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Origin of amphibian premelanosomes and their relation to microtubules.

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Origin of Amphibian Premelanosomes and
Their Relation to Microtubules
GARY E. WISE
Department of Molecular, Cellular and Developmental Biology,
University of Colorado, Boulder, Colorado
ABSTRACT
In amphibian melanophores, premelanosomes appear to be formed in
fenestrated cisternae of the Golgi complex. The premelanosomes may develop within
the cisternae or vesicles containing melanoprotein may bud from the cisternae to f o r m
premelanosomes. Some micrographs also demonstrate a close association between
microtubules and either premelanosomes or melanosomes. The microtubules themselves
may originate from electron dense particles in the centrosphere region of the cell.
Premelanosomes are nascent melanosomes or melanin granules. After full development they play a dominant role in the
physiology of melanophores, one of the
most important color cells in the skin of
amphibians. The fine structure of these
cells and ultrastructural changes that occur in response to variations in light and
certain hormones, such as melanophorestimulating hormone and melatonin, have
been described previously (Wise, '69). Although the developmental origin of premelanosomes has been studied in melanomas (Seiji et al., '63; Novikoff et al.,
'68) and in the pigment epithelium of the
eye (Moyer, '63), there is little information on their development in amphibian
melanophores.
Early stages of premelanosome development involve the synthesis of tyrosinase,
an enzyme which catalyzes two reactions :
( 1) tyrosine + L-dihydroxyphenylalanine
(dopa) and ( 2 ) dopa 3 dopa quinone.
Dopa quinone or a series of quinones
formed from it react with SH and NH,
groups of proteins to form melanoproteins
in premelanosomes (see review by Lerner
and Case, '59).
Seiji et al. ('63) studied in vivo incorporation of leucine-CI4into soluble tyrosinase of melanomas by examining the isolated fractions of the melanomas at various
times after labeling. Their results indicated
that tyrosinase was synthesized on ribosomes and then perhaps transported to the
Golgi apparatus where it was packaged into
vesicles which became premelanosomes as
the tyrosinase became active. On the basis
ANAT. REC., 165: 185-196.
of demonstrating tyrosinase activity histochemically, Novikoff et al. ('68) suggested
that the packaging of tyrosinase occurs in
GERL (Golgi, Endoplasmic Reticulum, Lysosome), a fenestrated region of smooth
endoplasmic reticulum associated or
merged with the innermost saccule of the
Golgi apparatus (Novikoff, '64, '67).
Moyer ('66) believed that concentration
on protein subunits was the primary event
in the formation of melanosomes. This
concentration could occur in the Golgi apparatus, as predicted by Seiji, or it could
occur at other sites in the cell where proteins could aggregate, For example, no
Golgi apparatus is present in the pigmented
epithelium of the eye of fetal and newborn
mice but melanosomes are synthesized, presumably in the endoplasmic reticulum
(Moyer, '63).
MATERIALS AND METHODS
Tadpoles of the South African Clawed
Toad, Xenopus laevis, stages 47 and 48
of Nieuwkoop and Faber ('56), were generously provided by Robert 0. Kelley. They
were fixed for 4-6 hours in 2% glutaraldehyde in either 0.1 M Sorenson's phosphate
buffer or 0.1 M cacodylate buffer, pH 7.4,
at 5 ° C . Then they were rinsed for 15 minutes in phosphate or cacodylate buffer and
placed in 1% Os04 in 0.1 M phosphate or
cacodylate buffer for one OF two hours.
Specimens were dehydrated in ethanol and
placed in a propylene oxide-Epon mixture
overnight. After transfer to Epon, pieces
Received Mar.ch 11, '69. Accepted May 9, '69.
185
186
GARY E. WISE
tubule is ending at the premelanosome.
Second, apparent micro tubule-premelanosome connections are more frequently observed than seems likely on the basis of
plane of section.
The microtubules themselves occasionally appear to form a reticular network
(fig. 8). As can be seen in figure 8, the
OBSERVATIONS
microtubules seem to be continuous with
Amphibian melanophores are stellate saccules and these saccules, in turn, are
cells which contain numerous melano- connected. I h addition, numerous microsomes. At the base of one of the processes tubules are observed in the region of the
a centrosphere region is present adjacent centrosphere. They appear to emanate
to the nucleus. Encircling the centrosphere from dense particles of 600-650 A situated
is an extensive Golgi complex which sorne- in this region (figs. 9, 10). Microtubules
times extends into the bases of the pro- were not observed passing through or past
cesses.
the dense particles.
Deposits of electron dense material, preDISCUSSION
sumably melanoprotein, frequently are
In amphibian melanophores the Golgi
seen in cisternae of the Golgi apparatus
(figs. 1, 2 ) . Vesicles containing this dense apparatus m,sy be implicated in the formamaterial are also seen adjacent to the Golgi tion of premelanosomes because it appears
to be the onlly organelle in which electron
complex (fig. 3 ) .
In figure 4, premelanosomes approach- dense mater la1 is observed. Vesicles coning maturity (possibly melanosomes) ap- taining this material may bud from sacpear to be within the cisternae of the Golgi cules of the Golgi apparatus and then fuse
apparatus. A premelanosome containing to form a melanosome or an entire saccule
electron dense fibers (e.g., see Moyer, '66) may enlarge to form a melanosome. In the
also is present adjacent to the Golgi cis- development of azurophil granules in polyternae. In addition, figure 4 also shows morphonuclear leukocytes (Bainton and
the presence of 700-790 A coated vesicles. Farquhar, '66) or granules of mammoThe Golgi cisternae often are fenestrated trophic hormone (Smith and Farquhar,
in the region of premelanosome formation. ' 6 6 ) , vesicles bud from the concave face
Figure 5 especially demonstrates this of the Golgi apparatus and then fuse to
marked fenestration and also shows a cross form the granules.
Although {heelectron dense material obsection of an adjacent premelanosome with
its fibrous substructure. Premelanosomes served in the Golgi apparatus was not
such as this often are 0.5 to 1 in length identified biochemically as melanoprotein,
the presence of premelanosomes in the
and 0.15 to 0.25 II in width.
Microtubules often were observed near melanophores suggests that melanopropremelanosomes and some profiles suggest tein is being synthesized. Apparently mela close association between microtubules anin synthesis can occur in melanophores
and premelanosomes (figs. 6, 7). Although after they are fully differentiated because
overlapping within the plane of section premelanosomes also were observed in the
can give a false appearance of connections mature melamophores of the adult lizard,
between microtubules and premelanosomes Lncertn viuipara (Breathnach and Poyntz,
or melanosomes, this may be unlikely for '67). Furthermore, in the amphibian i t is
the following reasons. First, in numerous unlikely that premelanosomes are transmicrographs the microtubule dilates ad- ferred from other cell types to the melanojacent to the developing melanosome (figs. phore there are no cells immediately ad6, 7). Since other research has shown that jacent to it in the dermis.
the ends of a microtubule are often dilated
The electron dense material is not obin neurons (de Iraldi and de Robertis, '68) served in the coated vesicles which are
and in plant cells (Hepler and Jackson, probably formed in the Golgi apparatus.
'68), this may indicate that the micro- These vesicles could be primary lysosomes
of skin were dissected from head and
trunk regions and embedded in fresh Epon.
Blocks were cured at 60°C, sectioned on a
Porter-Blum MT-2 ultramicrotome (silver
sections), stained with uranyl acetate and
lead citrate, and examined with an RCA-3G
electron microscope.
ORIGIN OF PREMELANOSOMES
(see Friend and Farquhar, '67) and the
absence of electron dense material within
them is indirect evidence that it is being
utilized for premelanosomes and not for
lysosomes.
It seems possible that the fenestrated
region of the Golgi apparatus of melanophores may be homologous to the GERL
complex in melanomas studied by Novikoff
('68). Their cyto-architecture is similar
in that both are fenestrated and both form
coated vesicles. Furthermore, Novikoff
('67) demonstrated that both the innermost saccule of the Golgi apparatus and
GERL contain acid phosphatase and nucleoside monophosphatase activity.
A heavily fenestrated Golgi complex has
been associated with pigment granule production in still another instance, the "test"
cells of the ovary of the tunicate, Styela
(Kessel and Beams, '65). Although the
biochemical nature of the pigment was unknown, vesicles containing the pigment appeared to bud from the Golgi apparatus.
The cyto-architecture of the Golgi apparatus in amphibian melanophores is
similar to the model of Mollenhauer and
Morrc? (see review of '66) which was based
upon observations of isolated Golgi from
plant cells. In their model the periphery of
the Golgi cisternae appeared as fenestrated
plates composed of tubules and only the
central region of the cisternae was flattened and disc-like. In melanophores, however, even the central regions of some of
the cisternae appear fenestrated. This is
similar to the Golgi apparatus of rat and
mouse epididymis m which the cisternae
of the concave face of the Golgi apparatus
are heavily fenestrated (Flickinger, '69).
Are there any other organelles in melanophores which may be involved in premelanosome formation? Although the
endoplasmic reticulum was considered in
the case of the pigmented epithelium of
the eye (Moyer, % 3 ) , it does not seem to
be involved in the final stages of melanosome formation in the amphibian melanophore because no electron dense material
was observed within it. Instead the endoplasmic reticulum :IS probably involved in
the synthesis and transport of protein utilized in melanosome formation. In the exocrine pancreas, for example, it has been
established that secretory proteins are
187
synthesized on the endoplasmic reticulum
and then move through the reticulum to
the condensing vacuoles on the inner face
of the Golgi apparatus (Caro and Palade,
'64; Jamieson and Palade, '68a,b).
What is the function of the microtubules
observed in melanophores? Since microtubules were recognized as organelles present in many cell types (Slautterback, '63),
there has been much speculation about
their function. Major theories include their
acting a s a cytoskeleton in the development
and maintenance of asymmetric cells or
their involvement in the movement of cytoplasmic inclusions (see review by Porter,
'66). Neither of these theories appears to
pertain to microtubules in amphibian melanophores (Wise, '69).
One of the first theories about microtubule function was that they transport
ions or small molecules such as water within the cell (Slautterback, '63). If this were
the case in melanophores, the close association of the microtubules to the premelanosomes might provide a means for
transporting materials for melanosome formation from sites of synthesis via the microtubules. It is also possible that the
microtubules are transporting materials
(water? ) from the premelanosomes.
Regardless of speculations about function, the close association between microtubules and premelanosomes or melanosomes is supported by other reports which
indicate that microtubules can connect to
inclusions. DuPraw ('65) utilized wholemount electron microscopy of honey bee
embryonic cells to demonstrate microtubules connected to cytoplasmic granules in
the cells. In addition, the apparent continuity between saccules and microtubules,
as seen in figure 8, is similar to the observations of continuity between pericentriolar vesicles and microtubules (Robbins
et al., '68) or to observations of connections between vesicles of the Golgi complex
and microtubules (Slautterback, '63 ) .
Whether this connection represents a conversion of the microtubule into a saccule
or vice versa is not known.
The greatest concentration of microtubules is in the region of the centrosphere
in the melanophore. Cells which form
granules usually have numerous microtubules in this region (e.g., Bainton and
188
GARY E. WISE
cytes. Two types derived from opposite faces
Farquhar, '66). Because of the abundance
of the Golgi complex in developing granuloof microtubules in this area, perhaps their
cytes. J. Cell Biol., 28: 277-301.
site of synthesis is here. The numerous, Breathnach, A. S., and S. V. Poyntz 1966 Elecspherical dense particles which surround
tron microscopy of pigment cells in tail skin of
Lacerta vivipera. J. Anat., 100: 549-569.
the centrioles in this area are the most
probable source of microtubules. The rea- Caro, L. G., and G. E. Palade 1964 Protein
synthesis, storage, and discharge i n the pansons for this are fourfold.
creatic exocrine cell: A n autoradiographic
First, some micrographs indicate a constudy. J. Cell Biol., 20: 473-495.
nection between the dense particles and Clark, A. W. 1967 Some aspect of spermiogenesis i n a lizard. Am. J. Anat., 121: 369-400.
the microtubules. Second, other workers
have stated that microtubules may orig- Dirksen, E. R.., and T. T. Crocker 1966 Centriole replication in differentiating ciliated cells
inate from electron dense material of variof mammalian respiratory epithelium. A n elecous sizes and shapes (see review by Porter,
tron microscopic study. J. Microscopie, 5: 629644.
'66; McManus and Roth, '67; Fuller and
Calhoun, '68). Third, numerous investi- DuPraw, E. J. 1965 The organization of honey
bee embryonic cells. 1. Microtubule and amoegators have shown that the microtubules
boid activity. Dev. Biol., 12: 53-71.
are not attached to the centrioles (de-The, Flickinger, C. J. 1969 Fenestrated cisternae i n
'64; Szollosi, '64; Murray et al., '65; Anderthe Golgi apparatus of the epididymis. Anat.
Rec., 163: 39-54.
son et al., '66; Clark, '67), but instead to
para-centriolar satellites which consist of Friend, D. S., and M. G. Farquhar 1967 Function of coated vesicles during protein absorpelectron dense material. Fourth, recent
tion i n the rat vas deferens. J. Cell Biol., 35:
work has demonstrated that similar dense
357-376.
particles also form centrioles in the trachea Fuller, M., and S. Calhoun 1968 Microtubulekinetosome relationships in the motile cells of
of the rat (Dirksen and Crocker, '66) and
the Blastocladiales. Z. Zellforsch., 87: 52C533.
in Xenopus laevis epidermis and trachea Hepler,
P. K., and W. T. Jackson 1968 Micro(Steinman, '68). Steinman also showed
tubules and early stages of cell-plate formation in the endosperm of Haemanthus Katherthat these spherical particles provide mainae Baker. J. Cell Biol., 38: 437446.
terial for synthesis of the axial microtubuIraldi, A. P., and E. de Robertis 1968 The
les of cilia. Therefore, it would seem prob- de neurotubu1a.r
system of the axon and the origin
ably that these dense particles are a source
of granulated and non-granulated vesicles in
regenerating nerves. Z. Zellforsch., 87: 330of microtubules in melanophores.
ACKNOWLEDGMENTS
This work was supported by United
States Public Health Service fellowships
1-F1-GM-32, 869-01 and 2-F1-GM-32, 86902 to the author, plus grant GM 10292 to
Professor Richard M. Eakin. This research
was conducted in Professor Eakin's laboratory at the University of California,
Berkeley, and the author is indebted to Dr.
Eakin for his guidance and generous assistance on this project. Appreciation also is
accorded to Dr. Charles J. Flickinger for his
critical reading of this manuscript and to
Dr. Daniel S . Friend for his invaluable advice pertaining to the functions of the
Golgi apparatus.
LITERATURE CITED
Anderson, W. A., A. Weissman and R. A. Ellis
1966 A comparative study of microtubules in
some vertebrate and invertebrate cells. Z. Zellforsch., 71: 1-13.
Bainton, D. F., and M. G. Farquhar 1966
Origin of granules in polymorphanuclear leuko-
344.
Jamieson, J. D.,arid G. E. Palade 1967a Intracellular transport of secretory proteins i n
the pancreatic exocrine cell, I. Role of the
peripheral (elements of the Golgi complex. J.
Cell Biol., :34: 577-596.
196713 Intracellular transport of secretory proteins i n the pancreatic exocrine cell. 11.
Transport of condensing vacuoles and zymogen
granules. J. Cell Biol., 34: 597-615.
Kessel, R. G., and H. W. Beams 1965 A n unusual configuration of the Golgi complex i n
pigment-praducing "test" cells of the ovary of
the tunicate, Styela. J. Cell Biol., 25: 55-68.
Lerner, A. B., and J. D. Case 1959 Pigment
cell regulatory factors. J. Invest. Dermatol., 32:
2 1 1-221.
McManus, Sister M. A., and L. E. Roth 1967
Microtubular structure in Myxomycete plasmodia. J. Ultrastruct. Res., 20: 260-266.
Mollenhauer, H. H., and D. J. Morre 1966 Golgi
apparatus and plant secretion. Ann. Rev. Plant
Physiol., 17': 27-46.
Moyer, F. H. 1963 Genetic ,effects on melanosome fine structure and ontogeny in normal
and malignant cells. Ann. N. Y. Acad. Sci.,
100: 584-606.
-- 1966 Genetic variations in the fine
structure and ontogeny of mouse melanin granule. Am. Zool., 6: 43-66.
ORIGIN OF PREMELANOSOMES
Murray, R. G., A. S. Murray and A. Pizzo 1965
The fine structure of mitosis in rat thymic
lymphocytes. J. Cell Biol., 26: 601-619.
Nieuwkoop, P. D., and J. Faber 1956 Normal
Table of Xenopus lacwis (Daudin). North Holland Publishing Co., Amsterdam.
Novikoff, A. B. 1964 GERL, its form and function in neurons of rat spinal ganglia. Biol.
Bull., 127: 358.
1967 Enzyme localization and ultrastructure of neurons. In: The Neuron, H.
HydCn, ed. Elsevier :Pub. Co., Amsterdam-London-New York, p. 2515-318,
Novikoff, A. B., A. Albala and L. Biempica 1968
Ultrastructural and cytochemical observations
o n B-16 and HardingPassey mouse melanomas:
The origin of premelanosomes and compound
melanosomes. J. Histochem. Cytochem., 16:
299-319.
Porter, K. R. 1966 Cytoplasmic microtubules
and their functions. In: Principles of Biomolecular Organization. Ciba Foundation Symposium Wolstenholme and O'Connor, ed. Little,
Brown, and Company, Boston, p. 308-356.
Robbins, E.. G. Jentzsch and A. Micali 1968
The centriole cycle i n synchronized HeLa cells.
J. Cell Biol., 36: 329-339.
189
Seiji, M., K. Shimao, M. S. C. Birbeck and T. B.
Fitzpatrick 1963 Subcellular localization of
melanin biosynthesis. Ann. N. Y. Acad. Sci.,
100: 497-533.
Slautterback, D. B. 1963 Cytoplasmic microtubules. I. Hydra. J. Cell Biol., 18: 367-388.
Smith, R. E., and M. G. Farquhar 1966 Lysosome function i n the regulation of the secretory process i n cells of the anterior pituitary
gland. J. Cell Biol., 31: 319-347.
Steinman, R. 1968 A n electron microscopic
study of ciliogenesis i n developing epidermis
and trachea in the embryo of Xenopus laevis.
Am. J. Anat., 122: 19-56.
Szollosi, D. 1964 The structure and function of
centrioles and their satellites i n the jellyfish,
Phiddidium gregarium. J. Cell Biol., 21: 465479.
de-ThC, G. 1964 Cytoplasmic microtubules in
different animal cells. J. Cell Biol., 23: 265275.
Wise, G. E. 1969 Ultrastructure of amphibian
melanophores after light-dark adaptation and
hormonal treatment. J. Ultrastruct. Res., 27:
472-485.
Note added in proof: After this paper was submitted for publication, a report by
G. G. Maul appeared i n the Journal of Ultrastructural Research (26: 163-176,
1969). The author reported that i n human melanomas premelanosomes appear to develop within smooth endoplasmic reticulum which is connected to
the Golgi apparatus.
PLATE 1
EXPLANATION OF FIGURES
190
1
Beginning stage of premelanosome formation showing electron dense
material ( a r r o w ) in the Golgi cisternae (gc). X 40,700.
2
Another deposit of electron dense material ( a r r o w ) , presumably melanoprotein, within the fenestrated cisternae ( F ) of the Golgi apparatus. x 78,900.
3
Numerous vesicles ( a r r o w s ) which may be utilized for premelanosome
formation are seen adjacent to the Golgi apparatus ( G ) . x 55,700.
ORIGIN OF PREMELANOSOMES
Gary E. Wise
PLATE 1
191
PLATE 2
EXPLANATION O F FIGURES
4
Premelanosomes ( P ) or possibly melanosomes which appear to be
developing within saccules of the Golgi apparatus (G). Adjacent to
the Golgi apparatus is a premelanosome (arrmu) with a fibrous substructure. Melanosomes ( M ) and coated vesicles ( c v ) also are present.
x 54,100.
5
Tangential section of a fenestrated cisterna (F) of the Golgi apparatus
with a premelanosome ( P ) adjacent to it. x 78,900.
6
Microtubule dilating (arrow) adjacent to a developing melanosome.
Note the close association between microtubule and premelanosome.
x 78,900.
7 Another microtubule which appears to dilate (arrow) before joining
the premelanosome.
192
x
78.900.
ORIGIN OF PREMELANOSOMES
Gary E. Wise
PLATE 2
193
PLATE 3
EXPLANATION OF FIGURES
8
A reticulum of microtubules ( m t ) that enter into saccules (s).
x 54,100.
9
Portion of centrosphere ( C ) illustrating dense particles ( d p ) of 600650 A and microtubules ( m t ) . Notice that some of the microtubules
appear to emanate from the dense particles (urrozus). X 78,900.
10 Centrosphere region ( C ) in which some microtubules ( m t ) extend to
the plasma membrane ( p m ) . Also, a microtubule appears to emanate
from a dense particle ( u r r o w ) . X 38,300.
194
ORIGIN OF PREMELANOSOMES
PLATE 3
Gary E. Wise
195
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