Organization Of Intermediate Filaments Research Articles

Different substrates influence the expression of intermediate filaments and the deposition of basement membrane proteins

A primary culture of serous cystadenocarcinoma of the ovary was used to study the expression of intermediate filament proteins and the deposition of basal lamina proteins. It was found that cells grown on type I and IV collagens or in collagen gels failed to express vimentin, which was readily demonstrable in cultures of the same cells grown on plastic or glass. Furthermore cells grown in collagen gels formed colonies demonstrating a cystic architecture. Unlike what is commonly observed on glass or plastic, where laminin and fibronectin are deposited as disorganized fibrils in the extracellular space, in or on collagen these proteins appear solely at the interface between the epithelial cells and matrix. The results suggest that the extracellular matrix influences the cytoskeletal organization of the intermediate filaments and determines cell polarity. They confirm that collagen substrates permit epithelial cell cultures to progress toward a more differentiated state.

Hexahydrocarbon effects on intermediate filament organization in human fibroblasts.

We reported previously that 2,5-hexanedione (2,5-HD), the neurotoxic metabolite of methyl-n-butylketone (MnBK) and n-hexane, induced aggregation of intermediate filaments of the vimentin type in cultured fibroblasts. To determine if these findings have relevance to the mechanism by which these hexacarbons induce their filamentous axonopathy, it was necessary to show that only those hexacarbon analogues that induce focal accumulation of neurofilaments in nerve fibers do aggregate intermediate filaments in fibroblasts. We report here that the nonneurotoxic hexacarbons, 1,6-hexanediol and 2,4-hexanedione (2,4-HD), had no primary effect on intermediate filament distribution in fibroblasts, although the profound, nonspecific cytotoxicity of the latter controverted comparisons with equimolar, effective concentrations of 2,5-HD. Fibroblasts did not metabolize MnBK to 2,5-HD sufficiently to induce reproducible aggregation of intermediate filaments in these cells in culture.

Metabolic inhibitors and intermediate filament organization in human fibroblasts

A number of metabolic inhibitors including the mRNA transcription inhibitor actinomycin D; the protein synthesis inhibitors emetine, cycloheximide, and puromycin; the energy metabolism inhibitors sodium azide and oligomycin; the amino acid analog l-azetidine-2-carboxylic acid; sodium fluoride; and acrylamide each cause the collapse of vimentin filament organization while leaving microtubule organization apparently unaffected in the human fibroblastic cell line MCH23. The protein kinase inhibitor N-[2-(methylamino) ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8) caused a partial collapse of vimentin organization but its effect was more difficult to discern, since it also induced a dramatic change in cellular morphology. Each of these drugs produced a significant inhibition of protein synthesis at concentrations that affected vimentin organization. The mechanisms by which these drugs affect intermediate filament organization are unclear, but our results demonstrate that intermediate filament organization in MCH23 cells is affected by a wide range of drugs and that such drugs cannot be used without great caution as reagents for the study of intermediate filament organization and function.

ヒト培養口腔粘膜上皮細胞に対する天ぽうそう患者血清の影響

Pemphigus is an antibody-mediated autoimmune skin disease that causes blistering of oral mucous membranes as well as skin, and it is generally considered that the blisters are induced by loss of cell to cell contact in the keratinocytes. However, the details are still unclear. In order to investigate the molecular mechanism of blister formation in oral mucous membranes, we observed the effects of pemphigus antibody on the organization of keratin intermediate filaments (KIFs) in cultured oral keratinocytes. After incubation with serum obtained from a patient with pemphigus vulgaris for 96 hrs at 37°C, the distribution of KIFs was changed from a fibrous structure to a dotted structure around the nucleus in some of the cells from normal gingiva and soft palate, but such changes were not observed in cells from normal tongue. These results suggest that the keratinocytes from the gingiva and soft palate may be more sensitive to the pemphigus antibody than cells from the tongue.

Childhood giant axonal neuropathy case report and review of the literature

Giant axonal neuropathy (GAN) is a rare autosomal recessive childhood disorder characterized by a peripheral neuropathy and features of central nervous system involvement. Typically seen are distal axonal swellings filled with 8–10 mm in diameter neurofilaments in central and peripheral axons, and intermediate filament collections in several other cell types. Many neurotoxins produce a morphologically similar neuropathy in humans and experimental animals. Defective nerve fiber energy metabolism has been postulated as a cause in these toxic neuropathies. It is possible that GAN represents an inborn error or metabolism of enzyme-linked sulfhydryl containing proteins, resulting in impaired production of energy necessary for the normal organization of intermediate filaments.

Heat shock gene expression and cytoskeletal alterations in mouse neuroblastoma cells

The cytoskeleton of neuroblastoma cells, clone Neuro 2A, is altered by two stress conditions: heat shock and arsenite treatment. Microtubules are reorganized, intermediate filaments are aggregated around the nucleus, and the number of stress fibers is reduced. Since both stress modalities induce similar cytoskeletal alterations, no thermic denaturation of one or more cytoskeletal components can be involved in this process. Heat shock proteins are induced both by heat and by arsenite. However, cells treated with arsenite synthesize hsp28 which is not detected in heat-treated cells. Synthesis of all hsps is prevented by addition of actinomycin D or cycloheximide. Under these conditions no alterations are observed in the organization of microtubules and intermediate filaments during heat or arsenite treatment. However, these drugs are not able to prevent the rapid loss of stress fibers. A re-formation of the cytoskeleton during the recovery period proceeds within 3 h and is also found to occur in the presence of a protein synthesis inhibitor. These data suggest that reorganization of microtubules and intermediate filaments during a stress treatment requires the synthesis of a new protein(s), probably hsp(s).

Characterization of established epithelioid cell lines derived from rat liver: expression of cytokeratin filaments

A number of epithelioid cell lines were established from livers of fetal and neonatal rats. The clear-epithelial cells of these lines are non-neoplastically altered, clonogenic, nearly-diploid and showed no hepatocyte-like properties. The distribution and organization of intermediate filaments were analysed by indirect immunofluorescence and immunoperoxidase techniques using polyclonal and monoclonal antibodies against cytokeratins and vimentin. The different keratin antibodies also react strongly with intermediate filaments of bile duct cells on frozen sections of fetal and neonatal liver. Several subcultures of all established cell lines contained cell populations which reacted positively with antibodies against prekeratin and broad-range cytokeratins in a different manner, but not with antibodies against cytokeratin No. 19 of the catalogue of MOLL. The intermediate filaments are arranged in predominantly stained pericellular rings as well as in fine filamentous networks more evenly distributed throughout the cytoplasm. In early passages almost all cells were found positive for pre- and cytokeratins whereas in later subcultures the number of positive cells decreases and the expression of cytokeratin polypeptides varies considerably as seen with the different antibodies. All cells of the different cell lines show a well developed fine filamentous vimentin network extending throughout the whole cell up to the outer cell margin. Our findings support the concept that the clear epithelioid cells in established cell lines are of epithelial nature but they are not derived from biliary epithelium, and therefore probably immature parenchymal liver cells.

Changes in the organization of non-epithelial intermediate filaments induced by triethyl lead chloride

The in vivo effect of triethyl lead chloride (TriEL) (10 −6-10 −8 M) on the organization of non-epithelial intermediate filaments (vimentin and desmin filaments) was studied by indirect immunofluorescence microscopy employing different mammalian cell lines. The in vitro effect of TriEL on filament formation as well as on the structure of preformed filaments was investigated by electron microscopy. TriEL induces perinuclear coil formation of intermediate filaments in SV40-transformed human fibroblasts and baby hamster kidney (BHK21) cells. The rearrangements observed are not correlated with significant changes in the microtubular system as tested by double labelling of both filament systems. The effect of TriEL is reversible. Assembly of intermediate filaments in vitro is disturbed in the presence of TriEL such that only short filaments and various kinds of fragments are formed. When preformed filaments are incubated in the presence of TriEL, unravelling of fibres into protofilamentous strands is observed. Possible mechanisms of TriEL-filament interaction are discussed.

Reorganization of Keratin Intermediate Filaments by the Drug-Induced Disruption of Microfilaments in Cultured Human Keratinocytes

It has been shown to date that a combined treatment with microtubule and microfilament inhibitors alters the cytoskeletal organization of keratin intermediate filaments in cultured HeLa, fetal mouse epidermal, and epithelial PtK2 cells, although neither of these inhibitors alone is able to do so. In the present study, we found that disruption of microfilaments with cytochalasin B induced a remarkable reorganization of keratin filaments in cultured human keratinocytes, while disruption of microtubules with colchicine did not affect keratin filaments. Keratin filament organization in the presence of cytochalasin B demonstrated a network of connecting star-like knots or foci. These foci coincided with actin aggregates that were formed by depolymerization of actin filaments as studied by double immunofluorescence using antiactin and antikeratin antibodies. Under these conditions, no change in microtubule arrangement was observed. Our observations suggest that the stability and architecture of keratin filament organization may be supported with the microfilament rather than the microtubule cytoskeleton in cultured human keratinocytes.

The organization and solubility properties of intermediate filaments and microtubules of cortical astrocytes in culture.

The organization of intermediate filaments (IF) and microtubules (MT) and the solubility of intermediate filament proteins and tubulin in astrocytes which develop from cerebral hemispheres of neonatal rats in culture were examined using immunocytochemical and immunochemical approaches. Results of immunocytochemical studies demonstrated that in flat astrocytes which develop after 3 weeks of culturing in serum-supplemented medium, the IF containing vimentin and glial fibrillary acidic protein (GFAP) are concentrated around the nucleus and dispersed in an irregular fashion throughout the cytoplasm. Astrocytes which develop in serum-free hormonally-defined medium irrespective of whether they are bipolar, multipolar or flattened, have IF organized as a fibrous network of filaments distributed from the nuclear regions to the cell periphery. Under both culture conditions, vimentin and GFAP are resistant to extraction with low salt buffer containing nonionic detergent, indicating that the different cytoplasmic distribution of IF is unrelated to the solubility properties of vimentin and GFAP. Double immunolabelling experiments with polyclonal antibody to GFAP and monoclonal antibody to each alpha-tubulin or beta-tubulin reveal an extensive codistribution and parallel organization of IF and MT in all morphological types of astrocytes studied. Stabilization of MT with taxol, or depolymerization of MT with colchicine, cause dramatic changes in the distribution of IF and inhibit the extension of astrocyte processes in response to dibutyryl cyclic AMP (dBcAMP). In early stages of treatment with dBcAMP, renewal of culture medium without dBcAMP produces a rapid and permanent retraction of astrocyte processes, whereas in later stages the processes only retract partially and are then restored and maintained for several days in the absence of dBcAMP. The retraction of processes is accompanied by changes of immunocytochemical staining of IF with antibody to GFAP, which appears more intense and diffuse. However, electrophoretic and immunoblot analyses of detergent-extracted proteins from parallel cultures demonstrate that neither the amount nor the solubility of GFAP and vimentin are changed. Detergent extraction in MT stabilizing conditions shows that a substantial proportion of tubulin in astrocytes cultured in serum-containing and serum-free media is assembled into MT, most of which depolymerize on treatment with low temperature and Ca2+. Following long exposure to dBcAMP the proportion of cold/Ca2+-stable MT increases. The results suggest that the IF of astrocytes in culture are dependent on MT with respect to their cytoplasmic distribution.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of mechanical tension on protrusive activity and microfilament and intermediate filament organization in an epidermal epithelium moving in culture.

Mechanical tension influences tissue morphogenesis and the synthetic, mitotic, and motile behavior of cells. To determine the effects of tension on epithelial motility and cytoskeletal organization, small, motile clusters of epidermal cells were artificially extended with a micromanipulated needle. Protrusive activity perpendicular to the axis of tension was dramatically suppressed. To determine the ultrastructural basis for this phenomenon, cells whose exact locomotive behavior was recorded cinemicrographically were examined by transmission electron microscopy. In untensed, forward-moving lamellar protrusions, microfilaments appear disorganized and anisotropically oriented. But in cytoplasm held under tension by micromanipulation or by the locomotive activity of other cells within the epithelium, microfilaments are aligned parallel to the tension. In non-spreading regions of the epithelial margin, microfilaments lie in tight bundles parallel to apparent lines of tension. Thus, it appears that tension causes alignment of microfilaments. In contrast, intermediate filaments are excluded from motile protrusions, being confined to the thicker, more central part of the cell. They roughly follow the contours of the cell, but are not aligned relative to tension even when microfilaments in the same cell are. This suggests that the organization of intermediate filaments is relatively resistant to physical distortion and the intermediate filaments may act as passive structural support within the cell. The alignment of microfilaments under tension suggests a mechanism by which tension suppresses protrusive activity: microfilaments aligned by forces exerted through filament-surface or filament-filament interconnections cannot reorient against such force and so cannot easily extend protrusions in directions not parallel to tension.

Cytoskeleton Organization Is Aberrantly Rearranged in the Cells of B Chronic Lymphocytic Leukemia and Hairy Cell Leukemia

The organization of actin-containing microfilaments and vimentin-containing intermediate filaments has been investigated in B chronic lymphocytic leukemia (B-CLL), hairy cell leukemia (HCL), and normal B cells cultured in vitro under basal conditions and after induction with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In uninduced B-CLL cells, F-actin was predominantly associated with dot-shaped structures scattered over the ventral membrane representing spotty close contact adhesion sites analogous to "podosomes" described in other cell types. On TPA induction, podosomes became clustered in sharply defined areas sitting in the cell center beneath the nucleus. In some cells, long actin-containing protrusions appeared. In HCL cells, F-actin was associated with thin microvilli responsible for the "hairy" appearance; occasional cells showed scattered podosomes. On TPA induction, HCL cells sprouted long dendritic processes rich in submembraneous F-actin, which made intertwined networks. Therefore, in both B-CLL and HCL cells, adhesion structures were present and the capacity for adhesion in vitro was marked, which might explain some peculiar clinical features of the diseases. Adhesion structures and adhesive properties never appeared in normal B cells. These data further support the notion that B-CLL and HCL, although clinically different, may share common biological features and suggest that in these disorders, cytoskeleton modifications may represent a hallmark of transformation.

Patterns of Expression and Organization of Cytokeratin Intermediate Filaments

Cytokeratins are a large multigene family comprising two polypeptide types, i.e. acidic (type I) and basic (type II) ones, which are distinguished on the basis of immunological, peptide mapping, mRNA hybridization, and primary amino acid sequence data. The acidic (type I) cytokeratins can be subdivided into at least two different subtypes on the basis of their carboxy-terminal sequences. Considerable interspecies conservation of sequences exists, even extending to the 3'-non-coding mRNA regions. Different pairs of type I and II cytokeratins show different resistance to dissociation in urea. Sequence differences of the type I cytokeratins containing functional domains may be an explanation of the observed preference of co-expression with certain type II cytokeratins. The distribution of the different type I and II cytokeratins in normal epithelia and in carcinomas is differentiation related and can be used for cell typing and identification. The cell type-specific expression of cytokeratin polypeptides is recognized at both the protein and the mRNA level. The building block of cytokeratin IFs is a heterotypic tetramer, consisting of two type I and two type II polypeptides arranged in pairs of laterally aligned coiled coils. This principle of tetrameric organization is thought to be generally applicable to IFs.

Dependence of Keratin Intermediate Filament Organization on Microtubules, Microfilaments, and Membrane Association Sitesa

Dependence of Keratin Intermediate Filament Organization on Microtubules, Microfilaments, and Membrane Association Sites^a

Are cross-bridging structures involved in the bundle formation of intermediate filaments and the decrease in locomotion that accompany cell aging?

Five different fibroblast strains derived from donors of a wide range of ages were used for investigation of senescence-associated changes in the organization of intermediate filaments (IFs) and the activity of cell locomotion. Results of immunofluorescence microscopy demonstrate that, in large and flat in vitro aged fibroblasts, vimentin-containing IFs are distributed as unusually organized large bundles. Electron microscopic examination shows that these large bundles are indeed composed of filaments of 8-10 nm. Such a profile of large bundles is rarely seen in young fibroblasts whose IFs are usually interdispersed among microtubules. Within the large filament bundles of senescent fibroblasts, cross-bridge-like extensions are frequently observed along the individual IFs. Immunogold labeling with antibody to one of the cross-bridging proteins, p50, further illustrates the abundance of interfilament links within the IF bundles. The senescence-related increase in interfilament association was also supported by the results of co-precipitation between vimentin and an associated protein of 50,000 D. Time-lapse cinematographic studies of cell locomotion reveal that accompanying aging, fibroblasts have a significantly reduced ability to translocate across a solid substratum. These results led me to suggest that the increased interfilament links via cross-bridges may in part contribute to the mechanism that orchestrates the formation of large filament bundles. The presence of enormous bundles in the cytoplasm may physically impede the efficiency of locomotion for these nondividing cells.

Organization of cytoskeleton and fibronectin matrix in rous sarcoma virus (RSV)-transformed fibroblast lines with different metastatic potential

Metastatic clones growing in 0.6% ‘hard’ agar were selected from the non-metastatic Rous sarcoma virus (RSV)-transformed tumorigenic B77-3T3 mouse fibroblast line. The incidence of spontaneous lung metastases varied among clones around 100%, while it was lower than 5% in the parental tumor line. The organization of microfilaments, microtubules and intermediate filaments as well as the pattern of extracellular fibronectin matrix were analyzed by immunofluorescence in two representative clones (B77-AA6 and B77-AA12) and was compared with the structural features displayed by a highly metastasizing RSV-induced mouse sarcoma line (SR-BALB). In the metastatic clones studied microtubules and intermediate filaments were similarly organized in a pattern not significantly different from that of the non-metastatic parental cell line. The major finding was a marked concentration of actin-containing structures in the periphery of cells and notably at the level of surface protrusions, suggesting a high surface motility. In the same lines the production of fibronectin and its distribution in the cell layer and culture medium were analyzed. Metabolic labelling and immunofluorescence experiments indicated that the nonmetastasizing cells (B77-3T3) retain higher amounts of fibronectin in the cell layer and organize this molecule in extracellular fibers, while the metastatic clones (B77-AA6 and B77-AA12) as well as the metastatic line (SR-BALB) are unable to retain and organize fibronectin at their surface. This paper shows that the progression of tumorigenic cell lines toward a metastatic phenotype involves a redistribution of cytoskeletal actin and a loss of organized fibronectin matrix.

A tumor promoter induces rapid and coordinated reorganization of actin and vinculin in cultured cells.

Treatment of epithelial African green monkey kidney (BSC-1) cells with the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a rapid and reversible redistribution of actin and vinculin that is detectable after only 2 min of treatment. Within 20-40 min, stress fibers disappear, while at the same time large actin-containing ribbons resembling ruffles develop both at the cell periphery and in more central regions. Vinculin is associated with these actin ribbons or bands in a punctate or patchy staining pattern. Adhesion to the substratum is changed from predominantly focal contacts associated with stress fiber ends in untreated cells to broad zones of close contact after TPA treatment. High voltage electron microscopic observations disclose the ribbons to consist of highly cross-linked actin filament networks. Thus, association of vinculin with filament networks, rather than (the ends of) filament bundles, is demonstrated. The integrity of microtubules and vimentin filaments is not affected by TPA treatment, but their distribution is altered to conform with the highly distorted cell shape. The response to TPA is neither prevented nor modified by nocodazole-induced depolymerization or taxol-induced stabilization of microtubules. An intact intermediate filament network seems not required either since colcemid-induced collapse of vimentin filaments towards the nucleus does not affect the cell's response to TPA. Rapid redistribution of actin and vinculin also takes place in enucleated cells and in the presence of cycloheximide, but is prevented by dinitrophenol or oligomycin. TPA-induced cytoskeletal alterations are independent of fibronectin expression and not mimicked, modified, or prevented by calmodulin inhibitors or experimentally elevated levels of calcium and cyclic AMP. Thus the morphological response to TPA involves rapid redistribution of actin and vinculin independent of transcription and translation, fluctuations in the levels of calcium or cyclic AMP, or changes in the organization of microtubules, intermediate filaments, and fibronectin.

Tumor promoters induce a specific morphological signature in the nuclear matrix-intermediate filament scaffold of Madin-Darby canine kidney (MDCK) cell colonies.

Tumor promoters such as phorbol 12-tetradecanoate 13-acetate (TPA), mezerein, teleocidin, aplysiatoxin, and benzoyl peroxide, although structurally unrelated, induce similar, profound changes in morphology in differentiated epithelial Madin-Darby canine kidney (MDCK) cell colonies. The alteration is evident in the organization of intermediate filaments in intact cells and in whole mounts of the nuclear matrix-intermediate filament (NM-IF) scaffold of the epithelial sheet. This substructure, obtained by salt extraction of the cytoskeletal framework, represents only 5% of the total cell protein but contains all of the intermediate filaments, nuclear matrix, and desmosomal core proteins arranged essentially as in the intact cell. The NM-IF is profoundly reorganized after exposure to TPA and retains the morphological changes observed in intact cells. These include bundling of the intermediate filaments, disruption of cell-cell borders, and marked deformation of the polygonal geometry of epithelia. Thus, TPA and all other complete or second-stage tumor promoters examined have a characteristic morphological signature that is not induced by mitogens, metabolic inhibitors, or agents known to disrupt microtubules or microfilaments. This signature, characteristic of tumor promoters, occurs in the absence of both protein and RNA synthesis. These results suggest that this response is prior to and independent of other biochemical markers for tumor promoters. Of the major filament systems, the cytokeratin network is implicated as an early or possibly primary site of tumor-promoter action because characteristics of the promoted cytoskeletal signature are observed in epithelial colonies after prior exposure to colchicine or cytochalasin D. Despite the massive reorganization of cytoskeletal morphology induced by TPA, the distribution of prelabeled proteins into structural fractions (i.e., cytoskeletal, chromatin, and the NM-IF) remains essentially unchanged. The sensitivity and specificity of the epithelial cell response suggest its possible use as a screen for promoting compounds.

The dynamics of nerve growth factor-induced neurofilament and vimentin filament expression and organization in PC12 cells

Monoclonal antibodies specific for neurofilament (NF) subunits or for vimentin filament (VF) protein were used to study nerve growth factor (NGF)-induced intermediate filament expression and organization in a rat pheochromocytoma cell line (PC12 cells). NGF induced increased amounts of NF subunits and VF protein, and they were, in part, differentially localized within PC12 cells. The relative expression of each of the NF subunits and of the VF protein was measured by an enzyme-linked immunosorbent assay which revealed that PC12 cells grown in the absence of NGF (PC12- cells) contained 3 times more VF than 68,000-dalton NF subunits and only barely detectable amounts of 150,000- and 200,000-dalton NF subunits. Exposure of these cells to NGF (PC12+ cells) increased the amount of 68,000-dalton NF subunits 8-fold, VF protein 3-fold, and 150,000- and 200,000-dalton NF subunits 2-fold. The ratio of VF to 68,000-dalton NF proteins in PC12- versus PC12+ cells decreased from 3.0 to 1.5. Both VF and 68,000-dalton NF subunits were arranged in juxtanuclear "ball-like" configurations and both were present in neurites of PC12+ cells. The distribution of 150,000- and 200,000-dalton NF subunits was diffuse with perinuclear stippling and only occasional, weakly fluorescent "balls." After perturbation of the PC12 cell cytoskeleton, 68,000-dalton NF subunits and VF redistributed together and, thus, may exist as heteropolymers. Finally, the increased expression of NF and VF proteins was specific for NGF since they could not be induced by other hormones or "growth" factors. We conclude that PC12 cells constitute a model system for studies of NF and VF expression, assembly, and interactions.

Intermediate filaments of the cytoskeleton in glandular cells of the rat fundic mucosa: immunofluorescence and electron microscopy study.

The organization of intermediate filaments (IF) in cells of the rat fundic mucosa was studied by electron microscopy and immunofluorescence microscopy using specific antiprekeratin antibodies on frozen sections and isolated cells. Our results suggest that mucous cells lining the gastric surface and the gastric pits, which appeared strongly decorated, are the most rich in IF. These cells displayed coarse bundles of IF oriented in all directions as well as desmosome-attached tonofibrils. Mucous neck cells contained fewer bundles of IF located preferentially toward the apical region. Zymogen cells showed a strong staining along the contour of the luminal border, together with a faint decoration of a fine meshwork extending throughout the cytoplasm. A poorly defined fibrillar cortex present underneath the secretory plasma membrane and sparse bundles of IF among the elements of the rough endoplasmic reticulum were seen in thin sections. In contrast, parietal cells appeared brightly stained and the prekeratinlike material formed a cortical polygonal meshwork especially visible in isolated cells. A developed system of IF formed by conspicuous bundles located underneath the secretory canaliculi, among the mitochondria and in the vicinity of the basal plasma membrane, was observed in the electron microscope.