Download A Difference between Rods and Cones in the Renewal of Outer

A difference between rods and cones in the
renewal of outer segment protein
Richard W. Young
The renewal of protein has been studied in the retinal rods and cones of the adult frog by
electron microscope autoradiography after injection of radioactive amino acids. In both classes
of photoreceptor cells, the synthesis of protein is concentrated in the niyoid zone of the inner
segment. Newly formed (radioactive) protein is then displaced past the mitochondria of the
ellipsoid zone and reaches the outer segment by flowing through the connecting structure.
In rods, the labeled protein accumulates at the base of the outer segment, apparently as a
component of newly assembled membranous discs, which are then gradually displaced
sclerally. In cones, however, no such concentration of radioactive protein has been observed.
On the contrary, protein delivered to the outer segment becomes diffusely distributed throughout that structure. TJuis, in the frog, there is a distinct difference between rods and cones
in the process of protein renewal.
Key words: retinal photoreceptor cells, protein synthesis,
amino acids, ultrastructure, histology.
I
pacted, stabilized, and precisely aligned.
The efficiency of this light-trapping device
is augmented by the exclusion of all other
cellular organelles from this part of the
photoreceptor.
Consequently, although protein is more
concentrated in the outer segment than
anywhere else in the visual cell,2 it is not
synthesized there. The protein synthetic
apparatus of the cell is located in the inner
segment, particularly its myoid portion,
and is separated from the outer segment
by mitochondrial aggregations in the ellipsoid, and by a narrow connecting structure (Fig. 1). Autoradiography has shown
that synthesis of protein is largely confined
to this inner segment.25
The first evidence that outer segment
protein might undergo renewal was presented by Droz,2 who observed by autoradiography that much of the protein
n all vertebrate photoreceptor cells
which have been studied so far, the outer
segment has proved to consist of a stack
of flattened saccules or discs, each consisting of a double layer of cell membrane.
The discs are oriented at right angles to
the long axis of the cell, and are enclosed
within the outer cell membrane. The result
is a regular, lamellar organization of unusual density, a sort of "membranous
crystal" constructed of oriented lipid and
protein.1 Within this highly organized structure the visual pigment molecules are com-
From the Department of Anatomy and the Jules
Stein Eye Institute, University of California
Medical School, Los Angeles, Calif.
Supported by Grant NB-03807 and by a Special
Fellowship, 1F11 NB 1658-01 VSN, United
States Public Health Service.
222
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Outer segment protein renewal 223
OS
formed in the inner segment was subsequently displaced to the outer segment in
the retinal rods of rats and mice. He suggested that it might be the visual pigment
(opsin) molecules which were being replaced. Transfer of protein to the outer
segment, shortly after its production in
rod inner segments in rats, mice, and frogs
was confirmed by the author.5 It was also
observed that the protein accumulated at
the base of the outer segment, then gradually moved sclerally as a discrete and stable unit which ultimately disappeared
when it reached the apical extremity of the
cell, in contact with the pigment epithelium. These findings led to the suggestion
that the entire rod outer segment might
be undergoing renewal by repeated addition of new membranous discs at the base
of the outer segment, in conjunction with
a balanced removal of disc material at its
apex.5 The retina of the frog contains cones
as well as rods. However, the cone outer
segments are so small that it was not possible to determine by conventional autoradiographic technique if a similar process
occurred in these cells. At best it could be
stated that some of the protein synthesized
in the cone myoids was later transferred
to the outer segments.5
The present report describes new observations made possible by the application of electron microscope autoradiography to the study of protein metabolism
in rods and cones. It will be shown that
in the frog there is a significant difference
in the mechanism of renewal of this basic
cell constituent in the two major classes
of retinal photoreceptor cells.
Methods
Fig. 1. Diagram depicting the two types of rods
and three types of cones found in the frog retina.
Abbreviations: SC, single cone; RR, red rod; PC,
principal cone; AC, accessory cone; GR, green
rod; 05, outer segment; cc, connecting cilium; e,
ellipsoid; m, myoid; p, paraboloid; od, oil droplet.
The ellipsoid and myoid together comprise the
inner segment.
Nine adult frogs (Rana esculenta), averaging
30 grams body weight, each received 10 me. of
a tritiated amino acid solution containing equal
amounts of radioactivity due to histidine, methionine, leucine, and phenylalanine." A mixture of
labeled amino acids was used to assure a gen°L-histidine-3H, g.l., S.A. 10.7 c. per millimole and Lmethionine-methyl-3H, S.A. 0~.3 c. per millimole, obtained
from the Commissariat a l'finergie Atomique, Saclay,
France. L-leucine-4,5-3H, S.A. 5.0 c. per millimole and
L-phenylalanine-3H, g.l., S.A. 2.8 c. per millimole obtained
from New England Nuclear Corp., Boston, Mass.
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Investigative Ophthalmology
April 1969
224 Young
eralized labeling of protein. These animals were
killed at 10 and 30 minutes, 1, 2, 4, and 8 hours,
and 1, 4, and 7 days after injection. The frogs
were maintained at 22.5° C. under conditions of
ordinary laboratory illumination. All were lightadapted at the time of death.
The eyes were fixed for 1 hour at 4° C. in a
solution of 4 per cent methanol-free formaldehyde, phosphate-buffered to pH 7.1. The anterior
portion of the globe, including the lens, was then
removed, and fixation continued for an additional
hour at room temperature. Next, the tissues were
cut into smaller pieces, rinsed in buffer, postfixed in 2 per cent osmic acid in the same buffer,
dehydrated, and embedded in epon. The retinal
fragments were then cut out and reoriented with
epoxy cement so that longitudinal sections of the
photoreceptor cells could be obtained.
Stiver sections, cut on an LKB ultrotome, were
deposited on glass microscope slides which previously had been coated with a thin layer of
celloidin. The sections were stained with uranyl
acetate and lead citrate, coated with carbon, then
dipped in Ilford L4 emulsion diluted 1:4 with
distilled water and maintained at 40° C. After
exposure in the dark under low humidity at room
temperature for 1 to 3 months, the preparations
were developed in Microdol X for 4 minutes at
17° C. and fixed in 30 per cent sodium thiosulfate.
The celloidin membrane was next separated
from the slide by floating on water, and grids
placed over the sections. The membrane and
grids were then removed from the surface of the
water by adhesion to wet filter paper. After drying, the grids were detached from the membrane
and placed in isoamyl acetate for 3 minutes to
diminish the thickness of the celloidin supporting
layer. The autoradiographic preparations were
examined and photographed in the Siemens
Elmiskop I electron microscope.
Results
In the frog retina there are two types
of rods (red and green), and three types
of cones (single, and double with both
Fig. 2. Two hours after injection. The outer segments of five cones (x) and a red rod (RR)
are shown. Radioactive protein, newly arrived from its site of synthesis in the myoid, is beginning to be incorporated into membranous discs at the base of the rod outer segment {arrow). No such localization is seen in the cones, od, Oil droplets in principal cones; e, mitochondria in the ellipsoid. The outer segments are separated from one another by pigment
epithelial processes (pe) containing pigment granules. (Electron micrograph, autoradiogram,
x7,100.)
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principal and accessory members). These
are well described by NilssonG and are depicted in Fig. 1.
In both rods and cones, 10 minutes after injection, the autoradiographic reaction
was largely restricted to the regions of
free and membrane-bound ribosomes concentrated in the myoid portion of the cell.
Labeling was somewhat heavier in rods
than in cones in this inner segment zone
of protein synthesis. The outer segments
of both were free of radioactivity. A shift
of labeled protein into the Golgi complex
was apparent by 30 minutes, and reached
a peak 1 to 2 hours after injection.7 Proteinbound radioactivity then moved rapidly
through the region of packed mitochondria
in the ellipsoid, accumulated momentarily
at the base of the ciliated connecting structure, then passed through the cilium into
Outer segment protein renewal 225
the outer segment.8 Up to this point the
sequence of events appeared to be similar
in rods and cones. However, at 2 hours a
distinct difference began to be revealed.
Rods. In rods, at this interval, incorporation of the newly synthesized protein into
the basal discs of the outer segment could
be detected (Fig. 2). During the next 2
hours, the amount of radioactive protein
contained in these membranous structures
was progressively increased as more was
delivered from the site of synthesis in the
inner segment. By 8 hours (Fig. 3), there
was evidence that the heavily labeled discs
were beginning to be displaced sclerally
within the outer segment, a process which
was obvious at 24 hours (Figs. 4 and 5).
During the following week, these discs continued to be gradually shifted from the
outer segment base toward its apex (Figs.
Fig. 3. Eight hours after injection. This field shows the outer segment of a principal cone
(right) and the base of the outer segment of a red rod (RR). In the rod, recently synthesized
(radioactive) protein has been assembled into discs which already appear to be slightly displaced from the outer segment base. There is no regional concentration of protein-bound
radioactivity in the cone outer segment, joe, Pigment epithelial processes; ods oil droplet; e,
mitochondria in the ellipsoid. (Electron micrograph, autoradiogram, x8,000.)
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Investigative Ophthalmology
April 1969
Young
6 to 8). The rate of displacement was
slightly greater in red rods than in green
rods, moving at a rate of about 36 discs
per day in the former and about 25 discs
per day in the latter. (This compares
closely with comparable measurements
made in the light microscope.5) A relatively weak, diffuse labeling behind the
moving radioactive discs resulted from
the continuing addition of newer protein
synthesized from precursor amino acids of
progressively decreasing specific activity
(Figs. 6 to 8).
Cones. In cones, a completely different
phenomenon was observed (Figs. 2 to 8).
No localized concentration of radioactive
I
;.«
^F;^
m
protein occurred in the outer segment. Instead, beginning at 2 to 4 hours after injection, a weak, diffuse labeling began to
appear throughout the dense layers of
membranous discs. After 8 hours there
was little or no further increase in- the
amount of this generally distributed, protein-bound radioactivity. Labeling in the
cone outer segments did not noticeably
change during the following week.
Discussion
The results indicate that there is a continual renewal of protein in the outer segments of both rods and cones in the retina
of the adult frog. Because the outer seg-
^
1 f ^ M x+##y***
If
Fig. 4. One clay after injection. Portions of two red rods (top, center) and the outer segments of three principal cones (x) are visible. The radioactive discs have been distinctly displaced sclerally in the rods. No discrete localization of newly formed protein has occurred in
the cone outer segments, pe, Pigment epithelial processes. (Electron micrograph, autoradiogram, x7;200.)
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ments of visual cells appear to be extremely
sensitive to damage from a variety of
sources, such renewal is clearly of value
in assuring the preservation of vision
throughout the life-span.
The renewal of protein in rod and cone
outer segments is achieved by mechanisms
which are strikingly different in these two
cell classes, at least in the frog. In rods,
there appears to be an unceasing formation of new membranous discs at the base
of the outer segment, incorporating protein
formed in the inner segment. These discs
are gradually displaced sclerally by the
Outer segment protein renewal 227
formation of newer discs. Old disc material
is probably removed at the apex of the rod
by the pigment epithelium.5' ° Recent
studies which show an increase in outer
segmentlike inclusions in the pigment epithelium during removal of damaged outer
segment material10 support this conclusion.
In cones, on the other hand, no evidence
to indicate a continued formation of outer
segment discs has been obtained. Protein
delivered through the connecting cilium
appears to become diffusely distributed
throughout the layers of the cone outer
segment.
Fig. 5. One day after injection. The diffuse distribution of renewal protein in the principal
cone outer segment (right) contrasts markedly with the discrete and heavy labeling of a
small group of discs near the base of the red rod outer segment (left). (Electron micrograph,
autoradiogram, x 14,000.)
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Investigative Ophthalmology
April 1969
228 Young
What might account for the dissimilarity
in the metabolic fate of such a basic cell
product as protein in these two classes of
photoreceptors? It seems doubtful that variations in functional state could produce
such an inflexible dichotomy between rods
and cones. A localized concentration of
labeled protein has never been seen in
any type of cone in any circumstance after
injection of radioactive amino acids. On
the contrary, evidence of an aggregation
of newly synthesized protein at the base
of the outer segment, or the subsequent
displacement of a group of heavily labeled
discs, has been observed without exception
in all types of rods in all animals examined,
whether these were light- or dark-adapted
at death, and whether they were raised
under conditions of normal, elevated, or
reduced illumination.5
Perhaps the differences in distribution
of renewal protein in the outer segment
arise more directly from differences in the
structure of the outer segments themselves.
In the frog, the cylindrically shaped outer
segments of rods are much larger than the
definitely conical outer segments of cones.
The thickness of individual discs is similar
in each, but in cones the spaces between
adjacent discs are 40 to 50 per cent larger
than in rods.11 In cones, all the disc membranes are continuous with one another,
and with the enclosing cell membrane. In
rods, only a few discs at the base of the
outer segment appear as infoldings of the
cell membrane. Above this level they lose
their connections and become "free-floatin j
In rods, then, the ultrastructural appearance is consistent with the autoradio-
Fig. 6. Four days after injection. The radioactive discs in the red rod (left) have continued
to be displaced sclerally by the continued formation of new discs at the base of the outer
segment. The weaker labeling of these new discs is an indication of the continued availability
of low levels of labeled protein precursors in the tissue fluids. Radioactive protein is diffusely
distributed in the cone outer segment. (Electron micrograph, autoradiogram, xl2,700.)
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graphic results which indicate that new
discs are continually formed by infolding
of the cell membrane at the outer segment
base, and that repeated basal invaginations
displace overlying discs sclerally. This renewal mechanism is a continuation of the
developmental process.5'12 During development, frog rods are initially cone-shaped.
That is, the first discs formed are smaller
than those which later displace them
sclerally.12'13 The fact that the mature rod
Outer segment protein renewal 229
is no longer conical at its apex is a further
indication of the removal of disc material
at this site.
In cones, the discs at the base of the
outer segment are of larger diameter than
those at the apex, whereas they would be
expected to have similar dimensions were
they simply being repeatedly assembled
below and then shifted upward. Thus, the
conical form of cone outer segments in the
frog is consistent with the observed ab-
Fig. 7. Four days after injection. The outer portions of the accessory and principal members
of a double cone, and part of a red rod outer segment (right) are shown. The radioactive
discs, significantly displaced from their site of assembly at the base of the outer segment, are
apparent in the rod (arrow). No such localization of labeled protein occurs in the cones.
e, Mitochondria in the cone ellipsoids; cc, connecting ciliuin; pe, pigment epithelial processes.
The lamellation in the oil droplet (od) is a sectioning artifact. (Electron micrograph, autoradiogram, xio.eoo.)
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230 Young
Investigative Ophthalmology
April 1969
Fig. 8. One week after injection. The radioactive discs in the red rod outer segment (left)
have continued to be displaced sclevally by the repeated addition of new discs at the base of
the outer segment. There is no evidence of new disc formation in the cone outer segment.
(Electron micrograph, autoradiogram, xll,200.)
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Outer segment protein renewal 231
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sence of continued production and scleral
displacement of discs.
It appears, then, that in mature, coneshaped outer segments, disc formation has
stopped. In mature, rod-shaped outer segments, disc formation may continue. Indeed, it has been found to continue in
every retina so far examined.5
Perhaps the small size of the cone outer
segments is compatible with a process of
protein renewal by simple diffusion. Possibly the relatively large spaces between
adjacent cone discs facilitate such molecular exchange. Such speculation has limited
value in view of the paucity of available
information.
However, should further research substantiate the generality of outer segment
renewal, it may not then be premature to
ask if a failure in the renewal mechanism
might lead to degeneration of visual cell
outer segments, and whether different renewal processes in rods and cones might
underlie conditions in which rod or cone
vision is selectively destroyed.
This project was carried out in the Department
cle Biologic, Centre d'fitudes Nucleaires, Saclay,
France, during the author's sabbatical leave from
UCLA, 1966-1967. The technical assistance of
Mrs. J. Boyenval and Mrs. M. Lucarain is acknowledged. Fig. 1 was drawn by Miss Jill Penkhus. Particular thanks are due to Dr. Bernard
Droz, for making available the full facilities of
his laboratory, for teaching the author the technique of electron microscope autoradiography and
for advice, encouragement, assistance, and hospitality.
REFERENCES
1. Schmidt, W. J.: Polarisationsoptische Analyse
der Verkniipfung von Protein- und Lipoid-
molekeln, erlautert am Aussenglied der
Sehzellen der Wirbeltiere, Pubbl. stazione
zool. Napoli. 23 (Suppl): 158, 1951.
2. Droz, B.: Dynamic condition of proteins in
the visual cells of rats and mice as shown
by radioautography with labeled amino acids,
Anat. Rec. 145: 157, 1963.
3. Maraini, G., Franguelli, R., and Peralta, S.:
Studies on the metabolism of the retina and
lateral
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
geniculate nucleus, INVEST. OPHTH.
2: 567, 1963.
Nover, A., and Schultze, B.: Autoradiographische Untersuchung iiber den Eiweissstoffwechsel in den Geweben und Zellen d'es
Auges, Arch. f. Ophth. 161: 554, 1960.
Young, R. W.: The renewal of photoreceptor.
cell outer segments, J. Cell. Biol. 33: 61,
1967.
Nilsson, S. E. C : An electron microscopic
classification of the retinal receptors of the
leopard frog (Rana pipiens), J. Ultrastruct.
Res. 10: 390, 1964.
Young, R. W., and Droz, B.: The renewal
of protein in retinal rods and cones, J. Cell
Biol. In press.
Young, R. W.: Passage of newly formed protein through the connecting cilium of retinal
rods in the frog, J. Ultrastruct. Res. In press.
Young, R. W.: The organization of vertebrate
photoreceptor cells, in Straatsma, B. R., Allen, R. A., Crescitelli, F., and Hall, M. O.,
editors: The retina, Los Angeles, 1968, University of California Press.
Kuwabara, T., and Corn, R. A.: Retinal damage by visible light, Arch. Ophth. 79: 69,
1968.
Nilsson, S. E. G.: Ultrastructure of the receptor outer segments in the retina of the
leopard frog (Rana pipiens), J. Ultrastruct.
Res. 12: 207, 1965.
Nilsson, S. E. G.: Receptor cell outer segment development and ultrastructure of the
disk membranes in the retina of the tadpole
(Rana pipiens), J. Ultrastruct. Res. 11: 581,
1964.
Cameron, J.: Further researches on the rods
and cones of vertebrate retinae, J. Anat. Physiol. 46: 45, 1911.
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