close

Вход

Забыли?

вход по аккаунту

?

38

код для вставкиСкачать
114
R.JOURNAL
ORTÍZ ET OF
AL. EXPERIMENTAL ZOOLOGY 286:114–119 (2000)
Small Ampullate Glands of Nephila clavipes
ROBERTO ORTÍZ, WAYCA CÉSPEDES, LUZ NIEVES, IRIS V. ROBLES,
ADOLFO PLAZAOLA, SHARON FILE, AND GRACIELA C. CANDELAS*
Department of Biology, University of Puerto Rico, San Juan,
Puerto Rico 00931-3360
ABSTRACT
The small ampullate glands of the orb-web spider, Nephila clavipes, have been
studied and compared to other of the silk producing glands from this organism. They exhibit the
same gross morphological features of the other glands. Electrophoretic analyses show that the
gland’s luminal contents migrate as a single band, while the contents of the secretory epithelium
reveal a step-ladder array of peptides in addition to the full size product. Previous studies from
our laboratory identified these peptides as products generated by translational pauses. This alternate mode of translation is typical of fibroin synthesis in all the spider glands thus far studied as
well as in those of the silkworm. The correlation of the peptides to the process of fibroin synthesis
is shown through experimental evidence in this paper. The gradual ultrastructural changes in
Golgi vesicles elicited by the fibroin synthesis stimulus can be seen in this paper. The response to
stimulation is of a higher magnitude in these glands than in any of those previously analyzed.
These studies show the small ampullate glands are a promising and certainly exploitable model
system for studies on the synthesis of tissue-specific protein product and its control. J. Exp. Zool.
286:114–119, 2000. © 2000 Wiley-Liss, Inc.
We have previously conducted studies on three
sets of fibroin producing glands of the orb-web spider, Nephila clavipes, the large ampullate (Candelas and Cintrón, ’81), the cylindrical (Candelas
et al., ’86) and the flagelliform glands (Rodríguez
and Candelas, ’95). Our investigations have confirmed that each of these sets of glands generate
one fibroin, the function(s) of which have been previously described (Lucas, ’64; Andersen, ’70).
From the three sets, we have exploited the large
ampullate glands as a model system for studies
on the synthesis of a tissue-specific protein product and its control. They have proven to be a fruitful model system. Simple manipulations have
offered us the unique opportunity to monitor the
process of elicited protein synthesis through time
sequence studies in the spider silk glands. In so
doing, we have detected a series of time and tissue-specific molecular syntheses events which prelude the production of fibroin by the glands. These
events optimize the gland’s protein synthesis machinery for the production of a huge fibroin with
an unusually high biased amino acid composition
(Candelas and Cintrón, ’81; Candelas et al., ’83,
’87, ’90; Candelas and López, ’83; Plazaola and
Candelas, ’91).
We have now turned our attention to another
set of the organism’s fibroin producing glands: the
small ampullate glands. This small pair of silk© 2000 WILEY-LISS, INC.
glands lies within the organism’s abdomen directly under the large ampullate glands. They
were originally described by Sekiguchi (’52) and
subsequently by Peters (’55). Andersen (’70)
claims that although the function of the small
ampullate’s product is not altogether clear, it is
definitely involved in the web structure and not
in the dragline as previously suggested by Warburton (1890). Andersen considered the possibility that the product of these glands might be used
in the production of the dry provisional spiral,
constructed as a temporary guiding line for the
sticky spiral thread produced by another set of
glands.
In this paper, we show that the product of these
glands migrates as a sole homogenous band, of
smaller size than the product of the large ampullate glands. We also provide evidence that the
process of translation occurs discontinuously. The
latter is made evident through the visualization
of the ladder of incomplete peptides in electrophoretic analyses of extracts of the gland’s secretory epithelium. This mode of elongation prevails
Grant sponsor: National Institutes of Health; Grant number:
5G12RR0364112; Grant sponsor: Institutional Funds.
*Correspondence to: Graciela C. Candelas, Department of Biology,
University of Puerto Rico, PO Box 23360, UPR Station, San Juan,
Puerto Rico 00931-3360. E-mail: gcandel@upracd.upr.clu.edu
Received 18 September 1998; Accepted 5 May 1999
SMALL AMPULLATE GLANDS OF NEPHILA CLAVIPES
in the other silk glands of the spider, as well as
in those of the silkworm Bombix mori (Lizardi et
al., ’79). We have also included the morphological
changes evoked by the fibroin synthesis stimulus
in the secretory epithelium at the ultrastructural
level. Lastly, the article contains data which confirms that the generation of incomplete fibroin
peptides correlates with the process of fibroin synthesis through a strategy previously used for
parallel studies in the large ampullate glands
(Candelas et al., ’83).
MATERIALS AND METHODS
Experimental animals
Adult female spiders collected from the field
were brought to the laboratory and kept unfed,
under high moisture conditions, in small containers to discourage web construction activity for a
minimum of five days. We have shown that under these conditions, the gland’s fibroin synthesis
is virtually abolished. Stimulation into a dramatic
level of synthesis is achieved through the mechanical depletion of the organism’s stored silks, also
as previously described (Candelas and Cintrón,
’81; Candelas and López, ’83).
115
at concentration of 300 µCi/ml. The incubations
were allowed to proceed for 90 min under gentle
shaking. The amino acids selected for these experiments were glycine (the most preponderant
amino acid of the small ampullate gland fibroin)
and leucine (a minor component), according to
Andersen (’70).
At the end of the incubation period, the glands
were transferred to unlabelled SSC and processed
for SDS-PAGE. Fluorography was conducted as
described by Bonner and Laskey (’74) without
modifications.
Electron microscopy
Small ampullate glands from stimulated organisms were excised and kept in 1× SSC for either
0, 30, 60 or 90 min prior to fixation. The same
procedure was followed with glands from unstimulated specimens. Tissues were fixed in 2.5%
glutaraldehyde buffered with 0.1 M PIPES, pH
7.4 (piperazine N-N′-bis (2-ethanesulfonic acid)
overnight at 4°C, post-fixed in 1% osmium tetrox-
Fibroin extracts
Two types of extracts were used in this work:
whole gland and luminal product extracts. In the
first case, the glands (large and small) were excised and handled as previously described in our
early publications (Candelas and Cintrón, ’81;
Candelas and López, ’83). To obtain the product
within the glands’ lumens, these are first excised,
the glands are then slit and their contents carefully removed and handled as previously described
in detail by Candelas and Cintrón (’81). This reference gives details on sample sizes and other
such pertinent details.
Solubilized gland extracts, as previously described, were loaded on gels bearing a 3% stack
with an analytical gel graded from 3.5% to 12.5%
acrylamide following Laemmli (’70) and Maizel
(’71), also previously described. Gels were imaged
and analyzed using a Bio Rad GS700 Imaging
Densitometer.
Gland culture and labeling
Glands were excised from unstimulated and
stimulated organisms and transferred to the incubation medium, 100 mM sodium citrate, 100
mM sodium chloride (1× SSC) supplemented with
either [3H] glycine (specific activity 16.2 Ci/mmol)
or [3H] leucine (specific activity 153.0 Ci/mmol)
Fig. 1. The small ampullate glands of Nephila clavipes.
T, tail; S, sac; D, duct.
116
R. ORTÍZ ET AL.
Fig. 3. SDS PAGE of whole gland extracts from the large
ampullate and small ampullate glands of Nephila clavipes.
Lanes 1–3, large ampullate gland extracts; lanes 4–6, small
ampullate gland extracts.
Fig. 2. Fluorograph of SDS electrophoresis of the luminal
protein content of the small ampullate glands labeled with
3
H-glycine. The analytical gel graded from 3.5 to 12.5%
acrylamide with a 3% stack.
ide aqueous solution for 1 hr in ice and subsequently dehydrated in a graded ethanol series, after which they were transferred to propylene oxide
and embedded in EmBed 812 (Electron Microscopy Sciences).
Silver sections were cut with a diamond knife,
mounted on copper grids. These were stained
with aqueous uranyl acetate and Reynold’s lead
citrate (Reynolds, ’63). The grids were examined
and micrographs taken using a Zeiss EM10 electron microscope.
RESULTS AND DISCUSSION
The small ampullate glands are seen in Figure
1. These glands are considerably smaller than the
large ampullates; however, they display identical
gross morphological features: a duct, a sac and a
highly convoluted tail. The latter, as is the case
of all known silk glands, bears the task of synthesizing fibroin product.
In Figure 2, we can see the luminal product of
the glands, extracted from glands of three differ-
ent organisms in the gel shown. These migrate
as a single band, seen in fluorography of a SDSPAGE. The small ampullates generate one fibroin,
as do the other spider glands thus far studied, an
unquestionable asset of a model system.
The gel shown in Figure 3 contains whole gland
extracts, including the secretory epithelium in the
preparations of both the small and large ampullates.
Lanes 1–3 were loaded with the extracts of the large
ampullates and 4–6 with those of small ampullate
glands. What we see in these gels, in addition to
the full size product (the uppermost band) is a reproducible step ladder array of peptides. Previous
studies have shown these to be incomplete fibroin
peptides generated by pauses made at reproducible
sites during the process of translational elongation.
This mode of elongation has been found to prevail
in fibroin synthesis, not only in the spider silk
glands, but also in those of the silkworm, Bombyx
mori (Lizardi et al., ’79; Candelas et al., ’83). This
figure also makes evident the difference in the size
of the two glands’ fibroin product.
Figure 4 shows a fluorograph of a gel loaded
with whole gland extracts of stimulated and
unstimulated glands. Lanes 1–6 are samples of
whole gland extracts incubated in the presence
SMALL AMPULLATE GLANDS OF NEPHILA CLAVIPES
117
Fig. 4. Fluorograph of SDS-PAGE of whole gland extracts
labeled [3H] glycine or [3H] leucine. Lanes 1–3, extracts from
stimulated spiders labeled with [3H] glycine; lanes 4–6, extracts from unstimulated spiders labeled with [3H] glycine;
lanes 7–8, extracts from stimulated spiders labeled with [3H]
leucine; lanes 9–10, extracts from unstimulated spiders labeled with [3H] leucine.
of labeled glycine, the most preponderant amino
acid of the small ampullate’s fibroin (Andersen,
’70). The first three lanes contain the extracts
of stimulated glands, while the other three are
from unstimulated ones. The effects of stimulation on the synthesis of fibroin are displayed in
these glands is very strongly in this experiment.
We have known from unpublished experiments
conducted by Cintrón that these glands response
to the stimulus is of greater magnitude than that
of the large ampullate glands (Cintrón, ’78). The
second set of lanes (7–10) contain the extracts
of glands incubated in labeled leucine, which according to Andersen (’70) comprises 0.96% of the
fibroin. This experiment, parallel to one conducted in the large ampullate glands (Candelas
et al., ’83), serves to confirm the correlation between the peptide ladders and the process of fibroin synthesis.
The last figure consists of three electromicrographs through the perinuclear region of the
secretory epithelium of the small ampullate
glands. The first plate is from a gland of an
unstimulated spider, while the other two are from
glands of stimulated spiders incubated for different time intervals, as indicated in the figure’s legend. Visible are the changes in the vesiculation
of the Golgi (G) as a function of stimulation. Observable is also the increase of mitochondria (M)
associated with the Golgi vesicles as fibroin synthesis activity progresses. Similar studies conducted in the large ampullates also revealed
similar gradual ultrastructural transitions in organelles of the glands in response to stimulation
(Plazaola and Candelas, ’91).
In conclusion, the small ampullate glands of this
orb-web spider generate a single secretory protein
product, of smaller molecular size than that generated by the large ampullates. Our data show
that translation occurs discontinuously, and generating, in so doing, the stepladder array of incomplete fibroin peptides, seen in all the other
glands previously studied. The intensity of the response to the fibroin synthesis stimulus is of a
higher magnitude than that of any of the previously studied glands. As previously mentioned,
118
R. ORTÍZ ET AL.
Fig. 5. Cross-section through the perinuclear region of the
secretory epithelium of the small ampullate glands (×10,400).
(A) Gland from unstimulated spider; (B) gland from stimu-
lated spider incubated for 30 min; (C) gland from stimulated
spider incubated for 60 min. G, Golgi complex; SG, secretory
granule; M, mitochondrion. Bar = 500 nm.
SMALL AMPULLATE GLANDS OF NEPHILA CLAVIPES
unpublished data from Cintrón (’78) who compared the rates of elicited fibroin synthesis in
these two sets of glands, show a two-fold difference to chemical stimulation and a four-fold to
mechanical stimulation between the small and
large ampullate glands. Since the latter have
proven to be what can be rightly called a lucrative model system (Arroyo et al., ’96), the exploitation of the small ampullates as a model system
for the synthesis of a tissue-specific protein product seems to be a promising venture.
ACKNOWLEDGMENTS
Supported by Institutional funds and NIH grant
5G12RR0364112 to G.C.C.
LITERATURE CITED
Andersen SO. 1970. Amino acid composition of spider silks.
Comp Biochem Physiol 35:705–711.
Arroyo G, Capó L, Cintrón I, Plazaola A, Vázquez E, Candelas GC. 1996. Las glándulas ampuladas mayores de
Nephila clavipes: un sistema modelo lucrativo. Ciencia y
Desarrollo 22:25–31.
Bonner WM, Laskey RA. 1974. A film detection method for
tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem 46:83–88.
Candelas GC, Cintrón J. 1981. A spider fibroin and its synthesis. J Exp Zool 216:1–6.
Candelas GC, Candelas T, Ortíz A, Rodríguez O. 1983. Translational pauses during a spider fibroin synthesis. Biochem
Biophys Res Commun 116:1033–1038.
Candelas GC, López F. 1983. Synthesis of fibroin in the cultured glands of Nephila clavipes. Comp Biochem Physiol
74B:637–641.
Candelas GC, Ortíz A, Molina C. 1986. The cylindrical or
119
tubiliform glands of Nephila clavipes. J Exp Zool 237:
281–285.
Candelas GC, Carrasco C, Arroyo G, Dompenciel R, Candelas
T. 1987. Strategies of fibroin synthesis. In: Ilan I, editor.
Translational control of gene expression. New York: Plenum
Publishers. p 209–228.
Candelas GC, Arroyo G, Carrasco C. 1990. Spider silkglands
contain a tissue-specific alanine tRNA that accumulates in
vitro in response to the stimulus for silk protein synthesis.
J Dev Biol 140:215–220.
Cintrón J. 1978. Aislamiento y caracterización parcial de la
proteína producida por las glándulas ampuladas mayores
de Nephila clavipes. MA thesis. Río Piedras: University of
Puerto Rico.
Laemmli UK. 1970. Cleavage of structural proteins during
the assembly of the head of bacteriophage T4. Nature
227:680–685.
Lizardi PM, Mahdavi V, Shields D, Candelas GC. 1979. Discontinuous translation of silk fibroin in a reticulocyte cellfree system and in intact glands. Proc Natl Acad Sci USA
76:6211–6215.
Lucas F. 1964. Spiders and their silks. Discovery 25:1–7.
Maizel JV Jr. 1971. Polyacrylamide gel electrophoresis of viral
proteins. In: Maramorosch K, Koprowski H, editors. Methods
in virology. vol. 5. New York: Academic Press. p 179–246.
Peters VHM. 1995. Uber der Spinneraparat Von Nephila
madagascariensis. Z Naturforsch 10B:395–404.
Plazaola A, Candelas GC. 1991. Stimulation of fibroin synthesis elicits ultrastructural modifications spider silk secretory cells. Tissue Cell 23:277–284.
Reynolds ES. 1963. The use of lead nitrate at high pH as an
electron opaque stain in electron microscopy. J Cell Biol 17:20.
Rodriguez R, Candelas GC. 1995. Flagelliform or coronata
glands of Nephila clavipes. J Exp Zool 272:257–280.
Sekiguchi K. 1952. On a new spinning gland found in geometric spiders and its functions. Annot Zoo Jan 25:394–399.
Warburton C. 1890. The spinning apparatus of geometric spiders. Q Microsc Sci 31:29–39.
Документ
Категория
Без категории
Просмотров
6
Размер файла
942 Кб
Теги
1/--страниц
Пожаловаться на содержимое документа