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: firstname.lastname@example.org 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. 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