scholarly journals Valosin-containing Protein (p97) Is a Regulator of Endoplasmic Reticulum Stress and of the Degradation of N-End Rule and Ubiquitin-Fusion Degradation Pathway Substrates in Mammalian Cells

2006 ◽  
Vol 17 (11) ◽  
pp. 4606-4618 ◽  
Author(s):  
Cezary Wójcik ◽  
Maga Rowicka ◽  
Andrzej Kudlicki ◽  
Dominika Nowis ◽  
Elizabeth McConnell ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Cell Receptor ◽  
Degradation Pathway ◽  
Detectable Effect ◽  
Independent Manner ◽  
Green Fluorescent ◽  
The Unfolded Protein Response ◽  
And Function

Valosin-containing protein (VCP; p97; cdc48 in yeast) is a hexameric ATPase of the AAA family (ATPases with multiple cellular activities) involved in multiple cellular functions, including degradation of proteins by the ubiquitin (Ub)–proteasome system (UPS). We examined the consequences of the reduction of VCP levels after RNA interference (RNAi) of VCP. A new stringent method of microarray analysis demonstrated that only four transcripts were nonspecifically affected by RNAi, whereas ∼30 transcripts were affected in response to reduced VCP levels in a sequence-independent manner. These transcripts encoded proteins involved in endoplasmic reticulum (ER) stress, apoptosis, and amino acid starvation. RNAi of VCP promoted the unfolded protein response, without eliciting a cytosolic stress response. RNAi of VCP inhibited the degradation of R-GFP (green fluorescent protein) and Ub-G76V-GFP, two cytoplasmic reporter proteins degraded by the UPS, and of α chain of the T-cell receptor, an established substrate of the ER-associated degradation (ERAD) pathway. Surprisingly, RNAi of VCP had no detectable effect on the degradation of two other ERAD substrates, α1-antitrypsin and δCD3. These results indicate that VCP is required for maintenance of normal ER structure and function and mediates the degradation of some proteins via the UPS, but is dispensable for the UPS-dependent degradation of some ERAD substrates.

scholarly journals A Novel Dynamin-like Protein Associates with Cytoplasmic Vesicles and Tubules of the Endoplasmic Reticulum in Mammalian Cells

1998 ◽  
Vol 140 (4) ◽  
pp. 779-793 ◽  
Author(s):  
Yisang Yoon ◽  
Kelly R. Pitts ◽  
Sophie Dahan ◽  
Mark A. McNiven
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Double Labeling ◽  
Subcellular Membrane ◽  
Morphological Studies ◽  
Cytoplasmic Vesicles ◽  
Membrane Compartment ◽  
Green Fluorescent

Abstract. Dynamins are 100-kilodalton guanosine triphosphatases that participate in the formation of nascent vesicles during endocytosis. Here, we have tested if novel dynamin-like proteins are expressed in mammalian cells to support vesicle trafficking processes at cytoplasmic sites distinct from the plasma membrane. Immunological and molecular biological methods were used to isolate a cDNA clone encoding an 80-kilodalton novel dynamin-like protein, DLP1, that shares up to 42% homology with other dynamin-related proteins. DLP1 is expressed in all tissues examined and contains two alternatively spliced regions that are differentially expressed in a tissue-specific manner. DLP1 is enriched in subcellular membrane fractions of cytoplasmic vesicles and endoplasmic reticulum. Morphological studies of DLP1 in cultured cells using either a specific antibody or an expressed green fluorescent protein (GFP)- DLP1 fusion protein revealed that DLP1 associates with punctate cytoplasmic vesicles that do not colocalize with conventional dynamin, clathrin, or endocytic ligands. Remarkably, DLP1-positive structures coalign with microtubules and, most strikingly, with endoplasmic reticulum tubules as verified by double labeling with antibodies to calnexin and Rab1 as well as by immunoelectron microscopy. These observations provide the first evidence that a novel dynamin-like protein is expressed in mammalian cells where it associates with a secretory, rather than endocytic membrane compartment.

scholarly journals Nox4-Derived H2O2 Mediates Endoplasmic Reticulum Signaling through Local Ras Activation

2010 ◽  
Vol 30 (14) ◽  
pp. 3553-3568 ◽  
Author(s):  
Ru-Feng Wu ◽  
Zhenyi Ma ◽  
Zhe Liu ◽  
Lance S. Terada
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluorescent Protein ◽  
Cell Fractionation ◽  
Protective Mechanisms ◽  
3 Dimensional ◽  
Binding Domains ◽  
Pathway Activation ◽  
Oxidant Production ◽  
Green Fluorescent ◽  
The Unfolded Protein Response

ABSTRACT The unfolded-protein response (UPR) of the endoplasmic reticulum (ER) has been linked to oxidant production, although the molecular details and functional significance of this linkage are poorly understood. Using a ratiometric H2O2 sensor targeted to different subcellular compartments, we demonstrate specific production of H2O2 by the ER in response to the stressors tunicamycin and HIV-1 Tat, but not to thapsigargin or dithiothreitol. Knockdown of the oxidase Nox4, expressed on ER endomembranes, or expression of ER-targeted catalase blocked ER H2O2 production by tunicamycin and Tat and prevented the UPR following exposure to these two agonists, but not to thapsigargin or dithiothreitol. Tat also triggered Nox4-dependent, sustained activation of Ras leading to ERK, but not phosphatidylinositol 3-kinase (PI3K)/mTOR, pathway activation. Cell fractionation studies and green fluorescent protein (GFP) fusions of GTPase effector binding domains confirmed selective activation of endogenous RhoA and Ras on the ER surface, with ER-associated K-Ras acting upstream of the UPR and downstream of Nox4. Notably, the Nox4/Ras/ERK pathway induced autophagy, and suppression of autophagy unmasked cell death and prevented differentiation of endothelial cells in 3-dimensional matrix. We conclude that the ER surface provides a platform to spatially organize agonist-specific Nox4-dependent oxidative signaling events, leading to homeostatic protective mechanisms rather than oxidative stress.

scholarly journals Kar2p availability defines distinct forms of endoplasmic reticulum stress in living cells

2012 ◽  
Vol 23 (5) ◽  
pp. 955-964 ◽  
Author(s):  
Patrick Lajoie ◽  
Robyn D. Moir ◽  
Ian M. Willis ◽  
Erik L. Snapp
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluorescent Protein ◽  
Secretory Protein ◽  
Living Cells ◽  
Secretory Proteins ◽  
High Throughput Analysis ◽  
Protein Levels ◽  
Unfolded Protein ◽  
Green Fluorescent ◽  
The Unfolded Protein Response

Accumulation of misfolded secretory proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) stress pathway. To enhance secretory protein folding and promote adaptation to stress, the UPR upregulates ER chaperone levels, including BiP. Here we describe chromosomal tagging of KAR2, the yeast homologue of BiP, with superfolder green fluorescent protein (sfGFP) to create a multifunctional endogenous reporter of the ER folding environment. Changes in Kar2p-sfGFP fluorescence levels directly correlate with UPR activity and represent a robust reporter for high-throughput analysis. A novel second feature of this reporter is that photobleaching microscopy (fluorescence recovery after photobleaching) of Kar2p-sfGFP mobility reports on the levels of unfolded secretory proteins in individual cells, independent of UPR status. Kar2p-sfGFP mobility decreases upon treatment with tunicamycin or dithiothreitol, consistent with increased levels of unfolded proteins and the incorporation of Kar2p-sfGFP into slower-diffusing complexes. During adaptation, we observe a significant lag between down-regulation of the UPR and resolution of the unfolded protein burden. Finally, we find that Kar2p-sfGFP mobility significantly increases upon inositol withdrawal, which also activates the UPR, apparently independent of unfolded protein levels. Thus Kar2p mobility represents a powerful new tool capable of distinguishing between the different mechanisms leading to UPR activation in living cells.

scholarly journals The Dynamin-like Protein DLP1 Is Essential for Normal Distribution and Morphology of the Endoplasmic Reticulum and Mitochondria in Mammalian Cells

1999 ◽  
Vol 10 (12) ◽  
pp. 4403-4417 ◽  
Author(s):  
K.R. Pitts ◽  
Y. Yoon ◽  
E.W. Krueger ◽  
M.A. McNiven
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Mitochondrial Morphology ◽  
Intimate Contact ◽  
Mammalian Tissues ◽  
Membrane Compartments ◽  
Green Fluorescent ◽  
Large Gtpases

The dynamin family of large GTPases has been implicated in vesicle formation from both the plasma membrane and various intracellular membrane compartments. The dynamin-like protein DLP1, recently identified in mammalian tissues, has been shown to be more closely related to the yeast dynamin proteins Vps1p and Dnm1p (42%) than to the mammalian dynamins (37%). Furthermore, DLP1 has been shown to associate with punctate vesicles that are in intimate contact with microtubules and the endoplasmic reticulum (ER) in mammalian cells. To define the function of DLP1, we have transiently expressed both wild-type and two mutant DLP1 proteins, tagged with green fluorescent protein, in cultured mammalian cells. Point mutations in the GTP-binding domain of DLP1 (K38A and D231N) dramatically changed its intracellular distribution from punctate vesicular structures to either an aggregated or a diffuse pattern. Strikingly, cells expressing DLP1 mutants or microinjected with DLP1 antibodies showed a marked reduction in ER fluorescence and a significant aggregation and tubulation of mitochondria by immunofluorescence microscopy. Consistent with these observations, electron microscopy of DLP1 mutant cells revealed a striking and quantitative change in the distribution and morphology of mitochondria and the ER. These data support very recent studies by other authors implicating DLP1 in the maintenance of mitochondrial morphology in both yeast and mammalian cells. Furthermore, this study provides the first evidence that a dynamin family member participates in the maintenance and distribution of the ER. How DLP1 might participate in the biogenesis of two presumably distinct organelle systems is discussed.

scholarly journals Macromolecular Uptake Is a Spontaneous Event during Mitosis in Cultured Fibroblasts: Implications for Vector-dependent Plasmid Transfection

2002 ◽  
Vol 13 (2) ◽  
pp. 570-578 ◽  
Author(s):  
Pierre Pellegrin ◽  
Anne Fernandez ◽  
Ned J. C. Lamb ◽  
René Bennes
Keyword(s):  
Plasmid Dna ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Fluorescein Isothiocyanate ◽  
Dna Encoding ◽  
Independent Manner ◽  
Cultured Fibroblasts ◽  
Amino Acid Peptide ◽  
Green Fluorescent ◽  
Mitotic Cells

The process through which macromolecules penetrate the plasma membrane of mammalian cells remains poorly defined. We have examined whether natural cellular events modulate the capacity of cells to take up agents applied extraneously. Herein, we report that during mitosis and in a cell type-independent manner, cells exhibit a natural ability to absorb agents present in the extracellular environment up to 150 kDa as assessed using fluorescein isothiocyanate-dextrans. This event is exclusive to the mitotic period and not observed during G0, G1, S, or G2 phase. During mitosis, starting in advanced prophase, oligonucleotides, active enzymes, and polypeptides are efficiently taken into mitotic cells. This uptake of macromolecules during mitosis still takes place in the presence of cytochalasin D or nocodazole, showing no requirement for intact microtubules or actin filaments in this process. However, cell rounding up, which still takes place in the presence of either of these drugs in mitotic cells, appears to be a key event in this process. Indeed, limited trypsinization of adherent cells mimics both the cell retraction and macromolecule uptake observed as cells enter mitosis. A plasmid DNA encoding green fluorescent protein (3.3Mda) coated with an 18 amino acid peptide is efficiently expressed when applied onto synchronized G2/M fibroblasts, whereas little or no expression is observed when the coated plasmid is applied onto asynchronous cell cultures. This shows that such coating peptides are only efficient for their encapsulating and protective effect on the plasmid DNA to be “vectorized” rather than acting as true vectors.

scholarly journals TCRα reporter mice reveal contribution of dual TCRα expression to T cell repertoire and function

2020 ◽  
Vol 117 (51) ◽  
pp. 32574-32583
Author(s):  
Letitia Yang ◽  
Burhan Jama ◽  
Huawei Wang ◽  
Lara Labarta-Bajo ◽  
Elina I. Zúñiga ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fluorescent Protein ◽  
Cell Receptor ◽  
Lymphocytic Choriomeningitis ◽  
Receptor Expression ◽  
Immune Memory ◽  
Cell Repertoire ◽  
Green Fluorescent ◽  
And Function

It is known that a subpopulation of T cells expresses two T cell receptor (TCR) clonotypes, though the extent and functional significance of this is not established. To definitively evaluate dual TCRα cells, we generated mice with green fluorescent protein and red fluorescent protein reporters linked to TCRα, revealing that ∼16% of T cells express dual TCRs, notably higher than prior estimates. Importantly, dual TCR expression has functional consequences, as dual TCR cells predominated response to lymphocytic choriomeningitis virus infection, comprising up to 60% of virus-specific CD4+and CD8+T cells during acute responses. Dual receptor expression selectively influenced immune memory, as postinfection memory CD4+populations contained significantly increased frequencies of dual TCR cells. These data reveal a previously unappreciated contribution of dual TCR cells to the immune repertoire and highlight their potential effects on immune responses.

Organization of transport from endoplasmic reticulum to Golgi in higher plants

2000 ◽  
Vol 28 (4) ◽  
pp. 505-512 ◽  
Author(s):  
A. V. Andreeva ◽  
H. Zheng ◽  
C. M. Saint-Jore ◽  
M. A. Kutuzov ◽  
D. E. Evans ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Higher Plants ◽  
Plant Cells ◽  
Yeast Cells ◽  
Green Fluorescent

In plant cells, the organization of the Golgi apparatus and its interrelationships with the endoplasmic reticulum differ from those in mammalian and yeast cells. Endoplasmic reticulum and Golgi apparatus can now be visualized in plant cells in vivo with green fluorescent protein (GFP) specifically directed to these compartments. This makes it possible to study the dynamics of the membrane transport between these two organelles in the living cells. The GFP approach, in conjunction with a considerable volume of data about proteins participating in the transport between endoplasmic reticulum and Golgi in yeast and mammalian cells and the identification of their putative plant homologues, should allow the establishment of an experimental model in which to test the involvement of the candidate proteins in plants. As a first step towards the development of such a system, we are using Sar1, a small G-protein necessary for vesicle budding from the endoplasmic reticulum. This work has demonstrated that the introduction of Sar1 mutants blocks the transport from endoplasmic reticulum to Golgi in vivo in tobacco leaf epidermal cells and has therefore confirmed the feasibility of this approach to test the function of other proteins that are presumably involved in this step of endo-membrane trafficking in plant cells.

scholarly journals Insulin targeting to the regulated secretory pathway after fusion with green fluorescent protein and firefly luciferase

1998 ◽  
Vol 331 (2) ◽  
pp. 669-675 ◽  
Author(s):  
Aristea E. POULI ◽  
Helen J. KENNEDY ◽  
J. George SCHOFIELD ◽  
Guy A. RUTTER
Keyword(s):  
Endoplasmic Reticulum ◽  
Green Fluorescent Protein ◽  
Laser Scanning ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Firefly Luciferase ◽  
Laser Scanning Confocal Microscopy ◽  
Detectable Effect ◽  
Scanning Confocal Microscopy ◽  
Green Fluorescent

We have prepared recombinant cDNAs encoding chimaeras between human preproinsulin (sp.B.C.A., for B-, Connecting- and A-peptides) and a thermostable mutant of green fluorescent protein (GFPS65T, V163A, GFP*). The subcellular localization of the expressed chimaeras was monitored in living insulin-secreting INS-1 β-cells by laser scanning confocal microscopy. When GFP* was fused at the immediate N-terminus of the B-chain (sp.[GFP*].B.C.A.myc) two distinct patterns of fluorescence were apparent. In 1530/1740 cells examined, fluorescence was confined to a reticular, exclusively extranuclear structure, and closely co-localized with the endoplasmic reticulum marker, calreticulin. However, 210/1740 (12.1%) of cells displayed punctate fluorescence, which partially co-localized with the trans-Golgi network marker, TGN 38, and with the dense core secretory granule marker, phogrin. Since secretion of GFP* fluorescence into the medium could not readily be measured, we prepared a chimaera in which firefly luciferase was fused at the C-terminus of proinsulin (sp.B.C.A.myc.[Luc]). This chimaera displayed a distribution closely similar to that of sp.[GFP*].B.C.A.myc, but with a lower proportion (15/310, 4.8%) of the cells showing clear punctate distribution. At substimulatory glucose concentrations (3 mM) secretion of sp.B.C.A.myc.[Luc] could not be detected (rate of release into the medium identical with that of the cytosolic Renilla reniformis luciferase), indicating that the chimaera did not enter the constitutive secretory pathway. However, elevated (30 mM) glucose stimulated the release of the sp.B.C.A.myc.[Luc] luciferase chimaera, without a detectable effect on R. reniformis luciferase release. These data suggest that fusion of insulin, and the much larger photoproteins GFP* and luciferase, leads predominantly to misfolding and retention in the endoplasmic reticulum. However, the properly folded chimaeras are apparently still correctly targeted to the regulated, rather than the constitutive, secretory pathway. These chimaeras should therefore be valuable tools to monitor the exocytosis of insulin in real time.

scholarly journals Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

2021 ◽  
Author(s):  
Noemi Ruiz-Lopez ◽  
Jessica Pérez-Sancho ◽  
Alicia Esteban del Valle ◽  
Richard P Haslam ◽  
Steffen Vanneste ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Abiotic Stress ◽  
Fluorescent Protein ◽  
Mechanical Damage ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

scholarly journals Rtn1p Is Involved in Structuring the Cortical Endoplasmic Reticulum

2006 ◽  
Vol 17 (7) ◽  
pp. 3009-3020 ◽  
Author(s):  
Johan-Owen De Craene ◽  
Jeff Coleman ◽  
Paula Estrada de Martin ◽  
Marc Pypaert ◽  
Scott Anderson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Cell Cortex ◽  
Proteomic Screen ◽  
Wide Range ◽  
Membrane Spanning ◽  
Green Fluorescent ◽  
Hydrophilic Loop ◽  
Yeast Mutants

The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Δ in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Δ cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.

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