Light and electron microscopic immunocytochemistry on the localization of 3β-hydroxysteroid dehydrogenase/isomerase in the bovine adrenal cortical cells

Summary The localization of 3-hydroxysteroid dehydrogenase/isomerase (3-HSD) was studied in bovine adrenal glands by light as well as electron microscopic immunocytochemistry, using anti-bovine adrenal 3-HSD antibody. With light microscopy the cytoplasm of the glomerulosa cells was weakly immunostained, while that of the fasciculata-reticularis cells was intensely immunostained though both the capsular connective tissue cells and the medullary cells were entirely negative for this reaction. Electron microscopic immunocytochemistry revealed that the positive reaction products for 3-HSD were present on the membrane of smooth endoplasmic reticulum of the cortical cells, especially that of the fasciculata and reticularis cells. Other cell organelles such as mitochondria and Golgi apparatus were entirely negative. The present results indicate that 3-HSD is present in the membrane of smooth endoplasmic reticulum of bovine adrenal cortical cells.Supported by grants from the Ministry of Education Science and Culture, Japan     

The ultrastructure of functional mouse adrenal cortical tumor cells in vitro.

Cells derived from a transplantable mouse adrenal cortical tumor maintain their differentiated function in vitro and secrete steroids in response to ACTH and other stimulatory agents. The cell line has been widely employed for various biochemical investigations but there have been few attempts to correlate this work with morphologic data. This communication describes the electron microscopic appearance of the tumor transplant in vivo and primary cultures derived from it at various intervals after the cells are placed in culture. Tumor cells in vivo bear considerable resemblance to normal adult mouse adrenal cortical cells. Organelles generally considered to be directly involved in steroid biosynthesis (mitochondria, smooth endoplasmic reticulum and lipid droplets) are not drastically altered. Certain modifications of the vasculature and cell membrane, seemingly related to steroidogenesis, are present in both the tumor and normal adrenal cortex. Within 2 days after the tumor cells are introduced to culture, their cytoplasm assumes a more simplified appearance. Smooth endoplasmic reticulum is less conspicuous and free ribosomes and polysomes are very abundant. Mitchondrial inner membranes are reorganized from a saccular arrangement in the cells in vivo into distinct lamellar cristae. The tumor cells now resemble undifferentiated embryonic adrenal cells, or cultured adrenal cells from various mammalian sources which have dedifferentiated in the absence of ACTH. In their morphologically unspecialized state, the normal cells are incapable of functional responses to ACTH. In contrast, the cultured, dedifferentiated tumor cells respond within minutes to this hormone and can demonstrate 5-20 fold increases in their basal steroid output. These data suggest that substantial steroidogenic activity can occur although the characteristic appearance of adrenal mitochondria is absent.     

The Ultrastructure of Functional Mouse Adrenal Cortical Tumor Cells in vitro

Cells derived from a transplantable mouse adrenal cortical tumor maintain their differentiated function in vitro and secrete steroids in response to ACTH and other stimulatory agents. The cell line has been widely employed for various biochemical investigations but there have been few attempts to correlate this work with morphologic data. This communication describes the electron microscopic appearance of the tumor transplant in vivo and primary cultures derived from it at various intervals after the cells are placed in culture. Tumor cells in vivo bear considerable resemblance to normal adult mouse adrenal cortical cells. Organelles generally considered to be directly involved in steroid biosynthesis (mitochondria, smooth endoplasmic reticulum and lipid droplets) are not drastically altered. Certain modifications of the vasculature and cell membrane, seemingly related to steroidogenesis, are present in both the tumor and normal adrenal cortex. Within 2 days after the tumor cells are introduced to culture, their cytoplasm assumes a more simplified appearance. Smooth endoplasmic reticulum is less conspicuous and free ribosomes and polysomes are very abundant. Mitochondrial inner membranes are reorganized from a saccular arrangement in the cells in vivo into distinct lamellar cristae. The tumor cells now resemble undifferentiated embryonic adrenal cells, or cultured adrenal cells from various mammalian sources which have dedifferentiated in the absence of ACTH. In their morphologically unspecialized state, the normal cells are incapable of functional responses to ACTH. In contrast, the cultured, dedifferentiated tumor cells respond within minutes to this hormone and can demonstrate 5–20 fold increases in their basal steroid output. These data suggest that substantial steroidogenic activity can occur although the characteristic appearance of adrenal mitochondria is absent.     

The inositol 1,4,5-trisphosphate-binding site in adrenal cortical cells is distinct from the endoplasmic reticulum

The distribution of binding sites for the calcium-mobilizing second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) was investigated in subcellular fractions of bovine adrenal cortex. The [3H]Ins(1,4,5)P3-binding capacity was enriched in the microsomal fraction, which contained a single class of high affinity binding sites with a Kd of 21.6 +/- 3.0 nM. The specific [3H]Ins(1,4,5)P3 binding appeared to be sharply pH dependent and was inhibited by millimolar concentrations of ATP. Upon fractionation of microsomes on sucrose density gradient there was a clearcut separation of the Ins(1,4,5)P3 receptor-containing fractions from those enriched in specific endoplasmic reticulum markers such as sulfatase C activity or RNA content. The microsomes enriched in Ins(1,4,5)P3-binding sites were of lower density than the endoplasmic reticulum and co-purified partly with the plasma membrane. In addition, Ins(1,4,5)P3-sensitive 45Ca2+ uptake into the microsomes was maximal in the lighter fractions. This distinction between Ins(1,4,5)P3-binding sites and endoplasmic reticulum-derived microsomes was confirmed upon fractionation according to their electrophoretic mobilities by free flow electrophoresis. These results indicate that in adrenal cortical cells, the source of Ca2+ mobilized by Ins(1,4,5)P3 upon stimulation with an agonist is not located in the endoplasmic reticulum. Our data support the hypothesis that a specialized vesicular organelle, distinct from endoplasmic reticulum and in close apposition with the plasma membrane, is involved in intracellular Ca2+ homeostasis.     

Ultrastructural and Morphometric Observations of Cortical Endoplasmic Reticulum in Arbacia,Spisula and Mouse Eggs*

Results of morphometric investigations indicate that Arbacia eggs possess a network of cortical endoplasmic reticulum equal in volume and surface area to that within the subcortex. The cortical endoplasmic reticulum surrounds individual cortical granules and forms associations with the plasma membrane reminiscent of junctions shared by the sarcolemma and sarcoplasmic reticulum. Mouse eggs, which also exhibit a cortical granule reaction, possess endoplasmic reticulum that is associated with cortical granules and the plasmalemma. The same relative volume of cortical endoplasmic reticulum is present in mouse eggs as in Arbacia. Significantly less cortical endoplasmic reticulum is present in Spisula eggs which do not undergo cortical granule discharge upon activation. These observations are discussed in light of the hypothesis that the cortical endoplasmic reticulum transduces the interaction of the gametes into an intracellular calcium release which initiates the cortical granule reaction and the activation of development.     

Ultrastructural pathology of the adrenal gland in Cushing's syndrome

The ultrastructural pathology of the adrenal glands was studied in fifteen cases of Cushing's syndrome. Some specific features correlated with the pathological aspects of adrenals were found. In the hyperplastic adrenal cortex the cytoplasms contained a rich smooth endoplasmic reticulum and many mitochondria. Increased lipid-pigment complexes were found especially in the compact cells. In adenomas, the clear cells showed large lipid vacuoles; the compact cells presented anisomorphous mitochondria, a well developed smooth endoplasmic reticulum and many pigment bodies. The nuclei of adenomatous cells were irregular, with deep invaginations. In adrenal carcinomas, the pleomorphism of nuclei, nucleoli, mitochondria, and smooth endoplasmic reticulum was more obvious. Absolutely reliable characteristics proving malignancy at ultrastructural level do not, however, exist. The steroidogenic activity of both hyperplastic and tumoral adrenal glands can be assessed using the agranular endoplasmic reticulum and the mitochondria as functional parameters.     

Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum

The distribution of calsequestrin and calreticulin in smooth muscle and non-muscle tissues was investigated. Immunoblots of endoplasmic reticulum proteins probed with anti-calreticulin and anti-calsequestrin antibodies revealed that only calreticulin is present in the rat liver endoplasmic reticulum. Membrane fractions isolated from uterine smooth muscle, which are enriched in sarcoplasmic reticulum, contain a protein band which is immunoreactive with anti-calreticulin but not with anti-calsequestrin antibodies. The presence of calreticulin in these membrane fractions was further confirmed by 45Ca2+ overlay and "Stains-All" techniques. Calreticulin was also localized to smooth muscle sarcoplasmic reticulum by the indirect immunofluorescence staining of smooth muscle cells with anti-calreticulin antibodies. Furthermore, both liver and uterine smooth muscle were found to contain high levels of mRNA encoding calreticulin, whereas no mRNA encoding calsequestrin was detected. We have employed an ammonium sulfate precipitation followed by Mono Q fast protein liquid chromatography, as a method by which calsequestrin and calreticulin can be isolated from whole tissue homogenates, and by which they can be clearly resolved from one another, even where present in the same tissue. Calreticulin was isolated from rabbit and bovine liver, rabbit brain, rabbit and porcine uterus, and bovine pancreas and was identified by its amino-terminal amino acid sequence. Calsequestrin cannot be detected in preparations from whole liver tissue, and only very small amounts of calsequestrin are detectable in ammonium sulfate extracts of uterine smooth muscle. We conclude that calreticulin, and not calsequestrin, is a major Ca2+ binding protein in liver endoplasmic reticulum and in uterine smooth muscle sarcoplasmic reticulum. Calsequestrin and calreticulin may perform parallel functions in the lumen of the sarcoplasmic and endoplasmic reticulum.     

Antibodies to the Golgi complex and the rough endoplasmic reticulum

Rabbits were immunized with membrane fractions from either the Golgi complex or the rough endoplasmic reticulum (RER) by injection into the popliteal lymph nodes. The antisera were then tested by indirect immunofluorescence on tissue culture cells or frozen, thin sections of tissue. There were may unwanted antibodies to cell components other than the RER or the Golgi complex, and these were removed by suitable absorption steps. These steps were carried out until the pattern of fluorescent labeling was that expected for the Golgi complex or RER. Electron microscopic studies, using immunoperoxidase labeling of normal rat kidney (NRK) cells, showed that the anti-Golgi antibodies labeled the stacks of flattened cisternae that comprise the central feature of the Golgi complex, many of the smooth vesicles around the stacks, and a few coated vesicles. These antibodies were directed, almost entirely, against a single polypeptide with an apparent molecular weight of 135,000. The endoplasmic reticulum (ER) in NRK cells is an extensive, reticular network that pervades the entire cell cytoplasm and includes the nuclear membrane. The anit-RER antibodies labeled this structure alone at the light and electron microscopic levels. They were largely directed against four polypeptides with apparent molecular weights of 29,000, 58,000, 66,000, and 91,000. Some examples are presented, using immunofluorescence microscopy, where these antibodies have been used to study the Golgi complex and RER under a variety of physiological and experimental condition . For biochemical studies, these antibodies should prove useful in identifying the origin of isolated membranes, particularly those from organelles such as the Golgi complex, which tend to lose their characteristic morphology during isolation.     

Thrombin stimulation of synthesis and secretion of fibronectin by human A549 epithelial cells and mouse LB fibroblasts

Thrombin, a serine protease generated at wound sites, takes part in multiple biological functions, including wound healing. The present report elucidates the effect of thrombin on fibronectin (FN) synthesis and secretion in fibroblasts and epithelial cells. Subconfluent cultures of mouse LB fibroblasts and human A549 epithelial cells were exposed to various concentrations of bovine plasma thrombin at 37 degrees C for 16 hr. After exposure, cells were processed for determination of cell-associated and secreted FN by metabolic labeling, immunoprecipitation, immunofluorescence, and peroxidase immunocytochemistry. The correlation of FN production with cell growth was studied by a combined procedure of peroxidase immunocytochemistry and light microscopic autoradiography. The amounts of cell-associated and secreted FN were significantly increased with dose increments of thrombin. The increases were most evident in secreted FN. The increase of cell-associated FN was also evidenced by results from immunofluorescence and immunocytochemical studies. Ultrastructurally, the intracellular FN was localized in rough endoplasmic reticulum, Golgi complexes, and secretory granules, whereas non-released extracellular FN was localized in the plasma membrane of cell-to-cell contacts and in the extracellular fibrils. More intense cytoplasmic FN staining was observed in cells that were not labeled with [3H]-thymidine, indicating that FN production may vary with different phases of cell growth. The results imply that thrombin may play an important role in the early phases of wound healing.     

Morphology and ultrastructure of the adrenal gland in Bactrian camels (Camelus bactrianus)

In the present study, we examined the morphological features of the adrenal gland in Bactrian camel by means of digital anatomy, light and electron microscopy. Our findings testified that the gland was divided into three parts, capsule, cortex and medulla from outside to inside as other mammals, and the cortex itself was further distinguished into four zones: zona glomerulosa, zona intermedia, zona fasciculate and zona reticularis. Notably, the zona intermedia could be seen clearly in the glands from females and castrated males, whereas it was not morphologically clear in male. There was a great deal of lipid droplets in the zona fasciculate, while it was fewer in the zona glomerulosa and zona reticularis. The cytoplasm of adrenocortical cell contained rich mitochondria and endoplasmic reticulum. The adrenal medulla was well-developed with two separations of external and internal zones. The most obvious histological property of adrenal medulla cells were that they contained a huge number of electron-dense granules enveloped by the membrane, and so medulla cells could be divided into norepinephrine cells and epinephrine cells. Moreover, the cortical cuffs were frequently present in adrenal gland. Results of this study provides a theoretical basis necessary for ongoing investigations on Bactrian camels and their good adaptability in arid and semi-arid circumstances.     

Steroidogenic characteristics of the adrenal cortex of the mare studied by electron microscopy

The three concentric zones of the horse adrenal cortex (zonae glomerulosa, fasciculata and reticularis) showed marked interpenetration and exhibited a different relative development according to their position in the gland. Whereas the three cortical zones each had a specific histological structure, the ultrastructure of their cells showed a certain qualitative homogeneity. The differences corresponded essentially to the relative abundance of the constituents which are generally considered typical of steroidogeneous cells: mitochondria with vesicular cristae, smooth endoplasmic reticulum and lipid droplets. Their importance increased progressively from the zona glomerulosa to the zona reticularis. In this zone, the presence of gap and septate-like cell junctions, and mitochondria with vesicular cristae in close proximity to a smooth endoplasmic reticulum with numerous dilated tubules, suggested that steroidogenesis may be the most active. Ultrastructural findings were indicative of only quantitative differences between the steroidogenic capacities of the three zones of the mare adrenal cortex.     

Monoclonal antibody 2H1 detects a 60-65 KD membrane polypeptide of the rough endoplasmic reticulum of neurons and selectively stains cells of several rat tissues

Monoclonal antibody (MAb) 2H1, raised in mice immunized with membrane fractions from cultured rat pheochromocytoma cells (clonal line PC-12), detects a polypeptide from rat brain and PC-12 cell membranes of 60-65 KD apparent molecular mass. The polypeptide has been localized by immunoelectron microscopy in the rough endoplasmic reticulum (RER) of neurons. By light microscopic immunocytochemistry, several rat tissues and two rat-derived cultured cell types show selective patterns of staining with 2H1. In the central nervous system, the antibody stains neuronal cytoplasm; in the spleen, staining is seen only in certain cells of the marginal zone of the white pulp, and in lymph nodes, in plasma cells, and in areas populated by monocytes and macrophages. Whereas astrocytes and adrenal medullary cells in situ are virtually unstained with 2H1, primary cultures of astrocytes and PC-12 cells, which are derived from adrenal medullary cells, stain intensely with 2H1. The strong staining of cultured astrocytes and PC-12 cells with 2H1 suggests that the levels of the 60-65 KD polypeptide are up-regulated during cell proliferation and growth. Only a few hepatocytes stain with 2H1; intestinal epithelial and pancreatic cells are not stained with 2H1. The organelle-specific antibody 2H1 may prove a useful probe in structural and functional studies of membranes of the rough endoplasmic reticulum in neurons, and in certain cells of the immune system.     

Cytochemistry of Ca(++)-dependent adenosine triphosphatase (Ca-ATPase) in rat anterior pituitary cells

In the present study, we demonstrate the localization of Ca(++)-ATPase in the anterior pituitary of the male rat. Ca(++)-ATPase was mainly distributed on the membrane system of the granular cells, which included the plasma membrane, the outer mitochondrial membrane, the enveloping membrane of secretory granules, the smooth endoplasmic reticulum and some components of the Golgi complex. No reaction product was detected on the membrane of the rough endoplasmic reticulum or that surrounding the lysosomes. A positive reaction was clearly observed on the membranes surrounding 'large' secretory granules, while that present on the membranes of the 'small' granules was comparatively weak. The cells which contained the 'large' granules were interpreted as growth hormone-secreting cells and those in which the 'small' granules were located as gonadotrophs. There were either no reaction or one that was barely detectable on the plasma membrane of the folliculo-stellate cells. These data along with our previous findings (Soji, 1982, 1984) suggest that the membranous enzymes are not uniformly distributed over all pituitary cells but rather are specific for a given cell population(s).     

Mechanisms of alpha-fetoprotein synthesis in hepatoma cells. Combined electron microscopic immunocytochemistry and autoradiography

Immunoreaction of alpha-fetoprotein (AFP) was detected not only in well-differentiated hepatocellular carcinoma but also in hepatocytes forming foci in livers with hyperplastic nodules during 3'-methyl-4-dimethylaminoazobenzene hepatocarcinogenesis. The subcellular location of AFP in hepatoma cells was in the rough endoplasmic reticulum, perinuclear space and well-developed Golgi apparatus around the nucleus. In livers with hyperplastic nodules it was also in some parts of the smooth endoplasmic reticulum and Golgi regions in hepatocytes in the vicinity of submembranous areas or bile canaliculi. These findings suggest that the Golgi apparatus in hepatoma cells acts mainly as an organelle for glycosylation of AFP and that the Golgi complexes in the hepatocytes in livers with hyperplastic nodules are organelles for secretion of AFP. Combined light microscopic immunoperoxidase study and autoradiography with 3H-thymidine revealed a higher cumulative labeling index in AFP-positive hepatoma cells than in non-tumorous areas. Combined electron microscopic immunoperoxidase study and autoradiography showed that hepatoma cells with AFP immunoreactivity only in the rough endoplasmic reticulum had a significantly higher labeling index than did cells with AFP immunoreactivity in both rough endoplasmic reticulum and Golgi apparatus. These findings suggest that AFP is synthesized in hepatoma cells before or during the stage of their DNA synthesis and is then transported to the Golgi apparatus.     

DIFFERENTIATION OF ENDOPLASMIC RETICULUM IN HEPATOCYTES : I. Glucose-6-Phosphatase Distribution In Situ

The distribution of glucose-6-phosphatase activity in rat hepatocytes during a period of rapid endoplasmic reticulum differentiation (4 days before birth-1 day after birth) was studied by electron microscope cytochemistry. Techniques were devised to insure adequate morphological preservation, retain glucose-6-phosphatase activity, and control some other possible artifacts. At all stages examined the lead phosphate deposited by the cytochemical reaction is localized to the endoplasmic reticulum and the nuclear envelope. At 4 days before birth, when the enzyme specific activity is only a few per cent of the adult level, the lead deposit is present in only a few hepatocytes. In these cells a light deposit is seen throughout the entire rough-surfaced endoplasmic reticulum. At birth, when the specific activity of glucose-6-phosphatase is approximately equal to that of the adult, nearly all cells show a positive reaction for the enzyme and, again, the deposit is evenly distributed throughout the entire endoplasmic reticulum. By 24 hr postparturition all of the rough endoplasmic reticulum, and in addition the newly formed smooth endoplasmic reticulum, contains heavy lead deposits; enzyme activity at this stage is 250% of the adult level. These findings indicate that glucose-6-phosphatase develops simultaneously within all of the rough endoplasmic reticulum membranes of a given cell, although asynchronously in the hepatocyte population as a whole. In addition, the enzyme appears throughout the entire smooth endoplasmic reticulum as the membranes form during the first 24 hr after birth. The results suggest a lack of differentiation within the endoplasmic reticulum with respect to the distribution of glucose-6-phosphatase at the present level of resolution.     

Evidence for a plasma membrane calcium pump in bovine adrenal medulla but not adrenal cortex.

Continuous sucrose density gradient subfractions from bovine adrenal medullary microsomes were found to accumulate 45-Ca-2+ in the presence of ATP and ammonium oxalate mainly in subfractions of intermediate density. (Na-++K-+)-ATPase (plasma membrane marker) and Ca-2+-ATPase activities were also concentrated in these intermediate subfractions but thiamine pyrophosphatase (Golgi apparatus marker) was not. NADH oxidase (endoplasmic reticulum marker) activity was distributed throughout all subfractions. 45-Ca-2+ accumulation in adrenal cortical microsomes was found to rise and fall in parallel with thiamine pyrophosphatase but not with (Na-++K-+)-ATPase or NADH oxidase activities. Accumulation of 45-Ca-2+ in membrane vesicles in these experiments suggests the existence of a calcium transfer mechanism in plasma membranes of the adrenal medulla but not adrenal cortex.     

Immunohistochemical localization of adrenal ferredoxin in bovine adrenal cortex.

Adrenal ferredoxin, the iron-sulfur protein associated with cytochrome P-450 in adrenocortical mitochondria, has been localized at the light microscopic level in bovine adrenal cortex. Localization was achieved through the use of rabbit antiserum to bovine adrenal ferrodoxin in an unlabeled antibody peroxidase-antiperoxidase method. When sections of bovine adrenal glands were exposed to the adrenal ferredoxin antiserum, intense staining was observed in parenchymal cells of the three cortical zones. Staining for adrenal ferredoxin was not detected in the medullary chromaffin cells. The presence of adrenal ferredoxin in the three cortical zones was verified by electron paramagnetic resonance spectrometry. These determinations also revealed that while the zona fasciculata and the zona reticularis contained approximately equal concentrations of adrenal ferredoxin, the concentration of the iron-sulfur protein in the zona glomerulosa was considerably lower. Similar results were obtained when the levels of cytochrome P-450 were determined in the three cortical zones. These results represent the first immunohistochemical localization within an intact tissue or cell of any component of an NADPH-dependent electron transport sequence which is responsible for the reduction of cytochrome P-450.     

The possible structural changes in the adrenal gland cortex after induction of hepatic ischemia–reperfusion injury in male albino rats: Light and electron microscopic study

The adrenal gland is an important endocrine gland in the body that secrets the adrenal hormones. One of the important clinical issues is the hepatic ischemia–reperfusion (IR) injury. Liver IR injury results in many distant organs dysfunctions such as lung, kidney, intestine, pancreas, and myocardium. The aim of the present study was to investigate the possible remote effects of hepatic IR on the structure of the adrenal cortex. Twenty healthy males, Sprague–Dawley albino rats aged 6–8 weeks were randomly divided into two groups (10 rats each): the sham control group (SC-group) and the ischemia–reperfusion group (IR-group). Sera were estimated for the following: aspartate transaminase (AST), alanine transaminase (ALT), lactic dehydrogenase (LDH), and corticosterone levels. Also oxidative markers such as malondialdehyde (MDA) and tumor necrosis factor-α (TNF-α), and the antioxidative enzyme, catalase were measured. Adrenal glands were processed for light and transmission electron microscopic study. The results showed a significant increase in serum liver enzymes (AST, ALT, and LDH), corticosterone, MDA, and TNF-α levels and a significant decrease in serum levels of catalase in IR-group compared with SC-group. Adrenal cortical tissue of IR-group showed the loss of normal appearance. Some cells of zona glomerulosa and most of the zona fasciculata cells appeared swollen and degenerated with highly vacuolated cytoplasm. Other cells were shrunken with deeply acidophilic cytoplasm and pyknotic nuclei. Degenerated mitochondria with disrupted cristae, lipid droplets were confluent and dilated smooth endoplasmic reticulum were seen. Few zona reticularis cells had the dark nucleus and cytoplasmic vacuolations. In the different zones, blood capillaries were markedly congested and some inflammatory cells infiltrations were observed. Liver IR affected the structure of the adrenal cortex.     

Autocrine motility factor receptor is a marker for a distinct membranous tubular organelle

Autocrine motility factor (AMF) is secreted by tumor cells and is capable of stimulating the motility of the secreting cells. In addition to being expressed on the cell surface, its receptor, AMF-R, is found within a Triton X-100 extractable intracellular tubular compartment. AMF-R tubules can be distinguished by double immunofluorescence microscopy from endosomes labeled with the transferrin receptor, lysosomes labeled with LAMP-2, and the Golgi apparatus labeled with beta-COP. AMF-R can also be separated from a LAMP-2 containing lysosomal fraction by differential centrifugation of MDCK cells and is found within a 100,000 g membrane pellet. By electron microscopic immunocytochemistry, AMF-R is localized predominantly to smooth vesicular and tubular membranous organelles as well as to a lesser extent to the plasma membrane and rough endoplasmic reticulum. AMF-R tubules have a variable diameter of 50-250 nm and can acquire an elaborate branched morphology. By immunofluorescence microscopy, AMF-R tubules are clearly distinguished from the calnexin labeled rough endoplasmic reticulum and AMF-R tubule expression is stable to extended cycloheximide treatment. The AMF-R tubule is therefore not a biosynthetic subcompartment of the endoplasmic reticulum. The tubular morphology of the AMF-R tubule is modulated by both the actin and microtubule cytoskeletons. In a similar fashion to that described previously for the tubular lysosome and endoplasmic reticulum, the linear extension and peripheral cellular orientation of the AMF-R tubule are dependent on the integrity of the microtubule cytoskeleton. The AMF-R tubule may thus form part of a family of microtubule- associated tubular organelles.     

The eta isoform of protein kinase C is localized on rough endoplasmic reticulum.

The eta isoform of protein kinase C, isolated from a cDNA library of mouse skin, has unique tissue and cellular distributions. It is predominantly expressed in epithelia of the skin, digestive tract, and respiratory tract in close association with epithelial differentiation. We report here that this isoform is localized on the rough endoplasmic reticulum in transiently expressing COS1 cells and constitutively expressing keratinocytes. By the use of polyclonal antibodies raised against peptides of the diverse D1 and D2/D3 regions, we found that immunofluorescent signals were strongest in the cytoplasm around the nucleus and became weaker toward the peripheral cytoplasm. Under immunoelectron microscopic examination, electron-dense signals were located on the rough endoplasmic reticulum and on the outer nuclear membrane which is continuous with the endoplasmic reticulum membrane. However, no signals were detected in the nucleus, inner nuclear membrane, smooth endoplasmic reticulum, Golgi apparatus, mitochondria, or plasma membrane. Treatment of the cells in situ with detergents suggested association of the isoform of protein kinase C with intracellular structures. By immunoblotting, a distinct single band with an M(r) of 80,000 was detected in whole-cell lysate and in rough microsomal and crude nuclear fractions, all of which contain outer nuclear membrane and/or rough endoplasmic reticulum. We further demonstrated the absence of a nuclear localization signal in the pseudosubstrate sequence. The present observation is not consistent with the report of Greif et al. (H. Greif, J. Ben-Chaim, T. Shimon, E. Bechor, H. Eldar, and E. Livneh, Mol. Cell. Biol. 12:1304-1311, 1992).