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Ultrastructural localization of concanavalin A-binding sites in the Golgi apparatus of various types of neurons in rat dorsal root ganglia Functional implications.

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THE AMERICAN JOURNAL OF ANATOMY 177%-95 (1986)
Ultrastructural Localization of Concanavalin A-Binding Sites in the
Golgi Apparatus of Various Types of Neurons in Rat Dorsal Root
Ganglia: Functional Implications
F. MALCHIODI, A. RAMBOURG, Y. CLERMONT, AND A. CAROFF
Dkpartment de Bwlogie du CEA, 91191 Ckdex, Saclay, France F: M., A.R.,
A. C.); and Department of Anatomy, McGill University, Montreal, Canada
H3A ZBZ (Y C.)
ABSTRACT
The localization of concanavalin A (con A) binding sites has
been determined at the electron-microscopic level in the six types of neurons
(Al, Az, As, B1, B2, C) of rat dorsal root ganglia. In all ganglion cells, con A
stained the plasma membrane, the nuclear envelope, the cisternae of the rough
endoplasmic reticulum, and the matrix of some multivesicular bodies. In contrast, the con A reactivity of the Golgi apparatus varied according to cell type.
In type B1 and Bz cells and possibly in type A3 cells, the lectin was exclusively
located in three or four saccules on the cis side of the Golgi stacks, whereas the
TPPase-positive saccules and the trans sacculotubular elements were unstained with con A. In type Al, A2, and C neurons, all Golgi saccules as well
as the trans sacculotubular elements were stained with the lectin. These
results suggest that different types of glycoproteins were produced in these
two groups of neurons. In the type Al, Az, and C cells, the persistence of the
lectin reactivity in the WPase-positive saccules or sacculotubular elements on
the trans side of the Golgi stacks suggests the presence of glycoproteins with
oligosaccharide side chains rich in a-D-mannosyl residues in terminal positions. In contrast, the disappearance of the con A reactivity in equivalent
elements of the Golgi stacks in type B1, B2, and A3 cells suggests the addition
a t this level of other sugar residues characteristic of complex oligosaccharide
side chains. The majority of the vesicular elements associated with the Golgi
apparatus, as well as lysosomes, were unstained with con A.
An analysis with the electron microscope
of the tridimensional structure of the components of the Golgi apparatus and the use of
phosphatase cytochemistry has permitted the
characterization of several distinct compartments within this organelle. A comparative
study of the Golgi apparatus of various cell
types also revealed that, although the general structure of this organelle was usually
similar, the relative development and cytochemical properties of its various compartments varied between cell types (Rambourg
et al., 1974, 1979, 1981, 1984; Hermo et al.,
1980; Clermont et al., 1981; Lalli, 1983; Rambourg and Clermont, 1986). In dorsal root
ganglia, differences in the localization of
phosphatases (Novikoff, 1967; Boutry and
Novikoff, 1975)or in the incorporation of sugars in the side chains of glycoproteins (Droz,
0 1986 ALAN R. LISS, INC.
1967) have been reported in the Golgi apparatus of the two main classes of neurons
found therein, i.e., the large and light type A
neurons and the small and dark type B neurons. Rambourg et al. (1983), on the basis of
the ultrastructural appearance and distribution of organelles within the perikarya of
neurons, proposed a classification of ganglion
cells into six types (Al, A2, As, B1, Bz, C).
The characteristic features of the various
types of ganglion cells are the following: Type
Al are large neurons in which Nissl bodies,
distributed throughout the cytoplasm, are
Dr. Fiorella Malchiodi’s present address is Istituto Superiore
di Sanit6, Viale Regina Elena 299,00161 Roma, Italia.
Address reprint requests to Dr. A. Rambourg, Departement de
Biologie du CEA, Gif-sur-Yvette,91191 CBdex, Saclay, France.
Received December 16, 1985. Accepted May 19, 1986.
82
F.MALCHIODI ET AL.
CON A-BINDING SITES IN NEURON GOLGI APPARATUS
separated from each other by narrow cytoplasmic spaces containing small stacks of
Golgi saccules and mitochondria. Type A2
are large neurons with Nissl bodies also distributed throughout the cytoplasm but separated from each other by wider strands of
neuroplasm containing Golgi stacks and mitochondria. Type A3 are the smallest of type
A cells and display densely packed Nissl bodies and long stacks of Golgi saccules which
form a perinuclear ring in the midportion of
the perikaryon. Type B1 are small neurons
with Nissl bodies showing parallel cisternae
located mainly in the outer cytoplasmic zone
and large, curved stacks of Golgi saccules
which form a broad perinuclear net. Type B2
are small neurons showing a cortical zone
composed mainly of Nissl substance, and a
Golgi apparatus forming a ring separated
from the nucleus by a zone of cytoplasm containing mitochondria and smooth ER. Type
C are the smallest of the ganglion cells and
contain small, poorly demarcated Nissl bodies and a juxtanuclear Golgi apparatus (Rambourg et al., 1983).
It thus became of interest to analyze at the
ultrastructural level the binding of concana-
CM
ER
G
L
m
MVB
NE
v
Abbreviations
cytoplasmic membrane
endoplasmic reticulum
Golgi apparatus
lysosome
mitochondria
multivesicular body
nuclear envelope
vesicles
Fig. 1. Low-power EM photograph of a type C cell.
Con A is seen along the cell-membrane surface of the
satellite cells (S)and of the neuron. In the perikaryon
itself, the lectin is located in cisternae of the rough
endoplasmic reticulum and in the Golgi apparatus. In
the latter, all saccules or sacculotubular elements are
reactive with the lectin. The nuclear envelope is also
positively stained. The rest of the nucleus (N) and mitochondria are unstained. x 18,000,
Fig. 2. EM photograph of the perikaryon of a type Bz
cell. The cytoplasmic membrane, the cisternae of rough
endoplasmic reticulum forming a Nissl body, as well as
the nuclear envelope are reactive to con A. Some saccules of a Golgi stack are also stained with the lectin,
but the cis (osmiophilic) element (arrowhead) and trans
saccular structures (arrow) are unreactive with the lectin. Vesicles in the Golgi region (asterisk), a multivesicular body, a dense granule identified as a lysosome, as
well as mitochondria, are unlabeled with the lectin.
~39,000.
83
valin A (con A) to the various types of neurons, to study their Golgi apparatus in
particular, using a con A-horseradish peroxidase complex to detect glycoconjugates containing a-D-mannopyranosyl residues (Gold
stein and Hayes, 1978). The results obtained
revealed marked differences in the distribution of the lectin in the Golgi apparatus of
the various types of neurons, the functional
implication of which will be examined closely.
MATERIALS AND METHODS
Adult rats were perfused through the left
ventricle with a 2.5% solution of glutaraldehyde in 0.1 M sodium cacodylate buffer. After
15 min of perfusion, the L4 and L5 dorsal root
ganglia were removed and postfixed for a n
additonal hour in the same fixative. After a
rinse in cacodylate buffer, they were impregnated in 30% glycerol and frozen in partly
solidified freon 22 cooled by liquid nitrogen.
The pieces of ganglia were transferred into
liquid nitrogen and 20- to 30-pm-thick sections were cut on a cryostat. The sections
were thawed in 30% glycerol and immersed
in a phosphate-buffered saline (PBS) solution
containing 0.05% saponin for 40 min at room
temperature. They were incubated overnight
a t room temperature in a solution of con A
(IBF, 200 pg/ml) in PBS, rinsed with PBS,
and treated with peroxidase (type I1 Sigma,
200 pg per ml in PBS) for 3 hr at room temperature. The bound peroxidase was then revealed by incubating sections €or 15 min a t
room temperature in a PBS solution containing diaminobenzidine (DAB, grade 11, Sigma,
1 mg per ml) and 0.5% H202. Control sections were incubated in a con A solution containing 0.1 M methyl mannoside, or were
treated with peroxidase or DAB without the
lectin pretreatment.
After a last rinse in PBS, treated or control
sections were postfixed €or 1 hr at room temperature in a 1:l mixture of 2% aqueous osmium tetroxide and 3% aqueous potassium
ferrocyanide (Karnovsky, 19711, dehydrated
in ethanol, and embedded in Epon. Thin sections were prepared and mounted on copper
grids. They were counterstained for 2 min in
lead citrate and examined at 80 kV with a
Philips 400 electron microscope.
For the demonstration of the thiamine pyrophosphatase (TPPase) activity the glutaraldehyde-fixed ganglia were kept overnight
at 4°C in a solution of 0.1 M sodium cacodylate containing 4% sucrose and 0.05% CaClz
a t pH 7.2. They were then sectioned a t a
84
F. MALCHIODI ET AL.
Figs. 3, 4. Sections treated with con A but not counterstained with lead acetate.
Fig. 3. Perikaryon of a type B1 neuron. Cisternae of
the rough endoplasmic reticulum as well as some elements of the Golgi stacks are stained with the lectin.
Other Golgi elements are unstained with con A (arrowheads). In the concavity of a Golgi stack, some trans
tubular elements also show a reactivity to con A (aster-
isk). Other cytoplasmic organelles such as mitochondria
are unstained. x 19,000.
Fig. 4. Perikaryon of a type A2 cell showing con A
binding to the rough endoplasmic reticulum and multivesicular bodies. The Golgi apparatus is also stained
with the lectin; but in this cell and in contrast to the
type B1 cell above (Fig. 3), all saccules or sacculotubular
elements of the stack are reactive. ~ 2 3 , 5 0 0 .
CON A-BINDING SITES IN NEURON GOLGI APPARATUS
thickness of 80-100 pm with an Oxford vibratome. The sections were incubated for 2
hr at 37°C in the medium (pH 7.2) described
by Novikoff and Goldfkcher (1961). After incubation the sections were washed in buffer,
rinsed in sodium cacodylate containing sucrose (4%)and CaClz (0.05%),postfixed and
stained with K-ferrocyanide-reduced osmium, and embedded in Epon. Thin sections
were counterstained with lead citrate before
examination with the electron microscope.
RESULTS
Reactivity of the various types of ganglion
cells to con A
In sections treated with con A and examined at the low magnifications of the EM,
several structures were sharply outlined in
the various types of ganglion cells. As illustrated in a C cell (Fig. 1) and a B2 cell (Fig.
2), the plasma membrane, nuclear envelope,
and cisternae of the rough endoplasmic reticulum (ER) were well stained with con A. In
addition several saccules and sacculotubular
elements of the Golgi apparatus were also
stained (Figs. 1, 2). In the type B2 cell, the
outer cortical zone of the perikaryon containing the con A-positive cisternae of Nissl bodies was easily distinguished from the ER-free
juxtanuclear region in which con A-positive
Golgi elements contrasted sharply with unstained mitochondria and profiles of the
smooth endoplasmic reticulum (Fig. 2). In
control sections no con A binding was observed. The reactivity of cellular structures
with con A was emphasized in sections that
were not counter-stained with lead citrate
(Figs. 3, 4). In such sections, the selective
staining of ER cisternae and of some components of the Golgi apparatus was evident;
but it also became apparent that, while in
type B1 and B2 cells and also possibly in the
rare type A3 cells only some of the Golgi
saccules were reactive with con A (Fig. 3), in
type Al, A2, and C neurons all saccules of
the Golgi stacks were reactive (Fig. 4). However, a better understanding of the con A
reactivity of the Golgi apparatus of the various types of neurons was obtained only when
sections counterstained with lead citrate
were examined at higher magnifications of
the electron microscope.
Con A reactivity of the Golgi apparatus of
type BI, B2,and A3 neurons
In type B1 and B2 cells, three or four lectinpositive elements were observed on the cis
85
side of the Golgi stacks (Figs. 5-7). The first
one, which corresponded to the so-called cis
(osmiophilic) element observed in the Golgi
apparatus of various cell types, consisted of a
network of tightly anastomosed tubules
which appeared as a series of interconnected
membranous profiles when the plane of section was perpendicular to the stack (Fig. 5).
This element reacted either positively or negatively with the lectin (Figs. 5-7). Subjacent
to it there were three parallel lectin-positive
saccules which showed a nodular appearance
when seen in transverse sections (Fig. 5). This
was due to the presence of small pores in the
saccules (Fig. 11). In addition, these labeled
saccules were interrupted by perforations in
register referred to as “wells” (Figs. 5-7, see
terminology in Rambourg and Clermont,
1986). Such wells in the stacks of saccules
were pan shaped with their mouth opening
on the cis face and their bottom closed by the
underlying saccules which were unreactive
with the lectin but thiamine-pyrophosphatase positive (see below). Such wells, which
involved only the lectin-positive saccules,
contained small vesicles (80 nm in diameter)
which were unstained with con A (Fig. 5-7,
10). On the trans side of the Golgi stacks,
under the lectin-positivesaccules, two closely
apposed unstained saccules were present
(Figs. 5,6,12). These saccules did not participate in the formation of wells. They showed
relatively few, small pores centrally and were
continuous at their edges with small anastomotic tubules (Fig. 10). Lastly, on the trans
face of the curved or cup-shaped stacks, two
or three sacculotubular elements, showing a
peeling off configuration, were usually unreactive with con A (Figs. 5, 7). The transmost element, however, which often assumed
the configuration of a network of anastomotic tubules with budding extremities, occasionally showed reactivity to the lectin
(Figs. 3,5,6). This reactivity was not uniform
and frequently involved only the tubular and
budding extremities of the element (Figs. 3,
5, 6). The large vesicles (120 nm) seen between the peeling off saccules or small (80
nm) vesicles at the edge of the saccules or in
the trans region of the Golgi apparatus were
usually unreactive with con A (Figs. 5-7, 10,
11). Some circular or vesicular profiles were
reactive in the trans region (Fig. 7, lo), but
they were likely to represent cross sections
through the con A-positive trans tubular network or ER cisternae. Indeed, terminal tubular extensions of con A-positive ER
86
F. MALCHIODI ET AL.
~~
Fig. 5. Perikaryon of a type B1 cell in a section of
tissue treated with con A-peroxidase complex and counterstained with lead citrate (as in the following Figures
6-13). Two stacks of Golgi saccules cut transversely are
seen in this field. While the cis (osmiophilic) element in
the Golgi stack on the left is stained with the lectin, the
cis element of the Golgi stack on the right is unstained
(vertical arrows). The underlying three saccules are
strongly reactive, while the next trans saccules are unstained with the lectin (arrowheads) in both stacks. The
trans sacculotubular element is partly stained with con
A in the concave region of the Golgi stack on the left
(asterisk). The vesicles associated with Golgi stacks are
generally unreactive to con A. Lysosomes and mitochon-
dria are negative while the rough ER cisternae are positive. ~32,500.
Fig. 6. Perikaryon of a type B1 cell showing transverse (left) and oblique (lower right) sections through
Golgi stacks. In the Golgi stack on the left, four elements
including the cis (osmiophilic) elements are positively
stained with con A. Other saccules on the trans side are
negative. In the obliquely cut Golgi stack, all small (80
nm) vesicles in a well are negative to the lectin (arrowhead) as well as most small and large vesicles associated
with the Golgi stacks (star). Some positively stained
rough ER cisternae approach the Golgi apparatus on all
sides. Mitochondria and lysosomes are unstained with
con A. ~ 4 4 , 2 0 0 .
CON A-BINDING SITES IN NEURON GOLGI APPARATUS
cisternae often entered or penetrated the
trans region of the Golgi stacks and were
sometimes seen in close apposition to unstained trans elements (Figs. 10-12).
Con A reactivity of the Golgi apparatus of
type Ab A% and C cells
In type Al, A2, and C cells, all the saccules
or the sacculotubular elements of the Golgi
stacks as well as the cis (osmiophilic) elements were reactive with con A (Figs. 1,4,8,
9). Occasionally, however, this cis element
was found to be unreactive (Fig. 13).In these
three cell types as for the type B1 and Bz
neurons, the small (80 nm) vesicles seen in
wells, at the edges of the Golgi stacks, or in
the trans region as well as the larger (120
nm) vesicles seen between the lectin-positive
peeling off saccules were all negative to the
lectin (Figs. 8,9,13).
Con A reactivity of Golgi-associated vesicles
and multivesicular bodies
In all cell types examined, clusters of small
vesicles seen in close contact with or close to
ER cisternae (Figs. 8,131or in the vicinity of
wells (Fig. 6) were always unreactive with
con A. Multivesicular bodies were present in
all ganglion cells; and, while some of them
were unstained with con A, others reacted
positively with the lectin (Figs. 2, 4, 12, 13).
In the latter, the matrix was reactive while
the enclosed vesicles were not (Fig. 12).
TPPase activity in the Golgi stacks of
neurons
In sections of spinal ganglia used to demonstrate TPPase activity, it was evident that
in type B1 and B2 cells the reactive saccules
were the two con A-negative saccules seen on
the trans side of the Golgi stack. These two
saccules were sandwiched between the con
A-positive saccules and the trans sacculotubular elements showing a peeling-off configuration (Fig. 14). In type A cells, these two
saccules were already shown to be TPPasepositive (Rambourg and Clermont, 1986)but,
as mentioned above, these two saccules were
also stained with con A.
Fig. 7. Photograph showing a Golgi stack of a type Bz
neuron. The cis (osmiophilic)element is not stained with
con A (arrow) while the three underlying saccules are
positively stained. The other trans saccules or “peeling
off’ trans sacculotubular elements are unreactive. The
small vesicles seen within the wells and most small and
large vesicles seen in the trans region are unreactive
with the lectin. A few large vesicular profiles are positive, however (arrowheads). X 35,750.
87
DISCUSSION
Staining properties of sugars with con A
Goldstein et al. (1965), using the technique
of hapten inhibition, established that the
binding sites of con A were most complementary to terminal a-D-mannopyranosyl residues but could also bind to a-D-glucopyranosy1 groups and their 2-acetamido-2-deoxy
derivatives. Since unmodified hydroxyl
groups at c3, c4, and c6 of the a-D-hexapyranosyl configuration were considered to be
the minimal structural features required for
the binding of con A to saccharides, internal
1-2-linked a-D-mannopyranosyl residues
were also shown to interact with the lectin
in conditions in which these residues were
sterically available Qoyoshima et al., 1972;
Goldstein et al., 1973; 1974). Finally, the
presence of at least two a-D-mannopyranosyl
residues with free hydroxyl groups at C3, C4,
c6 was found to be a prerequisite to obtain a
significant reaction with con A (Ogata et al.,
1975; Kornfeld and Ferris, 1975; Krusius et
al., 1976; Baenziger and Fiete, 19791, to retain glycopeptides in a con A sepharose column (Ogata et al., 1975), and to inhibit con
A binding to erythrocytes (Kornfeld and Ferris, 1975).
Various types of glycoproteins have been
isolated from the central nervous system and
characterized biochemically. Most of these
glycoproteins contain asparagine-linked oligosaccharide side chains (Margolis et al.,
1972; Krusius and Finne, 1978),although an
important fraction of them possess alkali-labile side chains 0-glycosidically linked to the
hydroxyl groups of serine or threonine (Margolis et al., 1972; Margolis and Margolis,
1973; Finne, 1975; Finne and Krusius, 1976).
Con A binding to glycoproteins of the brain
(Susz et al., 1973; Javaid et al., 1975;Zanetta
et al., 1975; Krusius and Finne, 1978; McIntyre et al., 1979; Gurd and Fu, 1982) has
been attributed to the presence of neutral
mannose-rich asparagine-linked oligosaccharides (Javaid et al., 1975)in which up to eight
mannose residues may be associated with a
single N acetylglucosamine (Krusius et al.,
1974; Gurd and Fu, 1982). A biochemical
analysis of glycoproteins in sensory ganglia
is not yet available. Nevertheless, the reactivity of ganglion cells to con A described in
the present study suggests that the same
types of oligosaccharides, particularly N-asparagine-linked carbohydrates, are also present in rat dorsal root ganglia.
88
F. MALCHIODI ET AL.
CON A-BINDING SITES IN NEURON GOLGI APPARATUS
Biosynthetic steps of glycoproteins side
chains us. con A reactivity of cytoplasmic
elements of ganglion cells
In the rough endoplasmic reticulum
Biochemical studies on the synthesis of asparagine-linked complex oligosaccharides
(see review by Kornfeld and Kornfeld, 1985)
have indicated that the processing of these
macromolecules takes place in various compartments of the cell. Thus, oligosaccharide
side chains containing terminal mannosyl
and glycosyl residues are added to the protein core in a dense (heavy) membrane fraction which corresponds to the rough ER.
Following the removal of terminal glycosyl
residues by glycosidase I and II in the same
fraction (Fig. 161, the oligosaccharide chains
will be processed in lighter membrane fractions containing elements of the Golgi apparatus (see below).
Since the ER
oligosaccharides are rich in glycosyl and
mannosyl terminal groups as well as in internal 2-0-substituted a-mannosyl residues,
as expected and stated above, con A was
found to bind consistently to the luminal side
of rough ER cisternae in various cell types
(Wood et al., 1974; Pinto da Silva et al., 1981;
Grifiths et a]., 1982; Roth, 1983; Tartakoff
and Vassali, 1983; Pavelka and Ellinger,
1985). This binding of con A to the rough ER
cisternae was confirmed on the present study
for all types of ganglion cells (Fig. 15).
In the Golgi apparatus
Labeling of the Golgi apparatus with con
A has been reported to occur, usually, in one
or two saccules located on the cis side of the
Golgi stacks moth, 1983; Tartakoff and Vassali, 1983; Pavelka and Ellinger, 1985), although Griffiths et al. (1982) observed a
binding of the lectin to all Golgi saccules in
frozen thin sections of BHK cells using an
antilectin antibody revealed by protein Agold complex. In ganglion cells, two main
Fig. 8. Stacks of Golgi saccules of a type A1 cell. All
elements of the Golgi stack react positively with con A
except the small or large vesicles (arrowheads) associated with the stack. Clusters of small vesicles (arrow)
seen between the con A-positive cisternae of the rough
ER and stack of saccules (on the right) are equally negative. x42,500.
Fig. 9. Stack of Golgi saccule of a type A2 cell. With
the exception of the vesicles (arrowheads)associatedwith
the stack, all saccular structures are positively stained
with con A. A positive ER cisterna (arrow) is closely
apposed to a trans saccule. ER, Cisternae of rough endoplasmic reticulum forming a Nissl body. ~ 4 2 , 8 0 0 .
89
types of reactions were observed with con A:
in type Al, A2, and C cells, all Golgi saccules
or sacculotubular elements were usually reactive to con A, whereas in type B1 and Bz
cells possibly in type A3 cells, only the cis
side of stacks stained positively (Fig. 15). In
the following discussion the latter group of
neurons will be considered first.
Golgi apparatus of type B1, Bz and C cells
The oligosaccharide side chains of glycoproteins elaborated in the ER are further
processed in the Golgi apparatus (Fig. 16).
Enzymes responsible for the trimming of CY 12-linked terminal mannosyl residues (Golgi
mannosidases I and IT) and the addition of
terminal P-N-acetylglucosaminyl residues
(glucosaminyl transferases I and IT) have
been isolated in a membrane fraction corresponding to the cis face elements of the Golgi
apparatus of Chinese hamster ovary cells
(Dunphy and Rothman, 1983) as well as in
mouse lymphoma cells and macrophages
(Goldberg and Kornfeld, 1983). In the latter
cells and in rat liver cells, the enzymes Nacetylglucosamine-1-phosphotransferaseand
N-acetylglucosamine-l-phosphodiester-a-Nacetyl-glucosaminidase (phosphodiesterglycosidase) involved in the phosphorylation of
high mannose oligosaccharides of lysosomal
enzymes were also localized in the same cellular fraction (Pohlman et al., 1982;Goldberg
and Kornfeld 1983; Fig. 16). As a result, in
this Golgi fraction there should be a loss of
con A-reactive terminal and 2-0-substituted
mannosyl residues and an almost concomitant addition of p 1-2-linkedN-acetylglucosaminyl residues to terminal mannosyl groups
of the core, a structural configuration which
is known to interact strongly with con A
(Toyoshima et al. 1972; Kornfeld and Ferris,
1975; Debray et al., 1981; see Fig. 16).
In type B cells, the erratic staining of the
cis (osmiophilic)element contrasted with the
strong and constant binding of con A to the
three subjacent saccules (Fig. 15). Since the
mannose-6-phosphate receptor for lysosomal
enzymes has been detected immunocytochemically in the cis element of several cell
types (Brown and Farquhar, 1984) and in
Golgi saccules of hepatocytes (Geuze et al.,
19841, the occasional lack of staining of the
cis element with con A may be attributed
partly to the phosphorylation at C6 of one or
several terminal or preterminal CY 1,2-linked
mannosyl residues in lysosomal oligosaccharide side chains and partly to the removal
90
F. MALCHIODI ET AL.
91
CON A-BINDING SITES IN NEURON GOLGI APPARATUS
REACTIVITY OF E R AND GOLGI ELEMENTS
TO CONCANAVALIN A
Type
Trans
sacculo - tubular
elements
+ +
+ +
Rough E R
C I S element ( O s + )
3 Sublocent saccules
( Pose - )
2 TPPase+ soccules
A3 Type A,. A,.C
B,. B.,
+
)
or
-
+
or
+ +
+ +
-
+ +
+-
+
-
Fig. 15. The structure of a Golgi stack is shown schematically on the left. The con A reactivity of the various
components of the stacks of saccules for the two groups
of ganglion cells, i.e., types B1, Bz, and A3 and types Al,
An, and C, is summarized in the table on the right.
of terminal mannosyl groups by mannosidase I in nonlysosomal oligosaccharides(Fig.
16; Kornfeld and Kornfeld, 1985).In contrast
in the subjacent con A-positive elements,
which in rabbit hepatocytes have been shown
to contain the N-acetylglucosaminetransferase I (Dunphy et al., 1984; Dunphy and Rothman, 1985),the removal of reactive mannosyl
residues by mannosidase I and 11was likely
to be counterbalanced by the addition at C2
of the terminal mannoses of N-acetylglucosaminyl residues (Fig. 16),thus explaining the
strong con A staining of these three saccules
(Fig. 15).
The lack of lectin reactivity in B cells of
the next two saccules of the stack (Fig. 15)
was attributed to the linkage of another terminal sugar residue to 0-N-acetylglucosamine (Fig. 16). Radioautographic studies by
Droz (1967), and the use of lectins binding
specifically to galactose (Streit et al., 19851,
revealed that terminal galactose residues are
present in the Golgi apparatus of type B neurons in rat spinal and trigeminal ganglia.
Furthermore, in ganglion cells as in other
cell types (Tartakoff and Vassali, 1983),these
two lectin-negative saccules show TPPase activity (Novikoff et al., 1971; Rambourg and
Clermont, 1986) which is usually colocated
within the same saccules as galactosyl transferase CRoth and Berger, 1982) and galactose
residues (Roth et al., 1983; Berger, 1985; Pavelka and Ellinger, 1985). It is likely, therefore, as indicated in Figure 16, that galactose
is the additional sugar on the oligosaccharide
side chains that prevents the binding of con
A to these two saccules. Lastly, in the
TPPase-positive saccules and in the trans
Fig. 10. Oblique section through a Golgi stack of a
type B1 cell. The saccules on the cis side of the stack
stain positively with con A (arrowhead)while the underlying saccules and the trans sacculotubular elements
(asterisk) are unstained with the lectin. One positive ER
cisterna penetrates the cis region of the stack and approaches the trans sacculotublar element. ~ 3 5 , 1 0 0 .
ledin. ER cisternae are reactive, and a cisterna is seen
in the trans region of stacks on the right (arrow). Multivesicular bodies are either positively or negatively
stained with con A. x 16,800.
Fig. 11. Face view of the lectin-positive elements of a
type B1 cell. Face views of wells containing unstained
small vesicles are indicated (arrowheads) in the lectinreactive saccules. A few positive large vesicular profiles
(arrows) are visible amongst unstained vesicles seen in
the trans region of the Golgi stack. Lectin-positiverough
ER cisternae are also visible. ~ 3 5 , 1 0 0 .
Fig. 12. Low magnification view of the perikaryon of
a type B1 cell showing stacks of saccules with a cupshaped arrangement. On the cis side or convex aspect of
the stacks, con A-stained elements are visible, while the
saccules and sacculotubular elements on the trans side
and concave aspect of the stacks are not stained with the
Fig. 13. Small portion of the perikaryon of a type Al
cell. In a stack of Golgi saccules at the upper left while
the saccules are positively stained with con A, a cis
element is negative (small arrowhead).Small vesicles in
the proximity of the Golgi stacks are unstained. A cluster of small vesicles (asterisk) seen next to ER cisternae
a t a distance from the Golgi stacks are also unstained
with con A. A multivesicular body is also unstained.
X35,750.
Fig. 14. Section of a type B1 cell stained to show
TPPase activity in a Golgi stack. Two saccules seen on
the trans side of the Gulgi stack do show the lead precipitate indicative of phosphatase activity. The other saccular elements and the Golgi-associatedvesicles do not
show TPPase activity. A similar reaction was observed
in type A neurons. ~ 3 5 , 0 0 0 .
92
F. MALCHIODI ET AL.
BlOSYNTHESlS OF N -ASPARAGIN€ -LINKED
OLlGOSACCHARlDE SlDE CHAINS
Rough ER
CIS-Element
( os+ 1
3
Su bjocent
Soccules
(Pase-1
2
TPPase +
Saccules
and
Trans Sacculotubular
Elements
OReactive with Con A
OStrongly reactlve with Con A
::::Possibly
reactive with Con A
Fig. 16. The steps in the biosynthesis of N-asparagine- indicates the biosynthetic steps and suggested localizalinked oligosaccharide side chains and their suggested tion of complex oligosaccharide side chains in type B1,
localization within the rough ER and the various com- Ba, and A, neurons. As indicated on the diagram, the
ponents of the Golgi stacks (&r Kornfeld and Kornfeld, circles around sugar residues indicate their degree of
1985). The shaded area indicates the steps in oligosac- reactivity with con A. See text for discussion. G, glucose;
charide processing and their intracellular location which M, mannose; GlcNAc, N-acetylglucosamine; Gal, galactake place in all neurons as suggested by the con A tose; SA, sialic acid; P, phosphorus; R, refers to the stem
reactivity. The stippled area on the lower right indicates of the oIigosaccharide side chain composed of two molethe presumed localization of mannose-rich oligosacchar- cules of GlcNAc N-linked to asparagine of the protein
ide side chains in type Al, AP, and C cells. The nonstip- chain.
pled and nonshaded area of the rest of the diagram
sacculotubular elements, sialic acid is added
to the galactose residues through the action
of a sialyl transferase (Bergeron et al., 1985;
Roth et al., 1985).
There is no explanation as yet for the slight
con A reactivity observed in the sacculotubular element seen in the trans region of the
Golgi stacks. It has been proposed by Rambourg and Clermont (1986) that trans ele-
ments peeling off from the trans face of the
Golgi stacks of type A neurons could eventually break up into vesicular or tubular elements. Such structural alterations must
possibly induce changes in the steric configuration of the membrane-bound oligosaccharides and thereby facilitate the binding of
mannocon A with the two ~~-2-O-substituted
syl residues of the core (Fig. 16).
CON A-BINDING SITES IN NEURON GOLGI APPARATUS
Golgi apparatus of type Al, A2, and C cells
In this group of ganglion cells, all saccules
and sacculotubular elements reacted with con
A (Fig. 15). A similar response to con A was
also observed in the Golgi apparatus of Purkinje cells, i.e., in neurons of the central nervous system, a tissue in which mannose-rich
glycoproteins have been isolated (Gombos et
al., 1972; Javaid et al., 1975; Gurd and Fu,
1982). Since the uptake of galactose (Droz,
1967) and the demonstration of this sugar
with lectins (Streit et al., 1985) was limited
to the small type B neurons, the relative
amount of galactose within the oligosaccharide side chains of glycoprotein should be reduced in the Golgi apparatus of type A
neurons. As discussed above, preterminal
galactose residues of complex oligosaccharides appear to be responsible for the reduction of con A binding to the TPPase-positive
elements of the Golgi apparatus of type B
cells; and thus the reactivity observed in the
same elements of the Golgi apparatus of type
A cells is likely due to the persistence of con
A-reactive mannose residues and to the presence of mannose-rich glycoproteins in this
class of cells (Figs. 15, 16).
In general, therefore, our results suggest
that the two groups of neurons in the spinal
ganglion, i.e., types Al, A2, and C vs. types
B1,B2, and A3, are possibly involved in the
biosynthesis
of
different
types
of
glycoproteins.
93
tent that their detection would be beyond the
sensitivity of the con A-staining technique.
Tartakoff and Vassali (1983) also observed
that in myeloma cells a considerable number
of vesicles located between the rough ER and
Golgi cisternae were not stained with con A.
They concluded that “although the permeabilization of such vesicles could not be directly evaluated, they may simply lack or
contain limited amounts of reactive oligosaccharides.” In fact, techniques which detect
material present in both endoplasmic reticulum and the cis (osmiophilic) element of the
Golgi apparatus, e.g., osmication method (unpublished observations) and glucose-6-phosphatase method (Broadwell and Cataldo,
19831, fail to stain these vesicles.
It has recently been postulated (Rambourg
and Clermont, 1986)that the small (80 nm)
vesicles seen in wells of the Golgi apparatus
of type A ganglion cells must form by budding from the saccules seen on the cis side of
the stacks to return to the endoplasmic reticulum. During the process of vesiculation,
conformational andor biochemical changes
might occur within the vesicular membranes
which would then lose the characteristic
properties of the saccular membrane from
which they originate. Such a possiblity,
which has to be confirmed by further studies,
could also account for the lack of reactivity
of these vesicles with con A.
ACKNOWLEDGMENTS
Absence of con A binding to vesicles
associated with the Golgi stacks
Small vesicles, also called intermediate or
transfer vesicles, seen in the proximity of ER
cisternae have usually been assumed to bud
from the cisternae and carry proteins to the
cis face of the Golgi apparatus (Palade, 1975;
Farquhar and Palade, 1981; Goldfscher,
1982). In the present study, con A-stained
vesicles, if any, were located in the trans
region of the Golgi stacks in all neurons. In
contrast, the small vesicles in the “wells”
and the clusters of vesicles observed close to
ER cisternae were never found to bind con
A. Several conditions may explain this lack
of reactivity. One possibility is that the steric
configuration of the oligosaccharide chains
in the restricted space of such small vesicles
did not allow the binding of the lectin. Another possibility is that the trimming of terminal mannosyl residues at this level reduces
the number of reactive sites to such a n ex-
The work done at McGill University was
supported by the Medical Research Council
of Canada. The help of Dr. L. Hermo in the
preparation of the manuscript is acknowledged. The drawings were prepared by Margo
Oeltzschner, McGill University.
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implications, site, dorsal, concanavalin, typed, rat, apparatus, roots, various, ultrastructure, localization, golgi, ganglia, neurons, function, binding
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