized by UV light at the same temperature
(Donohoe et al., 2007). This specimen preparation
method preserves antigenic sites very well as evidenced by the fact that more than 10 antibodies we
have tested recently (commercial monoclonal, as well
as anti-peptide, anti-protein, and anti-polysaccharide
polyclonal antibodies) gave excellent immunolabeling
results. Furthermore, considering the excellent structural preservation of the samples, this method is far
superior to the Tokyasu technique, which is still widely
used (Zeuschner et al., 2006).
ELECTRON TOMOGRAPHY CAN GENERATE 3D
IMAGES OF PLASTIC EMBEDDED SAMPLES WITH
NANOMETER-SCALE RESOLUTION
To comprehend the differences in imaging capabilities of different microscope techniques, it is instructive to compare the resolution that can be obtained
with different microscopes. At present, confocal light
microscopy is the most widely used microscopic technique in cell biology laboratories (Fig. 1A). Typically,
the x/y axis resolution of confocal micrographs is
approximately 200 nm, and the z axis resolution is
500 to 800 nm (Jakobs, 2006). Classical electron microscopy (Fig. 1B), which is based on the imaging of
60- to 80-nm-thick plastic sections, has an x/y axis
resolution of approximately 5 nm and a z axis resolution of 120 to 160 nm (approximately twice the section
thickness). Although this z axis resolution is approximately 4-fold better than what can be obtained with
confocal microscopy, it still limits the ability of researchers to determine the complete 3D architecture of
most cellular organelles and cytoskeletal systems at
high resolution. As discussed below, dual-axis electron
tomography (McIntosh et al., 2005) provides a means
for solving the z axis resolution problem of classical
electron microscopy as well as for producing 3D models of cellular structures with nanoscale resolution.
Electron tomography generates 3D structures of
objects by combining multiple 2D images of the objects
as they are systematically tilted from 160° to 260°
along two orthogonal axes. The desired 3D structure is
computed by back-projecting each 2D image with appropriate weighting (Supplemental Fig. S1; Koster
et al., 1997). The tomogram produced in this manner
constitutes a 3D block of data points that is represented
as an array of volume elements (voxels). Each voxel
corresponds to a cube (1–4 nm per side depending on
resolution), and each cube is assigned a gray scale that
corresponds to the mass density of that region of the
specimen. The voxel information can be displayed in
the form of 2D images of selected z axis levels of the
sample. The resulting voxel images resemble thin
section images (compare Figs. 1C and 2A). However,
because the displayed voxel layer is only approximately 2 nm thick versus 60 to 80 nm for thin sections,
many more details of the cellular structures can be
resolved.
The data presented in this review are from tomograms generated from 100- to 300-nm-thick plastic
sections of high-pressure-frozen/freeze-substituted cells
examined in 200- and 300-kV Tecnai intermediate
voltage EMs. The z axis resolution of the tomograms
is 6 to 8 nm and the x and y axis resolution approxFigure 1. Comparison of the spatial resolution of a
confocal and an EM image of plant Golgi stacks (A and
B). A, Tobacco BY-2 cell expressing a a-1,2 mannosidase I-GFP fusion protein that localizes to Golgi
stacks. In this confocal micrograph, the individual
Golgi stacks are seen as approximately 1 mm in
diameter fluorescent spots. B, Thin section electron
micrograph of a Golgi stack in a high-pressure-frozen
and freeze-substituted BY-2 cell. Note the differences
in architecture and staining of the cis-, medial-, and
trans-Golgi cisternae. Ribosomes (approximately 15-nm
black dots) and an ER cisterna adjacent to the Golgi
stack are also seen (courtesy of A. Nebenführ). C and
D, Enhancement of electron microscopy by tomography techniques. Two-nanometer-thick electron tomographic slice image (C) and a 3D reconstruction (D) of
an Arabidopsis Golgi stack are shown. The 3D model
is generated from tracings of 363 slice images, including the image in C.
Plant Physiol. Vol. 147, 2008
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Staehelin and Kang
Figure 2. Thin section electron micrographs of ER export sites with
budding COPII vesicle profiles (arrowheads) in a tobacco BY-2 cell (A; from
Ritzenthaler et al., 2002) and in an Arabidopsis root columella cell (B).
imately 4 nm. This z axis resolution is approximately
20-fold better than that of classical EM thin sections
and approximately 100-fold better than of typical
confocal images.
To generate a 3D reconstruction of a given cellular
structure from a tomographic data set, the structure
has to be traced in every serial slice image (typically
approximately 2 nm thick) and the tracings combined
into a 3D model that can be viewed from all sides with
the help of dedicated computer software programs
(Fig. 1D; Supplemental Movie S1). The tomographic
reconstructions (models) of ER and Golgi membrane
systems shown in this review involved the generation
of tracings of each membrane compartment in up to
570 sequential slice images! The validity of these
models can be verified by comparing the geometries
of the different membrane compartments with corresponding freeze-fracture micrographs of high-pressurefrozen cells (Craig and Staehelin, 1988). It is instructive
to compare the smoothness of the membranes in the
freeze-fracture micrographs and the corresponding
membrane smoothness seen in the best tomographic
reconstructions. To increase the overall size of a reconstruction, tomograms of serial sections and of montaged images can be joined together (Ladinsky et al.,
1999). The IMOD suite of image processing tools
employed in all of our studies can be downloaded
free of charge at the Web site of the Boulder Laboratory
of 3D Electron Microscopy of Cells (University of
Colorado, Boulder; http://bio3d.colorado.edu/). A more
detailed and practical guide to electron tomography
of membranous organelles is presented in a review
by Donohoe et al. (2006).
CURRENT STATUS OF PLANT SECRETORY
PATHWAY RESEARCH
During the past 10 years, research on secretory
pathway trafficking in plants, i.e. trafficking between
ER, Golgi, and TGN cisternae and beyond, has been
driven by studies using a combination of molecular
biology and confocal microscopy techniques. These
investigations have produced three types of advances:
(1) they have identified and characterized a multitude
of structural and regulatory proteins that operate at
different sites in the secretory pathway; (2) they have
produced insights into targeting and retention signals;
and (3) they have provided information on the dy1456
namic properties of the compartments with which
these proteins are associated. A number of excellent reviews summarizing progress in these fields of
research have been published in recent years (SaintJore-Dupas et al., 2004; Hawes, 2005; Hawes and
Satiat-Jeunemaitre, 2005; Aniento et al., 2006; Hanton
et al., 2006; Lam et al., 2007; Moreau et al., 2007;
Robinson et al., 2007). The purpose of this review is
2-fold: first, to provide an update of studies of the
structural organization of the secretory membrane
systems of plants and, second, to explain the implications of this new information in terms of functional
organization of these membrane compartments.
Research on the Golgi apparatus has a long history
of being controversial (see Farquhar and Palade [1998]
for a review of early studies and Robinson et al. [2007]
for a discussion of more recent controversies in the
plant field). Controversies are beneficial for science as
long as the focus remains on the facts, because it forces
the participants to develop better tests of competing
hypotheses. This competition has definitely aided
plant secretory pathway research. The following is a
current list of agreed-upon facts and controversial
features of this field.
Agreed-upon facts:
1. The plant Golgi apparatus consists of dispersed,
functionally independent Golgi stack units (Moore
et al., 1991; Staehelin and Moore, 1995; Boevink
et al., 1998; Nebenführ et al., 1999).
2. Each Golgi unit is comprised of cisternae that
exhibit a polar organization both with respect to
their architecture and to their function, and a TGN
type of cisterna on the trans-side of the stack
(Staehelin et al., 1990; Zhang et al., 1993).
3. The Golgi units retain their structural integrity and
remain functionally active during mitosis (Nebenführ
et al., 2000; Segui-Simarro et al., 2004; Reichardt et al.,
2007).
4. The Golgi units travel along actin tracks (not microtubules) through the cytoplasm with periods of
rapid translocational movements alternating with
slower and more wiggling motions (Boevink et al.,
1998; Nebenführ et al., 1999).
5. Coat protein complex I (COPI) vesicles bud from the
rims of Golgi cisternae, and secretory vesicles and
clathrin-coated vesicles from TGN cisternae (Staehelin
et al., 1990; Pimpl et al., 2000; Donohoe et al., 2007).
6. Plants produce COPII proteins and these proteins
are essential for ER export (Movafeghi et al., 1999;
Phillipson et al., 2001).
Controversies surround the following features:
1. The mechanism of ER-to-Golgi transport. Is this
transport mediated by vesicles formed at ER export
sites or by tubules (Hawes and Satiat-Jeunemaitre,
2005; Donohoe et al., 2007; Moreau et al., 2007;
Robinson et al., 2007)?
2. The nature of the relationship between ER export
sites and Golgi stacks. Are Golgi stacks permaPlant Physiol. Vol. 147, 2008
Electron Tomography Analysis of Plant Golgi Stacks
nently attached to ER export sites, or is the association with these sites transient (daSilva et al., 2004;
Yang et al., 2005)?
3. The mechanism of trafficking of cargo molecules
through Golgi stacks. Do cargo molecules traffic
according to the cisternal progression/maturation
model or the vesicle shuttle model (Nebenführ,
2003)?
4. The role of the TGN in endocytic trafficking/membrane recycling activities (Geldner, 2004; Lam et al.,
2007).
In the following, we demonstrate that new, highresolution structural information produced with the
help of electron tomography has provided both refining insights into the agreed-upon features of the plant
Golgi apparatus as well as clear answers to some of the
controversial questions.
PLANT ER EXPORT SITES BUD COPII VESICLES BUT
DO NOT PRODUCE TUBULES THAT CONNECT
DIRECTLY TO GOLGI CISTERNAE
One of the longest lasting controversies in plant
Golgi research surrounds the mechanism of transport
of cargo molecules from ER to Golgi. Do COPII-type
vesicles mediate this transport as in other eukaryotic
systems (Bonifacino and Glick, 2004), or does this
transport occur via tubular connections between ER
and Golgi membranes (Hawes and Satiat-Jeunemaitre,
2005; Moreau et al., 2007)? This controversy can be
traced to the use of chemical fixation and staining
methods, which are unable to preserve ER export sites,
termed ERES by some researchers (daSilva et al., 2004),
of plants in their natural state. For example, despite 30
years of efforts, no researcher to date has succeeded in
producing clear images of budding COPII vesicles in
chemically fixed higher plant cells (Robinson, 1980;
Aniento et al., 2006).
The case for tubular connections between ER and
Golgi membranes rests on just two publications (Juniper
et al., 1982; Harris and Oparka, 1983), which employed
chemical fixation and staining protocols that are now
known to produce major changes in the architecture of
ER and Golgi membranes. In particular, the harsh
chemical reaction conditions used during zinc iodine
osmium staining to generate distinctive but membranedistorting stain deposits in ER and Golgi cisternae not
only dramatically alter the fine structure of these
membrane systems, but also completely destroy all
ribosomes and other cytoplasmic proteins. In light of
these facts, and because tubular connections between
ER and Golgi membranes have never been observed in
cells preserved by other specimen preparation methods
(chemical fixation, cryofixation/freeze-substitution, or
cryofixation/freeze-etching), the heavy-metal-staininginduced tubular precipitates described by Juniper et al.
(1982) and Harris and Oparka (1983) should no longer
be used as evidence in support of claims that plants
employ tubules to transport cargo and membrane
Plant Physiol. Vol. 147, 2008
molecules from ER to Golgi cisternae (Brandizzi
et al., 2002; Moreau et al., 2007).
As reported by Yang et al. (2005), live cell imaging of
tobacco (Nicotiana tabacum) BY-2 cells expressing
Sec13:GFP, a COPII coat protein and ER export site
marker, produced ER-associated fluorescent punctate
structures that had an extremely short half-life of only
,10 s and seemed to form randomly over the ER. The
number of such structures was also greater than the
number of Golgi stacks, and the Golgi stacks were seen
to associate intermittently with such sites. These results, which partly contradict the findings of daSilva
et al. (2004; see below), suggest that COPII buds
produced by plant cells are unstable, transient membrane structures that are resorbed back into the ER
unless they are immediately transferred to a Golgi
stack. Considering the slow rate of chemical fixation
compared to the rate of COPII vesicle assembly/disassembly and the rapid translational movements of
the Golgi stacks, it is easy to understand why budding
COPII vesicles and their spatial relationship to Golgi
stacks have never been preserved in chemically fixed
cells. Nevertheless, considering that the first images of
high-pressure-frozen and freeze-fractured root tip
cells demonstrating ER cisternae budding vesicles in
close proximity to Golgi stacks were published 20
years ago (Craig and Staehelin, 1988) and have been
confirmed repeatedly since then (Ritzenthaler et al.,
2002; Donohoe et al., 2007; Robinson et al., 2007), it is
astonishing that the involvement of COPII vesicles in
ER-to-Golgi transport is still being questioned. Figure
2 shows two examples of COPII vesicles budding from
ER export sites as seen in high-pressure-frozen and
freeze-substituted plant cells. One example illustrates
a thin section image of budding COPII vesicles in a
tobacco BY-2 cell and the other a budding COPII
vesicle in a root columella cell of Arabidopsis (Arabidopsis thaliana; Fig. 2B).
One problem that has increased the difficulty of
obtaining unambiguous images of budding COPII
vesicles in high-pressure-frozen plant cells relates to
the 3D architecture of the ER export sites in many
cell types. The problem is illustrated in Figure 3, which
depicts an electron tomography-based 3D reconstruction of an ER export site in a root meristem cell. This
ER export site contains four budding COPII vesicles,
but the vesicles are seen budding from three different
tubular ER domains. This geometry makes it not only
difficult to obtain a thin section image of an ER export
site that clearly illustrates the budding configuration
of a COPII vesicle together with a clear image of an ER
membrane tubule (Fig. 3A), but also makes it nearly
impossible to show in a single thin section micrograph
two or more budding vesicles together with the originating ER membranes. Even in cell types such as
columella cells that contain more sheet-like ER cisternae, the budding COPII vesicles at any given ER
export site are seen to arise on different tubular membrane domains and not on one contiguous membrane
region (Figs. 2B and 3B). The identity of the budding
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Staehelin and Kang
Figure 3. 3D tomographic model images of ER export sites in a root
meristem cell (A) and in a root columella cell (B). A, Four budding
COPII vesicles (arrowheads) are located adjacent to the cis-side of a
Golgi stack (stippled structure). Note that these COPII buds are forming
on three different tubular ER domains and face in different directions. B,
Face-on view of an ER export site associated with the cortical ER of a
columella cell in which the cisternae are more sheet-like. Here too the
COPII buds (arrowheads) are seen budding from different ER domains
in close proximity to the cis-most cisterna (C1, stippled structure) of a
docked Golgi stack.
COPII vesicles seen in high-pressure-frozen cells has
been confirmed by EM immunolabeling with antiAtSar1 antibodies (Donohoe et al., 2007).
COPII VESICLES ARE BORN WITH A SCAFFOLD
LAYER THAT CAPTURES PASSING GOLGI STACKS
AND MEDIATES COPII VESICLE TRANSFER
TO THE GOLGI
Plant Golgi stacks are mobile organelles propelled
by molecular motor proteins along actin filaments
at speeds of up to 4 mm/s (Boevink et al., 1998;
Nebenführ et al., 1999). This mobility raises questions
as to how vesicular trafficking between ER and Golgi
is organized and regulated. Two models have been
proposed to explain this transport. Based on the observation that Golgi stacks alternate between phases of
fast (up to 4 mm/s), linear movements and slower
(,0.4 mm/s), wiggling-type motions at set locations,
the ‘‘stop-and-go’’ model (Nebenführ et al., 1999)
postulates that Golgi stacks are induced by a ‘‘signal’’
to stop at active ER export sites to pick up COPII
vesicles and then resume their fast movement (go
phase) after vesicle transfer. In contrast, and based on
live cell images of ER export sites and Golgi stacks
moving together, the ‘‘ER-Golgi secretory unit’’ model
(daSilva et al., 2004) proposes that ER export sites are
stably coupled to Golgi stacks. However, measurements of the rates of movement of the coupled structures shown in their video images indicate that these
ER-Golgi secretory units move at the speed of slower,
wiggling Golgi and not at the speed of fast Golgi.
We have tested these models by determining the
percentage of Golgi stacks that are closely associated
with an ER export site and the percentage that is
located at a distance from the closest ER cisterna. In
Arabidopsis root apical meristem cells, approximately
70% of the Golgi were associated with an ER export
site, whereas in the gravity-sensing columella cells,
only approximately 15% of the Golgi stacks were
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located in the vicinity of an ER cisterna (B.-H. Kang
and L.A. Staehelin, unpublished data). These results
contradict the prediction of the ER-Golgi secretory
unit model that close to 100% of the Golgi stacks
should be coupled to an ER export site, while supporting the contention of the stop-and-go model that Golgi
stacks associate transiently with ER export sites. This
begs the question as to the nature of the postulated
Golgi ‘‘stop signal’’ produced by ER export sites, and
how Golgi stacks become physically coupled to ER
export sites as observed by daSilva et al. (2004). Our
electron tomograms suggest that both of these activities
are mediated by scaffold-type molecules that assemble
on COPII vesicles and are transferred to the cis-side of
the Golgi matrix together with the vesicles.
The COPII-type vesicles that bud from ER export
sites in Arabidopsis are approximately 60 nm in diameter. In both thin sections and tomographic slice
images, their COPII coat proteins produce a darker
staining of the membrane around the buds compared
to the adjacent ER membrane (Figs. 2 and 4). In
addition to the COPII coat, budding COPII vesicles
also assemble a scaffold layer on their outer surface,
which is seen as a 40-nm-thick ribosome-excluding
zone (Fig. 4, A and B). This COPII scaffold, then, binds
directly to the cis-most region of the Golgi matrix,
which encompasses each Golgi stack in a cocoon-like
fashion (Figs. 4, C and D, and 5). In yeast, COPII
vesicles are tethered to the Golgi membrane by a long
coiled-coil protein, Uso1p (Cao et al., 1998), and the
Arabidopsis genome encodes a protein displaying
significant amino acid similarity with Uso1p
(At3g27530). Based on the affinity of the COPII scaffold to the cis-side of the Golgi matrix, the COPII
scaffold is likely to contain the Arabidopsis Uso1related protein. The COPII scaffold layer linked to the
cis-Golgi matrix merges with the matrix structure,
transferring enclosed COPII vesicles to the Golgi.
(Figs. 4, E–H, and 13A). These observations suggest
that the COPII scaffold proteins mediate both the
physical coupling between ER export sites and Golgi
stacks, noted by daSilva et al. (2004), as well as the
‘‘signaling’’ that has been postulated to induce passing
Golgi stacks to stop at active ER export sites (Nebenführ
et al., 1999). They also explain why drugs that perturb
the actin cytoskeleton do not inhibit ER-to-Golgi transport in plant cells (Brandizzi et al., 2002).
The functionality of the scaffold-mediated coupling
between ER export sites and Golgi stacks is supported
also by the finding that Golgi stacks docked to an ER
export site possessed 3 times more free COPII vesicles
in the vicinity of their cis-Golgi cisternae than Golgi
located .300 nm from an ER cisterna (B.-H. Kang and
L.A. Staehelin, unpublished data). Based on these and
other findings, we have formulated the ‘‘dock, pluck
and go’’ model of ER-to-Golgi trafficking shown in
Figure 6. The model postulates that the scaffold of
budding COPII vesicles captures passing Golgi by
binding to the cis-side of the Golgi matrix and that this
docking mechanism automatically orients the cis-side
Plant Physiol. Vol. 147, 2008
Electron Tomography Analysis of Plant Golgi Stacks
of the Golgi toward the ER export site. It also suggests
that the wiggling of the connected Golgi stacks provides the energy to pluck the budding COPII vesicles
from the ER and that, when the COPII vesicle harvesting is complete, the Golgi is free to resume its translational movement along the actin filaments. This
mechanical harvesting mechanism has yet to be tested
experimentally, but it would greatly increase the efficiency of ER-to-Golgi vesicle transport by ensuring
that COPII vesicles are released from an ER export site
only when a Golgi stack is available to pick up the
freed vesicles. It also explains why no COPII vesicle
fission machinery has been identified in biochemical,
molecular, or genetic studies of yeast or mammalian
cells (Kirchhausen, 2000).
ELECTRON TOMOGRAMS PROVIDE STRUCTURAL
EVIDENCE FOR ASSEMBLY INTERMEDIATES OF
CIS-GOLGI CISTERNAE
The proof for cis-to-trans vectorial transport of cargo
molecules through Golgi stacks was provided in the
1960s (for review, see Farquhar and Palade, 1981), but
how these molecules are moved from one side of a
stack to the other has remained controversial for several decades. The two principal hypotheses are the
‘‘vesicle shuttle’’ and the ‘‘cisternal progression/maturation’’ models of intra-Golgi transport (Farquhar
and Palade, 1981; Becker et al., 1995; Glick and
Malhotra, 1998; Nebenführ, 2003). The vesicle shuttle
model proposes that the stacked Golgi cisternae are
stable, long-lived structures that serve as repositories
of sets of processing enzymes and that the cargo
molecules are transported from one cisterna to the
next in an anterior direction by shuttling vesicles. In
contrast, the cisternal progression model postulates
that the secretory cargo never leaves individual cisternae, and that transport across the stack is brought
about by the assembly of new cisternae on the cis-side
of the stack and the fragmentation or shedding of old
cisternae on the trans-side. According to this model,
the function of the COPI-type vesicles that bud from
the rims of the cisternae is to transport the cisternal
enzymes in a retrograde direction to maintain the
characteristic polar distribution of those enzymes
across the stack. As discussed by Nebenführ (2003),
the preponderance of evidence now supports the
cisternal progression/maturation model, but several
major predictions of the two models have yet to be
tested experimentally.
Figure 4. Transfer of COPII vesicles and their scaffolds to the cis-Golgi
matrix. A and B, Tomographic slice image (A) and a corresponding
model (B) of a COPII vesicle budding from an ER membrane. The COPII
bud is surrounded by an approximately 40-nm-wide, ribosome-excluding
scaffold. C and D, Tomographic slice image (C) and a corresponding
model (D) of a COPII bud (arrowhead), which is still attached to the ER
but whose scaffold has become connected to the cis-side of the Golgi
Plant Physiol. Vol. 147, 2008
matrix. E and F, Tomographic slice image (E) and a corresponding
model (F) of a released COPII vesicle (arrow) that is still close to the ER
membrane. The COPII scaffold is linked to the Golgi matrix. G and H,
Tomographic slice image (G) and a corresponding model (H) illustrating a COPII vesicle (arrow) that has been transferred to the cis-face
of the Golgi stack together with its COPII scaffold. The distal side of
the COPII vesicle is completely surrounded by the Golgi matrix. Scale
bars 5 300 nm.
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Staehelin and Kang
Figure 5. Electron tomographic model of a Golgi stack and its
encompassing, ribosome-excluding scaffold (Golgi matrix). A TGN
cisterna is also contained within the trans-side extension of the Golgi
matrix.
To date, the four major lines of experimental evidence that support the cisternal progression/maturation model are (1) bulky secretory products such as
algal scales (Becker et al., 1995), collagen complexes
(Bonfanti et al., 1998), and protein aggregates (Hillmer
et al., 2001) never leave the cisternae during intra-Golgi
transport; (2) immunolabeling and biochemical fractionation experiments have demonstrated that COPItype vesicles transport primarily Golgi enzymes in a
retrograde direction and not cargo molecules in an
anterograde direction (Martinez-Menarguez et al.,
1999; Lanoix et al., 2001); (3) live cell imaging has
demonstrated maturational changes in individual
Golgi cisternae in Saccharomyces cerevisiae (Losev et al.,
2006; Matsuura-Tokita et al., 2006); and (4) live cell
imaging and correlative electron tomography studies
of Pichia pastoris cells have shown that trans-Golgi
cisternae are shed from Golgi stacks (Bevis et al., 2002;
Mogelsvang et al., 2003). One question that has yet to be
answered unambiguously is the type of compartment
COPII vesicles fuse with upon arriving at the Golgi.
According to the cisternal progression model, cis-Golgi
cisternae should be highly variable in size and shape,
reflecting the de novo assembly of such cisternae from
COPII vesicles. In contrast, the vesicle shuttle model
predicts cis-Golgi cisternae to possess a more uniform
and mature architecture.
In thin section electron micrographs of high-pressurefrozen/freeze-substituted plant Golgi stacks, cis-, medial-,
and trans-Golgi cisternae can be positively identified by
means of three structural criteria: position in a stack,
lumenal staining, and cisternal geometry (Staehelin
et al., 1990). Golgi stacks in Arabidopsis and tobacco
are typically composed of five to seven cisternae, the
first two of which are usually classified as cis-type. cis1460
Type cisternae are smaller than medial- and transcisternae, they have a variable shape, and their lumen
are not only wider but also very lightly stained (Fig. 1,
B and C).
The tremendous structural variability of plant cisGolgi cisternae is illustrated in the three tomographic
models of Figure 7. These models depict cis-side, faceon views of three different root apical meristem Golgi
stacks, with the cis-most (C1) cisterna colored orange
and the second cis-cisterna (C2) colored green. The
most striking difference between the C1 cisternae
shown in Figure 7 is the huge variability in their size
and shape. Thus, whereas the first stack displays only
one tiny cisternal initial and the second one two
branched, tubular cisternal initials, the third stack
has a C1 cisterna that is more mature and disc-shaped
and has tubular extensions along its margins. The
models also suggest that fusion of the COPII vesicles
with growing C1 cisternae occurs primarily at the ends
of the tubular domains. In all instances, the growing
C1 cisternae appear to use the C2 cisternae as a growth
template, as evidenced by the fact that C1 cisternae
never extend beyond the edge of the C2 cisterna on
which they are growing. Furthermore, C2 cisternae are
always smaller than the C3 cisternae and associated
with both COPII and COPIa-type but not COPIb-type
vesicles (see below), consistent with the idea that they
too are growing cisternae. Together, these models
provide evidence for the de novo assembly of cisGolgi cisternae as postulated by the cisternal progression/maturation model.
Figure 6. Dock, pluck, and go model of ER-to-Golgi vesicle trafficking.
A, Go-phase: Golgi stacks travel along actin filaments propelled by
myosin motors. B, Dock- and Pluck-phase: The COPII scaffold of a
budding vesicle attaches to the cis-side of the Golgi matrix and pulls the
passing Golgi off the actin track. Once the COPII scaffold binds to the
Golgi matrix, the wiggling motion of the Golgi stack facilitates release
of the COPII vesicles by plucking. After COPII vesicle harvesting, the
Golgi is free to resume its movement along the actin track.
Plant Physiol. Vol. 147, 2008
Electron Tomography Analysis of Plant Golgi Stacks
Figure 7. Face-on views of three Golgi stack models in which the cismost (C1) cisternae are colored orange (arrows) and the underlying C2
cis-cisternae are colored green. The cis-most cisternae vary in size from
a small blob (A), to two intermediate-sized, branched tubules (B), and
to a slightly larger disc with tubular extensions around its margins (C).
These highly variable shapes are consistent with the de novo assembly
of cis-Golgi cisternae from COPII vesicle (yellow spheres) as postulated
by the cisternal progression model of Golgi trafficking.
CIS-GOLGI CISTERNAE ARE BIOSYNTHETICALLY
INACTIVE DURING THEIR ASSEMBLY BUT ENABLE
PROTEIN AGGREGATE/COMPLEX FORMATION
Another basic question that has not been addressed
in the plant Golgi literature is whether cis-Golgi cisternae are functionally active while they are being
assembled or whether they only become functionally
active after their assembly is complete. One of the
characteristic structural features of cis-Golgi cisternae
in high-pressure-frozen/freeze-substituted plant cells
is the lack of luminal staining of cis-Golgi cisternae
(Figs. 1, B and C, and 15D), i.e. they appear devoid of
stainable materials except for seed storage protein
aggregates in cisternal margins (Otegui et al., 2006)
and cell wall scale backbone complexes in Scherffelia
dubia (Donohoe et al., 2006, 2007). Indeed, the structural transition between cis- and medial-cisternae is
marked by the sudden appearance of stained luminal
contents in the medial-cisternae (Fig. 1B; Staehelin
et al., 1990; Donohoe et al., 2007).
To determine if the lack of luminal staining of cisGolgi cisternae reflects a lack of biosynthetic activities,
we have mapped the distribution of a-1,2 mannosidase I, the enzyme that mediates the first reaction of
the N-linked glycan-processing pathway in the Golgi
(Moremen et al., 1994), using an antibody that detects
the two native a-1,2 mannosidase I isotypes of Arabidopsis (At1g51590 and At3g21160). Our data demonstrate (Fig. 8) that native a-1,2 mannosidase I localizes
nearly exclusively to medial-Golgi cisternae and is
essentially absent from cis-Golgi cisternae and the
ER. This labeling pattern contrasts with studies of
transgenic cells expressing a-1,2 mannosidase I-GFP
constructs in which the labeled protein is seen to
accumulate in both cis- and medial-Golgi cisternae
(Nebenführ et al., 1999) and even ER cisternae (SaintJore-Dupas et al., 2006), and highlights the pitfalls
of using GFP fusion proteins for such localization
studies.
The abrupt change in luminal staining from cis- to
medial-Golgi cisternae suggests that the accumulation
Plant Physiol. Vol. 147, 2008
of sugar nucleotides, the activation of biosynthetic
enzymes, and the appearance of cargo molecules with
modified glycans only begins in medial-Golgi cisternae, i.e. only after the assembly of the cis-Golgi cisterna is complete. This finding is also consistent with
the demonstration that the backbone epitopes of pectic polysaccharides and xyloglucans can be detected
by immunological means only in medial- and transbut not in cis-Golgi cisternae (Zhang and Staehelin,
1992).
In electron micrographs of high-pressure-frozen and
freeze-substituted cells of the alga S. dubia (Donohoe
et al., 2007) and of the yeast P. pastoris (Mogelsvang
et al., 2003), the cis-Golgi cisternae display the same
type of unstained lumen as in plants. This suggests
that the cis-Golgi cisternae in these organisms are also
biosynthetically inactive during their assembly. In
support of this hypothesis, the cis-Golgi cisternae of
the scale-forming alga Scherffelia only contain wispy
cell wall scale complexes, and these complexes only
become more heavily glycosylated and stained once
they reach the medial-cisternae (L.A. Staehelin, unpublished data). Taken together, these results suggest
that in walled cells, i.e. cells that employ H1-gradients
to create turgor pressure and to drive their plasma
membrane transport systems, cisternal assembly and
biosynthesis of secretory products are mutually incompatible Golgi activities and therefore occur sequentially during cisternal maturation.
Figure 8. Immunoelectron tomography localization of native a-1,2
mannosidase I (ManI) in an Arabidopsis meristem Golgi stack. A to C,
Electron micrographs of three serial thin sections labeled with antiManI 15-nm immunogold particles. D, 3D tomographic model of the
Golgi stack and immunogold particles seen in A to C. Note that nearly
all of the immunogold particles are associated with the C3 and C4
medial-cisternae (arrowheads). The Golgi cisternae models were rendered semitransparent to make all of the gold particles visible. Scale
bars 5 100 nm.
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Staehelin and Kang
Figure 9. COPII-, COPIa-, and COPIb-type vesicles of S. dubia. A to C,
A gallery of tomographic slice images showing COPII-type vesicles. D
to F, A gallery of tomographic slice images showing COPIa-type
vesicles. G to I, A gallery of tomographic slice images depicting
COPIb-type vesicles. In C, F, and I, models of COPII, COPIa, and
COPIb vesicles are overlaid on tomographic slices, respectively. J, 3D
model of Golgi associated vesicles superimposed on a single slice of an
S. dubia Golgi stack illustrating the nonoverlapping distribution of
COPIa-type (light green spheres) and COPIb-type vesicles (light purple
spheres). COPII-type vesicles (gold spheres) colocalize with COPIatype vesicles, though. Light blue and pink spheres in the trans-side of
the Golgi represent secretory and clathrin-coated vesicles (Donohoe
et al., 2007). Scale bar 5 100 nm.
GOLGI STACKS PRODUCE TWO TYPES OF COPI
VESICLES, COPIA AND COPIB
As already discussed in preceding sections, COPIItype vesicles transport membrane and cargo molecules
from ER to Golgi. In contrast, COPI vesicles originate
on Golgi cisternae and transport cisternal membrane
1462
proteins in a retrograde direction (Malhotra and Mayor,
2006). The Golgi-to-ER, COPI vesicle-mediated transport is responsible for returning escaped ER proteins to
the ER, whereas the COPI vesicles involved in retrograde intra-Golgi transport help maintain the steady
state of enzyme distribution within Golgi stacks as the
individual cisternae traverse the stacks in an anterograde direction. This latter type of retrograde transport has been postulated to operate between the TGN
and cis-Golgi cisternae and to possibly extend back to
the ER. Here we address the question whether COPI
vesicles derived from medial- and trans-Golgi cisternae can recycle molecules directly back to the ER, or if
they are limited to intra-Golgi transport activities.
To answer this question, we took advantage of the
increased resolution of electron tomographic images of
high-pressure-frozen/freeze-substituted Golgi stacks
in which COPII and COPI vesicles can be identified
by means of six structural criteria: coat architecture,
coat thickness, membrane structure, cargo staining,
cisternal origin, and spatial distribution (Donohoe
et al., 2007). In addition, COPI and COPII buds and
vesicles were positively identified by immunolabeling
techniques. Using this multi-parameter approach and
the ability to map the distribution of all vesicles in 3D
space, we demonstrated that COPII and COPI vesicles
are confined to well-defined regions around ER export
sites and Golgi cisternae and that Golgi stacks produce
two types of COPI vesicles, COPIa and COPIb.
COPII vesicles bud from ER export sites and localize
to the space between the ER and cis-Golgi cisternae
(Fig. 9, A–C). COPIa vesicles bud exclusively from cisGolgi cisternae and, like these cisternae, they have
lightly stained contents (Fig. 9, D–F). They are also
confined to the ER-cis-Golgi interface, colocalizing
with COPII vesicles. In contrast, the COPIb vesicles
bud exclusively from medial, trans, and early TGN
cisternae, have darkly stained luminal contents like
the cisternae from which they originate (Fig. 9, G–I),
and are confined to the space around these latter
cisternae. To determine the generality of this distribution pattern, we compared the distribution of COPIa
and COPIb vesicle types in Arabidopsis and in Scherffelia, which contains two Golgi stacks, each of which
generates a new cis-Golgi cisterna every 20 to 30 s
(McFadden et al., 1986). Due to the large size of those
Golgi stacks and the high rate of cisternal turnover, the
number of COPII and COPI vesicles produced by
Scherffelia is very large, which allows for the clear
demarcation of the domains in which the COPIa and
COPIb operate (Fig. 9J). In Arabidopsis, the distribution patterns of COPIa- and COPIb-type vesicles are
the same as in Scherffelia, but due to the much smaller
number of vesicles, the patterns can be confirmed only
by means of statistical analysis of the vesicle distribution around many Golgi stacks. A model summarizing
the trafficking patterns of COPII, COPIa, and COPIb,
as well as of clathrin-coated and secretory vesicles
around ER, Golgi, and TGN cisternae (Fig. 10), is illustrated in Figure 10. Based on these vesicle distribution
Plant Physiol. Vol. 147, 2008
Electron Tomography Analysis of Plant Golgi Stacks
Figure 10. Schematic diagram illustrating the sites of origin and the
trafficking routes of five types of ER/Golgi/TGN-associated vesicles.
COPIa-type vesicles bud from assembling cis-cisternae and recycle
molecules back to the ER, whereas the COPIb-type vesicles are
produced by medial, trans, and early TGN cisternae and recycle
molecules between these cisternae.
patterns, the COPIa vesicles are postulated to recycle
escaped ER proteins from forming cis-Golgi cisternae
back to the ER, and the COPIb vesicles to mediate
retrograde transport of Golgi enzymes between early
TGN, trans-, medial-, and late-stage cis-Golgi cisternae.
PLANT GOLGI STACKS DIVIDE BY FISSION
Three mechanisms of Golgi division have been
reported in the literature (Munro, 2002): (1) the ‘‘disintegration and reassembly model,’’ which appears to
be the principal mechanism in mammalian cells; (2)
the ‘‘de novo construction model’’ employed by the
yeast P. pastoris (Bevis et al., 2002) and by mammalian
cells treated with microtubule-disrupting drugs such
as nocodazole (Cole et al., 1996); and (3) the ‘‘fission
model’’ used in plants, algae, and protozoa (Morre
et al., 1971; Benchimol et al., 2001; Pelletier et al., 2002).
In Arabidopsis shoot meristem cells, the number of
Golgi stacks has been shown to double from approximately 34 to approximately 66 during the G2 stage of
the cell cycle, i.e. just prior to mitosis (Segui-Simarro
and Staehelin, 2006). Golgi stack duplication can also
occur in interphase cells during cell growth. The
fission model of Golgi multiplication arose in the
1960s based on electron micrographs of Golgi stacks
that exhibited two partial stacks attached to one or
several larger trans-cisternae (for review, see Morre
et al., 1971), but the mechanism of division has
remained a mystery. Assuming that Golgi stacks traffic
according to the cisternal progression/maturation
model discussed above, the fission model predicts
that duplication starts with the assembly of two halfsized cis-cisternae instead of just one on the surface of
Plant Physiol. Vol. 147, 2008
a laterally expanded C2 cisterna. Each of these new,
separate cisternae then serves as an independent template for the assembly of the next cisterna. As this
process is repeated and as the older trans-side cisternae are shed, the two new stacks increase in size,
eventually giving rise to two complete and separate
daughter Golgi stacks when the last joining transcisterna is released.
The tomographic model of a dividing Golgi stack
illustrated in Figure 11 provides several additional
lines of evidence for the fission model. First, the
dividing Golgi is seen to be docked to an active ER
export site, and several released COPII vesicles are
located adjacent to the cis-Golgi cisternae. Second, the
two cis-most Golgi cisternae (C1, C2) are also smaller
than the underlying older cisternae and display morphological features of assembling cis-Golgi cisternae
(compare Fig. 11B with Fig. 7). Finally, the model
shows three TGN cisternae at different stages of maturation and moving away from the Golgi stack, which
supports the interpretation that this Golgi stack was
actively shedding trans-cisternae at the time of cryofixation. Together, these structural features provide
strong direct support for the fission model of plant
Golgi multiplication.
TRANSFORMATION OF A TRANS-MOST GOLGI
CISTERNA INTO A TGN CISTERNA INVOLVES A
REDUCTION IN SURFACE AREA AND
RECRUITMENT OF THE GTPASE RABA4B
By definition, the TGN sorts and packages Golgi
products into secretory and clathrin-coated vesicles for
transport to the cell surface and to vacuoles/lysosomes,
respectively (Griffiths and Simons, 1986). In plants,
TGN cisternae arise from the trans-most Golgi cisternae
by means of a cisternal peeling process that leads to the
separation of the TGN cisterna from the stack and to the
formation of independent TGN compartments (Figs.
11C, 12, 13, and 14). These independent TGN compartments continue to undergo maturational changes until
the entire cisterna fragments into vesicles and small,
residual membrane fragments (Figs. 12 and 14). TGN
cisternae that are in the process of peeling away from a
Golgi stack are referred to as early TGN cisternae (Fig.
12B), and those that have completed this separation
are referred to as late TGN cisternae (Fig. 12C).
In small meristem cells, where Golgi movements
and cytoplasmic streaming are restricted during interphase (B.-H. Kang and L.A. Staehelin, unpublished
data) and stop during cell division (Nebenführ et al.,
2000), electron tomographic reconstructions often
show multiple TGN cisternae at different distances
from the trans-side of a Golgi stack (Figs. 11C and 13).
In contrast, in large interphase suspension-cultured
cells that exhibit fast Golgi movements and vigorous
cytoplasmic streaming (Nebenführ et al., 1999), very
few Golgi stacks possess a late TGN cisterna near their
trans-face, and many stacks even seem to lack a
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Staehelin and Kang
originating Golgi stacks. Free-floating TGN cisternae
have been observed in high-pressure-frozen and
freeze-substituted pine cambial cells, which also exhibit rigorous streaming (Rensing et al., 2002).
Transformation of a trans-Golgi cisterna into an
early TGN cisterna starts with cisternal peeling, a
reduction in surface area (approximately 30% in Arabidopsis root meristem cells) by COPI vesicle-mediated
recycling, the formation of secretory vesicles outside
the plane of the cisterna, and the binding of antiRabA4b and anti-phosphatidyl inositol-4-kinase b antibodies (Preuss et al., 2006; B.-H. Kang and L.A.
Staehelin, unpublished data). In contrast, clathrincoated vesicle formation is not a reliable early TGN
marker, because the production of such vesicles can be
delayed until most secretory vesicles have formed on
late TGN cisternae (Figs. 11 and 13). Both RabA4b
and phosphatidyl inositol-4-kinase b appear to be
components of the coat protein complexes of budding secretory vesicles that contain mostly complex
carbohydrate-type cell wall matrix polysaccharides
(B.-H. Kang and L.A. Staehelin, unpublished data).
LATE TGN CISTERNAE CONTINUE TO MATURE AS
INDEPENDENT ORGANELLES AND RELEASE THEIR
VESICLES BY MEANS OF FRAGMENTATION
The maturational steps of TGN cisternae can be
deduced from the sequence of structural and compositional changes seen in reconstructed meristem GolgiTGN complexes in which the older TGN cisternae are
displaced further from the trans-side of the Golgi
stacks (Figs. 11C and 13). The sequence of morphological changes associated with TGN maturation
shown in Figure 14 is typical for meristematic cells,
except that clathrin-coated buds often form already on
early TGN cisternae (Fig. 13, D–E).
Early TGN cisternae display a central, flat domain
where they were attached to the Golgi stack. After
their release from the stack, the late-type TGN cisternae
continue to generate budding secretory and clathrincoated vesicles, yielding mature cisternae with a
Figure 11. 3D tomographic model of a dividing Golgi stack docked to an
ER export site in an Arabidopsis root meristem cell. A, The two sets of cisand medial-cisternae are held together by a larger trans-Golgi cisterna
(pink). The Golgi stack is connected to budding COPII vesicles via
interactions between the COPII scaffolds and the cis-Golgi matrix (arrowheads). B, Face-on view of the dividing Golgi stack shown in A. The C1
(orange) and C2 (green) cisternae of the two cis-side stacks (C1, C2, C1’,
and C2’ marked with arrows) display varying shapes resembling the
assembling cis-most cisternae in Figure 7. C, On the trans-side, three TGN
cisternae (TGN1–TGN3) at different stages of maturation are seen.
distinct TGN cisterna (Zhang and Staehelin, 1992).
These observations suggest that shearing forces generated by Golgi movements and cytoplasmic streaming can dislodge and then quickly disperse late and
sometimes even early TGN cisternae away from the
1464
Figure 12. 3D models (face-on views) of a trans-most Golgi, an early
TGN, and a late TGN cisterna of meristem cell Golgi stacks illustrating
the TGN maturation process. CCV, Clathrin-coated vesicle; SV, secretory vesicle.
Plant Physiol. Vol. 147, 2008
Electron Tomography Analysis of Plant Golgi Stacks
different ratios of these proteins will be locally produced and then delivered to individual Golgi stacks,
thereby leading to constantly changing mixtures of
cargo molecules in Golgi and TGN cisternae. These
findings highlight the inherent plasticity of the TGN
compartments of plant cells and their ability to quickly
adapt their sorting and packaging systems to the type
of cargo molecules present.
Electron tomography has also enabled us to address
the question as to what happens to TGN cisternae at
the end of their life. Do they release completed secretory and clathrin-coated vesicles one at a time, withering away as this loss of membrane and contents
occurs, or do they release all of their vesicles simultaneously by means of cisternal fragmentation? Based
on a quantitative analysis of large cytoplasmic volumes surrounding Golgi stacks, we estimate that
,10% of the secretory and clathrin-coated vesicles
are released prior to the fragmentation of late TGN
cisternae. Thus, cisternal fragmentation appears to the
principal mechanism for releasing TGN vesicles (Fig.
14A). Mollenhauer (1971) was the first to describe the
fragmentation of what he called mature dictyosome
cisternae. In the model depicted in Figure 14, 20
secretory vesicles and eight clathrin-coated vesicles
together with four membrane fragments of variable
size can be discerned. Yet to be determined is the fate
of the residual membrane fragments.
CONCLUDING REMARKS
Figure 13. A, Tomographic slice image of an Arabidopsis meristem
cell with two Golgi stacks (Golgi 1, Golgi 2). Golgi stack 1 (B and C) is
associated with a late TGN cisterna that is budding mostly secretory
vesicles (SV). In contrast, the late TGN cisterna associated with Golgi
2 (D and E) is producing mostly clathrin-coated buds (CCV). This
latter type of TGN is also known as partially coated reticulum
(Pesacreta and Lucas, 1985). Scale bar in A 5 250 nm, scale bars in
B to E 5 200 nm.
grape-like architecture (Figs. 12C and 13, B and C).
Interestingly, the ratio of complex carbohydratecontaining secretory vesicles to clathrin-coated vesicles on TGN cisternae can vary from 5:1 to 1:3 within a
single cell (Fig. 13, A–E). The clathrin-coated, bud-rich
TGN cisternae appear to correspond to membrane
compartments known as partially coated reticulum
(Pesacreta and Lucas, 1985). The variation in vesicle
type ratios is most likely due to the presence of varying
ratios of vacuolar to secretory cargo molecules within
individual TGN cisternae. Obviously, depending on
when and where the translation of mRNAs coding for
vacuolar and secretory proteins occurs on the ER,
Plant Physiol. Vol. 147, 2008
The principal goals of this Update were to develop a
coherent framework of nanoscale structural information on ER export sites, Golgi stacks, and TGN cisternae of higher plants and to demonstrate how this
information can be used to address questions related
to Golgi trafficking and Golgi function. The review
started out by highlighting the importance of integrating several new, cutting-edge research tools to gain
paradigm-shifting insights into these dynamic membrane systems. It then followed the path taken by
Figure 14. 3D model images of a Golgi stack and its TGN cisternae. A,
The late TGN cisterna has fragmented into a cluster of secretory vesicles
(SV) that surround small, residual membrane fragments. CCV, Clathrincoated vesicle. B, Face-on view of the late TGN model shown in A in
which the secretory and clathrin-coated vesicles have been omitted to
illustrate the morphologies of the residual fragments of the cisternal
membrane (arrowheads).
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Staehelin and Kang
cargo molecules though these membrane systems,
describing the 3D architecture of ER export sites, the
mechanism of ER-to-Golgi transport, the assembly of
new cis-Golgi cisternae, the immunolocalization of
native Golgi enzymes, trafficking of cargo and membrane enzymes, the importance of the Golgi matrix, the
mechanism of Golgi stack division, and the structural
and functional plasticity of TGN cisternae. Together,
this new information should aid other cell biologists in
not only interpreting their confocal microscope images
of secretory pathway organelles but also appreciating
the limits of the confocal microscopy technique. In
addition, the new nanoscale database should be a
valuable resource for helping molecular biologists,
biochemists, and geneticists plan novel types of studies of the plant secretory pathway.
Where might these advanced EM and EM tomography techniques be employed next? The greatest
need would appear to be in the field of plant cell endocytosis, where confusion reigns supreme with respect to the role of the TGN in endocytosis (Geldner,
2004; Lam et al., 2007). In that field, confocal microscopy studies have reached a resolution-based ceiling,
and major progress will require comprehensive,
correlative confocal and EM studies of entire cells or
large cellular volumes together with the quantitative
accounting of all of the membrane compartments,
including vesicles, that traffic endocytosed and secretory molecules. A critical challenge of these studies will be to distinguish compartments that are
involved in transporting the actual endocytic cargo
molecules to the lytic vacuoles from those that serve
as recipients of recycled molecules from early endosomal compartments. This analysis will also require
a much more detailed characterization of the physicochemical properties and uptake mechanisms of the
tracer molecules used in endocytosis experiments
such as cationized ferritin and the FM4-64-type dye
molecules. Last but not least, these next generation
studies will have to distinguish within the TGN
which proteins and polysaccharides are new and
being packaged for secretion and which molecules
have been brought to this and other compartments
via endocytosis. The following articles provide examples of the type of comprehensive 3D EM/EM
tomography studies needed in the field of endocytosis research: Golgi region of a liver cell (amazing
models!; Marsh et al., 2001); membranes and microtubules involved in syncytial-type cytokinesis
(Otegui et al., 2001); membranes and microtubules
involved in somatic cell cytokinesis (Segui-Simarro
et al., 2004); and cell cycle-dependent changes in
Golgi, TGN, vacuole, and multivesicular body
compartments in entire cells (Segui-Simarro and
Staehelin, 2006).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Principles of electron tomography.
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Supplemental Movie S1. 3D model of the Arabidopsis Golgi stack in
Figure 1.
Received April 4, 2008; accepted May 22, 2008; published August 6, 2008.
LITERATURE CITED
Aniento F, Matsuoka K, Robinson DG (2006) ER-to-Golgi transport: the
COPII pathway. In DG Robinson, ed, The Plant Endoplasmic Reticulum.
Springer Verlag, New York, pp 99–124
Austin JR II, Segui-Simarro JM, Staehelin LA (2005) Quantitative analysis
of changes in spatial distribution and plus-end geometry of microtubules involved in plant-cell cytokinesis. J Cell Sci 118: 3895–3903
Becker B, Bolinger B, Melkonian M (1995) Anterograde transport of algal
scales through the Golgi complex is not mediated by vesicles. Trends
Cell Biol 5: 305–307
Benchimol M, Ribeiro KC, Mariante RM, Alderete JF (2001) Structure and
division of the Golgi complex in Trichomonas vaginalis and Tritrichomonas foetus. Eur J Cell Biol 80: 593–607
Bevis BJ, Hammond AT, Reinke CA, Glick BS (2002) De novo formation of
transitional ER sites and Golgi structures in Pichia pastoris. Nat Cell
Biol 4: 750–756
Boevink P, Oparka K, Santa Cruz S, Martin B, Betteridge A, Hawes C
(1998) Stacks on tracks: the plant Golgi apparatus traffics on an actin/ER
network. Plant J 15: 441–447
Bonfanti L, Mironov AA Jr, Martinez-Menarguez JA, Martella O, Fusella
A, Baldassarre M, Buccione R, Geuze HJ, Mironov AA, Luini A (1998)
Procollagen traverses the Golgi stack without leaving the lumen of
cisternae: evidence for cisternal maturation. Cell 95: 993–1003
Bonifacino JS, Glick BS (2004) The mechanisms of vesicle budding and
fusion. Cell 116: 153–166
Brandizzi F, Snapp EL, Roberts AG, Lippincott-Schwartz J, Hawes C
(2002) Membrane protein transport between the endoplasmic reticulum
and the Golgi in tobacco leaves is energy dependent but cytoskeleton
independent: evidence from selective photobleaching. Plant Cell 14:
1293–1309
Buckley IK (1973) Studies in fixation for electron microscopy using
cultured cells. Lab Invest 29: 398–410
Cao X, Ballew N, Barlowe C (1998) Initial docking of ER-derived vesicles
requires Uso1p and Ypt1p but is independent of SNARE proteins.
EMBO J 17: 2156–2165
Cole NB, Sciaky N, Marotta A, Song J, Lippincott-Schwartz J (1996)
Golgi dispersal during microtubule disruption: regeneration of Golgi
stacks at peripheral endoplasmic reticulum exit sites. Mol Biol Cell 7:
631–650
Craig S, Staehelin LA (1988) High pressure freezing of intact plant tissues. Evaluation and characterization of novel features of the endoplasmic reticulum and associated membrane systems. Eur J Cell Biol 46:
81–93
daSilva LL, Snapp EL, Denecke J, Lippincott-Schwartz J, Hawes C,
Brandizzi F (2004) Endoplasmic reticulum export sites and Golgi bodies
behave as single mobile secretory units in plant cells. Plant Cell 16:
1753–1771
Donohoe BS, Kang BH, Staehelin LA (2007) Identification and characterization of COPIa- and COPIb-type vesicle classes associated with plant
and algal Golgi. Proc Natl Acad Sci USA 104: 163–168
Donohoe BS, Mogelsvang S, Staehelin LA (2006) Electron tomography of
ER, Golgi and related membrane systems. Methods 39: 154–162
Farquhar MG, Palade GE (1981) The Golgi apparatus (complex)-(19541981)-from artifact to center stage. J Cell Biol 91: 77s–103s
Farquhar MG, Palade GE (1998) The Golgi apparatus: 100 years of progress
and controversy. Trends Cell Biol 8: 2–10
Geldner N (2004) The plant endosomal system: its structure and role in
signal transduction and plant development. Planta 219: 547–560
Glick BS, Malhotra V (1998) The curious status of the Golgi apparatus. Cell
95: 883–889
Griffiths G, Simons K (1986) The trans Golgi network: sorting at the exit
site of the Golgi complex. Science 234: 438–443
Hanton SL, Matheson LA, Brandizzi F (2006) Seeking a way out: export of
proteins from the plant endoplasmic reticulum. Trends Plant Sci 11:
335–343
Plant Physiol. Vol. 147, 2008
Electron Tomography Analysis of Plant Golgi Stacks
Harris N, Oparka K (1983) Connections between dictyosomes, ER and
GERL in cotyledons of mung bean. Protoplasma 114: 93–102
Hawes C (2005) Cell biology of the plant Golgi apparatus. New Phytol 165:
29–44
Hawes C, Satiat-Jeunemaitre B (2005) The plant Golgi apparatus: going
with the flow. Biochim Biophys Acta 1744: 93–107
Hillmer S, Movafeghi A, Robinson DG, Hinz G (2001) Vacuolar storage
proteins are sorted in the cis-cisternae of the pea cotyledon Golgi
apparatus. J Cell Biol 152: 41–50
Jakobs S (2006) High resolution imaging of live mitochondria. Biochim
Biophys Acta 1763: 561–575
Juniper GE, Hawes C, Horne JC (1982) The relationships between the
dictyosomes and the forms of endoplasmic reticulum in plant cells with
different export programs. Bot Gaz 143: 135–145
Kirchhausen T (2000) Three ways to make a vesicle. Nat Rev Mol Cell Biol
1: 187–198
Kiss JZ, Staehelin LA (1995) High pressure freezing. In DM Shotton, ed,
Rapid Freezing, Freeze Fracture and Deep Etching. Wiley-Liss, New
York, pp 89–104
Koster AJ, Grimm R, Typke D, Hegerl R, Stoschek A, Walz J, Baumeister
W (1997) Perspectives of molecular and cellular electron tomography.
J Struct Biol 120: 276–308
Ladinsky MS, Mastronarde DN, McIntosh JR, Howell KE, Staehelin LA
(1999) Golgi structure in three dimensions: functional insights from the
normal rat kidney cell. J Cell Biol 144: 1135–1149
Lam SK, Tse YC, Robinson DG, Jiang L (2007) Tracking down the elusive
early endosome. Trends Plant Sci 12: 497–505
Lanoix J, Ouwendijk J, Stark A, Szafer E, Cassel D, Dejgaard K, Weiss M,
Nilsson T (2001) Sorting of Golgi resident proteins into different
subpopulations of COPI vesicles: a role for ArfGAP1. J Cell Biol 155:
1199–1212
Losev E, Reinke CA, Jellen J, Strongin DE, Bevis BJ, Glick BS (2006) Golgi
maturation visualized in living yeast. Nature 441: 1002–1006
Malhotra V, Mayor S (2006) Cell biology: the Golgi grows up. Nature 441:
939–940
Marsh BJ, Mastronarde DN, Buttle KF, Howell KE, McIntosh JR (2001)
Organellar relationships in the Golgi region of the pancreatic beta cell
line, HIT-T15, visualized by high resolution electron tomography. Proc
Natl Acad Sci USA 98: 2399–2406
Martinez-Menarguez JA, Geuze HJ, Slot JW, Klumperman J (1999) Vesicular tubular clusters between the ER and Golgi mediate concentration
of soluble secretory proteins by exclusion from COPI-coated vesicles.
Cell 98: 81–90
Matsuura-Tokita K, Takeuchi M, Ichihara A, Mikuriya K, Nakano A
(2006) Live imaging of yeast Golgi cisternal maturation. Nature 441:
1007–1010
McFadden GI, Preisig HR, Melkonian M (1986) Golgi apparatus activity
and membrane flow during scale biogenesis in the green flagellate
Scherffelia dubia (Prasinophyceae). 2. Cell wall secretion and assembly.
Protoplasma 131: 174–184
McIntosh R, Nicastro D, Mastronarde D (2005) New views of cells in 3D:
an introduction to electron tomography. Trends Cell Biol 15: 43–51
Mersey B, McCully M (1978) Monitoring the course of fixation of plant
cells. J Microsc 166: 43–56
Mogelsvang S, Gomez-Ospina N, Soderholm J, Glick BS, Staehelin LA
(2003) Tomographic evidence for continuous turnover of Golgi cisternae
in Pichia pastoris. Mol Biol Cell 14: 2277–2291
Mollenhauer HH (1971) Fragmentation of mature dictyosome cisternae.
J Cell Biol 49: 212–214
Moore PJ, Swords KM, Lynch MA, Staehelin LA (1991) Spatial organization of the assembly pathways of glycoproteins and complex polysaccharides in the Golgi apparatus of plants. J Cell Biol 112: 589–602
Moreau P, Brandizzi F, Hanton S, Chatre L, Melser S, Hawes C, SatiatJeunemaitre B (2007) The plant ER-Golgi interface: a highly structured
and dynamic membrane complex. J Exp Bot 58: 49–64
Moremen KW, Trimble RB, Herscovics A (1994) Glycosidases of the
asparagine-linked oligosaccharide processing pathway. Glycobiology
4: 113–125
Morphew MK (2007) 3D immunolocalization with plastic sections. In JR
McIntosh, ed, Cellular Electron Microscopy, Vol 79. Elsevier, San Diego,
pp 494–511
Morre JD, Mollenhauer HH, Bracker CE (1971) Origin and Continuity of
Golgi Apparatus. Springer-Verlag, New York, pp 82–126
Plant Physiol. Vol. 147, 2008
Movafeghi A, Happel N, Pimpl P, Tai GH, Robinson DG (1999) Arabidopsis Sec21p and Sec23p homologs. Probable coat proteins of plant
COP-coated vesicles. Plant Physiol 119: 1437–1446
Munro S (2002) More than one way to replicate the Golgi apparatus. Nat
Cell Biol 4: E223–224
Nebenführ A (2003) Intra-Golgi transport: escalator or bucket brigade? In
DG Robinson, ed, The Golgi Apparatus and the Plant Secretory Pathway, Vol 9. Blackwell Publishing, Oxford, pp 76–89
Nebenführ A, Frohlick JA, Staehelin LA (2000) Redistribution of Golgi
stacks and other organelles during mitosis and cytokinesis in plant cells.
Plant Physiol 124: 135–151
Nebenführ A, Gallagher LA, Dunahay TG, Frohlick JA, Mazurkiewicz
AM, Meehl JB, Staehelin LA (1999) Stop-and-go movements of plant
Golgi stacks are mediated by the acto-myosin system. Plant Physiol 121:
1127–1142
Otegui MS, Herder R, Schulze J, Jung R, Staehelin LA (2006) The
proteolytic processing of seed storage proteins in Arabidopsis embryo
cells starts in the multivesicular bodies. Plant Cell 18: 2567–2581
Otegui MS, Mastronarde DN, Kang BH, Bednarek SY, Staehelin LA
(2001) Three-dimensional analysis of syncytial-type cell plates during
endosperm cellularization visualized by high resolution electron tomography. Plant Cell 13: 2033–2051
Pelletier L, Stern CA, Pypaert M, Sheff D, Ngo HM, Roper N, He CY, Hu
K, Toomre D, Coppens I, et al (2002) Golgi biogenesis in Toxoplasma
gondii. Nature 418: 548–552
Pesacreta TC, Lucas WJ (1985) Presence of a partially-coated reticulum in
angiosperms. Protoplasma 123: 173–184
Phillipson BA, Pimpl P, daSilva LL, Crofts AJ, Taylor JP, Movafeghi A,
Robinson DG, Denecke J (2001) Secretory bulk flow of soluble proteins
is efficient and COPII dependent. Plant Cell 13: 2005–2020
Pimpl P, Movafeghi A, Coughlan S, Denecke J, Hillmer S, Robinson DG
(2000) In situ localization and in vitro induction of plant COPI-coated
vesicles. Plant Cell 12: 2219–2236
Preuss ML, Schmitz AJ, Thole JM, Bonner HK, Otegui MS, Nielsen E (2006) A
role for the RabA4b effector protein PI-4Kb1 in polarized expansion of root
hair cells in Arabidopsis thaliana. J Cell Biol 172: 991–998
Reichardt I, Stierhof YD, Mayer U, Richter S, Schwarz H, Schumacher K,
Jürgens G (2007) Plant cytokinesis requires de novo secretory trafficking
but not endocytosis. Curr Biol 17: 2047–2053
Rensing KH, Samuels AL, Savidge RA (2002) Ultrastructure of vascular
cambial cell cytokinesis in pine seedlings preserved by cryofixation and
substitution. Protoplasma 220: 39–49
Ritzenthaler C, Nebenführ A, Movafeghi A, Stussi-Garaud C, Behnia L,
Pimpl P, Staehelin LA, Robinson DG (2002) Reevaluation of the effects
of brefeldin A on plant cells using tobacco Bright Yellow 2 cells
expressing Golgi-targeted green fluorescent protein and COPI antisera.
Plant Cell 14: 237–261
Robinson DG (1980) Dictyosome-endoplasmic reticulum associations
in higher plant cells? A serial-section analysis. Eur J Cell Biol 23:
22–36
Robinson DG, Herranz MC, Bubeck J, Pepperkok R, Ritzenthaler C
(2007) Membrane dynamics in the early secretory pathway. Crit Rev
Plant Sci 26: 199–225
Saint-Jore-Dupas C, Gomord V, Paris N (2004) Protein localization in the
plant Golgi apparatus and the trans-Golgi network. Cell Mol Life Sci 61:
159–171
Saint-Jore-Dupas C, Nebenführ A, Boulaflous A, Follet-Gueye ML,
Plasson C, Hawes C, Driouich A, Faye L, Gomord V (2006) Plant
N-glycan processing enzymes employ different targeting mechanisms
for their spatial arrangement along the secretory pathway. Plant Cell 18:
3182–200
Segui-Simarro JM, Austin JR II, White EA, Staehelin LA (2004) Electron
tomographic analysis of somatic cell plate formation in meristematic
cells of Arabidopsis preserved by high-pressure freezing. Plant Cell 16:
836–856
Segui-Simarro JM, Staehelin LA (2006) Cell cycle-dependent changes in
Golgi stacks, vacuoles, clathrin-coated vesicles and multivesicular bodies in meristematic cells of Arabidopsis thaliana: a quantitative and
spatial analysis. Planta 223: 223–236
Staehelin LA, Giddings TH Jr, Kiss JZ, Sack FD (1990) Macromolecular
differentiation of Golgi stacks in root tips of Arabidopsis and Nicotiana
seedlings as visualized in high pressure frozen and freeze-substituted
samples. Protoplasma 157: 75–91
1467
Staehelin and Kang
Staehelin LA, Moore I (1995) The plant golgi appratus: structure, functional organization and trafficking mechanisms. Annu Rev Plant Physiol
Plant Mol Biol 46: 261–288
Yang YD, Elamawi R, Bubeck J, Pepperkok R, Ritzenthaler C, Robinson
DG (2005) Dynamics of COPII vesicles and the Golgi apparatus in
cultured Nicotiana tabacum BY-2 cells provides evidence for transient
association of Golgi stacks with endoplasmic reticulum exit sites. Plant
Cell 17: 1513–1531
Zeuschner D, Geerts WJ, van Donselaar E, Humbel BM, Slot JW, Koster
AJ, Klumperman J (2006) Immuno-electron tomography of ER exit sites
1468
reveals the existence of free COPII-coated transport carriers. Nat Cell
Biol 8: 377–383
Zhang GF, Driouich A, Staehelin LA (1993) Effect of monensin on plant
Golgi: re-examination of the monensin-induced changes in cisternal
architecture and functional activities of the Golgi apparatus of sycamore
suspension-cultured cells. J Cell Sci 104: 819–831
Zhang GF, Staehelin LA (1992) Functional compartmentation of the Golgi
apparatus of plant cells: immunocytochemical analysis of high-pressure
frozen- and freeze-substituted sycamore maple suspension culture cells.
Plant Physiol 99: 1070–1083
Plant Physiol. Vol. 147, 2008