Brefeldin A Affects Growth, Endoplasmic Reticulum, Golgi Bodies, Tubular Vacuole System, and Secretory Pathway in Pisolithus tinctorius

2000 ◽  
Vol 29 (2) ◽  
pp. 95-106 ◽  
Author(s):  
Louise Cole ◽  
Danielle Davies ◽  
Geoffrey J Hyde ◽  
Anne E Ashford
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Pisolithus Tinctorius ◽  
Golgi Bodies

Leptin biosynthetic pathway in white adipocytes

2006 ◽  
Vol 84 (2) ◽  
pp. 207-214 ◽  
Author(s):  
Philippe G Cammisotto ◽  
Ludwik J Bukowiecki ◽  
Yves Deshaies ◽  
Moise Bendayan
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
De Novo ◽  
Brefeldin A ◽  
Mrna Levels ◽  
De Novo Synthesis ◽  
Cellular Content ◽  
Constitutive Secretion

The aim of this study was to determine through morphological and biochemical means the biosynthetic and secretory pathway followed by leptin in adipocytes. Immunocytochemistry revealed the presence of leptin in the rough endoplasmic reticulum, the Golgi apparatus, and in numerous small vesicles along the plasma membrane of white adipo cytes. In vitro, isolated adipocytes under nonstimulated conditions (basal) continuously secreted leptin while their intra cellular content remained unchanged. When adipocytes were stimulated with insulin, leptin cellular content and secretion increased in parallel and were significantly different from basal secretion only after 45 min. L-leucine and L-glutamate also strongly stimulated leptin synthesis and secretion. These stimulating effects were abolished by cycloheximide and brefeldin A. The transcriptional inhibitor actinomycin D did not have any effects in either basal or stimulated conditions. Leptin mRNA levels were not affected by any stimulating or inhibiting agents. Finally, norepinephrine, isoproterenol, CL316243, and palmitate inhibited the effects of insulin, L-leucine, and L-glutamate on leptin synthesis. We thus conclude that (i) adipocytes continuously synthesize and secrete leptin along a rough endoplasmic reticulum–Golgi secretory vesicles pathway, (ii) an increase in leptin secretion requires increased de novo synthesis, and (iii) short-term leptin secretion does not involve changes in mRNA levels.Key words: leptin, vesicles, constitutive secretion, de novo synthesis, transcription.

scholarly journals The Cargo Receptors Surf4, Endoplasmic Reticulum-Golgi Intermediate Compartment (ERGIC)-53, and p25 Are Required to Maintain the Architecture of ERGIC and Golgi

2008 ◽  
Vol 19 (5) ◽  
pp. 1976-1990 ◽  
Author(s):  
Sandra Mitrovic ◽  
Houchaima Ben-Tekaya ◽  
Eva Koegler ◽  
Jean Gruenberg ◽  
Hans-Peter Hauri
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Matrix Proteins ◽  
Anterograde Transport ◽  
Early Secretory Pathway ◽  
Partial Redistribution ◽  
Intermediate Compartment ◽  
Human Orthologue ◽  
Golgi Matrix

Rapidly cycling proteins of the early secretory pathway can operate as cargo receptors. Known cargo receptors are abundant proteins, but it remains mysterious why their inactivation leads to rather limited secretion phenotypes. Studies of Surf4, the human orthologue of the yeast cargo receptor Erv29p, now reveal a novel function of cargo receptors. Surf4 was found to interact with endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-53 and p24 proteins. Silencing Surf4 together with ERGIC-53 or silencing the p24 family member p25 induced an identical phenotype characterized by a reduced number of ERGIC clusters and fragmentation of the Golgi apparatus without effect on anterograde transport. Live imaging showed decreased stability of ERGIC clusters after knockdown of p25. Silencing of Surf4/ERGIC-53 or p25 resulted in partial redistribution of coat protein (COP) I but not Golgi matrix proteins to the cytosol and partial resistance of the cis-Golgi to brefeldin A. These findings imply that cargo receptors are essential for maintaining the architecture of ERGIC and Golgi by controlling COP I recruitment.

scholarly journals A Novel Quality Control Compartment Derived from the Endoplasmic Reticulum

2001 ◽  
Vol 12 (6) ◽  
pp. 1711-1723 ◽  
Author(s):  
Shiri Kamhi-Nesher ◽  
Marina Shenkman ◽  
Sandra Tolchinsky ◽  
Sharon Vigodman Fromm ◽  
Rachel Ehrlich ◽  
...  
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Degradation Process ◽  
Transmembrane Conductance Regulator ◽  
Heavy Chains ◽  
Tightly Coupled ◽  
Ubiquitin Proteasome ◽  
Control Compartment

Degradation of proteins that, because of improper or suboptimal processing, are retained in the endoplasmic reticulum (ER) involves retrotranslocation to reach the cytosolic ubiquitin-proteasome machinery. We found that substrates of this pathway, the precursor of human asialoglycoprotein receptor H2a and free heavy chains of murine class I major histocompatibility complex (MHC), accumulate in a novel preGolgi compartment that is adjacent to but not overlapping with the centrosome, the Golgi complex, and the ER-to-Golgi intermediate compartment (ERGIC). On its way to degradation, H2a associated increasingly after synthesis with the ER translocon Sec61. Nevertheless, it remained in the secretory pathway upon proteasomal inhibition, suggesting that its retrotranslocation must be tightly coupled to the degradation process. In the presence of proteasomal inhibitors, the ER chaperones calreticulin and calnexin, but not BiP, PDI, or glycoprotein glucosyltransferase, concentrate in the subcellular region of the novel compartment. The “quality control” compartment is possibly a subcompartment of the ER. It depends on microtubules but is insensitive to brefeldin A. We discuss the possibility that it is also the site for concentration and retrotranslocation of proteins that, like the mutant cystic fibrosis transmembrane conductance regulator, are transported to the cytosol, where they form large aggregates, the “aggresomes.”

scholarly journals Assembly and Maturation of the Flavivirus Kunjin Virus Appear To Occur in the Rough Endoplasmic Reticulum and along the Secretory Pathway, Respectively

2001 ◽  
Vol 75 (22) ◽  
pp. 10787-10799 ◽  
Author(s):  
Jason M. Mackenzie ◽  
Edwin G. Westaway
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rough Endoplasmic Reticulum ◽  
Envelope Protein ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Immunogold Labeling ◽  
Virus Particles ◽  
Infected Cells ◽  
Assembly Site

ABSTRACT The intracellular assembly site for flaviviruses in currently not known but is presumed to be located within the lumen of the rough endoplasmic reticulum (RER). Building on previous studies involving immunofluorescence (IF) and cryoimmunoelectron microscopy of Kunjin virus (KUN)-infected cells, we sought to identify the steps involved in the assembly and maturation of KUN. Thus, using antibodies directed against envelope protein E in IF analysis, we found the accumulation of E within regions coincident with the RER and endosomal compartments. Immunogold labeling of cryosections of infected cells indicated that E and minor envelope protein prM were localized to reticulum membranes continuous with KUN-induced convoluted membranes (CM) or paracrystalline arrays (PC) and that sometimes the RER contained immunogold-labeled virus particles. Both proteins were also observed to be labeled in membranes at the periphery of the induced CM or PC structures, but the latter were very seldom labeled internally. Utilizing drugs that inhibit protein and/or membrane traffic throughout the cell, we found that the secretion of KUN particles late in infection was significantly affected in the presence of brefeldin A and that the infectivity of secreted particles was severely affected in the presence of monensin and N-nonyl-deoxynojirimycin. Nocodazole did not appear to affect maturation, suggesting that microtubules play no role in assembly or maturation processes. Subsequently, we showed that the exit of intact virions from the RER involves the transport of individual virions within individual vesicles en route to the Golgi apparatus. The results suggest that the assembly of virions occurs within the lumen of the RER and that subsequent maturation occurs via the secretory pathway.

scholarly journals Myosin driven Actin Filament Sliding is Responsible for Endoplasmic Reticulum and Golgi Movement

2018 ◽  
Author(s):  
Joseph F McKenna ◽  
Stephen E D Webb ◽  
Verena Kriechbaumer ◽  
Chris Hawes
Keyword(s):  
Endoplasmic Reticulum ◽  
Actin Cytoskeleton ◽  
Actin Filament ◽  
Secretory Pathway ◽  
Motor Proteins ◽  
In Planta ◽  
Cell Dynamics ◽  
Myosin Motor ◽  
Golgi Bodies ◽  
Early Secretory Pathway

AbstractThe plant secretory pathway is responsible for the production of the majority of proteins and carbohydrates consumed on the planet. The early secretory pathway is composed of Golgi bodies and the endoplasmic reticulum (ER) and is highly mobile in plants with rapid remodelling of the ER network. The dynamics of the ER and Golgi bodies is driven by the actin cytoskeleton and myosin motor proteins play a key role in this. However, exactly how myosin motor proteins drive remodelling in plants is currently a contentious issue. Here, using a combination of live cell microscopy and over-expression of non-functional myosins we demonstrate that myosin motor proteins drive actin filament sliding and subsequently the dynamics of the secretory pathway.SummaryIn plants, the actin cytoskeleton and myosins are fundamental for normal dynamics of the endomembrane system and cytoplasmic streaming. We demonstrate that this is in part due to myosin driven sliding of actin filaments within a bundle. This generates, at least in part, the motive force required for cell dynamics in planta.

scholarly journals Cytokinin fluoroprobe and receptor CRE1/AHK4 localize to both plasma membrane and endoplasmic reticulum

2019 ◽  
Author(s):  
Karolina Kubiasová ◽  
Juan Carlos Montesinos ◽  
Olga Šamajová ◽  
Jaroslav Nisler ◽  
Václav Mik ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Cell Plate ◽  
Root Apical Meristem ◽  
Transcriptional Responses ◽  
Lateral Organs ◽  
Gfp Reporter ◽  
New Perspective

The plant hormone cytokinin regulates various cell and developmental processes, including cell division and differentiation, embryogenesis, activity of shoot and root apical meristems, formation of shoot and root lateral organs and others 1. Cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Based on the subcellular localization of cytokinin receptors in various transient expression systems, such as tobacco leaf epidermal cells, and membrane fractionation experiments of Arabidopsis and maize, the endoplasmic reticulum (ER) membrane has been proposed as a principal hormone perception site 2–4. Intriguingly, recent study of the cytokinin transporter PUP14 has pointed out that the plasma membrane (PM)-mediated signalling might play an important role in establishment of cytokinin response gradients in various plant organs 5. However, localization of cytokinin HK receptors to the PM, although initially suggested 6, remains ambiguous. Here, by monitoring subcellular localizations of the fluorescently labelled natural cytokinin probe iP-NBD 7 and the cytokinin receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin fluoroprobes and receptors can enter the secretory pathway and reach the PM. We demonstrate that in cells of the root apical meristem, CRE1/AHK4 localizes to the PM and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. Our results provide a new perspective on cytokinin signalling and the possibility of multiple sites of perception at PM and ER, which may determine specific outputs of cytokinin signalling.

Ultrastructural changes in the rough endoplasmic reticulum and its contents following Brefeldin A treatment of human chondrocytes in vitro

1991 ◽  
Vol 49 ◽  
pp. 256-257
Author(s):  
H. E. Gruber
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Brefeldin A ◽  
Ultrastructural Level ◽  
Human Chondrocytes ◽  
Ultrastructural Changes ◽  
Ultrastructural Studies

The rough endoplasmic reticulum (rER) is now recognized as a major organelle responsible for ensuring that only structurally correct and properly folded proteins are allowed to enter the cellular secretory pathway. We are especially interested in the behavior of the chondrocyte rER since ultrastructural studies of many skeletal dysplasias have revealed that electron dense material accumulates or is not degraded within the rER of chondrocytes from patients. Remodelling of the rER in chick chondrocytes has also been evaluated at the ultrastructural level and the rER found to play a role in procollagen export from the cell. We have utilized normal human chondrocytes grown in culture to investigate the role of brefeldin A, an antiviral antibiotic, which has been shown to primarily block protein transport from the ER to the Golgi complex.

scholarly journals The chondrodystrophy, nanomelia: biosynthesis and processing of the defective aggrecan precursor

1994 ◽  
Vol 301 (1) ◽  
pp. 211-216 ◽  
Author(s):  
B M Vertel ◽  
B L Grier ◽  
H Li ◽  
N B Schwartz
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Basis ◽  
Secretory Pathway ◽  
Chondroitin Sulphate ◽  
Genetic Diseases ◽  
Brefeldin A ◽  
Chondroitin Sulphate Proteoglycan ◽  
Skeletal Defects ◽  
Membrane Compartment ◽  
Do So

The lethal chicken mutation nanomelia leads to severe skeletal defects because of a deficiency of aggrecan, which is the largest aggregating chondroitin sulphate proteoglycan of cartilage. In previous work, we have demonstrated that nanomelic chondrocytes produce a truncated aggrecan precursor that fails to be secreted, and is apparently arrested in the endoplasmic reticulum (ER). In this study, we investigated the biosynthesis and extent of processing of the abnormal aggrecan precursor. The truncated precursor was translated directly in cell-free reactions, indicating that it does not arise post-translationally. Further studies addressed the processing capabilities of the defective precursor. We found that the mutant precursor was modified by N-linked, mannose-rich oligosaccharides and by the addition of xylose, but was not further processed; this is consistent with the conclusion that it moves no further along the secretory pathway than the ER. Using brefeldin A we demonstrated that the defective precursor can function as a substrate for Golgi-mediated glycosaminoglycan chains, but does not do so in the nanomelic chondrocyte because it fails to be translocated to the appropriate membrane compartment. These studies illustrate how combined cell biological/biochemical and molecular investigations may contribute to our understanding of the biological consequences and molecular basis of genetic diseases, particularly those involving errors in large, highly modified molecules such as proteoglycans.

scholarly journals Chitinase Secretion by Encysting Entamoeba invadensand Transfected Entamoeba histolytica Trophozoites: Localization of Secretory Vesicles, Endoplasmic Reticulum, and Golgi Apparatus

1999 ◽  
Vol 67 (6) ◽  
pp. 3073-3081 ◽  
Author(s):  
Sudip K. Ghosh ◽  
Jessica Field ◽  
Marta Frisardi ◽  
Benjamin Rosenthal ◽  
Zhiming Mai ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Entamoeba Histolytica ◽  
Secretory Pathway ◽  
Signal Sequence ◽  
Gene Promoter ◽  
Brefeldin A ◽  
Host Cells ◽  
Secretory Vesicles ◽  
Fluorescent Substrate

ABSTRACT Entamoeba histolytica, the protozoan parasite that phagocytoses bacteria and host cells, has a vesicle/vacuole-filled cytosol like that of macrophages. In contrast, the infectious cyst form has four nuclei and a chitin wall. Here, anti-chitinase antibodies identified hundreds of small secretory vesicles in encysting E. invadens parasites and in E. histolytica trophozoites overexpressing chitinase under an actin gene promoter. Abundant small secretory vesicles were also identified with antibodies to the surface antigen Ariel and with a fluorescent substrate of cysteine proteinases. Removal of an N-terminal signal sequence directed chitinase to the cytosol. Addition of a C-terminal KDEL peptide, identified on amebic BiP, retained chitinase in a putative endoplasmic reticulum, which was composed of a few vesicles of mixed sizes. A putative Golgi apparatus, which was Brefeldin A sensitive and composed of a few large, perinuclear vesicles, was identified with antibodies to ADP-ribosylating factor and to ɛ-COP. We conclude that the amebic secretory pathway is similar to those of other eukaryotic cells, even if its appearance is somewhat different.

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