A novel basal lamina matrix of the stratified epithelium of the ovarian follicle.

Basal laminas are important sheets of specialized extracellular matrix that underlie and surround groups of cells, such as epithelia or endothelia, enabling the cells to orientate their basal/apical polarity and creating a microenvironment for them. Basal laminas can also individually encapsulate whole cells, such as muscle cells, thereby forming a microenvironment but not polarizing the enclosed cells. Other mesenchymal or stromal cells exist with no basal lamina. In the course of studying the bovine follicular basal lamina which underlies the multilayered epithelium of the ovarian follicle, we identified a developmentally regulated novel extracellular matrix (which we call focimatrix for focal intra-epithelial matrix). Focimatrix is composed of basal lamina-like material deposited as plaques or aggregates between the multilayers of the epithelial granulosa cells. The focimatrix does not encapsulate individual or groups of cells and therefore does not form a microenvironment for them. Focimatrix contains collagen type IV subunits alpha1 and alpha2 (but not alpha3-alpha6), and laminin chains alpha1, beta2 and gamma1 (but not alpha2 or beta1), and nidogen-1 and perlecan (but not versican). The amount of focimatrix increases with increasing follicular size, and its appearance precedes the expression by granulosa cells of the enzymes for steroid hormone synthesis, cholesterol side-chain cleavage cytochrome P450 (SCC) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD), in the days preceding ovulation. The expression in granulosa cells of two components examined, nidogen-1 and perlecan, also increases substantially when follicles enlarge to a sufficient size capable of ovulating. Following ovulation the follicular basal lamina is degraded, and presumably focimatrix is too since it is not detected in corpora lutea that develop from the ovulating follicles. During this development the granulosa cells undergo an epithelial-mesenchymal transition (EMT) into luteal cells following ovulation, and substantially increase their expression of steroidogenic enzymes in the process. During EMT epithelial cells lose polarity. Since focimatrix exists on more than one side of the granulosa cells, we propose that it disrupts the polarity induced by the follicular basal lamina in the lead up to ovulation. Hence focimatrix maybe a key part of the follicular/luteal EMT.     

Microdissection by ultrasonication: porosity of the intestinal epithelial basal lamina

The porosity of the epithelial basal lamina of normal rat intestine was studied by SEM. Epithelial removal was accomplished by prolonged fixation of tissue samples in OsO4 or immersion in aqueous H3BO3, followed by dehydration in acetone and microdissection by ultrasonic vibration. The underlying basal lamina of intestinal epithelium reveals numerous pores of variable size. These pores are more numerous in small than in large intestine and penetrate the entire thickness of the basal lamina. Within the basal lamina overlying lymph nodules, they are numerically increased. Their occurrence is evident in fixed and unfixed, sonicated and unsonicated tissue samples. Microprojections of epithelial cytoplasm are often observed within these pores. The results of this study suggest that migrating cells or epithelial-cell processes induce pore formation in epithelial basal laminae and that these pores may be eventually repaired.     

The basal lamina of the postnatal mammary epithelium contains glycosaminoglycans in a precise ultrastructural organization

The mammary epithelium was investigated to determine whether glycosaminoglycans (GAG) are components of the basal lamina of epithelia undergoing postnatal morphogenesis. Isolated epithelial tissues from midpregnant mice produce substantial amounts of GAG, consisting predominantly of hyaluronic acid and heparan sulfate. The basal surfaces of mammary epithelia at various postnatal developmental stages show GAG, as demonstrated by histochemistry and by autoradiography coupled with enzyme susceptibility. Electron microscopy using ruthenium red staining reveals polyanionic components, presumably GAG, within the epithelial basal lamina. Detailed ultrastructural analyses of tannic acid-treated and ruthenium red-stained material demonstrate that the lamina contains a two-dimensional symmetrical array of tetragonally ordered components colsely associated with the basal plasma membrane. This array is similar to that found in the hyaluronate-containing lamina of embryonic epithelia. A structurally ordered complex of GAG-containing macromolecules may characterize the basal lamina of all epithelia which undergo morphogenetic changes in cell shape.     

The role of Plasmodium berghei ookinete proteins in binding to basal lamina components and transformation into oocysts.

The ookinete is a motile form of the malaria parasite that travels from the midgut lumen of the mosquito, invades the epithelial cells and settles beneath the basal lamina. The events surrounding cessation of ookinete motility and its transformation into an oocyst are poorly understood, but interaction between components of the basal lamina and the parasite surface has been implicated. Here we report that interactions occur between basal lamina constituents and ookinete proteins and that these interactions inhibit motility and are likely to be involved in transformation to an oocyst. Plasmodium berghei ookinetes bound weakly to microtitre plate wells coated with fibronectin and much more strongly to wells coated with laminin and collagen IV. A 1:1 mixture of collagen and laminin significantly enhanced binding. Binding increased with time of incubation up to 10 h and different components showed different binding profiles with time. Two parasite molecules were shown to act as ligands for basal lamina components. Western blots demonstrated that the surface molecule Pbs21 bound strongly to laminin but not to collagen IV whereas a 215 kDa molecule (possibly PbCTRP) bound to both laminin and collagen IV. Furthermore up to 90% inhibition of binding of ookinetes to collagen IV/laminin combination occurred if parasites were pre-incubated with anti-Pbs21 monoclonal antibody 13.1. Some transformation of ookinetes to oocysts occurred in wells coated with laminin or laminin/collagen IV combinations but collagen IV alone did not trigger transformation. No binding or transformation occurred in uncoated wells. Our data support the suggestion that ookinete proteins Pbs21 and a 215 kDa protein may have multiple roles including interactions with midgut basal lamina components that cause binding, inhibit motility and trigger transformation.     

Mesenchyme cells degrade epithelial basal lamina glycosaminoglycan

We investigated whether turnover of basal lamina glycosaminoglycan (GAG), an active process during epithelial morphogenesis, involves the mesenchyme. Fixed, prelabeled, isolated mouse embryo submandibular epithelia were prepared retaining radioactive surface components, as determined by autoradiographic and enzymatic studies, and a basal lamina, as assessed by electron microscopy. Recombination of mouse embryo submandibular mesenchyme with these epithelia stimulates the release of epithelial radioactivity when the labeled precursor is glucosamine or glucose but not when it is amino acid. The release is linear with time during 150 min incubation. Augmented release of epithelial label requires living mesenchyme which must be close proximity with the epithelia. Although heterologous mesenchymes, including lung, trachea, and jaw, stimulate the release of submandibular epithelial label, epithelial tissues do not. The label released by intact submandibular mesenchyme from prelabeled epithelia is in GAG and in two unique fractions: heterogeneous materials of tetrasaccharide or smaller size and N-acetylglucosamine. Enzymatic treatment of the heterogeneous materials revealed the presence of glycosaminoglycan-derived oligosaccharides. These unique products were not obtained by incubating prelabeled epithelia with a mesenchymal cell extract, suggesting that intact mesenchymal cells are required. N-Acetylglucosamine was also released when mesenchyme was recombined with living prelabeled epithelia which contained labeled basal laminar GAG. Our results establish that submandibular epithelial basal lamina GAGs are degraded by submandibular mesenchyme. We propose that one mechanism of epithelial-mesenchymal interaction is the degradation of epithelial basal laminar GAG by mesenchyme.     

A simple mechanical method to isolate the basal lamina of insect midgut epithelial cells

In mealworms (Tenebrio molitor) the midgut epithelium is surrounded by a 1.6mum thick basal lamina of low electron density with a framework of high electron density imbedded in a part of it. The lamina can be isolated by ultrasonication followed by repeated filtrations and high-speed centrifugations, making large-scale preparation of the lamina for further analyses possible. The isolation of the basal lamina is confirmed by electron microscopy.     

Morphologic changes of the basal lamina in the small intestine of Xenopus laevis during metamorphosis

Further investigations of the epithelial and mesothelial basal lamina of the duodenum of Xenopus laevis during metamorphosis were performed by means of scanning electron microscopy (SEM) and histochemical techniques using polyethyleneimine (PEI) to demonstrate anionic sites as well as light- and transmission-electron-microscopic methods involving morphometric analysis. The basal lamina of the duodenal epithelial cells was smooth, and it was occasionally curved along the processes of the epithelial cells (stages 56-59). The basal lamina became thicker by folding, and the thickness of the folded basal lamina exceeded 1 micron (stages 60-62). Subsequently, the folded basal lamina disappeared gradually and became almost smooth again and consisted of only one layer (stages 63-66). After removing the epithelium by boric acid, SEM revealed that the small ridges of the basal lamina protruded like a mesh-work into the luminal side, and the luminal surface of the basal lamina became smooth at later stages of the metamorphic climax. The electron-dense granules of PEI-positive material were localized at both sides of the lamina densa at regular intervals (80-100 nm). The basal lamina of the mesothelial cells was almost smooth at stages 56-59 and started to show occasional slight folding. This folding became continuous and deeper (stages 60-62). The folded mesothelial basal lamina disappeared except for the cell-associated basal lamina and became smooth again at later stages of the metamorphic climax (stages 63-66). These morphologic changes of the basal lamina observed in the epithelium and mesothelium may be induced by common factors. We suggest that physical changes in the small intestine involving the shortening and narrowing should be a main factor to cause these changes in the basal lamina. Furthermore, morphometric analysis proposed that the basal lamina becomes more complex by adding newly synthesized basal lamina material, especially in the epithelium.     

Hemocytes contribute to both the formation and breakdown of the basal lamina in developing wings of Manduca sexta

Monoclonal antibodies (MAbs) raised against wing tissues of Manduca sexta recognize epitopes shared by both hemocytes and basal laminae. During the last larval stadium, the basal lamina of moth wing epithelium forms after hemocytes have migrated into the space adjacent to basal surfaces of epithelial cells. As adult development commences, hemocytes participate in phagocytosis of the same basal lamina; and as dissolution of the basal lamina proceeds (day 2-day 5 post-pupation), wing epithelial cells send forth long basal processes and rearrange within the plane of the epithelium. During this period of cell rearrangement, the immunoreactivity of the basal lamina decreases in concert with an increase in immunoreactive vesicles within hemocytes; and at the ultrastructural level, hemocytes have been observed to engulf fragments of basal lamina. The distribution of immunolabel in the developing moth wing suggests that hemocytes contribute not only to the formation of the wing's basal lamina but also to its breakdown. Since basal laminae are probably important determinants of epithelial form and pattern, hemocytes also contribute to the shaping of epithelial populations.     

Biosynthesis of the basal lamina as a result of interaction between thyroid and mesenchymal cells in culture

Porcine thyroid cells were cultured alone or in mixed cultures with mesenchymal cells. The formation of a basal lamina in vitro was investigated ultrastructurally. Follicular reassociation of thyroid cells occurred in both types of culture; however, it was followed by formation of the basal lamina only when mesenchymal cells were present. The present findings suggest an epithelial origin of the basal lamina resulting from an interaction with mesenchymal cells.     

Ultrastructural changes in the midgut epithelium in Podura aquatica L. (Insecta, Collembola, Arthropleona) during regeneration

Data considering the degeneration and regeneration of the midgut epithelium in the primitive wingless insects, such as Collembola, are rather poor. Also information, which treats the regenerative cells as the primordial cells, is poorly known. The midgut epithelium of Podura aquatica L. (Insecta, Collembola, Arthropleona) is formed by the epithelial and regenerative cells. The epithelial cells show distinct regionalisation in the organelles distribution. The ultrastructure of the basal, perinuclear and apical regions of the epithelial cells is described. As in insects without Malpighian tubules, structures which resemble urospherites occur in the cytoplasm of the epithelial cells. After degeneration of the entire midgut epithelium, a new epithelium is formed from regenerative cells. During the process of regeneration, the degenerated epithelium gradually is separated from the basal lamina by the newly formed one. Finally, the detached epithelium is moved into the midgut lumen. Regenerative cells play a role of primordial cells during epithelial regeneration.     

Type I collagen reduces the degradation of basal lamina proteoglycan by mammary epithelial cells

When mouse mammary epithelial cells are cultured on a plastic substratum, no basal lamina forms. When cultured on a type I collagen gel, the rate of glycosaminoglycan (GAG) synthesis is unchanged, but the rate of GAG degradation is markedly reduced and a GAG-rich, basal lamina-like structure accumulates. This effect of collagen was investigated by comparing the culture distribution, nature, and metabolic stability of the 35S-GAG-containing molecules produced by cells on plastic and collagen. During 48 h of labeling with 35SO4, cultures on collagen accumulate 1.4-fold more 35S-GAG per microgram of DNA. In these cultures, most of the extracellular 35S-GAG is immobilized with the lamina and collagen gel, whereas in cultures on plastic all extracellular 35S-GAG is soluble. On both substrata, the cells produce several heparan sulfate-rich 35S-proteoglycan fractions that are distinct by Sepharose CL-4B chromatography. The culture types contain similar amounts of each fraction, except that collagen cultures contain nearly four times more of a fraction that is found largely bound to the lamina and collagen gel. During a chase this proteoglycan fraction is stable in cultures on collagen, but is extensively degraded in cultures on plastic. Thus, collagen-induced formation of a basal lamina correlates with reduced degradation and enhanced accumulation of a specific heparan sulfate-rich proteoglycan fraction. Immobilization and stabilization of basal laminar proteoglycan(s) by interstitial collagen may be a physiological mechanism of basal lamina maintenance and assembly.     

Retention of epithelial basal lamina allows isolated mandibular mesenchyme to form bone

The initiation of bone formation in the avian mandible requires that neural crest-derived cells undergo an inductive interaction with mandibular epithelium. To examine the role of the epithelial basal lamina in that interaction, mandibles were separated into their epithelial and mesenchymal components following exposure to the chelating agent, EDTA. Transmission and scanning electron microscopy was used to show that the basal lamina was retained as a continuous layer over the mesenchyme. Osteogenesis was initiated when such EDTA-isolated mesenchyme was grafted to the chorioallantoic membranes of host embryos. In contrast, mesenchyme isolated using trypsin and pancreatin failed to form bone. It is concluded that the property of mandibular epithelium which permits osteogenesis resides within the basal lamina.     

Structure and regeneration of the planarian basal lamina: An ultrastructural study

The structure and regeneration of the planarian subepidermal basement membrane or basal lamina have been electron microscopically examined, particularly in relation to the changes of extracellular products at the wounded area. The intact basal lamina consists of three structural elements; namely, an electron-lucent zone, a limiting layer and a microfibrillar layer. Ultrastructural changes during wound healing have suggested that the amorphous material secreted in the interspace between the epidermal cells and blastema contains precursors of the basal lamina. Within the amorphous zone two distinct phases of the basal lamina regeneration are observed: one is a reconstitution of the limiting layer and the other is a polymerization of the microfibrils. The limiting layer arises from areas subjacent to newly developed hemidesmosomes of epidermal cells. The unit microfibrils are formed from an accumulation of the precursors through transitional smaller microfibrils. At the late stage, individual mature microfibrils are regularly lined with the limiting layer and cell membranes of the newly differentiated muscle fibres. On the basis of these observations we suggest that the planarian basal lamina is regenerated by the interaction between epidermal cells and myoblasts.     

Effects of acid on the basal lamina of the rat stomach and duodenum

Rapid restitution of the gastric and intestinal epithelium after acute injury involves emigration of cells from the gastric glands and basal half of the intestinal villi. An intact basal lamina is prerequisite to the restitution process. The present study was performed to determine the effects of acid on the rat gastric and duodenal basal lamina. The basal lamina was denuded in vitro by ultrasonic vibration. The tissue was then immersed in 0.2 M mannitol (control) or in HCl (5-50 mM) for 10 min. Samples of the tissues were examined by transmission and scanning electron microscopy. Some samples were stained with ruthenium red to demonstrate glycosaminoglycans. The lower concentrations of acid (5 and 10 mM) had little or no effect on the structure of the basal lamina. However, exposure to 20 and 50 mM HCl caused extensive damage to the basal lamina and exposed the underlying connective tissue matrix of the lamina propria. Ruthenium red staining demonstrated differences in size and location of glycosaminoglycans within the basal laminae of stomach and intestine. Exposure to acid at concentrations of 20 or 50 mM caused total loss of ruthenium red staining in both intestinal and gastric basal laminae. Exposure to 10 mM acid resulted in loss of the outermost (luminal) layer of anionic sites from the gastric basal lamina. These studies demonstrate that brief exposure to acid, in concentrations which are necessary for the formation of hemorrhagic erosions in the stomach, caused damage to the basal lamina. This damage may impair epithelial restitution and thus account, in part, for the role of acid in ulcerogenesis.     

Death is the major fate of medial edge epithelial cells and the cause of basal lamina degradation during palatogenesis

During mammalian development, a pair of shelves fuses to form the secondary palate, a process that requires the adhesion of the medial edge epithelial tissue (MEE) of each shelf and the degeneration of the resulting medial epithelial seam (MES). It has been reported that epithelial-mesenchymal transformation (EMT) occurs during shelf fusion and is considered a fundamental process for MES degeneration. We recently found that cell death is a necessary process for shelf fusion. These findings uncovered the relevance of cell death in MES degeneration; however, they do not discard the participation of other processes. In the present work, we focus on the evaluation of the processes that could contribute to palate shelf fusion. We tested EMT by traditional labeling of MEE cells with a dye, by infection of MEE with an adenovirus carrying the lacZ gene, and by fusing wild-type shelves with the ones from EGFP-expressing mouse embryos. Fate of MEE labeled cells was followed by culturing whole palates, or by a novel slice culture system that allows individual cells to be followed during the fusion process. Very few labeled cells were found in the mesenchyme compartment, and almost all were undergoing cell death. Inhibition of metalloproteinases prevented basal lamina degradation without affecting MES degeneration and MEE cell death. Remarkably, independently of shelf fusion, activation of cell death promoted the degradation of the basal lamina underlying the MEE ('cataptosis'). Finally, by specific labeling of periderm cells (i.e. the superficial cells that cover the basal epithelium), we observed that epithelial triangles at oral and nasal ends of the epithelial seam do not appear to result from MEE cell migration but rather from periderm cell migration. Inhibition of migration or removal of these periderm cells suggests that they have a transient function controlling MEE cell adhesion and survival, and ultimately die within the epithelial triangles. We conclude that MES degeneration occurs almost uniquely by cell death, and for the first time we show that this process can activate basal lamina degradation during a developmental process.     

Composition, synthesis, and assembly of the embryonic chick retinal basal lamina

To study the biology of basal laminae in the developing nervous system the protein composition of the embryonic retinal basal lamina was investigated, the site of synthesis of its proteins in the eye was determined, and basal lamina assembly was studied in vivo in two assay systems. Laminin, nidogen, agrin, collagen IV, and XVIII are major constituents of the retinal basal lamina. However, only agrin is synthesized by the retina, whereas the other matrix constituents originate from cells of the ciliary body, the lens, or the optic disc. The synthesis from extraretinal tissues infers that the retinal basal lamina proteins must be shed from their tissues of origin into the vitreous body and from there bind to receptor proteins provided by the retinal neuroepithelium. The fact that all proteins typical for the retinal basal lamina are abundant in the vitreous body and a new basal lamina is only formed when the vitreous body was directly adjacent to the retina is consistent with the contention of the vitreous body having a function in retinal basal lamina formation. Basal lamina assembly was also studied after disrupting the retinal basal lamina by intraocular injection of collagenase. The basal lamina regenerated after chasing the collagenase with Matrigel, which served as a collagenase inhibitor. The basal lamina was reconstituted within 6 h. However, the regenerated basal lamina was located deeper in the retina than normal by reconstituting along the retracted neuroepithelial endfeet demonstrating that these endfeet are the preferred site of basal lamina assembly.     

The role of the basal lamina in mouth formation in the embryo of the starfish Pisaster ochraceus

Details of mouth formation in normal and exogastrulated Pisaster ochraceus larvae have been studied by light microscopy and transmission and scanning electron microscopy. As the archenteron begins to bend, the cells in the presumptive mouth region dissociate and migrate into the blastocoele where they become mesenchyme cells. This leaves a defect in the “blind” endodermal tube, which is covered by a basal lamina. Subsequently this exposed basal lamina bulges to form a blister which appears to extend across the blastocoele to make contact with spikelike projections from the future stomodeal region of the ectoderm. Mesenchyme cell processes are associated with both the basal lamina blister and the ectoderm in this region and may provide both motive power and guidance for contact. Shortly after contact is made the blister of basal lamina from the endoderm fuses with the basal lamina of the ectodermal cells and the ectoderm begins to invaginate. At this time the lateral walls of the presumptive oesophagus are largely formed of naked basal lamina with some loosely associated cells on the endodermal side. Eventually the lateral walls of the proximal part of the oesophagus become cellular, giving rise to an epithelium. A cell plug located between the stomodeum and oesophagus persists for some time before finally breaking down to complete the larval digestive tract. Experiments with exogastrulae suggest that many of these developmental patterns are determined before gastrulation.     

Fibronectin and its relation to the basal lamina and to the cell surface in the chicken blastoderm

Summary The ultrastructural distribution of fibronectin immunoreactivity was investigated in the chicken embryo during late gastrulation. Sites of binding of anti-fibronectin antibodies were ascribed to the basal lamina and associated structures, and to the cell surface. The fibronectin-rich basal lamina was resolved into (1) a lamina densa, which appears as a continuous, dense sheet, (2) a lamina lucida, consisting of anchoring cords between lamina densa and epithelial cells, and (3) a lamina intima, closely juxtaposed to the cell surface. Cell-surface labelling was also observed in mesoblast cells, and along the dorsal side of the deep-layer cells. The ventral side of the latter cells was poorly stained in the endophyllic crescent, except in coated pits, and more regularly stained at the level of definitive endoblast. Some structures associated with the basal lamina reacted intensely with anti-fibronectin antibodies. These are (1) the interstitial bodies, which are aggregates of extracellular material, and (2) a kind of fibril or tubule, embedded in a fibronectin matrix and mainly found in the endophyllic crescent. Some intracellular labelling was found in most deep-layer cells, in few epiblast cells, never in mesoblast cells. These results extend previous studies on the localization of fibronectin, and correlate its presence and surface topology with its postulated role in migration of mesoblast cells on the basal lamina which, chemically, constitutes an appropriate substrate.     

Neuron-schwann cell interaction in basal lamina formation

The availability of tissue culture systems that allow the growth of nerve cells, Schwann cells, and fibroblasts separately or in various combinations now makes possible investigation of the role of cell interactions in the development of the peripheral nervous system. Using these systems it was earlier found that basal lamina is formed on the Schwann cell surface in cultures of sensory ganglion cells and Schwann cells without fibroblasts. It is here reported that the presence of nerve cells is required for the generation of basal lamina on the Schwann cell plasmalemma. Utilizing nerve cell-Schwann cell preparations devoid of fibroblasts, this was found in the following ways. (1) When nerve cells are removed from 3- to 5-week-old cultures, the basal lamina disappears from Schwann cells. (2) If nerve cells are added back to such Schwann cell populations, Schwann cell basal lamina reappears. (3) Removal of nerve cells from older (3–4 months) cultures does not lead to basal lamina loss; areas presumed not to have been coated with lamina before neurite degeneration remain so, suggesting that the lamina persists but is not reformed. (4) If basal lamina is removed with trypsin, it is reformed in neuron plus Schwann cell cultures but not in Schwann cell populations alone. Thus, the formation but not the persistence of Schwann cell basal lamina requires the presence of nerve cells.     

Isolation of separate apical,lateral and basal plasma membrane from cells of an insect epithelium. A procedure based on tissue organization and ultrastructure

The tissue used in this study was the midgut of the tobacco hornworm larva, Manduca sexta. The midgut epithelium is a single layer of cells resting on a thin basal lamina and underlying discontinuous muscle layer. The epithelial cells are of two main types, goblet and columnar cells, joined together by the septate junctions characteristic of insect epithelia. From this tissue we were able to isolate four distinct plasma membrane fractions; the lateral membranes, the columnar cell apical membrane, the goblet cell apical membrane and a preparation of basal membranes from both cell types. The lateral membranes were isolated by density gradient centrifugation following gentle homogenization of the midgut hypotonic medium, which caused the cells to rupture at their apical and basal surfaces, releasing long segments of lateral membranes still joined by their septate junctions. For isolation of apical and basal membranes the tissue was disrupted by ultrasound, based on the light microscopic observation that carefully controlled ultrasound can be used to disrupt each cell in layers starting at the apical surface. The top layer contained the columnar cell apical membrane, which consists of microvilli forming a brush border covering the lumenal surface of the epithelium. The second layer contained the goblet cell apical membrane, which is invaginated to form a cavity occupying the apical half of the cell, and the third layer contained the basal membranes. As each layer was stripped off the epithelium it was collected and the plasma membrane purified by differential or density gradient centrifugation. For all four membrane fractions, the isolation procedure was designed to preserve the original structure of the membrane as far as possible. This allowed electron microscopy to be used to follow each step in the isolation procedure, and to identify the constituents of each subcellular preparation. Although developed specifically for M. sexta midgut, these techniques could readily be modified for use on other epithelia.