Cytoskeletal F-actin patterns in whole-mounted larval and adult salivary glands of the fleshfly, Sarcophaga bullata

TISSUE AND CELL, 199123 (2) 285-290 0 1991 Longman Group UK Ltd.

W. MEULEMANS

and A. DE LOOF

CYTOSKELETAL F-ACTIN PATTERNS IN WHOLEMOUNTED LARVAL AND ADULT SALIVARY GLANDS OF THE FLESHFLY, SARCOPHAGA BULLATA Keywords:

Salivary

glands,

actin, insect, phalloidin,

Sarcophaga

ABSTRACT. The patterns of filamentous actin were analysed in different larval, pupal and adult stages in the salivary glands of the fleshfly Sarcophaga bullata. Using the rhodamine labelled phalloidin staining method in combination with detergent extraction specific actin filament distribution was detected. The salivary glands which are histolysed during the process of metamorphosis show distinct cellular morphology and actin filament patterns in larvae and adults. The large third instar larval salivary gland cells contain a well developed apicolateral microvillar zone. In third instar larvae this microvillar zone invaginates and expands in the basal part of the lateral membranes. Larval salivary gland cells also contain numerous parallel basal actin bundles. The larval glands arc histolysed during metamorphosis and adult glands are formed out of the imaginal cell group. At the onset of metamorphosis these basal actin bundles form a network of crossing bundles. The filamentous actin patterns of the proximal part of adult gland cells is confined to the apicolateral microvillar membranes. The cells in the distal, tubular pait of the adult salivary glands show intense staining of their folded lateral membranes.

skeleton is known (Faulstich et al., 1988). Furthermore, using the RLP staining method we could observe the three dimensional organisation of the microvillar brush border in this transportive epithelium, as we could observe in the Malpighian tubules of the same insect (Meulemans and De Loof, 1990). We also describe the general morphology of the salivary glands of Sarcophaga bullata since the adult glands are quite different from the extensively investigated glands of the related fleshfly Culliphoru (Oschman and Berridge , 1970).

Introduction

The organisation of the salivary glands of the fleshfly Calliphora in tubules which are closed at one end allows easy measurement of their in vitro fluid transport (Berridge, 1977). Many physiological experiments could be conducted with this system (Berridge, 1977; Berridge, 1981, Berridge and Patel, 1968; Oschman and Berridge, 1970) leading to the elucidation of the intracellular pathways by which salivary secretion takes place. Our study focused on the cytoskeletal F-actin patterns in Sarcophuga bdlutu salivary gland cells. We used the fluorescent dye rhodamine labelled phalloidin (RLP) to label the cytoskeletal actin. This labelled mushroom toxin is a bicyclic heptapeptide which binds selectively to filamentous actin (Wieland, 1977). No association with other cytoskeletal proteins or components associated with the cytoZoological Naamsestraat Received:

Institute of the University 59, B-3000 Leuven, Belgium.

2 November

Materials and Methods Animals

Flies are reared as described by Huybrechts and De Loof (1977). Adults were collected at 24 hourly intervals. Three larval stages were discerned by means of their posterior stigmata as described by Roberts (1976). Flies are briefly anaesthetised with COz prior to dissection.

of Leuven,

1990. 285

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Whole-mount Feulgen staining method

This method was adapted from Wieschaus and Niisslein-Volhard (1986) and has been described by Meulemans and De Loof (1990). Phalloidin staining procedure

After dissection in Ringer solution (Chan and Gehring, 1971) the salivary glands (SG) are immediately fixed at room temperature in a 4% solution of paraformaldehyde in l/l cytoskeleton extraction buffer/distilled water (made fresh, pH 6.8) for 1.5min. The composition of this cytoskeleton extraction buffer (CEB) and further handling have been described by Meulemans and De Loof (1990). Briefly, the SG were permeabilised in 0.1% Triton X-100 in CEB. After a short wash in CEB the tissue was then stained with a 33 nanomolar solution of the rhodamine labelled phalloidin (Molecular Probes Inc., Pitchford Ave., Eugene, Oregon) in CEB for 60 min. Finally the SG were mounted in 60-70% glycerol in CEB on a slide. Epifluorescence

microscopy

Slides are immediately examined under a Leitz Laborlux S microscope, equipped for epifluorescence in the rhodamine mode. Photomicrographs were taken with KODAK T max 400 ASA black and white film or with KODAK Ektachrome 400 ASA colour slide film, of which black and white negatives were prepared on an Elinchrom dia duplicator. Results General morphology

and metamorphosis

Sarcophaga bullata has two salivary glands,

which join in a common salivary duct. Larvae have two tubular glands, built up by one layer of large cells with large polytene nuclei (Figs la, 2). These tubular glands are very large and swollen in the postfeeding third instar larvae, due to an increase in cell size. During the metamorphosis the SG cells are histolysed. Only the imaginal cells (Figs la, 2), which lie at the transition of the salivary duct and the gland, are retained and out these cells the adult SG will develop (Berridge et al., 1976). These consist of small cylindrical cells with small nuclei and contain a small tubular (probably secretory) distal part lead-

AND DE LOOF

ing to a large (probably reabsorptive) reservoir in which the salivary duct begins (Fig. lb). Unlike the SG of the related fleshfly Calliphora (Oschman and Berridge, 1970), the SG of Sarcophaga do not extend into the abdomen. The coiled distal part of the adult Sarcophaga glands is located just before the transition of the thorax to the abdomen. F-actin distribution Larval salivary glands. Larval SG consist of

large, round cells which show a smooth actin filament pattern at their apical membranes (Figs 3,4). This staining is due to the microvillar pattern present at these membranes. The apical staining pattern in first instar larvae is confined to the upper cell part (about one third of the cell height). In second instar larvae this apical pattern descends along the lateral membranes (Fig. 3) and it reaches the deepest point in third instar larvae (about one fifth of the cell height, Fig. 4). These third instar larval SG cells have a large apicolateral microvillar surface and are only connected to one another by a small lateral zone. Sometimes one or two infoldings of the upper part of the apical membrane are found but these are rather seldom (i’ in Fig. 4). In these third instar larval SG cells large infoldings are formed in the lowest part of their apical membrane (Figs 4-6). These infoldings lie underneath the nucleus and above a small less stained basal zone. They increase in size during the third larval instar, giving postfeeding larvae SG cells with a highly developed pattern of lateral infoldings (Fig. 5 shows the SG of a feeding third instar larva and Fig. 6 of a postfeeding larva). These infoldings are in fact deep invaginations of the apical microvillar zone. Remarkably in the most proximal cells of the SG, nearest to the SG duct, no infoldings of the apicolateral membrane are found. Cells distal to these few cells contain some basal infoldings and cells further away show many basal infoldings. At the basal membrane of all larval SG a pattern of small parallel actin bundles is present. These bundles which are always oriented perpendicularly to the SG length form a basal actin pattern around the entire SG (Fig. 7). In early pupae, larval SG are still present but they are gradually histolysed. The apical microvillar zone decreases and the inva-

F-ACTIN

PATTERNS

IN SALIVARY

287

GLANDS

Fig. la: Larval salivary gland. Note the group of small imaginal cells (i) at the proximal side of the gland. Associated with the SG are cells resembling larval fat cells (f) and cell of nephrocytic origin (n). The salivary duct (du) is lined with cuticular ridges. x12. Fie. lb: Adult salivarv eland. The sac-like oroximal Dart (D) and the coiled and tubular distal part yd) are built up by bmall cylindrical cells: The sali;ary &it (du) is also lined with cuticular ridges. x24.

ginations of the apicolateral membranes shrink. So the inverse process takes place: microvillar surface decreases and the volume of infoldings shrinks. The basal actin bundles in this stage are thicker and they form a basal network of crossing bundles now (unpublished results), instead of the parallel bundles in the larval stages. Adult salivary glands

The large proximal part of the adult SG contains cells associated in a tight epithelium, in which only the lateral membranes and an apical protrusion stain markedly (Fig. 8). In older flies the lateral membranes show some wrinkles, but no deep infoldings are present (inset in Fig. 8). In the distal tubular part of the adult SG the cells are still closely aligned but here the lateral membranes show a pattern of diverse lateral infoldings which stain much brighter than the lateral membranes in the proximal part of the SG (Fig. 9). This staining might be caused by microvilli-containing canaliculi, like in the secretory part of Culliphora SG (Oschman and Berridge, 1970; Skaer et al., 1975), but due to the extensive staining of this part of the SG, no separate canaliculi can be discerned.

Discussion

Our results clearly indicate increasing transportive capabilities are associated with an increase in microvillar surface. Indeed, we find an increase in microvillar surface at times when saliva secretion increases and vice versa. Poels et al. (1971) and Harrod and Kastritsis (1972) showed an increase in mucopolysaccharide synthesis in Drosophila larval SG cells prior to pupariation. This enormous increase in secretion is also triggered by administration of 20-OH-ecdysone in middle third instar larvae of Drosophila (Poels, et al., 1971). A similar increase in secretory processes might occur in Sarcophaga bullata, accompanied by an increase in microvillar surface. However the significance of such an increase in (mucopolysaccharide) secretion is unknown, since Sarcophaga pupae do not use any ‘glue substance’ to adhere to a substrate. Drosophila and other flies for example use such a salivary gland ‘glue’ (Harrod and Kastritsis, 1972; Fraenkel, 1952). In adult SG cells less actin filaments are found. The much smaller cells remain cylindrical and closely aligned in a tight

F-ACTIN PATTERNS IN SALIVARY

GLANDS

Fig. 8. Side-view of the proximal part of an adult SG with cylindrical cells, each with an apical protrusion. x800. The inset represents a top view of the SG cells of a S-day-old fly which show some folding of their lateral membranes (a: apical protrusion, b: basal membrane). x800.

Fig. 9. Distal part of an adult SG showing the highly folded lateral membranes. These are more heavily stained than the proximal part. x800.

Fig. 2. Larval SG in which the imaginal cell group (i) is clearly recognised after Feulgen staining. Note the very large polytene nuclei of the SG cells (N), by comparison with the nuclei of the imaginal cells (i) and the cells of the salivary duct (d). x250.

Fig. 3. (and following: stained with the RLP method): Second instar larval SG, in which the apical microvihar zone (m) is restricted to the apical half of the cell. No infoldings of the microvillar zone are found at either lateral or apical membranes. x450.

Fig. 4. Third instar postfeeding larval SG. At the basal cell surface (b) large and flat infoldings (i) of the apical membrane are found, while the apical membrane (a) shows one small infolding (i’). x600. Fig. 5. Third instar feeding larval SG showing fingerlike infoldings of the lateral part of the apical membrane (top-view of the cell). x800. Fig. 6. Third instar postfeeding larval SG showing large flat infoldings of the lateral part of the apical membrane. These develop out of the fingerlike infoldings shown in Figures 4 and 5. The cuticular lining of a tracheole is included (t). x500.

Fig. 7. Outer part of a third instar larval SG showing parallel basal actin bundles which are oriented perpendicularly to the length of the SG. x900.

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epithelium. The cells in the distal part of the glands show high concentrations of actin filaments, probably representing microvillar canaliculi in which the salivary fluid is formed. The small actin bundles at the basal cell side of larval glands might give cells greater adhesiveness. These bundles seem to run from cell to cell without limitation to cell boundaries. In our earlier work, similar actin filaments were also found at the basal membrane of Sarcophaga bullata ovariolar follicle cells (unpublished results). These actin bundles at the basal membrane of different types of insect epithelial cells might increase adhesiveness to the basal lamina. At least

in the Malpighian tubules of Sarcophaga, indications are found that thick basal actin bundles may give actively transporting cells structural support (Meulemans and De Loof, 1990).

Acknowledgements

The authors acknowledge Professor M. Quaghebeur for his continuous interest and for the use of the fluorescence microscope. Meulemans W. is supported by a junior research fellowship of the I.W.O.N.L. of Belgium. J. Puttemans is acknowledged for advice and assistance in photography.

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