scholarly journals Comparative studies of intracellular transport of secretory proteins.

1978 ◽  
Vol 79 (3) ◽  
pp. 694-707 ◽  
Author(s):  
A Tartakoff ◽  
P Vassalli ◽  
M Détraz
Keyword(s):  
Intracellular Transport ◽  
Cell Types ◽  
Secretory Protein ◽  
Pancreatic Acinar Cell ◽  
Secretory Cells ◽  
Secretory Proteins ◽  
Cell Fractionation ◽  
Secretory Product ◽  
Electron Microscopic ◽  
Secretory Rate

The physiology of protein intracellular transport and secretion by cell types thought to be free from short-term control has been compared with that of the pancreatic acinar cell, using pulse-chase protocols to follow biosynthetically-labeled secretory products. Data previously obtained (Tartakoff, A.M., and P. Vassalli. J. Exp. Med. 146:1332-1345) has shown that plasma-cell immunoglobulin (Ig) secretion is inhibited by respiratory inhibitors, by partial Na/K equilibration effected by the carboxylic ionophore monensin, and by calcium withdrawal effected by the carboxylic ionophore A 23187 in the presence of ethylene glycol bis (beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and absence of calcium. We report here that both inhibition of respiration and treatment with monensin slow secretion by fibroblasts, and also macrophages and slow intracellular transport (though not discharge per se) by the exocrine pancreatic cells. Attempted calcium withdrawal is inhibitory for fibroblasts but not for macrophages. The elimination of extracellular calcium or addition of 50 mM KCl has no major effect on secretory rate of either fibroblasts or macrophages. Electron microscopic examination of all cell types shows that monensin causes a rapid and impressive dilation of Golgi elements. Combined cell fractionation and autoradiographic studies of the pancreas show that the effect of monensin is exerted at the point of the exit of secretory protein from the Golgi apparatus. Other steps in intracellular transport proceed at normal rates. These observations suggest a common effect of the cytoplasmic Na/K balance at the Golgi level and lead to a model of intracellular transport in which secretory product obligatorily passes through Golgi elements (cisternae?) that are sensitive to monensin. Thus, intracellular transport follows a similar course in both regulated and nonregulated secretory cells up to the level of distal Golgi elements.

scholarly journals SYNTHESIS, INTRACELLULAR TRANSPORT, AND DISCHARGE OF SECRETORY PROTEINS IN STIMULATED PANCREATIC EXOCRINE CELLS

1971 ◽  
Vol 50 (1) ◽  
pp. 135-158 ◽  
Author(s):  
James D. Jamieson ◽  
George E. Palade
Keyword(s):  
Protein Synthesis ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Secretory Proteins ◽  
Cell Fractionation ◽  
Secretory Product ◽  
Zymogen Granules ◽  
Pancreatic Exocrine ◽  
Storage Granules

Our previous observations on the synthesis and transport of secretory proteins in the pancreatic exocrine cell were made on pancreatic slices from starved guinea pigs and accordingly apply to the resting, unstimulated cell. Normally, however, the gland functions in cycles during which zymogen granules accumulate in the cell and are subsequently discharged from it in response to secretogogues. The present experiments were undertaken to determine if secretory stimuli applied in vitro result in adjustments in the rates of protein synthesis and/or of intracellular transport. To this intent pancreatic slices from starved animals were stimulated in vitro for 3 hr with 0.01 mM carbamylcholine. During the first hour of treatment the acinar lumen profile is markedly enlarged due to insertion of zymogen granule membranes into the apical plasmalemma accompanying exocytosis of the granule content. Between 2 and 3 hr of stimulation the luminal profile reverts to unstimulated dimensions while depletion of the granule population nears completion. The acinar cells in 3-hr stimulated slices are characterized by the virtual complete absence of typical condensing vacuoles and zymogen granules, contain a markedly enlarged Golgi complex consisting of numerous stacked cisternae and electron-opaque vesicles, and possess many small pleomorphic storage granules. Slices in this condition were pulse labeled with leucine-3H and the route and timetable of intracellular transport assessed during chase incubation by cell fractionation, electron microscope radioautography, and a discharge assay covering the entire secretory pathway. The results showed that the rate of protein synthesis, the rate of drainage of the rough-surfaced endoplasmic reticulum (RER) compartment, and the over-all transit time of secretory proteins through the cells was not accelerated by the secretogogue. Secretory stimulation did not lead to a rerouting of secretory proteins through the cell sap. In the resting cell, the secretory product is concentrated in condensing vacuoles and stored as a relatively homogeneous population of spherical zymogen granules. By contrast, in the stimulated cell, secretory proteins are initially concentrated in the flattened saccules of the enlarged Golgi complex and subsequently stored in numerous small storage granules before release. The results suggest that secretory stimuli applied in vitro primarily affect the discharge of secretory proteins and do not, directly or indirectly, influence their rates of synthesis and intracellular transport.

Correlative transmission and high-resolution scanning electron microscopic studies on pituitary cells

1990 ◽  
Vol 48 (3) ◽  
pp. 20-21
Author(s):  
S. Tai
Keyword(s):  
Cell Types ◽  
Depth Of Field ◽  
Secretory Cells ◽  
Specific Cell ◽  
Pituitary Cells ◽  
Electron Microscopic ◽  
3 Dimensional ◽  
Microscopic Studies ◽  
Structure And Activity ◽  
Intracellular Structure

Extensive cytological and histological research, correlated with physiological experimental analysis, have been done on the anterior pituitaries of many different vertebrates which have provided the knowledge to create the concept that specific cell types synthesize, store and release their specific hormones. These hormones are stored in or associated with granules. Nevertheless, there are still many doubts - that need further studies, specially on the ultrastructure and physiology of these endocrine cells during the process of synthesis, transport and secretion, whereas some new methods may provide the information about the intracellular structure and activity in detail.In the present work, ultrastructural study of the hormone-secretory cells of chicken pituitaries have been done by using TEM as well as HR-SEM, to correlate the informations obtained from 2-dimensional TEM micrography with the 3-dimensional SEM topographic images, which have a continous surface with larger depth of field that - offers the adventage to interpretate some intracellular structures which were not possible to see using TEM.

scholarly journals Glycoprotein transport in the surface mucous cells of the rat stomach.

1977 ◽  
Vol 73 (3) ◽  
pp. 533-547 ◽  
Author(s):  
M F Kramer ◽  
J J Geuze
Keyword(s):  
Golgi Complex ◽  
Intracellular Transport ◽  
Tritiated Thymidine ◽  
Secretory Protein ◽  
Secretory Cells ◽  
Rat Stomach ◽  
Gastric Glands ◽  
Electron Microscope Autoradiography ◽  
Glycoprotein Transport

The intracellular transport of glycoproteins pulse-labeled in vitro with tritiated leucine and galactose in the surface mucous lining cells (SMC) of the fundus of the rat stomach was studied by electron microscope autoradiography. The SMC survive for several hours in pieces of the fundus incubated in a bicarbonate-buffered medium. The SMC have a normal ultrastructure for at least 4 h of incubation. Kinetic activity is normal for at least 5 h, as demonstrated by the normal nuclear incorporation of tritiated thymidine; The SMC incorporate labeled leucine and galactose at normal rates up to 4 h and 6 h, respectively. In contrast to the SMC, the cells of the gastric glands show signs of degeneration within 1 h after the start of incubation. In the SMC the secretory protein forms a smaller part of the total protein synthesized than in other secretory cells studied. The intracellular tranpsort of the leucine-labeled moiety of the glycoproteins follows the normal pathway. The RER loses 35% of its transportable labeled protein within 30 min. The Golgi complex is maximally labeled at 40 min and the mucous granules after 120 min. Galactose is attached to the glycoproteins mainly in the Golgi complex. Glycoproteins are not secreted within 2 h after synthesis of their protein moiety.

scholarly journals Vesicular stomatitis virus glycoprotein, albumin, and transferrin are transported to the cell surface via the same Golgi vesicles.

1983 ◽  
Vol 97 (6) ◽  
pp. 1815-1822 ◽  
Author(s):  
G J Strous ◽  
R Willemsen ◽  
P van Kerkhof ◽  
J W Slot ◽  
H J Geuze ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Intracellular Transport ◽  
Intracellular Localization ◽  
Protein A ◽  
Secretory Protein ◽  
Hepatoma Cells ◽  
Secretory Proteins ◽  
Human Hepatoma Cells ◽  
Transmembrane Glycoprotein ◽  
Vesicular Stomatitis

Human hepatoma cells, infected by vesicular stomatitis virus, offer a good system to study simultaneously the intracellular localization of a well defined transmembrane glycoprotein (VSV-G), a secretory glycoprotein (transferrin), and a nonglycosylated secretory protein (albumin). We used monospecific antibodies in combination with 5- and 8-nm colloidal gold particles complexed with protein A to immunolabel these proteins simultaneously in thin frozen sections of hepatoma cells. VSV-G, transferrin, and albumin are present in the same rough endoplasmic reticulum cisternae, the same Golgi compartments, and the same secretory vesicles. In the presence of the ionophore monensin intracellular transport is blocked at the trans cisternae of the Golgi complex, and VSV-G, transferrin, and albumin accumulate in dilated cisternae, which are apparently derived from the trans-Golgi elements. Glycoproteins, synthesized and secreted in the presence of monensin, are less acidic than those in control cultures. This is probably caused by a less efficient contact between the soluble secretory proteins and the membrane-bound glycosyltransferases that are present in the most monensin-affected (trans) Golgi cisternae.

scholarly journals ER-to-Golgi Trafficking and Its Implication in Neurological Diseases

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 408 ◽  
Author(s):  
Bo Wang ◽  
Katherine R. Stanford ◽  
Mondira Kundu
Keyword(s):  
Protein Localization ◽  
Neurological Diseases ◽  
Cell Types ◽  
Cellular Function ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Complex Cell ◽  
Final Destination ◽  
And Function ◽  
Spatiotemporal Control

Membrane and secretory proteins are essential for almost every aspect of cellular function. These proteins are incorporated into ER-derived carriers and transported to the Golgi before being sorted for delivery to their final destination. Although ER-to-Golgi trafficking is highly conserved among eukaryotes, several layers of complexity have been added to meet the increased demands of complex cell types in metazoans. The specialized morphology of neurons and the necessity for precise spatiotemporal control over membrane and secretory protein localization and function make them particularly vulnerable to defects in trafficking. This review summarizes the general mechanisms involved in ER-to-Golgi trafficking and highlights mutations in genes affecting this process, which are associated with neurological diseases in humans.

scholarly journals Identification of ERGIC-53 as an intracellular transport receptor of α1-antitrypsin

2008 ◽  
Vol 180 (4) ◽  
pp. 705-712 ◽  
Author(s):  
Beat Nyfeler ◽  
Veronika Reiterer ◽  
Markus W. Wendeler ◽  
Eduard Stefan ◽  
Bin Zhang ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Er Export ◽  
Transport Receptor

Secretory proteins are exported from the endoplasmic reticulum (ER) by bulk flow and/or receptor-mediated transport. Our understanding of this process is limited because of the low number of identified transport receptors and cognate cargo proteins. In mammalian cells, the lectin ER Golgi intermediate compartment 53-kD protein (ERGIC-53) represents the best characterized cargo receptor. It assists ER export of a subset of glycoproteins including coagulation factors V and VIII and cathepsin C and Z. Here, we report a novel screening strategy to identify protein interactions in the lumen of the secretory pathway using a yellow fluorescent protein–based protein fragment complementation assay. By screening a human liver complementary DNA library, we identify α1-antitrypsin (α1-AT) as previously unrecognized cargo of ERGIC-53 and show that cargo capture is carbohydrate- and conformation-dependent. ERGIC-53 knockdown and knockout cells display a specific secretion defect of α1-AT that is corrected by reintroducing ERGIC-53. The results reveal ERGIC-53 to be an intracellular transport receptor of α1-AT and provide direct evidence for active receptor-mediated ER export of a soluble secretory protein in higher eukaryotes.

scholarly journals Differential arrest of secretory protein transport in cultured rat hepatocytes by azide treatment.

1988 ◽  
Vol 107 (6) ◽  
pp. 2503-2510 ◽  
Author(s):  
R Persson ◽  
E Ahlström ◽  
E Fries
Keyword(s):  
Intracellular Transport ◽  
Protein Transport ◽  
Rat Hepatocytes ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Atp Content ◽  
Isolated Rat Hepatocytes ◽  
Atp Level ◽  
Atp Levels ◽  
Endoglycosidase H

The effect of reduced cellular ATP content on intracellular transport of two secretory proteins, albumin and haptoglobin, in isolated rat hepatocytes was studied. The cells were labeled with [35S]methionine and the cellular ATP content was then rapidly reduced to different stable levels by incubation with azide at different concentrations (2.0-10 mM). The amount of the radioactively labeled secretory proteins in the cells and in the medium after 150 min of incubation was determined by immunoprecipitation followed by gel electrophoresis, fluorography, and densitometry. At progressively lower ATP levels, down to 50% of normal, the protein secretion was unaffected, whereas at even lower levels an increasing portion of the proteins remained in the cells; at 30 and 10% of normal ATP level, 25 and 75% of albumin, respectively, was arrested intracellularly. Analysis of the carbohydrate structure of intracellularly arrested haptoglobin showed that in cells with an ATP level of approximately 30% of normal, the majority of haptoglobin molecules (55%) were fully or partially resistant to endoglycosidase H. This result indicates that exit from the medial and/or the trans part of the Golgi complex (GC) was inhibited under these conditions. It also shows that the protein had accumulated in the GC, since under normal conditions the fraction of the intracellular haptoglobin that is endoglycosidase H resistant is approximately 10%. By similar criteria it was found that at ATP levels below 10% of normal transport of haptoglobin from the endoplasmic reticulum to the medial GC (and possibly also to the cis GC) as well as from the trans GC to the medium were blocked.

scholarly journals BiP Availability Distinguishes States of Homeostasis and Stress in the Endoplasmic Reticulum of Living Cells

2010 ◽  
Vol 21 (12) ◽  
pp. 1909-1921 ◽  
Author(s):  
Chun Wei Lai ◽  
Deborah E. Aronson ◽  
Erik Lee Snapp
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Protein Misfolding ◽  
Single Cells ◽  
Cell Types ◽  
Secretory Protein ◽  
Misfolded Proteins ◽  
Secretory Proteins ◽  
Molecular Events ◽  
The Unfolded Protein Response

Accumulation of misfolded secretory proteins causes cellular stress and induces the endoplasmic reticulum (ER) stress pathway, the unfolded protein response (UPR). Although the UPR has been extensively studied, little is known about the molecular changes that distinguish the homeostatic and stressed ER. The increase in levels of misfolded proteins and formation of complexes with chaperones during ER stress are predicted to further crowd the already crowded ER lumen. Surprisingly, using live cell fluorescence microscopy and an inert ER reporter, we find the crowdedness of stressed ER, treated acutely with tunicamycin or DTT, either is comparable to homeostasis or significantly decreases in multiple cell types. In contrast, photobleaching experiments revealed a GFP-tagged variant of the ER chaperone BiP rapidly undergoes a reversible quantitative decrease in diffusion as misfolded proteins accumulate. BiP mobility is sensitive to exceptionally low levels of misfolded protein stressors and can detect intermediate states of BiP availability. Decreased BiP availability temporally correlates with UPR markers, but restoration of BiP availability correlates less well. Thus, BiP availability represents a novel and powerful tool for reporting global secretory protein misfolding levels and investigating the molecular events of ER stress in single cells, independent of traditional UPR markers.

Secretory product expression during Clara cell differentiation in the rabbit and rat

1993 ◽  
Vol 264 (6) ◽  
pp. L543-L552 ◽  
Author(s):  
W. V. Cardoso ◽  
L. G. Stewart ◽  
K. E. Pinkerton ◽  
C. Ji ◽  
G. E. Hook ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Secretory Granules ◽  
Close Association ◽  
Secretory Protein ◽  
Clara Cell ◽  
Secretory Proteins ◽  
Secretory Product ◽  
Secretory Function ◽  
Clara Cells ◽  
Clara Cell Secretory Protein

One function of the nonciliated (Clara) cells of bronchiolar epithelium is to synthesize, store, and release small-molecular-mass (6–12 kDa) secretory proteins or Clara cell secretory protein (CCSP). This study compares the emergence of this secretory function during Clara cell differentiation in rabbits and rats. Lungs of fetal and postnatal animals were evaluated by ultrastructural morphometry and immunohistochemistry. Secretory granules were rarely seen in perinatal animals and increased to adult levels of abundance earlier in rats (1 wk postnatal) than in rabbits (3–4 wk). In contrast, rough endoplasmic reticulum was abundant in perinatal animals and decreased with age. Antibodies raised against CCSP revealed little CCSP in fetal animals; however, after birth CCSP increased to adult levels earlier in rats (1 wk postnatal) than in rabbits (3 wk). We conclude that the maturation of Clara cell secretory function 1) occurs postnatally, 2) involves a decrease in biosynthetic organelles, 3) shows close association between CCSP expression and secretory granule abundance, and 4) varies by species in timing and cellular abundance of biosynthetic machinery.

scholarly journals Immunocytochemical localization of BiP to the rough endoplasmic reticulum: evidence for protein sorting by selective retention.

1989 ◽  
Vol 37 (12) ◽  
pp. 1817-1823 ◽  
Author(s):  
D G Bole ◽  
R Dowin ◽  
M Doriaux ◽  
J D Jamieson
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Membrane System ◽  
Myeloma Cell ◽  
Secretory Protein ◽  
Electron Microscopic Level ◽  
Immunoperoxidase Staining ◽  
Secretory Proteins ◽  
Electron Microscopic ◽  
Exocytic Pathway

Immunoglobulin heavy chain binding protein (BiP) (also known as GRP 78) is a protein of the endoplasmic reticulum (ER) which has been shown to be involved in post-translational processing of nascent membrane and secretory proteins. To determine BiP's location in the exocytic pathway, we localized BiP at the electron microscopic level in mouse myeloma cell lines by immunoperoxidase cytochemistry. BiP was found to be present within the cisternal spaces of the RER and nuclear envelope but was not detected in the cisternae of the Golgi complex. BiP reaction product was also found within transitional elements of the RER but was absent from smooth-surfaced vesicles found between the ER and the Golgi complex. Immunoperoxidase staining of BiP was reduced or absent in regions of a smooth ER membrane system in myelomas that contained endogenous murine retrovirus A particles. All compartments of the exocytic pathway, including the virus-containing smooth ER, stained for IgG, a secretory protein. These observations suggest that BiP is selectively retained in the cisternae of the ER and is not free to enter Golgi-directed transport vesicles. These studies suggest that BiP's subcellular localization may occur by selective interaction with component(s) of the ER.

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