Evidence in vivo of asynchronous intracellular transport of rat pancreatic secretory proteins

1986 ◽  
Vol 2 (2) ◽  
Author(s):  
Volker Keim ◽  
Gerhard Rohr
Keyword(s):  
Intracellular Transport ◽  
Secretory Proteins

A possible role for stable microtubules in intracellular transport from the endoplasmic reticulum to the Golgi apparatus

1994 ◽  
Vol 107 (5) ◽  
pp. 1321-1331 ◽  
Author(s):  
M. Mizuno ◽  
S.J. Singer
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Transfer Process ◽  
Intracellular Transport ◽  
Early Stage ◽  
Critical Role ◽  
Treatment Protocol ◽  
Secretory Proteins ◽  
Random Diffusion

The intracellular transport of secretory proteins involves at an early stage the formation of vesicles from transitional elements of the endoplasmic reticulum (ER) containing these proteins and the transfer of these vesicles to the cis-face of the Golgi apparatus. We propose that the latter transfer process does not occur by random diffusion, but is instead mediated by tracking along stable microtubules. To test this proposal, we have carried out double immunoelectron microscopic labeling experiments on frozen sections of HepG2 hepatoma cells secreting the protein human serum albumin (HSA). By a cycloheximide treatment protocol, the stage during which the transfer of newly synthesized HSA from the ER to the Golgi apparatus occurs in vivo was determined. Sections of the cells were then double immunolabeled using primary antibodies to HSA and to glu-tubulin, the latter specifically detecting stable microtubules. We observed a significantly high frequency of HSA-containing structures between the ER and the Golgi apparatus with which stable microtubules were closely associated. These results support the proposal that stable microtubules may play a critical role in directing the transfer process from the ER to the Golgi apparatus.

Pancreatic effects of ethionine: blockade of exocytosis and appearance of crinophagy and autophagy precede cellular necrosis

1982 ◽  
Vol 242 (4) ◽  
pp. G297-G307 ◽  
Author(s):  
H. Koike ◽  
M. L. Steer ◽  
J. Meldolesi
Keyword(s):  
Intracellular Transport ◽  
Pancreatic Necrosis ◽  
Young Female ◽  
Secretory Proteins ◽  
Zymogen Granules ◽  
Luminal Membrane ◽  
Sequence Of Events ◽  
Complete Disruption ◽  
Cellular Necrosis

Young female mice fed a choline-deficient, ethionine-supplemented (CDE) diet for 24 h develop hemorrhagic pancreatic necrosis with a 5-day mortality rate of approximately 50%. At the end of the diet administration, the in vivo discharge of digestive enzymes is blocked, images of exocytosis and luminal membrane recycling disappear, and zymogen granules accumulate within acinar cells. The general ultrastructure, however, remains well preserved, protein synthesis is normal, and intracellular transport of secretory proteins is only slightly retarded. Thus, the CDE diet does not affect the general phenomenon of membrane fusion-fission but specifically inhibits that associated with exocytosis. Twenty-four hours after withdrawal of the CDE diet, discharge of zymogen granules into lysosomes (crinophagy) can be observed, and, 24 h latr, autophagocytosis is noted. Finally, shortly before the onset of pancreatic necrosis, cells of nearly normal appearance are noted to be scattered among cells showing varying degrees of lesion up to complete disruption. Thus, the CDE-induced pancreatic necrosis results from a sequence of events: blockade of exocytosis evolves to crinophagy and autophagy, which might lead to lysosomal activation of zymogens.

The adrenal paraneurone: tubulin organization

1984 ◽  
Vol 62 (5) ◽  
pp. 502-511 ◽  
Author(s):  
M. F. Bader ◽  
F. Bernier-Valentin ◽  
B. Rousset ◽  
D. Aunis
Keyword(s):  
Cell Body ◽  
Intracellular Transport ◽  
Cultured Cells ◽  
Specific Binding ◽  
Secretory Granules ◽  
Immunofluorescent Staining ◽  
Chromaffin Granules ◽  
Two Dimensional Gel Electrophoresis ◽  
Granule Membrane

When chromaffin cells from the bovine adrenal medulla are maintained in culture, they develop neuritelike processes which end with growth-cone-like structures. Chromaffin granules were found to migrate from the cell body to the neurite endings. Thus, the intracellular transport of secretory granules, existing in vivo, seems to occur in an exaggerated way in the cultured cells. These cells offer an excellent model for studying the mechanism of transport, particularly the role of microtubules. By immunofluorescent staining, we observed that tubulin antibodies decorate a complex network visible along the neurites. Colchicine treatment induced the disappearance of this network followed by a return of granules in the cell body and a retraction of neurites. To test the presence of tubulin in the chromaffin granule membrane, we used two-dimensional gel electrophoresis and a radioimmunoassay. Our results indicate that tubulin is not a significant component of chromaffin granules. However, binding experiments show that granule membranes are able to bind tubulin through high affinity binding sites. These results show that microtubules appear involved in neurite formation and probably in granule transport. Tubulin is not an integral constituent of the granule membrane, but is present as a result of a reversible specific binding. This insertion of tubulin into the membrane might represent a step in the association between microtubules and secretory granules.

scholarly journals The ER v-SNAREs are required for GPI-anchored protein sorting from other secretory proteins upon exit from the ER

2003 ◽  
Vol 162 (3) ◽  
pp. 403-412 ◽  
Author(s):  
Pierre Morsomme ◽  
Cristina Prescianotto-Baschong ◽  
Howard Riezman
Keyword(s):  
Golgi Complex ◽  
Protein Sorting ◽  
Secretory Proteins ◽  
Rab Gtpase ◽  
Cargo Protein ◽  
Conserved Oligomeric Golgi Complex ◽  
Sorting Process ◽  
Conserved Oligomeric Golgi

Glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event requires the Rab GTPase Ypt1p, tethering factors Uso1p, and the conserved oligomeric Golgi complex. Here we show that proper sorting depended on the vSNAREs, Bos1p, Bet1p, and Sec22p. However, the t-SNARE Sed5p was not required for protein sorting upon ER exit. Moreover, the sorting defect observed in vitro with bos1–1 extracts was also observed in vivo and was visualized by EM. Finally, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in a bos1–1 mutant at semirestrictive temperature. Therefore, we propose that v-SNAREs are part of the cargo protein sorting machinery upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins.

scholarly journals Dynamic tracking and identification of tissue-specific secretory proteins in the circulation of live mice

2020 ◽  
Author(s):  
Jae Myoung Suh ◽  
Kwang-eun Kim ◽  
Isaac Park ◽  
Jeesoo Kim ◽  
Myeong-Gyun Kang ◽  
...  
Keyword(s):  
Blood Plasma ◽  
Mouse Liver ◽  
Secretory Pathway ◽  
Secretory Protein ◽  
Protein Labeling ◽  
Secretory Proteins ◽  
Tissue Specific ◽  
Dynamic Tracking

Abstract Here we describe iSLET (in situ Secretory protein Labeling via ER-anchored TurboID) which labels secretory pathway proteins as they transit through the ER-lumen to enable dynamic tracking of tissue-specific secreted proteomes in vivo. We expressed iSLET in the mouse liver and demonstrated efficient in situ labeling of the liver-specific secreted proteome which could be tracked and identified within circulating blood plasma. iSLET is a versatile and powerful tool for studying spatiotemporal dynamics of secretory proteins, a valuable class of biomarkers and therapeutic targets.

scholarly journals Mesenchymal stem cells versus their extracellular vesicles in treatment of liver fibrosis: Is it possible to compare?

2021 ◽  
Vol 9 (2) ◽  
Author(s):  
Noha Attia ◽  
Yasmine Khalifa ◽  
Dina Rostom ◽  
Mohamed Mashal
Keyword(s):  
Liver Fibrosis ◽  
Extracellular Vesicles ◽  
Therapeutic Strategy ◽  
Secretory Proteins ◽  
Apoptotic Bodies ◽  
Limited Evidence ◽  
In Vivo Models ◽  
Dose Determination

Liver fibrosis (LF) is a worldwide health problem that is associated with a range of complications and high mortality. Due to the scarcity of liver donors, mesenchymal stem cell (MSC) therapy emerged as an alternative therapeutic strategy. However, it is widely accepted that most of the transplanted MSCs exhibit their therapeutic impact mainly via a bystander paracrine (medicinal) capacity. In addition to their secretory proteins, MSCs also produce various types of extracellular vesicles (EVs) that are classified into three main subtypes: microvesicles, exosomes and apoptotic bodies. Thanks to their peculiar cargo composition (e.g., proteins, lipids, and nucleic acids), EVs serve as an advantageous candidate for cell-free therapy. Recently, MSC-derived EVs (MSC-EVs) have gained the podium due to their regenerative and immunomodulatory effect. In mitigation/treatment of LF, a plethora of recent studies have shown the anti-inflammatory, anti-fibrotic and cytoprotective effects of both MSCs and MSC-EVs in various in vitro and in vivo models of LF. However, despite the limited evidence, we sought in this mini review to sort out the established data and formulate several challenging questions that must be answered to pave the way for further clinical applications. One of the major questions to ask is “Which is the best therapeutic approach, MSCs or MSC-EVs?” We tried to highlight how difficult it might be to compare the two approaches while our understanding of both candidates is still deficient. Among the major obstacles against such comparison is the inaccurate equivalent dose determination, the unknown in vivo behavior, and the undetermined lifespan/fate of each. Currently, the fields of MSCs and MSC-EVs seem to be rich in ideas but lacking in appropriate technologies to test these ideas. Nevertheless, continuous efforts are likely to help resolve some of the challenges listed here.

scholarly journals Proteomic Profiling of Mammalian COPII Vesicles

2018 ◽  
Author(s):  
Frank Adolf ◽  
Manuel Rhiel ◽  
Bernd Hessling ◽  
Andrea Hellwig ◽  
Felix T. Wieland
Keyword(s):  
Intracellular Transport ◽  
Spectrometric Analysis ◽  
Secretory Proteins ◽  
Proteomic Profiling ◽  
Vesicular Carriers ◽  
Copii Vesicle ◽  
Cargo Proteins ◽  
Core Proteome ◽  
Copii Vesicles

AbstractIntracellular transport and homeostasis of the endomembrane system in eukaryotic cells depend on formation and fusion of vesicular carriers. COPII vesicles export newly synthesized secretory proteins from the endoplasmic reticulum (ER). They are formed by sequential recruitment of the small GTP binding protein Sar1, the inner coat complex Sec23/24, and the outer coat complex Sec13/31. In order to investigate the roles of mammalian Sec24 isoforms in cargo sorting, we have combined in vitro COPII vesicle reconstitutions with SILAC-based mass spectrometric analysis. This approach enabled us to identify the core proteome of mammalian COPII vesicles. Comparison of the proteomes generated from vesicles with different Sec24 isoforms confirms several established isoform-dependent cargo proteins, and identifies ERGIC1 and CNIH1 as novel Sec24C‐ and Sec24A-specific cargo proteins, respectively. Proteomic analysis of vesicles reconstituted with a Sec24C mutant, bearing a compromised binding site for the ER-to-Golgi QSNARE Syntaxin5, revealed that the SM/Munc18 protein SCFD1 binds to Syntaxin5 prior to its sorting into COPII vesicles. Furthermore, analysis of Sec24D mutants implicated in the development of a syndromic form of osteogenesis imperfecta showed sorting defects for the three ER-to-Golgi QSNAREs Syntaxin5, GS27, and Bet1.

scholarly journals In vivo cell penetration and intracellular transport of anti-Sm and anti-La autoantibodies

2000 ◽  
Vol 12 (4) ◽  
pp. 415-423 ◽  
Author(s):  
Sophie X. Deng ◽  
Elaine Hanson ◽  
Iñaki Sanz
Keyword(s):  
Intracellular Transport ◽  
Cell Penetration

Internalization and intracellular transport of folate-binding protein in rat kidney proximal tubule

1993 ◽  
Vol 264 (2) ◽  
pp. C302-C310 ◽  
Author(s):  
H. Birn ◽  
J. Selhub ◽  
E. I. Christensen
Keyword(s):  
Plasma Membrane ◽  
Proximal Tubule ◽  
Brush Border ◽  
Intracellular Transport ◽  
Brush Border Membrane ◽  
Specific Binding ◽  
Binding Protein ◽  
Rat Kidney ◽  
Folate Binding Protein

Folate-binding protein (FBP) is involved in folate reabsorption in the renal proximal tubule. Immunocytochemical studies have located FBP to the brush-border membrane, endocytic vacuoles, and dense apical tubules. We applied the same polyclonal antibody (anti-FBP) against FBP to investigate the dynamic relationship between FBP in the different compartments by microinjecting the antibody into rat kidney proximal tubules in situ. Specific binding of anti-FBP in vivo to the brush-border membrane was followed by fixation at various times. Protein A-gold labeling shows that anti-FBP is transported from endocytic invaginations into vacuoles followed by transport into dense apical tubules within 15 s. Thus FBP is rapidly internalized, and together with previous studies this study strongly suggests recycling of FBP back to the luminal plasma membrane through dense apical tubules. The results are consistent with reabsorption of folate through endocytosis of the FBP-folate complex followed by dissociation and recycling of FBP. When time is allowed there is a steady accumulation of FBP in dense apical tubules combined with an increase in surface density of the same compartment. A possible explanation involves partial inhibition of the fusion between dense apical tubules and plasma membrane because of the anti-FBP labeling of the receptor.

Proteins are secreted from heterogeneous prestored sources in the exocrine pancreas

1987 ◽  
Vol 252 (6) ◽  
pp. G768-G775
Author(s):  
P. E. Miller ◽  
J. W. Adelson
Keyword(s):  
Digestive Enzymes ◽  
Exocrine Pancreas ◽  
Secretory Proteins ◽  
Pancreatic Ducts ◽  
Regulated Secretion ◽  
Correlation And Regression Analysis ◽  
Label Method ◽  
A Cell ◽  
Partial Depletion

Recent studies demonstrating nonparallel regulated secretion of prestored digestive enzymes in tightly linked groups consistent with the exocytosis mechanism led us to predict that digestive enzymes would be found to be secreted from heterogeneous sources within the exocrine pancreas (J. W. Adelson, and P.E. Miller, Science Wash. DC 228: 993-996, 1985). We explored whether the gland was heterogeneous with respect to its sources of prestored secretory proteins with a double isotopic label method not dependent on activity of secreted digestive enzymes. Rabbit pancreatic proteins were double labeled in vivo by injection of each animal with chemically identical but isotopically distinct mixtures of 3H- and 14C-labeled amino acids, which were administered separately or together on consecutive days after partial depletion of prestored proteins by administration of cholecystokinin (CCK), methacholine chloride, or saline in a protocol in which order of both isotope and secretagogue administration was varied. Three days after labeling, proteins were recovered by collection from cannulated pancreatic ducts of anesthetized animals after stimulation with alternating increasing doses of CCK and methacholine chloride. Pooled secretory data were analyzed to determine whether secretagogue pretreatment resulted in specific and heterogeneous sequestration of proteins after synthesis; data after final secretory stimulation with methacholine chloride and CCK were individually analyzed to determine whether presequestered proteins were mobilized from heterogeneous compartments during secretion. Correlation and regression analysis of isotopic outputs and variance analysis of specific radioactivities of secreted proteins showed sequestration into and secretion from heterogeneous pools of secretory proteins, directly confirming out hypothesis. These results provide a cell biological mechanism explaining regulated nonparallel secretion of digestive enzymes.

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