Jan 29, 1985 - We have used for comparison major ampul- late glands from the same spider with one gland serving as control for the second (experimental).
0022-l9lO/X6 $3.00+ 0.00
J. Inserffhpiol. Vol. 32. No. 2, pp.
I 17-123. 1986 Printed in Great Britain. All rights reserved
Copyright
(1’ 1986 Pergamon Press Ltd
THE INDEPENDENT REGULATION OF PROTEIN SYNTHESIS IN THE MAJOR AMPULLATE GLANDS OF ARANEUS CAVATICUS (KEYSERLING) E. K. Department
and M. A.
TILLINGHAST
TOWNLEY
of Zoology, University of New Hampshire, Durham, NH 03824, U.S.A. (Received 29 January 1985)
Ahatract-The effect of mechanical silking on the rate of protein synthesis in the major ampullate glands of the spider Aruaeur cuvaricushas been investigated. Silking of one of the paired glands results in a greater rate of protein synthesis than in the unpulled control gland; in both in viuo and in vitroexperiments. Our data indicate that the rate of protein synthesis can be regulated independently in the two silk glands. Radioisotopic label appears in the orb web more rapidly than one would expect were older silk drawn before newly synthesized silk.
Key Word Index: Protein synthesis, mechanical silking, spider web INTRODUCTION
The major ampullate gland of orb-weaving spiders is the source of the frame, radii, and hub spiral of the orb web (Andersen, 1970). It is composed of three regions (Fig. 1): the tail, which is the major site of protein synthesis (Bell and Peakall, 1969); the ampulla, the function of which includes both storage of progenitive silk and protein synthesis in its posterior region (Bell and Peakall, 1969); and the duct, in which water is withdrawn from the progenitive silk during its transport to the spinnerets (Witt et al., 1968); a process that appears to be accompanied by an exchange of Na+ and K+ ions (Tillinghast et al., 1984). It may be observed in Fig. 1 that a sac enshrouds the major ampullate duct. Mullen (1969) had earlier observed this structure in tissue sections and identified it as a sinus, but as it is a closed structure, it might be more accurately described as a sac or bursa. Two coexisting mechanisms have been postulated to promote the synthesis of protein in the major ampullate silk glands of orb-weaving spiders: (1) an acetylcholine-cholinesterase mechanism which is blocked by atropine and (2) a gland depletioninduced mechanism which is not blocked by atropine (Peakall, 1964, 1965). Candelas and Cintron (1981) have provided confirmatory evidence for both mechanisms. Both Peakall (1964, 1965) and Candelas and Cintron (1981) based their conclusions upon comparison of data from different individual spiders. In the present study, we have re-examined the second of ‘these mechanisms in order to (1) confirm or negate that the removal of silk from the gland is a stimulus to protein synthesis and (2) to ascertain if the paired silk glands are regulated independently of each other or not. We have used for comparison major ampullate glands from the same spider with one gland serving as control for the second (experimental). MATERIALS
AND METHODS
Large female Aruneus cuvaticus (Keyserling) were collected locally and maintained individually in 117
100 ml vials to prevent them from building webs until studied. Typically, the spiders were studied between 1 and 5 days after capture. Because this species would not build webs regularly under the prevailing laboratory conditions, large female Argiope trifarciata were utilized for those experiments requiring web construction. The spiders were mechanically silked as previously described (Tillinghast et al., 1984), with frequent inspections made with an Olympus, Model X-Tr, stereo dissecting microscope to assure that only major ampullate fibres were drawn. In vivo studies Group I. To observe the effect of silking upon the rate of protein synthesis, one gland of each spider was mechanically silked for 8 h. This gland will henceforth be referred to as “pulled”, the second gland as “unpulled”. Immediately following silking, the spiders were either fed 1 PCi [“C(U)]glycine (sp. act. 116mCi/mmol) or 5&i [2-‘Hlglycine (sp. act. 1 Ci/mmol). The spiders were confined so as not to allow construction of webs for a period of about 4, 16 or 40 h, depending on the experiment. They were then killed by severing the pedicel, and the major gland tail and ampulla were removed intact and in their entirety using spider saline (1.5 g NaCl/ 100 ml) during dissection. In our experience, this operation takes 2 h to perform. The glands were placed in 1 ml 10% trichloroacetic acid in a new culture tube (Kimax 16 x 125 mm) and placed on a rotary wheel for 1 h to remove unincorporated isotope. The trichloroacetic acid was then removed and replaced with 0.5 ml 6N hydrochloric acid and the glands were hydrolyzed at 110°C for 18 h. Following the transfer of the hydrolysate to a Falcon 2002 RIA tube, the hydrolysis vessel was rinsed with 0.5 ml distilled water which was also added to the RIA tube. The hydrolysate was brought to dryness on a Savant Speed-Vat evaporator and the residue was resolubilized in 500 ~1 of distilled water. A 100~1 sample was assayed for radioactivity in a
E. K.
and M. A.
TILLINGHAST
Incorporation into 450
glucose
silk
(A)
r
100
of [‘“Cl pulled
TOWNLEY
-
50J 1
I
I
I
I
I
I
2
3
4
5
6
7
1
450
-
100
I, 50 a
Hours
Incorporation into 450
j-
50 c 1
of f4C] pulled
glycine
silk 450
(8)
1
I 2
I
I
I
I
I
3
4
5
6
7
1
50
8
hours
Fig. 3. The appearance of radioactivity in silk drawn from the major ampuliate gland immediately following (A) the feeding of I PCi D-[‘4C(U)]glucoseand (B) 1 PCi [‘C(U)]glycine. Amino acid content is in micrograms. Beckman Beta-Mate II scintillation counter using Beckman Ready-Solv HP as the scintillation medium. Three different volumes of resolubilized residue were assayed for their amino acid content by the method of Moore and Stein (1948). Group 2
To observe the effect of unequal deficits on the rate of silk gland protein synthesis, both glands were silked for 4 h whereupon the silk from one gland was severed and the second gland was silked for an additional 4 h. The spiders were then fed 1 pCi of [Y(U)]glycine. Two hours were allowed to elapse before killing the spiders and assaying both glands for radioactivity and amino acid content by the above methods. Group 3A
To measure how rapidly radioactivity
and, there-
fore, new silk, would appear during an 8-h period of mechanical silking, the spiders were fed 1 PCi [‘4C(U)]glycine or D-[W_l)]glucose (sp. act. 3.9 mCi/mmol) just prior to the start of silking. The silk was drawn and collected at hourly intervals for 8 h. The samples were hydrolyzed in 6N hydrochloric acid and assayed for radioactivity and amino acid content by the procedures described above. Group 38
To compare mechanical silking to natural silking, three large female A. rrifaciata which had constructed webs that day were each fed I PCi of either of the isotopes used in Group 3A. The spiders’ existing webs were then removed and the first 3 webs constructed by each spider were collected on glass plates for autoradiography by methods previously described (Tillinghast and Kavanagh, 1977). All webs were exposed to X-ray film for 90 h in order to compare
Fig. 1. A complete major ampullate gland of A. cauoticus showing the tail, ampulla (AMP), and duct. Bar equals I mm. Insert A shows the end of the tail in detail; insert B shows the major ampullate duct bursa.
119
Fig. 2. An autoradiogram
of the first web constructed by A. tr(@ciuta of D-[‘4c(u)]glucose.
120
following the feeding of I pCi
Regulation of protein synthesis in silk gland Table I. The effect of mechanical silking on the in vbo rate of protein synthesis in the major ampullate .gkxnd (Group -I samples) Time after pull (h)
Isotope incorp. @pm/gland)
Weight of silk
% Silk
Gland
Time pulled (h)
pulled (cg)
from glandt
L R
0.0 8.0
4.0 4.0
20.314’ 32,044
5823 2443
0 2810
0 49
L R
8.0 0.0
4.0 4.0
90.766. 36.726
3276 5842
2550 0
44 0
L R
0.0 8.0
16.9 16.9
6571 19,716
3156 I324
0 1710
0 54
L R
0.0 8.0
15.5 15.5
4734 15,944
4013 3493
0 1440
0 36
L R
8.0 0.0
16.6 16.6
27,340 14,230
2512 3525
1350 0
38 0
L R
0.0 8.0
IS.8 15.8
133,865’ 206,300
6234 3415
0 2810
0 45
L R
0.0 8.0
16.0 16.0
90,897’ 243,017
5432 3741
0 2900
0 53
L R
8.0 0.0
37.9 37.9
21,957 13,447
4065 4429
1900 0
43 0
L R
0.0 8.0
41.1 41.1
9795 25,650
6108 5706
0 1710
L R
8.0 0.0
40.8 40.8
30,386 9336
2448 2395
980 0
41 0
L R
8.0 8.0
41.0 41.0
10,954 10,799
2636 2506
1330t 133oi
50 0, 53
Amino acid M/gland)
WlllOVCd
*For reasons of availability, [“Clglycine was used in thase experiments. In all others, [3Hklycine was used. tThesc values were obtained by dividing the weight of silk pulled by the amino acid content of the unpulled gland. $lt is assumed that the silk drawn came equally from both glands.
isotope incorporation in a semi-quantitative manner. For that autoradiogram which we present (Fig. 2), the web was later scraped from the plate and washed in 1.0 ml distilled water. The water insoluble fibroin was hydrolyzed in 6N hydrochloric acid, and following the removal of the acid, the radioactivity and amino acid content of both water soluble and waterinsoluble fibroin fractions were assayed. In vitro studies Group 4. To observe the effects of mechanical silking on the rate of protein synthesis in vitro, the silk was mechanically drawn from the major ampullate glands for a period of between 20 min and 8 h depending upon the experiment. The spiders were then killed and both major ampullate glands were removed intact and in their entirety. These were placed in 1.0 ml incubation medium (140 mM NaCl; 20 mM KH,PO,; 10 mM CaCl,; and 7 mM MgCl,) in a new culture tube (Kimax, 16 x 125 mm) and checked to assure that the tissue was completely submerged. Five microcuries of [2-3H]glycine or 1 PCi [‘4C(U)]glycine- were added to each vessel, which then were capped with parafilm and placed on a rotary wheel for 3 h of incubation. Following the incubation period, the incubation fluid was removed, the glands were washed in 10% trichloroacetic acid for 1 h, hydrolyzed in 6N hydrochloric acid for 18 h, and assayed for radioactivity and amino acid content as described above. All radioactive compounds employed in these studies were the products of New England Nuclear.
RESULTS
Table 1 illustrates the effect of mechanical silking of one gland on protein synthesis in uivo in both major ampullate glands of Group-l animals. In all cases, isotope incorporation was higher in the pulled gland than in the unpulled gland. And whereas a protein deficit remained in the pulled gland 4-16 h after pulling, the two glands were essentially equal in their protein content after 40 h. The effect of unequal silk deficits, created by silking one major ampullate gland for 4 h and the second for 8 h, on protein synthesis is presented in Table 2 (Group 2 samples). In all cases, the gland pulled for the greater length of time and, therefore, having the greater deficit, incorporated larger amounts of isotope. When isotopically labelled compounds were administered prior to pulling silk from the major ampullate gland (Group 3A), low levels of radioactivity were observed at all time intervals for up to 8 h of silking (Fig. 3); although at no time was a surge of radioactivity observed such as to suggest that radiolabelled fibroin was being drawn. When spiders were fed [‘“CJglucose or [“CJglycine (Group 3B) and allowed to construct webs, radioactive label was evident on all fibres of the first-constructed web. Subsequent webs built by the same given spider contained an apparently decreased amount of isotope. The web shown in the autoradiogram in Fig. 2 had a sp. act. of 45 cpm/pg amino acid and 31 cpm/pg amino acid for the water-soluble and water-insoluble orb-web components, respectively.
122
and M. A.
E. K. TILLINGHAST
TOWNLEY
Table 2. The effect of unequal deticits on the rate of protein synthesis in rice in the major ampullate gland (Group 2 samples) Time pulled
Time elapsed after 8-h
(h)
pull (h)
Gland
Isotope incorp. @pm/gland)
Wt silk pulled (Mg) Amino acid (pg/gland)
1st 4 h
2nd 4 h 1310
L R
4.0 8.0
2.0 2.0
10,349 14,129
3760 1961
1965’ 1965
L R
8.0 4.0
2.0 2.0
7570 5340
5440 6880
1o&o*
L R
4.0 8.0
2.0 2.0
8122 Il.857
3658 2192
15858 1585
I060
760 II70
*It is assumed that equal amounts of silk were pulled from each of the two silk glands during the first 4 h. The value presented for each gland is, therefore, half of the total weight of silk collected for the first 4 h.
Mechanical silking was also observed to increase the rate of protein synthesis in vitro (Group 4, Table 3). However, whereas both glands, experimental and unpulled control, incorporated [“Clglycine or [‘Hlglycine, the stimulating effect of silking was only observed following silking for 8 h. DISCUSSION
The results presented in Table 1 confirm the observation made earlier by Peakall (1964) and Candelas and Cintron (1981) that mechanical silking of the major ampullate gland stimulates the synthesis of protein. In all cases, mechanical silking resulted in a greater incorporation of isotope into the pulled gland compared to the unpulled control gland. These data indicate, at least a degree of, independent regulation in the two glands. One might have expected that if silking resulted in the release of a hormone from endocrine tissue, then both glands would have responded to it equally. As this was not the case, either (1) there is no haemolymph-borne hormone controlling protein synthesis in the major ampullate glands or (2) the response to such a hormone is modulated by the state of fullness of the major ampullate gland. This asymmetry of response was also observed when unequal deficits were created by pulling silk for 4 h from one gland and 8 h from the second gland
(Group 2, Table 2). These data would suggest that either regulation is intrinsic to the glands, or that they are regulated independently by nerves rather than by a circulating hormone, which would most likely stimulate protein synthesis equally in both glands. Again, however, a circulating hormone cannot be ruled out, as it is possible that the state of fullness of the major ampullate gland somehow influences the hormone’s ability to promote protein synthesis. The presence of radioactive label in the unpulled glands (Tables 1 and 3) is problematical. Whether this (1) reflects a turnover of protein in the silk gland epithelium, (2) indicates that silk synthesis is a continuous process at all times, to some extent, in the major ampullate gland, or (3) indicates that silking of one gland is a stimulus to both pulled and unpulled glands, cannot be distinguished from our data. The second possibility seems unlikely, for we know of no report where spiders, confined so as to be unable to construct webs, have been found to contain exceptionally large major ampullate glands. Yet under such conditions, there is an enlargement of the ftagelliform glands (Andersen, 1970). The third possibility cannot be dismissed, but it does raise again the question of why there was an unequal response between pulled and unpulled glands. Again, the state of fullness of the gland may govern its response to endocrine or neuroendocrine factors.
Table 3. The effect of mechanical silking on the in vim rate of protein syntheses in the major ampullate gland (Group 4 samples) Weight of silk pulled (pg)
% Silk removed from glandt
Gland
Time pulled (mitt)
Isotope incorp. @pm/gland)
Amino acid @g/gland)
L R
20 0
IO.272 10,627
2338 2572
L R
0 I52
15.242 14.887
2960 2671
-
-
L R
226 0
10.576 12,086
2106 2000
560 0
28 0
:
480 0
IO.784 5634
3592 I552
I7400
48 0
L R
480 0
74.340’ 32.450
257 I 5195
2150 0
41 0
L R
0 480
22,321. 30,712
5108 3108
0 I780
0 35
*For reasons
60 0
2 0
0
0
of availability. [“C)glycine was used in these experiments. In all others. [‘H!glycine was used. *These values were obtained by dividing the weight of silk pulled by the amino acid content of the unpulled gland.
123
Regulation of protein synthesis in silk gland We have observed that the dry weight of the combined ampullae of the major ampullate glands of Aruneus cavaficus is approx. 4mg. There being only a thin layer of tissue associated with this structure, the bulk of the weight must be progenitive silk. In previous studies (Tillinghast et al., 1984), we observed that the scaffold (orb web minus sticky spiral) of Argiope aurantia weighs about 0.3 mg. For A. cavweighing at&is, this must be larger; perhaps 0.5-l .Omg; and derives mostly from the major ampullate glands. If the scaffold weight for A. cuvaticus is in this range, then radioactively labelled, newly synthesized silk, would not be expected to be evident for several days after the feeding of isotope. This is not the case, however, since (1) radioactive silk appears in the scaffold of the first orb web constructed following the feeding of isotope (Fig. 2, Group 3B) and (2) low, but detectable, levels of radioactivity were observed for all 8 h of silking immediately following the feeding of isotope (Fig. 3, Group 3A). There are at least two explanations for these observations, and they are not mutually exclusive: (1) the radioactive label represents material synthesized and added to the progenitive silk during web construction and (2) the progenitive silk may mix within the ampulla in a manner not previously considered. A comparison of the specific activities of the silk samples obtained from Group 3A and Group 3B spiders clearly reveals that the level of radioactivity seen in the Group 3A silk samples (taken in hourly intervals for 8 h immediately following isotope feeding) is much too low to account for the amount of radioactivity which produced the autoradiograms of Group 3B spiders. For example, while the web in Fig. 2 had a sp. act. of 31 cpm/pg amino acid for the water-insoluble fraction (which would contain the major ampullate gland silk), the specific activities of the silk samples plotted in Fig. 3 ranged from 0.28 cpm/pg amino acid to 0.44 cpm/pg amino acid and from 0.47 cpm/pg amino acid to 1.12 cpm/pg amino acid for [‘4C]glycine fed and [‘4C]glucose fed spiders, respectively. Thus both of the above possibilities seem plausible and are worthy of further investigations.
Finally, it should be pointed out that when silk is mechanically drawn at a constant rate of speed, the actual amount of silk drawn tends to decrease with time (Fig. 3, Table 2). However, only rarely did we observe the silk to break during the 8 h of silking. The exact significance of this decrease in fibre size is unknown, although these observations are in keeping with Peakall’s (Witt et al., 1968) conclusion that the legs may be necessary to actually terminate a fibre. Acknowledgements-We thank Dr Herbert Levi (Harvard University) and Dr Marcel Reeves (University of New Hampshire) for identifying the spiders used in these experiments. We thank Dr Robert Work (North Carolina State University) for his constructive review of an earlier draft of the manuscript. This material is based upon work supported by the National Science Foundation under Grant No. PCM-8202807. REFERENCES
Andersen S. 0. (1970) Amino acid composition of spider silks. Comp. Biochem. Physiol. 35, 705-71 I. E&UA. L. and Peakall D. B. (1969) Changes in fine structure during silk protein production in the ampullate gland of the spider Aroneus sericatus. J. Cell. Biol. 42, 284-295. Candelas G. C. and Cintron L. (1981) A spider fibroin and its synthesis. J. exp. Zool. 216, l-6. Moore S. and Stein W. H. (1948) Photometric ninhydrin method. J. biol. Chem. 176, 367-388. Mullen G. R. (1969) Morphology and histology of the silk glands in Araneus sericafus Cl. Trans. Rm. microsc. Sot. 88, 232-240. Peakall D. B. (1964) Effects of chohnergic and anticholine@ drugs on the synthesis of silk fibroins of spiders. Comp. Biochem. Physiol. 12, 465-470. Peakall D. B. (1965) Regulation of the synthesis of silk fibroins of spiders at the glandular level. Camp. Biochem. Physiol. 15, 509-5 15. Tillinghast E. K. and Kavanagh E. J. (1977) The alkaline proteases of Argiope and their possible role in web digestion. J. exp. Zool. 202, 213-222. Tiliinghast E. K., Chase S. F. and Townley M. A. (1984) Water extraction by the major ampullate duct during silk formation in the spider, Argiope aurantioLucas. J. Insect Physiol. 30, 591-596. Witt P. N., Rctd C. F. and Peakall D. B. (1968) A Spider’s Web. Problems in Reguiarory Biology. Springer, New York.
