Large populations (up to 600/cell) of spherical, electron-opaque granules approximately 0.3 to 0.4 micro in diameter are characteristically found in muscle fibers of mammalian atria. They are absent in muscle fibers of the ventricles. The granules are concentrated in the sarcoplasmic core and occur in lesser numbers in the sarcoplasmic layers between myofibrils and under the plasma membrane. Their intimate association with a central voluminous Golgi complex and the frequent occurrence of material reminiscent of the granular content within the cisternae of the Golgi complex suggest that the latter is involved in the formation of the atrial granules. Atrial granules are larger and more numerous in smaller species (rat, mouse), and generally smaller and less numerous in larger mammals (dog, cat, human); they are absent from the atrial fibers of very young fetuses (rat) but are present in those of newborn animals. A small population of bodies containing glycogen particles and remnants of the endoplasmic reticulum and mitochondria occurs in the sarcoplasmic cores of atrial as well as ventricular muscle fibers in the rat; they contain acid phosphatase and thus appear to be residual bodies of autolytic foci. Their frequency increases with the age of the animal. Typical lipofuscin pigment granules, which are known to contain acid phosphatase and are found in the sarcoplasmic cores in old animals (cat, dog and human), are presumed to arise by progressive aggregation and fusion of small residual bodies.
A series of representative cell types including avian fibroblasts, and macrophages; rabbit mesothelia, endothelia, and nephron epithelia; and rat glandular epithelia (parotid) were studied comparatively in vitro and in situ with the electron microscope. Cells in vitro were examined in whole mounts and in sections whereas cells in situ were observed exclusively in sections. It was found that an endoplasmic reticulum similar to that previously described in cultured material is present in situ in all cell types examined. Modifications in its appearance introduced by the sectioning technique were discussed and explained. The observations showed in addition that the endoplasmic reticulum is a network of cavities which may enlarge into relatively vast, flattened vesicles here described as cisternae.
To better understand the function of Rab1a, we have immunoisolated Rab1a-associated transport vesicles from rat liver using affinity-purified anti-Rab1a-coated magnetic beads. A fraction enriched in endoplasmic reticulum (ER) to Golgi transport vesicles (CV2, ρ = 1.158) was subjected to immunoisolation, and proteins of the bound and non-bound subfractions were analyzed by Western blotting. To our surprise, we found that immunoisolated vesicles contained not only ER markers (105-kDa form of the polymeric IgA receptor (pIgAR)) but also transcytotic markers (dIgA and the 120-kDa form of pIgAR), suggesting that Rab1a is associated with transcytotic vesicles in rat liver. To investigate this possibility, we used an antibody to the cytoplasmic domain of pIgAR to immunoisolate transcytotic vesicles from a fraction (CV1, ρ = 1.146) known to be enriched in these vesicles. Rab1a was detected in the immunoadsorbed subfractions. The composition of the vesicles immunoisolated from the CV1 fraction on anti-Rab1a was similar to that of transcytotic vesicles immunoisolated from the same fraction on pIgAR. Both were enriched in transcytotic markers and depleted in ER and Golgi markers. The main difference between the two was that those isolated on anti-Rab1a appeared to be enriched in postendosomal transcytotic vesicles, whereas those isolated on pIgAR contained both pre- and postendosomal elements. Analysis of anti-Rab1a isolated vesicles using [α-32P]GTP overlay demonstrated the presence of multiple GTP-binding proteins. Some of these were identified by immunoblotting as epithelia-specific Rab17 and ubiquitous Rabs1b, −2, and −6. Taken together, these results indicate that: 1) Rab1a is associated with both ER to Golgi and postendosomal transcytotic vesicles, and 2) multiple GTP-binding proteins are associated with each class of isolated vesicle. To better understand the function of Rab1a, we have immunoisolated Rab1a-associated transport vesicles from rat liver using affinity-purified anti-Rab1a-coated magnetic beads. A fraction enriched in endoplasmic reticulum (ER) to Golgi transport vesicles (CV2, ρ = 1.158) was subjected to immunoisolation, and proteins of the bound and non-bound subfractions were analyzed by Western blotting. To our surprise, we found that immunoisolated vesicles contained not only ER markers (105-kDa form of the polymeric IgA receptor (pIgAR)) but also transcytotic markers (dIgA and the 120-kDa form of pIgAR), suggesting that Rab1a is associated with transcytotic vesicles in rat liver. To investigate this possibility, we used an antibody to the cytoplasmic domain of pIgAR to immunoisolate transcytotic vesicles from a fraction (CV1, ρ = 1.146) known to be enriched in these vesicles. Rab1a was detected in the immunoadsorbed subfractions. The composition of the vesicles immunoisolated from the CV1 fraction on anti-Rab1a was similar to that of transcytotic vesicles immunoisolated from the same fraction on pIgAR. Both were enriched in transcytotic markers and depleted in ER and Golgi markers. The main difference between the two was that those isolated on anti-Rab1a appeared to be enriched in postendosomal transcytotic vesicles, whereas those isolated on pIgAR contained both pre- and postendosomal elements. Analysis of anti-Rab1a isolated vesicles using [α-32P]GTP overlay demonstrated the presence of multiple GTP-binding proteins. Some of these were identified by immunoblotting as epithelia-specific Rab17 and ubiquitous Rabs1b, −2, and −6. Taken together, these results indicate that: 1) Rab1a is associated with both ER to Golgi and postendosomal transcytotic vesicles, and 2) multiple GTP-binding proteins are associated with each class of isolated vesicle.
Subfractionation of preparations of rat liver microsomes with a suitable concentration of sodium deoxycholate has resulted in the isolation of a membrane fraction consisting of smooth surfaced vesicles virtually free of ribonucleoprotein particles. The membrane fraction is rich in phospholipids, and contains the microsomal NADH-cytochrome c reductase, NADH diaphorase, glucose-6-phosphatase, and ATPase in a concentrated form. The NADPH-cytochrome c reductase, a NADPH (or pyridine nucleotide unspecific) diaphorase, and cytochrome b(5) are recovered in the clear supernatant fraction. The ribonucleoprotein particles are devoid of, or relatively poor in, the enzyme activities mentioned. Those enzymes which are bound to the membranes vary in activity according to the structural state of the microsomes, whereas those which appear in the soluble fraction are stable. From these findings the conclusion is reached that certain enzymes of the endoplasmic reticulum are tightly bound to the membranes, whereas others either are loosely bound or are present in a soluble form within the lumina of the system. Some implications of these results as to the enzymic organization of the endoplasmic reticulum are discussed.
Abstract The turnover of various constituents of the membranes of the endoplasmic reticulum has been measured by injecting 14C-leucine and 14C-acetate, or in some cases 14C-leucine and 14C-glycerol, into a series of 12 to 15 rats (weighing 150 to 200 g). At various times, up to 2 weeks after injection, three rats were killed, their livers were pooled, microsomes were obtained and divided into rough and smooth fractions, and purified membranes were isolated from each fraction. The constituents examined were the following: total membrane proteins; two specific membrane components, purified reduced-NADP: ferricytochrome c oxidoreductase and purified cytochrome b5; total membrane lipids; and the nonpolar (fatty acid) and polar (glycerol backbone) moieties of the lipid. The two enzymes were purified 100- to 150-fold by trypsin digestion of KCl-washed microsomes, followed by chromatography on Sephadex G-100 and diethylaminoethyl cellulose. The purity of both proteins was close to 100%, in yields of 25 to 35% from original microsomes. Both total proteins and total lipids of rough microsomal membranes had the same half-lives as their counterparts from smooth microsomal membranes. In the various experiments, the total membrane proteins had half-lives of from 75 to 113 hours, while in the same experiments, total membrane lipids have 10 to 30% shorter half-lives. The half-life of the NADPH-cytochrome c reductase was, in two experiments, almost exactly that of total membrane proteins, while the half-life of cytochrome b5 was significantly (about 50%) longer. The polar and nonpolar components of the lipids had different apparent half-lives, that of fatty acids being much longer than that of the glycerol backbone. The longer half-life of fatty acids may be connected with the presumed presence of transacylating enzymes in microsomal membranes. The results are discussed in relation to current concepts on membrane structure and biogenesis.
Intracellular transport of secretory proteins has been studied in the parotid to examine this process in an exocrine gland other than the pancreas and to explore a possible source of less degraded membranes than obtainable from the latter gland. Rabbit parotids were chosen on the basis of size (2–2.5 g per animal), ease of surgical removal, and amylase concentration. Sites of synthesis, rates of intracellular transport, and sites of packaging and storage of newly synthesized secretory proteins were determined radioautographically by using an in vitro system of dissected lobules capable of linear amino acid incorporation for 10 hr with satisfactory preservation of cellular fine structure. Adequate fixation of the tissue with minimal binding of unincorporated labeled amino acids was obtained by using 10% formaldehyde-0.175 M phosphate buffer (pH 7.2) as primary fixative. Pulse labeling with leucine-3H, followed by a chase incubation, showed that the label is initially located (chase: 1–6 min) over the rough endoplasmic reticulum (RER) and subsequently moves as a wave through the Golgi complex (chase: 16–36 min), condensing vacuoles (chase: 36–56 min), immature granules (chase: 56–116 min), and finally mature storage granules (chase: 116–356 min). Distinguishing features of the parotid transport apparatus are: low frequency of RER-Golgi transitional elements, close association of condensing vacuoles with the exit side of Golgi stacks, and recognizable immature secretory granules. Intracelular processing of secretory proteins is similar to that already found in the pancreas, except that the rate is slower and the storage is more prolonged.
The distribution of the three glycosyltransferases synthesizing the terminal trisaccharide sialic acid yields D-galactose yields N-acetylglucosamine present in many glycoproteins was determined in Golgi fractions prepared from rat liver homogenates by a modification of the procedure of Ehrenreich et al. (1973, J. Cell Biol. 70:671--684). The enzymes were assayed with asialofetuin, ovomucoid, and Smith-degraded ovomucoid as sugar acceptors. Careful adjustment of the pH of all sucrose solutions to 7.0 +/- 0.1 prevented enzyme inactivation, and allowed quantitative recoveries at every isolation step. The three morphologically and functionally different Golgi fractions GJ1, GF2, and GF3 showed (in that order) decreasing specific activities of all three enzymes, but the relative amounts and relative specific activities of the three transferases in any given fraction were nearly identical. Two marginal fractions, one extra heavy (collected on the gradient below GF3) and the other extra light (isolated by flotation from the postmicrosomal supernate) were found to contain recognizable Golgi elements. An enrichment of any transferase over the two others was not detected in either preparation. A partial release of content from a combined GF1+2 was achieved by treatment with the nonionic detergent Triton X-100. Low Triton/phospholipid ratios (less than 2 mg/mg) led to lysis of the vesicles and cisternae and loss of very low density lipoprotein particles (ascertained by electron microscopy), but failed to separate the transferases from each other; the three enzymes sedimented together with a population of empty vesicles to a density of approximately 1.08 g/ml.
ABSTRACT In rat hepatocytes, transcytotic vesicular carriers transport the mature 120× 103Mr form of the polymeric IgA receptor (plgA-R), with or without its ligand, plgA, from the sinusoidal to the biliary plasmalemma, where the ectodomain of the receptor is cleaved to produce an 80×103Mr fragment that is secreted into the bile. Here we show that cholestasis induced by bile duct ligation results in the accumulation of transcytotic carriers, identified by the 120 ×103Mr plgA-R and plgA, in the pericanalicular cytoplasm of hepatocytes. To determine the extent of plgA-R accumulation, hepatic total microsomes (TM) were prepared from control and cholestatic rats. Solubilized TM proteins were separated by SDS-PAGE and receptor forms were detected by immunoblotting and autoradiography. Quantitative densitometry of these autoradiograms showed that after duct ligation the 120 × 103Mr receptor accumulated to a level ∼ threefold higher than the control. Concomitantly, immunologically related, novel 124, 90 and 80 × 103Mr proteins (cholestatic antigens) became detectable. Immunoblot analyses of biliary and serum proteins showed that cholestasis resulted in: (1) a marked decrease in the concentrations of the 80 × 103Mr receptor and plgA in the bile, whereas albumin concentrations remained at control levels; and (2) a marked increase in the concentration of the 80 × 103Mr receptor in the serum. Positive sites for plgA-R were localized to the pericanalicular cytoplasm of hepatocytes by indirect immunofluorescence on semithin frozen sections in cholestatic hepatocytes. The sites were more numerous and the positive signal stronger than in controls. One day post-ligation, plgA-positive sites were located to the same pericanalicular cytoplasm of hepatocytes; by three days, however, most plgA appeared in sinusoidal endothelia and Kupffer cells. To validate the vesicular character of the receptor-positive sites, sham-operated and cholestatic livers were processed for either transmission electron microscopy (TEM) or immunogold localization of receptors on thin frozen sections. TEM verified the accumulation of pericanalicular vesicles in cholestatic hepatocytes. Immunogold tests localized plgA-R to pleiomorphic, pericanalicular vesicles, which were increased in number, size and concentration of antigenic sites in cholestatic hepatocytes. These findings indicate that bile duct ligation provides a method for manipulating the in vivo transcytotic pathway and for accumulating previously unstudied transcytotic carriers in hepatocytes.
A fraction of secretion granules has been isolated from rabbit parotid by a procedure which was found to be especially effective in reducing contamination resulting from aggregation and/or cosedimentation of granules with other cell particulates. The fraction, representing 15 percent (on the average) of the total tissue amylase activity, was homogeneous as judged by electron microscopy and contaminated to exceedingly low levels by other cellular organelles as judged by marker enzymatic and chemical assays. Lysis of the granules was achieved by their gradual exposure to hypotonic NaHCO3, containing 0.5 mM EDTA. The content and the membranes separated by centrifugation of the granule lysate were characterized primarily by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis which indicated that the content was composed of a limited number of molecular weight classes of polypeptides of which three bands (having approximate mol wt 58,000, 33, 000, and 12,000) could be considered major components. The gel profile of the membrane subfraction was characterized by 20-30 Coomassie brilliant blue-staining bands of which a single species of mol wt 40,000 was the conspicuous major polypeptide. Two types of experiments employing gel electrophoretic analysis were carried out for identifying and assessing the extent of residual secretory protein adsorbed to purified granule membranes: (a) examination of staining and radioactivity profiles after mixing of radioactive secretion granule extract with nonradioactively labeled granule membranes and (b) comparison of gel profiles of secretion granule extract and granule membranes with those of unlysed secretion granules and secretory protein dischraged from lobules in vitro or collected by cannulation of parotid ducts, the last two samples being considered physiologic secretory standards. The results indicated that the membranes were contaminated to a substantial degree by residual, poorly extractable secretory protein even though assays of membrane fractions for a typical secretory enzyme activity (amylase) indicated quite through separation of membranes and content. Hence, detailed examination of membrane subfractions for residual content species by gel electrophoresis points to the general unity and sensitivity of this technique as a means for accurately detecting a defined set of polypeptides occurring as contaminants in cellular fractions or organelle subfractions.
