J. Cell Set. 2, 137-144 (1967) 137Printed in Great Britain

FINE STRUCTURE OF THE CYTOPLASM

IN SALIVARY GLANDS OF SIMULIUM

H. C. MACGREGOR AND J. B. MACKIEDepartment of Zoology, The University, St Andrews, Fife

SUMMARY

The salivary glands of 3rd or 4th instar larvae of Simulium niditifrons are about 5 mm long andup to 400 fi wide. They have a capacious lumen which is normally filled with secretion.

The apical (luminal) plasmalemma of the gland cells is thrown into numerous microvilli. Thebasal plasmalemma is usually straight but is infolded in places. The infoldings may be complexnear to cell junctions. There is a thick, uniform basement membrane. Contact surfaces ofadjacent cells often interdigitate. A septate junction extends inwards from the lumen for one-quarter the depth of the cells. Rough endoplasmic reticulum is distributed evenly throughoutthe cytoplasm. Many Golgi complexes with dark membrane-bounded granules are scatteredthroughout the cytoplasm. Solitary granules, often more than 1 fi in diameter, lie in the apicalcytoplasm, especially near the apical border of the cell. These granules resemble the largerGolgi granules and the contents of the lumen. Solitary granules consisting of 2 componentshave been seen in various stages of passage through the cell membrane. The 2 components arepresent in roughly constant proportions and can be identified in the larger Golgi granules andin the secretion in the lumen. The nucleus is spherical. The nuclear envelope is smooth in thelarger cells of a gland but may be folded in the smaller cells. There are 80-100 pores//*2 ofnuclear envelope. Each pore appears to have a small granule at its centre. Microtubules,about 180 A thick, are numerous in the apical cytoplasm, particularly near the luminal border.Tubules which lie deep in the cytoplasm are flanked by a clear area 100—200 A wide.

The fine structure of a salivary gland cell of Simulium appears to indicate that the majorcomponents of the salivary secretion are synthesized in association with the ribosomes on therough endoplasmic reticulum, concentrated in the Golgi regions, formed into secretion granules,and passed out of the cell into the lumen of the gland by reverse phagocytosis.

INTRODUCTION

It is logical to look for a relationship between the activity of specific genes and thesynthesis of specific proteins in the cytoplasm of a cell. There is equivocal evidencefor such a relationship in the salivary glands of dipteran larvae. The cells of theseglands secrete a silky substance which normally fills the lumen of the gland. Thissecretion serves a variety of purposes during larval life and is used in the constructionof a capsule prior to pupation. It is made up of a number of proteins (Laufer &Nakase, 1965; Phillips & Swift, 1965), and some polysaccharide material (Phillips &Swift, 1965). It can be identified in the cytoplasm of salivary gland cells in the form ofnumerous small granules. In Sciara 3 types of secretion granule have been de-scribed (Phillips & Swift, 1965). One of these appears at a specific developmentalstage, another is present throughout development, and the presence of a third isunpredictable. This pattern of secretion is not yet known to be related to changes inthe appearance or particular regions of the chromosomes of salivary gland cells, but

138 H. C. Macgregor and J. B. Mackie

such a relationship may be anticipated if chromosomal changes in Sciara resemble thosewhich have been described in Chironomus (Beerman, 1952). In Chironomus some puffsappear at one developmental stage, others persist over long periods of development,and some come and go independently of the developmental stage of the larva. In cellsof a special lobe (' Sonderzellen') of the salivary gland in Chironomus palUdivittatus thepresence of one specific Balbiani-ring (BR 4 (SZ)) on the fourth chromosome isclearly related to the production by these cells of a particular kind of secretion granule(Beerman, 1961). The Sonderzellen of C. tentans lack the special secretion granulesand show no Balbiani-ring at the locus corresponding to BR 4 (SZ) in C. palUdivittatus.One might therefore suppose that in C. palUdivittatus the Balbiani-ring at BR 4 (SZ)is making an informational RNA which serves as a template for the synthesis of amajor component of the salivary secretion.

Some other studies, however, point to a more complex mechanism for the produc-tion of salivary secretion. Laufer & Nakase (1965) state that the secretion from salivaryglands of Chironomus thuntmi includes 3 enzymes, trehalase, hyaluronidase, and pro-tease, and 6 antigenic components. None of these enzymes nor the antigens are con-fined to the salivary glands. All are present in the haemolymph.

The activity of trehalase and hyaluronidase in salivary secretion is depressed byexposure of larvae to actinomycin D (Laufer, Nakase & Vandenberg, 1964). The con-centration of actinomycin D which produces a significant depression is different foreach enzyme. Likewise the dosage of actinomycin D which causes regression ofchromosomal puffing is not the same for all puffs. On the basis of this evidence Lauferet al. (1964) suggest that trehalase and hyaluronidase are synthesized in the cells of thesalivary glands, and that the continued synthesis and secretion of these enzymes isdependent upon the production of a template RNA. The 6 antigenic components of thesecretion on the other hand are supposed to be transported intact from the haemolymphacross the cells of the gland and passed into the lumen of the gland as part of the formedsecretion (Laufer & Nakase, 1965). Blood proteins labelled with 14C are transported inthis way and included in the secretion, as are human serum albumin and ferritin. It hasbeen argued from these observations that at least some of the major components ofthe secretion are synthesized outside the salivary glands and that the glands functionin their uptake, transport and secretion.

Laufer's observations raise two questions concerning the structure of salivary glandcells in dipteran larvae. First, does the cytoplasm of a salivary gland cell show all thefeatures which one might expect to find in a cell which is actively synthesizing andsecreting large amounts of a few specific proteins? These features have been welldocumented for the exocrine cells of the mammalian pancreas (Sjostrand & Hanzon,1954a, b, c; Ekholm, Zelander & Edlund, 1962; Ekholm & Edlund, 1959; Herman,Sato & Fitzgerald, 1964), which undoubtedly make their own exportable proteins(Warshawsky, Leblond & Droz, 1963; Caro & Palade, 1964). Secondly, does a dipteransalivary gland cell show any features which might betray its supposed role as a trans-porter cell?

The answer to the first of these questions is already known. Salivary gland cellsfrom larvae of a variety of Diptera have been shown to possess a highly organized

Cytoplasmic structure in Simulium salivary glands 139

endoplasmic reticulum studded with ribosomes, numerous Golgi regions, varioustypes of secretion granule, and many conspicuous mitochondria (Jacob & Jurand,1963, 1965; Phillips & Swift, 1965). The same authors have also remarked upon themicrovillate luminal border of the glands, and a particularly clear account of the glandcell junction and the basement membrane has been given by Loewenstein & Kanno(1964) and Wiener, Spiro & Loewenstein (1964). Some other features, includingcytoplasmic microtubules, have been identified by Jacob & Jurand (1965) in thesalivary glands of Smittia.

In the present paper we shall describe the cytoplasmic fine structure of salivaryglands from larvae of the black fly Simulium niditifrons (Edwards), and we shalldiscuss our observations in the light of current ideas concerning the origin of thesalivary secretion.

Simulium larvae live in fast-flowing water and use some of their salivary secretion toform silky threads which act as 'life lines' during local excursions downstream.Immediately before pupation the larva weaves a stout pupal case entirely from threadsof the salivary secretion. Both of these functions demand an intense production ofsecretion, and it was for this reason that we chose Simulium for our investigations.

MATERIALS AND METHODS

Larvae of Simulium niditifrons were collected from the bed of a stream runningthrough the Lade Braes in St Andrews. Salivary glands were dissected from 3rd or4th instar larvae which measured 3-5 mm in length. Dissections were performed withcare to avoid displacement of the secretion filling the lumen of the gland. Glands wereimmediately drawn into a pool of 1 % osmium tetroxide buffered with veronal acetateat pH 7-4 (Palade, 1952) and cooled to 2 °C. They were fixed for 1 h, dehydrated inacetone, and embedded in Vestopal W. Silver-to-grey sections were cut with glassknives on a Cambridge Ultra-Microtome (A. F. Huxley pattern), and mounted onAthene 483 grids without supporting films. Sections were double-stained with2 % uranyl acetate for 5 min and lead citrate (Reynolds, 1963) for 2 min, and examinedwith a Siemens Elmiskop 1 (80 kV) at negative magnifications of between 5 coo and40000.

OBSERVATIONS

The larvae of S. niditifrons are club-shaped. They are about | mm long athatching and 6-7 mm long immediately before pupation. The wider rear portion ofthe larva accommodates 2 long tubular salivary glands. Each gland is folded back uponitself once and is connected to the head by a long thin duct (Fig. 1). In transversesection the glands show a wide lumen filled with secretion and surrounded by flatrelatively narrow cells (Fig. 2). The nuclei of these cells are spherical and in the middleregion of the gland they occupy more than half the width of the cells.

Along the apical (luminal) border of the cell there are numerous microvilli project-ing into the lumen of the gland (Fig. 3). These are bounded by a continuation of thecell membrane. They are more numerous in cells from older larvae than in cells from

140 H. C. Macgregor andj. B. Mackie

corresponding regions of the gland in young larvae. They are also more numerous incells near the base of the gland than in cells of the neck region.

The contact surfaces of adjacent cells are folded and interdigitated. A septatejunction (Fig. 4) extends inwards from the lumen of the gland for about one-quarterthe depth of the cells. The septa are 80-90 A thick and are regularly spaced at intervalsof 40-60 A. We think that in such junctions the septa connect uninterrupted surfacemembranes.

The plasma membrane at the base of the cell is generally straight (Fig. 5). It does,however, fold inwards in some places, and in cells from older larvae these infoldingsmay be deep and complex (Fig. 6). The more complex infoldings are usually foundnear to a junction between adjacent cells. Sometimes there are membrane-boundedvesicles, which are relatively empty, inside the folds of the cell membrane (Fig. 6);such vesicles are extracellular. Similar vesicles may also be enclosed in the underlyingbasement 'membrane' (Fig. 6). Some care is needed in interpreting the profiles ofmembrane complexes near the base of the cell since these are often the result ofsectioning near to and in the plane of a cell junction, and thereby cutting through thetips of interdigitations between adjacent cells. In such cases all the compartments ofthe complex must necessarily be filled with cytoplasm and bounded by 2 unitmembranes.

Endoplasmic reticulum is distributed evenly throughout the cell (Figs. 3, 7) but islacking from regions near the basal and apical borders. The appearance of the endo-plasmic reticulum varies from gland to gland. Generally it appears as round, oval, orelongated profiles. Near the nucleus the elongated profiles lie more or less parallel tothe nuclear envelope (Fig. 7); in the apical regions of the cell they tend to be orientatedradially with respect to the lumen of the gland. The elements of the endoplasmicreticulum are heavily studded with ribosomes and interspersed with free ribosomes.

In a thin section of a single cell there may be as many as 100 Golgi complexes. Theseare scattered uniformly throughout the cell. Each consists of a disorderly collection ofsmooth membranes, round vesicles, and granules of dark material (Fig. 8). Each darkgranule is bounded by a membrane. In some cases the membrane compartmentsoccupied by 2 or more granules are confluent.

Large solitary granules (Figs. 3, 10) are characteristic of the cytoplasm on theapical side of the nucleus. These resemble the granules in the Golgi regions. Each isbounded by a membrane. Solitary granules vary in size; the smallest compare withthe larger granules of the Golgi regions, whereas the largest may be more than 1 /i indiameter. Large solitary granules are most common near the apical border of the cell.The fate of these granules can be reconstructed from the following observations.They consist of material which precisely resembles the contents of the lumen of thegland (Figs. 3, 9). We have seen granules immediately inside and outside the cellmembrane. We have also seen situations similar to that illustrated in Fig. 9. In suchcases the membrane surrounding the granule has fused with the cell membrane andthe granule has been ejected from the confines of the cytoplasm into the lumen. There-after the granule probably joins the main mass of secretion in the lumen.

Most solitary granules show 2 components (Fig. 10). The bulk of the granule con-

Cytoplasmic structure in SimuUum salivary glands 141

sists of a compact matrix which appears dark after staining with lead citrate. Embeddedin this matrix are numerous small round light areas which give the whole granule aspongy appearance. The light areas are not bounded by a membrane. They areuniformly about 500 A in diameter and spaced 900-1000 A apart. In sections theyoften seem to be arranged in a series of concentric rings. The light areas are not confinedto solitary granules; there are usually a few of them in the larger granules of the Golgiregions (Figs. 7 8).

The nucleus is spherical. The nuclear envelope is often folded in the smaller cells ofa gland but always smooth in the larger cells. The outer and inner membranes of thenuclear envelope are spaced about 200 A apart. The nuclear pores have an insidediameter of between 450 and 550 A. Distances between the centres of adjacent poresrange from 850 to 1400 A. There are 80-100 pores//*2 of nuclear envelope. When seenin surface view, as in sections tangential or oblique to the nuclear envelope, each poreappears to have a small granule in its centre (Fig. 11). These granules are rarely evidentin transverse sections through the nuclear envelope.

Cytoplasmic microtubules (Slautterback, 1963; Ledbetter & Porter, 1963; Porter,Ledbetter & Badenhausen, 1964) are common in the salivary glands of SimuUumlarvae. They appear in side view as 2 parallel dark lines and have an average thicknessof 180 ±30 A (Figs. 12, 13). They usually appear straight, never branch, and aremost numerous in the apical half of the cell, particularly near the luminal border.Elsewhere they are sparse. Unlike Jacob & Jurand (1965) we have never seen micro-tubules near the adjoining membranes of neighbouring cells. Microtubules which liedeep in the cytoplasm are particularly easy to see on account of a clear area whichextends for 100-200 A on either side of the tubule (Fig. 13). This area contrastssharply with the surrounding congestion of ribosomes and endoplasmic reticulum.Such clear areas are less evident around microtubules near the luminal border.

DISCUSSION

In the exocrine cells of the mammalian pancreas, digestive enzymes are synthesizedon ribosomes.attached to the limiting membranes of the rough endoplasmic reticulum(Siekevitz & Palade, i960). They are then transported across these membranes, segre-gated within the cisternae of the endoplasmic reticulum (Redman, Siekevitz & Palade,1966), and move to small peripheral vesicles of the Golgi region (Jamieson & Palade,1966). Thereafter they are concentrated into the condensation vacuoles of the Golgiregions and formed into individual secretion granules (Caro & Palade, 1964). Thegranules move towards the apical border of the cell and are thence discharged into thelumen of the acinus.

In SimuUum salivary glands, formed secretion granules lying near to the apicalborder of the cell consist of material which, in our electron micrographs, looks exactlylike the material occupying the lumen of the gland. We therefore think that the lumencontents are wholly derived from the visible intracellular secretion granules. If anyother material is secreted by the cells into the lumen then it is not detectable in ourelectron micrographs and can only be a minor component of the final secretion.

142 H. C. Macgregor and J. B. Mackie

The material of the solitary secretion granules looks exactly like the material in thecondensing vacuoles of the Golgi regions; and these condensing vacuoles are sur-rounded first by smooth vesicles and then by rough endoplasmic reticulum which isoften highly organized. Accordingly it seems reasonable to regard the salivary glandcell of a Simulium larva as a large version of an exocrine pancreas cell, making largequantities of one or a few specific materials and exporting them, by reverse phago-cytosis, to the lumen of the gland.

In our opinion there would appear no reason for supposing that any of the major pro-teins of the secretion are regularly imported from the haemolymph and merely trans-ported across the cells of the gland. The structure of the salivary gland cells inSimulium gives no indication of a transporting function beyond that of transportingsecretion granules from their site of formation to the apical border of the cell. Laufer &Goldsmith (1965) have reported 'specializations' of the basal plasmalemma in Chiro-nomus salivary glands which suggest that these cells are engaged in micropinocytosis.The salivary glands of Simulium show no such specializations. In these cells the basalplasmalemma is occasionally infolded and in glands from older larvae such infoldingsmay be deep and complex. Infoldings of the basal plasmalemma in secretory cells arenot uncommon, however, and they need not be interpreted as indications of pinocytoticactivity. They have been described in mammalian pancreas (Ekholm et al. 1962), inBrunner's gland in the mouse (Friend, 1965), and in certain cells of mammaliangastric mucosa (Ito & Winchester, 1963); yet there is nothing to suggest that any ofthese cells function by transporting rather than synthesizing their respective products.

The salivary glands of Simulium make only one type of secretion granule. Thesegranules consist of 2 microscopically distinct components, the dark material and thelight areas in our electron micrographs, and these components are present in roughlyconstant proportions. These observations contrast with the findings of Phillips & Swift(1965) who found, in Sciara, 3 different types of secretion granule. However, we havenot yet been able to look at the salivary glands of a larva which is in the act of makingits pupal capsule, and the secretion used to make this capsule may well differ from thatwhich is produced intermittently throughout the larval life.

We think that the microtubules in salivary gland cells of Simulium larvae may beconcerned with movement of materials through the cytoplasm of these cells. Such arole has already been suggested for microtubules in cultured mammalian cells byFreed (1965). The clear area surrounding microtubules which lie deep in the cyto-plasm can be interpreted as a passage through the dense endoplasmic reticulumcreated by a flow of non-particulate material along the outside of the tubule. The mostlikely role for tubules near the luminal border is that of propelling the larger secretorygranules towards the lumen. This idea is upheld by the fact that some secretorygranules have microtubules clustered around them (Fig. 10).

We are indebted to Dr Lewis Davies for his generous help in identifying the species and thestages of the larvae used in this work.

Cytoplasmic structure in Simulium salivary glands 143

REFERENCESBKERMAN, W. (1952). Chromomerenkonstanz und spezifische Modifikationen der Chromoso-

menstruktur in der Entwicklung und Organ-differenzierung von Chironomus tentans. Chromo-soma 5, 139-198.

BEEKMAN, W. (1961). Ein Balbiani-Ring als Locus einer Speicheldriisen-Mutation. Chromosoma12, 1-25.

CARO, L. G. & PALADE, G. E. (1964). Protein synthesis, storage, and discharge in the pancreaticexocrine cell. J. Cell Biol. 20, 473-495.

EKHOLM, R. & EDLUND, Y. (1959). infrastructure of the human exocrine pancreas. J. Ultra-struct. Res. 2, 453-481.

EKHOLM, R., ZELANDBR, T. & EDLUND, Y. (1962). The ultrastructural organization of the ratexocrine pancreas. J. Ultrastruct. Res. 7, 61-72.

FREED, J. J. (1965). Microtubules and saltatory movements of cytoplasmic elements in culturedcells. Jf. Cell Biol. 27, 29A.

FRIEND, D. S. (1965). The fine structure of Brunner's glands in the mouse. J. Cell Biol. 25,563-576.

HERMAN, L., SATO, T. & FITZGERALD, P. J. (1964). The pancreas. In Electron MicroscopeAnatomy (ed. S. M. Kurtz), pp. 59-95. New York and London: Academic Press.

ITO, S. & WINCHESTER, R. J. (1963). The fine structure of the gastric mucosa in the bat. J. CellBiol. 16, 541-577-

JACOB, J. & JURAND, A. (1963). Electron microscope studies on salivary gland cells of Bradysiamycorum Frey (Sciaridae). III. The structure of the cytoplasm. J. Insect Physiol. 9, 849—857-

JACOB, J. & JURAND, A. (1965). Electron microscope studies on salivary gland cells. V. Thecytoplasm of Smittia parthenogenetica (Chironomidae). J. Insect Physiol. u , 1337-1343.

JAMIESON, J. D. & PALADE, G. E. (1966). Role of the Golgi complex in the intracellular trans-port of secretory proteins. Proc. natn. Acad. Sci. U.S.A. 55, 424-431.

LAUFER, H. & GOLDSMITH, M. (1965). Ultrastructural evidence for a protein transport systemin Chironomus salivary glands and its implication for chromosomal puffing, jf. Cell Biol. 27,57 A.

LAUFER, H. & NAKASE, Y. (1965). Salivary gland secretion and its relation to chromosomalpuffing in the dipteran, Chironomus ihummi. Proc. natn. Acad. Sci. U.S.A. 53, 511-516.

LAUFER, H., NAKASE, Y. & VANDENBEKG, J. (1964). Developmental studies of the dipteransalivary gland. I. The effects of actinomycin D on larval development, enzyme activity, andchromosomal differentiation in Chironomus ihummi. Devi Biol. 9, 367-384.

LEDBETTER, M. C. & PORTER, K. R. (1963). A microtubule in plant cell fine structure. J. CellBiol. 19, 239-250.

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PALADE, G. E. (1952). A study of fixation for electron microscopy. J. exp. Med. 95, 285-298.PHILLIPS, D. M. & SWIFT, H. (1965). Cytoplasmic fine structure of Sciara salivary glands. I.

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SJOSTRAND, F. S. & HANZON, V. (19546). Membrane structures of cytoplasm and mitochondriain exocrine cells of mouse pancreas as revealed by high resolution electron microscopy.Expl Cell Res. 7, 393-414.

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{Received 7 September 1966)

Fig. 1. Photomicrograph showing a salivary gland from a 4th instar Simulium larvafolded as it is when inside the larva. Only the rear portion of the salivary duct (d) isincluded in the picture.Fig. 2. Photomicrograph of a 1-/1 section through a salivary gland in the region im-mediately behind the fold in the gland (n, nucleus; s, secretion filling the lumen of thegland).Fig. 3. Electron micrograph of the apical border of a salivary gland cell and the lumenof the gland (/, lumen; mv, microvilli; s, secretion; sg, solitary granules).

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Fig. 4. Part of a septate junction between adjacent cells. The arrows indicate regionswhere the septa are visible.Fig. 5. The basal edge of a salivary gland cell showing the basement membrane (b),the plasma membrane (p), and 2 relatively simple folds (/) in the plasma membrane.Fig. 6. A complex infolding (/) of the basal plasma membrane at a junction between2 adjacent cells. Membrane-bounded vesicles (v), which are relatively empty comparedwith the cytoplasm, lie within the basement membrane (b) and between adjacentcell membranes in the infolded region. The area marked sd and similar areas to thelower left of it are interpreted as sections through the tips of interdigitations betweenadjacent cells.

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Fig. 7. An area of cytoplasm on the apical side of the nucleus showing the arrangementof rough endoplasmic reticulum and several Golgi regions (G). The larger granulesof the Golgi regions have a speckled appearance due to the presence of small regularlyarranged light areas within them (ch, chromosomes; nc, nucleolus; mn, nuclearmembrane).Fig. 8. A typical Golgi region. The largest granules in this region show some of thelight patches which characterize the solitary secretion granules.Fig. 9. Section through a secretion granule (sg) as it leaves the confines of the cyto-plasm and is discharged into the lumen of the gland (s, secretion in the lumen).

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Fig. 10. A region near the apical border of a cell showing 3 large secretion granules(sg) and several smaller ones. Immediately above the large granule on the right arenumerous short profiles of microtubules; /, lumen.Fig. 11. Oblique section through part of a nuclear envelope. The arrows indicatepores which have granules visible in their centres.Fig. 12. A region near the apical border of a cell showing numerous profiles of micro-tubules. The more distinct profiles are indicated by arrows.Fig. 13. An enlarged view of a microtubule with a clear area on either side of it.

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