Mining the human genome using microarrays of open reading frames.

To test the hypothesis that the human genome project will uncover many genes not previously discovered by sequencing of expressed sequence tags (ESTs), we designed and produced a set of microarrays using probes based on open reading frames (ORFs) in 350 Mb of finished and draft human sequence. Our approach aims to identify all genes directly from genomic sequence by querying gene expression. We analysed genomic sequence with a suite of ORF prediction programs, selected approximately one ORF per gene, amplified the ORFs from genomic DNA and arrayed the amplicons onto treated glass slides. Of the first 10,000 arrayed ORFs, 31% are completely novel and 29% are similar, but not identical, to sequences in public databases. Approximately one-half of these are expressed in the tissues we queried by microarray. Subsequent verification by other techniques confirmed expression of several of the novel genes. Expressed sequence tags (ESTs) have yielded vast amounts of data, but our results indicate that many genes in the human genome will only be found by genomic sequencing.

High-throughput quantitative histological analysis of Alzheimer's disease pathology using a confocal digital microscanner.

To develop a rapid method of quantifying immunohistochemical information in tissue sections, we tested a confocal laser fluorescence microscanner initially designed for DNA microarray analysis. This instrument collects digital images at multiple wavelengths, scans entire sections at a resolution of 5 or 10 microm in less than 10 min, and quantifies structures labeled with fluorescent or nonfluorescent probes. We assessed the microscanner by studying immunostained amyloid plaques in the Alzheimer's disease (AD) brain and in the brain of a transgenic mouse model of AD amyloidosis, as efforts to correlate measures of amyloid plaques in brain sections with behavioral impairments are impeded by limitations in current morphometric methods. Microscanner analysis was used to determine amyloid burden in the occipital and entorhinal cortices of the mouse (3.7%) and human AD brain (1.6%). We also quantified the colocalization of plaque beta-amyloid (Abeta) with glial fibrillary acidic protein, a marker of gliosis (mouse 0.9%, human AD 3.7%). The microscanner may be generally applicable to a wide variety of human histopathologies and their animal models, wherever rapid unbiased quantitative analysis is needed.

Analysis of mitochondrial morphology and function with novel fixable fluorescent stains.

Investigation of mitochondrial morphology and function has been hampered because photostable, mitochondrion-specific stains that are retained in fixed, permeabilized cells have not been available. We found that in live cell preparations, the CMXRos and H2-CMXRos dyes were more photostable than rhodamine 123. In addition, fluorescence and morphology of mitochondria stained with the CMXRos and CMXRos-H2 dyes were preserved even after formaldehyde fixation and acetone permeabilization. Using epifluorescence microscopy, we showed that CMXRos and H2-CMXRos dye fluorescence fully co-localized with antibodies to subunit I of cytochrome c oxidase, indicating that the dyes specifically stain mitochondria. Confocal microscopy of these mitochondria yielded colored banding patterns, suggesting that these dyes and the mitochondrial enzyme localize to different suborganellar regions. Therefore, these stains provide powerful tools for detailed analysis of mitochondrial fine structure. We also used poisons that decrease mitochondrial membrane potential and an inhibitor of respiration complex II to show by flow cytometry that the fluorescence intensity of CMXRos and H2-CMXRos dye staining responds to changes in mitochondrial membrane potential and function. Hence, CMXRos has the potential to monitor changes in mitochondrial function. In addition, CMXRos staining was used in conjunction with spectrally distinct fluorescent probes for the cell nucleus and the microtubule network to concomitantly evaluate multiple features of cell morphology.

Using dyes and filters in a fluorescent imaging system.

The FluorImager fluorescence imaging system uses monochromatic 488-nm laser light to excite fluorochromes. It contains a built-in 515-nm long-pass filter that rejects excitation laser light, but allows emission light with wavelengths longer than 515 nm to pass through. A fluorochrome appropriate for use in the system is excitable by 488-nm light and emits at least some of its fluorescence at wavelengths longer than 515 nm. Two types of optical filters, long-pass and band-pass, are used in the system. Long-pass filters reject shorter wavelengths and transmit longer wavelengths. The number in the filter name denotes the cutoff wavelength (midpoint of the transition between rejected and transmitted light) for the filter. Band-pass filters transmit a band of wavelengths and reject both shorter and longer wavelengths. The numbers in the filter name denote the center wavelength of the passed band and the width of the band at half maximum transmission. Generally, when scanning for a single fluorochrome, only the built-in 515-nm long-pass filter is needed. An interchangeable filter can be added to decrease the contribution from a broad-spectrum background signal and to attenuate strong fluorochrome signals.

Brefeldin A causes structural and functional alterations of the trans-Golgi network of MDCK cells.

The trans-Golgi network (TGN) of MDCK cells is exquisitely sensitive to the fungal metabolite brefeldin A (BFA), in contrast to the refractory Golgi stack of these cells. At a concentration of 1 microgram/ml, BFA promoted extensive tubulation of the TGN while the medical Golgi marker alpha-mannosidase II was not affected. Tubules emerging minutes after addition of the drug contained both the apical marker influenza hemagglutinin (HA), previously accumulated at 20 degrees C, and the fusion protein interleukin receptor/TGN38 (TGG), a TGN marker that recycles basolaterally, indicating that, in contrast to TGN vesicles, TGN-derived tubules cannot sort apical and basolateral proteins. After 60 minutes treatment with BFA, HA and TGG tubules formed extensive networks widely spread throughout the cell, different from the focused centrosomal localization previously described in non-polarized cells. The TGG network partially codistributed with an early endosomal tubular network loaded with transferrin, suggesting that the TGG and endosomal networks had fused or that TGG had entered the endosomal network via surface recycling and endocytosis. The extensive structural alterations of the TGN were accompanied by functional disruptions, such as the extensive mis-sorting of influenza HA, and by the release of the TGN marker gamma-adaptin. Our results suggest the involvement of BFA-sensitive adaptor proteins in TGN-->surface transport.

Structural reorganization of the rough endoplasmic reticulum without size expansion accounts for dexamethasone-induced secretory activity in AR42J cells.

A striking reorganization of the rough endoplasmic reticulum (RER) from a tubulo-vesicular (TV-RER) to a stacked cisternal (SC-RER) configuration was observed when the secretory activity of AR42J cells, a cell line derived from a rat pancreatic acinar carcinoma, was induced by dexamethasone. Treatment with 10 nM dexamethasone resulted in a 6.6-fold increase in the intracellular and a 4.6-fold increase in the secreted amylase activity, respectively. On the basis of the morphometric analysis of thin-section electron micrographs it has been previously reported that this increase in secretory activity is accompanied by a 2.4-fold or 30-fold increase in the size of the RER. We have developed a new biochemical method to determine the size of the RER by quantifying the membrane-bound ribosomes. Using this procedure we did not detect any change in the size of the RER after induction of an active secretory state in AR42J cells. Electron microscopic observation showed the predominance of SC-RER in dexamethasone-treated cells compared to the abundance of TV-RER in control cells. Laser scanning confocal microscopy showed a patchy distribution of ER staining in dexamethasone-treated cells compared to more basal localization in control cells. On the basis of our observations we conclude that in AR42J cells the increase in secretory activity induced by dexamethasone is accompanied by a reorganization of the RER rather than by an increase in ER surface area, as reported by others. Our results suggest that SC-RER is a biosynthetically more efficient form of the RER, which is found predominantly in actively secreting cells.

Characterization of membrane and cytoskeletal compartments in cultured parietal cells: immunofluorescence and confocal microscopic examination.

Primary cultures of rabbit gastric parietal cells respond to various gastric secretagogues as evidenced by morphological alterations and [14C]aminopyrine uptake. The availability of cultures of > 95% purity has allowed us to utilize immunofluorescence and confocal microscopy to observe the direct effect of histamine upon the distribution of membrane and cytoskeletal proteins in parietal cells. Cells cultured for 3 days were incubated for 45 min with or without 10(-4) M histamine, washed, and fixed with 3% paraformaldehyde. Immunofluorescence was performed with antibodies against H+/K(+)-ATPase, Na+/K(+)-ATPase, ezrin, and beta-tubulin, as well as with Bodipy-phallacidin. Anti-H+/K(+)-ATPase antibody stained resting cells in a vesicular cytoplasmic pattern. Stimulation with histamine resulted in the development of a well-defined linear pattern, outlining the expanded secretory canaliculi. The Na+/K(+)-ATPase was restricted to predominantly the lateral surface in both the resting and stimulated cells, suggesting that the cultured parietal cells retain membrane polarity. Ezrin was visualized outlining the intracellular canaliculi in the resting state, and surrounding the large secretory canaliculi in the stimulated cell. Phallacidin labeling of F-actin localized to an area tightly surrounding the intracellular canaliculi in the resting cell, and was comparable with the staining observed with ezrin. In the stimulated cells this fluorescence pattern became more diffuse and surrounded the expanded secretory surface. In both the resting and stimulated cells, antibodies to beta-tubulin revealed a microtubular pattern located predominantly in the basal portion of the cell. These results demonstrate that the cells are capable of translocating the H+/K(+)-ATPase-containing tubulovesicles to a secretory surface, and that they exhibit organization and maintenance of basolateral and canalicular membrane domains. Furthermore, these studies demonstrate the directed movement of membrane and cytoskeletal proteins upon stimulation of the cultured parietal cells.

Membrane and protein recycling associated with gastric HCl secretion.

Stimulation of the gastric parietal cell requires massive membrane transformations as H(+)-pumps from the domain of cytoplasmic tubulovesicles are recruited into the apical plasma membrane domain. The recycling of membrane pools, through fusion and fission processes that accompany stimulation and inhibition of HCl secretion, also involves highly selective events of protein incorporation and segregation. This manuscript describes several proteins that have been identified with the apical plasma membrane from maximally stimulated parietal cells, and broadly characterizes them either as permanent resident proteins of the apical membrane, or transient proteins that move into and out of the apical membrane as the cell progresses through the secretory cycle. A typical example of transient association with the apical membrane concerns the pump proteins, including the 94 kDa catalytic alpha-subunit of the H+K(+)-ATPase and its newly discovered beta-subunit glycoprotein, which move between tubulovesicles. Proteins that remain associated with the apical plasma membrane during rest and secretion include actin, and an 80-kDa phosphoprotein, which has been variously called 80 K, ezrin, p81 and cytovillin, and whose phosphorylation is increased by the histamine/cAMP pathway of parietal cell stimulation. An example of a cytosolic protein that becomes associated with the apical plasma membrane after stimulation is a 120-kDa protein, which appears to have protein kinase activity. Note that the identification, localization and characterization of the K+ and Cl- transport proteins, which participate in net HCl secretion, are of immediate importance.

Acid secretion and membrane reorganization in single gastric parietal cell in primary culture.

A digitally-enhanced videomicroscopy study of rabbit gastric parietal cells in primary culture was performed using alternate observations with differential interference contrast and fluorescence optics of cells mounted and perfused on a temperature-controlled microscope stage. The effect of histamine, a physiological effector of acid secretion, was followed. Isolated parietal cells possess an internal apical vacuole, which kept the cell in a pseudopolarized state. This apical vacuole is a site of acid secretion. This was demonstrated by the direct visualization of the uptake of the fluorescent weak base 9-amino acridine and of the concomitant enormous swelling of the acid vacuole which reached an estimated size of 3-7 times the normal cell volume. This morphological change of shape and acidification of apical vacuoles was fully reversible and cells could respond to successive stimulations. A quantitative study of these events provided a value of the acid accumulation index for each single cell in response to histamine. Individual cell response varied within a factor of 7. The cellular localization of the proton pump complex responsible for acid secretion and of the major components of the secretory microvilli, actin and ezrin, a histamine-dependent phosphorylation target of protein kinase A, were detected by indirect immunofluorescence microscopy in resting and stimulated cells. Both actin and ezrin colocalized at the apical vacuole membrane in resting and stimulated cells, whereas the proton pump shifted from an intracytoplasmic pool to the apical vacuole membrane upon stimulation.

Immunological localization of an 80-kDa phosphoprotein to the apical membrane of gastric parietal cells.

Monoclonal antibodies were raised against an 80-kDa phosphoprotein (80K) that is phosphorylated upon stimulation of gastric acid secretion and that copurifies with the acid-forming H+-K+-ATPase isolated from stimulated tissue. These antibodies were used to demonstrate that in the gastric mucosa 80K is limited to parietal cells and not found in surface, mucous neck, or chief cells. 80K was also found in other transporting epithelia, including intestine and kidney, but was not found in brain, liver, red blood cells, or colon. Immunohistological localization of 80K in resting glands revealed a fine network, projecting from the gland lumen and anastomosing throughout the parietal cell. This network is quite similar to the staining pattern for F-actin contained in microvilli that line the apical membrane of parietal cells. Stimulation of acid secretion rearranges 80K to a more rugose pattern filling the entire cell. In stimulated cells the distribution pattern of 80K is indistinguishable from that stained with antibodies against the H+-K+-ATPase. These data strongly suggest that 80K is an apical membrane protein of the parietal cell.

Characterization of an 80-kDa phosphoprotein involved in parietal cell stimulation.

When isolated rabbit gastric glands were stimulated with histamine plus isobutylmethylxanthine, a redistribution of H+-K+-ATPase, from microsomes to a low-speed pellet, occurred in association with the phosphorylation of an 80-kDa protein (80K) in the apical membrane-rich fraction purified from the low-speed pellet. Histamine alone or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), but not carbachol, also stimulated both the redistribution of H+-K+-ATPase and phosphorylation of 80K. Under stimulated conditions, 80K copurified in the apical membrane fraction along with H+-K+-ATPase and actin; whereas purified microsomes from resting stomach were highly enriched in H+-K+-ATPase but contained neither 80K nor actin. Treatment of the apical membranes with detergents, salts, sonication, and so on, led us to conclude that 80K is a membrane protein, unlike actin; however, the mode of association of 80K with membrane differed from H+-K+-ATPase, an integral membrane protein. Isoelectric focusing and peptide mapping revealed that 80K consists of six isomers of slightly differing pI, with 32P occurring only in the three most acidic isomers and exclusively on serine residues. Moreover, stimulation elicited a shift in the amount of 80K isomers, from basic to acidic, as well as phosphorylation. We conclude that 80K is an apical membrane protein in the parietal cell and an important substrate for cAMP-dependent, but not calcium-dependent, pathway of acid secretion.

Pumps and pathways for gastric HCl secretion.

Data reviewed herein show that the HCl-secreting parietal cell is an exaggerated example of dynamic membrane transformation. Recruitment and recycling of membrane provide the means for the massive redistribution of the gastric proton pump, the H,K-ATPase, from one membrane domain (cytoplasmic tubulovesicles) to another (apical plasma membrane) as a function of parietal cell activation and inactivation. Functional activation of HCl secretion requires not only the redistribution of pump protein, but also the participation of pathways for the rapid flux of K+ and Cl- across the apical membrane. In apical plasma membrane vesicles from stimulated cells these pathways appear to be conductive and can operate independently. Thus, our model for the parietal cell proposes that K+ and Cl- flux from cell to lumen, operating in parallel and in concert with ATP-driven H+/K+ exchange, provides the concentration and osmotic forces required for net HCl secretion. Whether and how the K+ and Cl- pathways are activated by stimulation and/or how they get to the apical membrane domain remain important questions. With respect to mechanisms of parietal cell activation, secretagogue-coupled elevation of cAMP and activation of protein kinase A form the basis of a well-established second messenger pathway. Several laboratories have identified various proteins that are phosphorylated concomitant with parietal cell stimulation, representing numerous candidates for effectors in stimulus-secretion coupling. Here, we emphasized the possible involvement of an 80-kDa protein whose phosphorylation was correlated with the cAMP pathway of HCl secretion. Immunocytolocalization of the 80-kDa phosphoprotein to the apical membrane and associated actin microfilaments prompted our suggestion that this protein might serve as a linkage between plasma membrane and cytoskeleton. Search for a possible role for the 80-kDa phosphoprotein in apical surface organization, stability, and turnover should represent an important thrust of research. Further understanding of the mechanism of cell activation will require a more complete elaboration of the functional role of many activation-related proteins.

Protein phosphorylation associated with stimulation of rabbit gastric glands.

Changes in protein phosphorylation associated with stimulation of acid secretion were investigated using isolated rabbit gastric glands labeled with 32P. The glands were stimulated by 100 microM histamine plus either 10 microM forskolin or 50 microM isobutylmethylxanthine, homogenized, and fractionated into a series of pellets: 40 X g, 5 min; 4000 X g, 10 min; 14,500 X g, 10 min; 48,200 X g, 90 min (microsomes); and supernatant. Stimulation induced a redistribution of H+/K+-transporting ATPase among the membrane fractions, i.e., a reduction in activity of the microsomal fraction, and a compensatory increase in the 4000 X g fraction. Further subfractionation of the 4000 X g pellet by Ficoll density gradient produced an 18% Ficoll layer, greatly enriched in the H+/K+-ATPase, and which is thought to be rich in apical membranes of parietal cells. SDS-polyacrylamide gel electrophoresis showed that the amount of 94 kDa peptide (the molecular size of the H+/K+-ATPase) was increased in the 18% Ficoll layer and decreased in the microsomal fraction by stimulation. Analysis of autoradiograms of the gels revealed that apparent changes in level of phosphorylation occurred in the 120, 94 and 80 kDa regions of the 18% Ficoll layer, and in the 94 kDa region of the microsomal fraction. The phosphorylation changes in the 94 kDa region may not reflect changes in specific activity of a single peptide but may be due to the heterogeneity of proteins in this region, which was demonstrated by selective heat treatment of the samples as well as two-dimensional electrophoresis. Phosphorylation of 120 kDa protein in the 18% Ficoll layer was clearly increased by stimulation, and this appeared to be associated with protein distribution changes as well as phosphorylation. The 80 kDa protein in the 18% Ficoll layer showed marked increased phosphorylation by stimulation, with little change in protein distribution. This 80 kDa protein was focused on two-dimensional gels as several sequential spots, with the most radioactive peptide focused toward the acidic side; thus, we propose isomeric forms of an 80 kDa protein with sequential phosphorylation sites. The phosphorylation changes observed in this study are considered to be important to the process of gastric acid secretion because they occurred in the putative apical membrane fractions in which biochemical and functional changes with stimulation have been demonstrated.