1 Department of Molecular and Cell Biology, University of California, Berkeley,
CA 94720, USA
2 Electron Microscope Laboratory, University of California, Berkeley, CA 94720,
USA
3 Boulder Laboratory for 3-D Fine Structure, Department of MCD Biology,
University of Colorado, Boulder, CO 80309, USA
* Author for correspondence (e-mail: jforte{at}uclink.berkeley.edu )
Accepted 19 December 2001
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Summary |
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To address this controversy we used high-pressure, rapid freezing techniques to fix non-stimulated (resting) rabbit gastric glands for electron microscopy. Ultra-thin (60-70 nm) serial sections were used for conventional TEM; 400-500 nm sections were used for tomography. Images were digitized and models constructed using Midas and Imod software (http://bio3d.colorado.edu ). Images were aligned and contours drawn on specific cellular structures. The contours from a stack of serial sections were arranged into objects and meshed into 3D structures. For resting parietal cells our findings are as follows: (1) The apical canaliculus is a microvilli-decorated, branching membrane network that extends into and throughout the parietal cell. This agrees well with a host of previous studies. (2) The plentiful mitochondria form an extensive reticular network throughout the cytoplasm. This has not previously been reported for the parietal cell, and the significance of this observation and the dynamics of the mitochondrial network remain unknown. (3) H,K-ATPase-rich membranes do include membrane tubules and vesicles; however, the tubulovesicular compartment is chiefly comprised of small stacks of cisternae. Thus a designation of tubulocisternae seems appropriate; however, in the resting cell there are no continuities between the apical canaliculus and the tubulocisternae or between tubulocisternae. These data support the recruitment-recycling model of parietal cell stimulation.
Key words: Tubulovesicles, Membrane trafficking, H,K-ATPase, Exocytosis, Tomography
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Introduction |
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At the heart of the tubulovesicle controversy lies the question of whether
tubulovesicles are a distinct membrane compartment or whether they are
continuous with the apical plasma membrane. These two views lead to very
different models of morphological transformation. The view that tubulovesicles
are a distinct membrane compartment leads to the conclusion that parietal cell
stimulation and consequent relaxation are exocytic and endocytic events,
respectively. This so-called membrane recruitment/recycling hypothesis holds
that proton pump-rich tubulovesicles are recruited to the plasma membrane as
the cells stimulate and then recycle it back to the cytoplasm as food leaves
the stomach and the cells return to a resting state (Forte and Yao, 1996;
Forte et al., 1977). Proponents
of the view that the tubulovesicle system is one membrane continuous with the
plasma membrane posit that the apparent expansion of the apical canaliculus is
actually the result of ions and water moving into the highly collapsed
`tubulovesicular' continuum as a result of osmotic forces generated by the
activated pump (Berglindh et al.,
1980
). This view is called the osmotic expansion hypothesis.
Although there is a wide assortment of structural and functional evidence
to support the membrane recycling hypothesis (Forte and Yao, 1996;
Agnew et al., 1999;
Peng et al., 1997
;
Duman et al., 1999
), arguments
favoring the osmotic expansion view persist. Most of these latter arguments
are based on morphological evidence. For example, Pettitt et al.
(Pettitt et al., 1995
;
Pettitt et al., 1996
) have
contested earlier electron microscopic evidence, noting that glutaraldehyde
fixatives can fragment intracellular membrane compartments and asserting that
the model of resting parietal cell morphology embraced by the
recruitment/recycling hypothesis was based on artefact. These authors employed
rapid-freeze fixation and suggested that the tubulovesicular compartment might
be arranged as a series of tightly coiled membranes, which they alleged were
continuous with each other and with the apical canaliculus. To deal with
functional studies, such as those involving the penetration of electron dense
tracers and measurements of electrical capacitance, they proposed small sized
openings for the `connections' and tight packing of the membrane coils. Using
rapid freeze fixation and high-resolution scanning electron microscopy, Ogata
and his colleagues have offered an alternative interpretation of
tubulovesicular morphology (Ogata
1997
; Ogata and Yamasaki,
2000a
; Ogata and Yamasaki,
2000b
). They proposed that the compartment is actually composed of
multiple flattened membrane sacs, or cisternae. They also proposed that the
cisternae intercommunicate with each other by means of very narrow tubular
connections that are present even in the resting cell, but the communication
with the surface only occurs with stimulation. Thus, it is clear that
important structural details must be elucidated for function of
gastric-acid-secreting cells to be fully appreciated.
In the present work, we used high pressure rapid freezing techniques to fix
samples for electron microscopy and tomography. We sought to investigate
whether the tubulovesicular membranes are, in fact, distinct from the apical
canaliculus, determine the actual morphology of the tubulovesicular membranes
and appreciate the tubulovesicular compartment in the context of other
cellular membranes within the parietal cell. Our data provide an exciting view
of the parietal cell interior. They fully support the claim that
tubulovesicles are distinct from the canalicular membrane and provide evidence
consistent with the tubulocisternal morphology promoted by Ogata
(Ogata, 1997). However, there
is no evidence of permanent continuities between the individual elements of
the tubulocisternal system of the resting parietal cell.
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Materials and Methods |
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Glands were next freeze-substituted on dry ice at -78°C with 2% osmium
tetroxide and 0.1% uranyl acetate in acetone for 3 days and then warmed to
room temperature over 12 hours (McDonald,
1999). After three 10-minute rinses with pure acetone, glands were
infiltrated and embedded in Epon-Araldite resin. Resting gastric gland
preparations were made from three rabbits. Each preparation was embedded in
multiple blocks, and sections were examined in three to five blocks from each
gland preparation.
Image collection
Ultra-thin (60-70 nm thick) serial sections were cut from the embedded
glands with a Reichert Ultracut E microtome and poststained with lead citrate
and uranyl acetate. We visualized the sections in a JEOL 100CX (JEOL USA)
transmission electron microscope operating at 80 kV. Electron micrographs were
collected for the same regions of parietal cell interiors, usually including
at least one canaliculus, in each of the series of sections. Negatives of all
images were subsequently digitized by scanning.
The tomogram was collected as previously described
(Ladinsky et al., 1999;
Marsh et al., 2001
;
McIntosh, 2001
). The tomogram
was generated at the Laboratory for Three-Dimensional Fine Structure at the
University of Colorado, Boulder. Briefly, 400 nm thick sections of the
embedded material were cut on a Leica Ultracut UCT microtome (Leica, Inc.) and
placed on formvar-coated, copper-rhodium slot grids. Following staining as per
above, 15 nm colloidal gold particles were placed on both surfaces of the grid
to serve as fiducial markers for subsequent image alignment. The grid was
stabilized with carbon and placed on a stage capable of high tilt and
rotation. It was visualized with a JEM-1000 (JEOL USA) operating at 750 KeV.
The section was rotated from +60° to -60° with images being captured
at 1.5° intervals; the section was then rotated 90° and a similar
series of images taken. Tomograms calculated from each tilt-series were
combined into a single high-resolution tomogram using a warping procedure
(Mastronarde, 1997
).
Model construction
Models were constructed on Silicon Graphics computers running MIDAS and
IMOD software (Kremer et al.,
1996). Image stacks of particular areas were aligned using MIDAS.
Because the electron beam mildly distorts ultra-thin sections, images had to
be aligned with respect to specific structures of interest and re-aligned to
model structures that were distant from these. This step was not necessary for
the tomograms. We used IMOD to stack the aligned images and to draw contours
(outlines) on specific cellular structures. Contours of the same structure
from different serial sections were arranged into objects using IMOD. Using
the IMODmesh feature of IMOD, we joined the contours of each object to form a
3D model.
Movies of these models rotating in space were made using Mediarecorder and are available at jcs.biologists.org.
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Results |
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|
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In order to appreciate the three-dimensional organization and arrangement
of parietal cell organelles, three-dimensional models were created from serial
ultra-thin sections of gastric glands. Fig.
3 shows the steps in the modeling process applied to a region of
the canaliculi extending through 29 sections accounting for a depth of 2
µm (approximately 120 µm3 volume). Morphological complexity
made it difficult to follow the exact outline of the entire canalicular
surface including each microvillar extension, so we first included only the
surface that outlined the canaliculus at the base of the microvilli.
Fig. 3A represents one section
in the stack and shows contours outlining two canaliculi (in blue); a series
of 29 unconnected contours are assembled into an object in
Fig. 3B; and
Fig. 3C represents the final
meshed model in which three canaliculi wending through the cytoplasm can be
seen. The canaliculi clearly form a branching network, demonstrated by the
joining of two of these structures; we have seen many such interconnections.
In order to appreciate the microvillar relationships at the apical surface, we
first modeled a single segment of a canaliculus as one object, then modeled
the inherent microvilli as a separate series of objects and finally put them
together in a reconstruction. Fig.
4 highlights the interior of an individual canaliculus through
cutaway views of two orientations: a longitudinal
(Fig. 4A) and a cross-sectional
view (Fig. 4B). When one looks
at an individual ultrathin section, a large number of microvilli are typically
seen in cross section, but there is little information about their orientation
within the canaliculus. This reconstructed model, in which only
50% of
the microvilli have been modeled, shows a random distribution of microvilli
projecting up, down or in line with the normal axis of the canaliculus. This
random arrangement is clearly different from what is typically observed in
other microvilli-rich epithelial cells.
|
|
Fig. 5 is a model
reconstructed from a series of mitochondrial profiles from a region of 70
µm3 within the parietal cell cytoplasm. The model clearly
demonstrates that the mitochondria do not resolve into discrete units. In
fact, they form an extensive reticular network coursing throughout the
cytoplasm. The tight radii of some of the interconnections suggest the
possibility of transient connections that may open and close in time, although
this cannot be confirmed within these data.
|
A three-dimensional model of many tubulovesicular structures from a region near a canaliculus is shown in Fig. 6A. This reconstruction from serial sections reveals that the tubulovesicles exist primarily in the form of small cisternal stacks: each stack including from three to six cisternae. Often, small tubular or vesicular structures are apposed to these stacks. Careful review of many sections did not reveal any connections between membranes within the stack; each tubulovesicle (or tubulocisterna) appears to be a discreet membranous unit. Furthermore, orientation of the axis for a given tubulocisternal stack appears to be independent of nearby stacks. Fig. 6B shows a model of several stacks of tubulocisternae juxtaposed with the apical canalicular membrane. Although the membrane stacks are packed quite close to one another and frequently are subadjacent to the canaliculi, we did not observe any visible connections among tubulocisternae or between tubulocisternae and the canaliculus. Thus, while our data clearly show membrane interconnections between canalicular segments, and among mitochondria, they argue against intercompartmental connections in the resting parietal cell.
|
A possible artefact in these models is the distortion of thin section geometry that might be caused by the electron beam, as mentioned above. Also, the modeling data are resolved only between ultrathin sections that are about 70 nm in thickness. To examine the system at a higher point to point resolution, we employed tomography, which involves recording and deconvoluting a series of images at different tilt angles through thick sections of tissue. The tomographic procedure yields a stack of images similar to those obtained through serial sections but without the motional distortions between different sections. Thus the image stack thus possess extraordinary continuity with a distance of only 2.5 nm between successive sections. Individual image quality is also quite good, as seen in Fig. 7.
|
Tomograms taken from 0.4 µm sections were used to model the tubulovesicles/tubulocisternae, employing the same methods with which we modeled the ultra-thin sections. Fig. 7 shows the result of this process. The insert to Fig. 7A represents one section from the stack in which we have outlined the contour of many tubulocisternal profiles. Fig. 7A depicts contoured stacks of tubulovesicles and tubulocisternae within a section about 0.3 µm thick. A stack of contours for a nearby mitochondrion is shown for comparison of size and orientation. In Fig. 7B the profiles have been meshed and the entire reconstruction is rotated. Individual colors were assigned to each membrane structure and maintained through each part of the figure; the red contour lines outline a vicinal mitochondrion. This model is in excellent agreement with Figs 6A and 6B, which were reconstructed from serial sections. Close apposition favors some sort of stacking of the tubulocisternae, and small vesicles and tubules cluster around the stacks. As for the analysis of serial section data, interconnections between individual cisternae, vesicles and tubules are not observed.
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Discussion |
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Ever since the ultrastructure of the parietal cell was first examined more
than 40 years ago, the system of extensive intracellular membranes has been of
great interest, especially with respect to its possible role in the HCl
secretory process. The morphological form of these membranes has variously
been called vesicles, tubules, bulbotubules, tubulovesicles, coiled tubules
and, most recently, tubulocisternae. Although not always conceding to the
specific morphological form, most authors have referred to the system
generically as `tubulovesicles'. These descriptive differences have been due
in part to the animal species examined and in part to the methods of tissue
preservation (Forte and Forte,
1971; Helander,
1981
; Ito, 1987
).
The discovery that H,K-ATPase, the gastric proton pump, was the predominant
protein in tubulovesicles (Forte et al.,
1975
), coupled with secretion-related interconversion between
tubulovesicles and apical canalicular membrane, led to the membrane recycling
hypothesis of acid secretion (Forte et al.,
1977
). However, as noted in the introduction, other authors have
offered alternative views for secretion-related changes in membrane morphology
that very much depend upon the specific form of the tubulovesicular system in
the resting cell (Pettitt et al.,
1995
). Thus the need to examine parietal cell ultrastructure in
three dimensions and with superior fixation techniques is readily
apparent.
From the structural analyses presented here, we conclude that the
H,K-ATPase-rich intracellular membranes in rabbit parietal cells are
predominantly comprised of flattened cisternae, although tubules and vesicles
can also be found. Earlier work using the same high pressure freezing method,
but with freeze-substitution fixation more suitable to immunocytochemistry, we
showed that these membranes are in fact the loci of H,K-ATPase
(Okamoto et al., 2000).
Moreover, there are no apparent permanent connections among the
tubulocisternal components or between the tubulocisternae and the apical
plasma membrane.
We are not the first group to apply rapid freezing methods to the problem
of parietal cell ultrastructure; however, our conclusions are at variance with
several other authors. From their analysis of micrographs and 3D modeling,
Pettitt et al. (Pettitt et al.,
1995) concluded that the H,K-ATPase-rich membrane system was
composed of densely packed helical coils of tubule, each having an axial core
of cytoplasm and joined together by connecting straight tubules. Furthermore
they predicted that the coiled tubules were interconnected with the apical
canalicular membrane and that the secretagoguemediated resting/stimulated
transition was the result of unwinding of the coils, thus exposing the
cytoplasmic cores as shaped elements resembling extended microvilli of the
stimulated apical surface. Although their model is interesting, it is
difficult to reconcile with the raw data. Micrographs of unstimulated parietal
cells in Pettitt et al. (Pettitt et al.,
1995
) reveal a preponderance of elongated membrane profiles,
sometimes straight but often curved. In nine of their published micrographs of
unstimulated parietal cells we counted 75.6% (SD=10.8) of the membrane
profiles as `elongate' (for simplicity we define `elongate' as any profile
whose length is >2x the width; `circular' as a profile where length
is <2x width). For an ideal thin section through a system of straight
tubules we would predict that at least 60% of the image profiles should appear
as `circular'; the percentage would be even higher for curved tubules. Thus a
model of coiled tubules cannot be the predominant form for the H,K-ATPase-rich
intracellular membrane system of mammalian parietal cells. That is not to say
that coiled tubules do not exist or that tubules and vesicles are not among
the H,K-ATPase-rich membranes, but that there would probably be a significant
number of cisternal elements in the pool, as presented by Pettitt et al.
(Pettitt et al., 1995
). We
would also like to point out that while tubulocisternae form the preponderance
of the H,K-ATPase-rich membranes in these mammalian cells, the same is not
true for amphibian cells where the principal morphological form is that of
relatively straight tubular structures
(Forte and Forte, 1971
).
We are also not the first group to suggest that the H,K-ATPase-rich
membranes occur in the form of tubulocisternae. Ogata et al. came to this
conclusion independently using alternate techniques, that is, scanning
electron microscopy of macerated gastric glands
(Ogata and Yamasaki, 2000a,
Ogata and Yamasaki, 2000b
;
Ogata 1997
). However, our
picture of the resting cell cytoplasm is not in complete agreement with
theirs. Ogata's group observed a number of connections, in the form of slender
tubules, between the various tubulocisternae. They also reported that tubules
could also occasionally be seen connecting the apical canaliculus and
tubulovesicles. Although Ogata generally does not favor the osmotic expansion
hypothesis, he does propose a schematic model that includes extensive
interconnections among the elements of the H,K-ATPase-rich membranes. Thus,
despite our similar conclusions about a tubulocisternal structure, there
remain substantive differences in observation and interpretation. The models
we developed from the present data are based on structures that have received
a maximum care in structural preservation, and we find no evidence for
connections within and among the tubulocisternae. Even if connectivities were
difficult to see in ultra-thin sections, they should have been readily
apparent in the high-resolution tomograms.
A functional implication of our model is that it clearly adds support to the membrane recruitment/recycling hypothesis. If the membranes containing the pump are not continuous with the plasma membrane, they surely must be at the time of acid secretion. Aside from this obvious consequence, a number of questions remain. How does the cisternal morphology of H,K-ATPase-rich membranes play a role in the process of acid secretion? Are the tubules and vesicles that we do observe intermediates in a sorting or general housekeeping pathway? How are the cisternal stacks regenerated upon cell relaxation? All of these questions present challenges for the future.
The structure of the apical canaliculus brought few surprises. In agreement
with a host of previous work from light microscopy, the canalicular membranes
were shown to form a network wending through the cytoplasm. Microvilli were
plentiful, even in the resting cell. Although there were no fixed continuities
between the canalicular membrane and tubulocisternae in the resting cell,
there was abundant evidence for endocytic activity of clathrin-rich membranes
as documented in an earlier study (Okamoto
et al., 2000).
Another interesting observation regards the nature of parietal cell
mitochondria. The modeling data clearly show branching and interconnections
among many of the mitochondria, supporting the evolving idea of a large
dynamic mitochondrial network within cells. Using 3D reconstruction of thin EM
sections, Hoffmann and Avers (Hoffmann and
Avers, 1973) reported that mitochondria in Sacchromyces
cerevisiae were arranged in a single large reticular network and
predicted that this might be a common arrangement in eukaryotic cells. Many
subsequent studies suggested that cellular mitochondrial networks are
frequently dynamic. Experimental evidence, both from mitochondrial and
membrane-potential tracers, provides support for the model of a large dynamic
mitochondrial organelle undergoing constant remodeling
(De Giorgi et al., 2000
;
Amchenkova et al., 1998). Moreover, there is evidence for a variety of
specialized mitochondrial fusion/fission proteins
(Otsuga et al., 1998
;
Smirnova et al., 1998
). These
observations raise some fascinating questions about how its structure and
dynamism contribute to the function of mitochondria. In a review of these data
Skulachev has developed an hypothesis whereby mitochondrial connectivities
contribute to power transmission in the form of a transmembrane electrical
potential difference over long distances within (or even between) cells
(Skulachev, 1990
). He pointed
out that mitochondrial reticula are often associated, or appear to be, under
conditions of hard work or energy deficiency, for example, in skeletal muscle
mitochondrial these structures are associated with red rather than white
muscle (Ogata and Yamasaki,
1997
). This may have special relevance to the parietal cell where
there is a prominent mitochondrial presence and energy requirements owing to
the proton pump are enormous. The huge demand of a functioning H,K-ATPase
drives ATP conversion to ADP + Pi at the apical plasma membrane. Oxygen and
substrates to regenerate ATP come into the cell at the basal surface. Even
with extensive canalicular invaginations there are substantial distances, up
to 20 µm, between apical and basal surfaces. The opportunity for power
transmission along mitochondrial networks could reduce the time constraints of
3D diffusion for nucleotides and substrates, providing an efficient and well
coupled use of the entire mitochondrial compartment in the face of a localized
energy sink. Dynamic mitochondrial networks, and their apposition to
Ca2+ storage compartments, have also been implicated in the
regulation of cell signaling (Rizzuto et
al., 1998
). Because of its abundance of mitochondria, marked
difference in energy usage between resting and stimulated states and
modulation of secretory activity by calcium, the parietal cell may present
itself as an ideal native model to address issues of mitochondrial
networks.
The current work offers a new 3D view of parietal cell structures. It also prescribes a number of future directions. We will seek to model the stimulated cells to better appreciate the nature of the stimulated canaliculus. Hopefully we will answer the question as to whether there is an increase in number, as well as length, of apical microvilli in the rest/stimulated transition. Even more exciting, we will model cells transiting both to the stimulated and to the resting state. These observations should greatly assist in addressing the questions that we have posed in this paper regarding the particular advantages and consequences of the model that we have proposed for the resting parietal cell.
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Footnotes |
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