scholarly journals GRASP55 restricts early-stage autophagy and regulates spatial organization of the early secretory network

Biology Open ◽  
2021 ◽  
Vol 10 (10) ◽  
Author(s):  
Jennifer Y. Liu ◽  
Yu-Hsiu Tony Lin ◽  
Andrew M. Leidal ◽  
Hector H. Huang ◽  
Jordan Ye ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Spatial Organization ◽  
Early Stage ◽  
Prior Work ◽  
Autophagosome Formation ◽  
Cellular Mechanisms ◽  
Early Secretory Pathway ◽  
Stress Response Pathway ◽  
Lc3 Puncta ◽  
A Site

ABSTRACT There is great interest in understanding the cellular mechanisms controlling autophagy, a tightly regulated catabolic and stress-response pathway. Prior work has uncovered links between autophagy and the Golgi reassembly stacking protein of 55 kDa (GRASP55), but their precise interrelationship remains unclear. Intriguingly, both autophagy and GRASP55 have been functionally and spatially linked to the endoplasmic reticulum (ER)­­-Golgi interface, broaching this compartment as a site where GRASP55 and autophagy may intersect. Here, we uncover that loss of GRASP55 enhances LC3 puncta formation, indicating that GRASP55 restricts autophagosome formation. Additionally, using proximity-dependent biotinylation, we identify a GRASP55 proximal interactome highly associated with the ER-Golgi interface. Both nutrient starvation and loss of GRASP55 are associated with coalescence of early secretory pathway markers. In light of these findings, we propose that GRASP55 regulates spatial organization of the ER-Golgi interface, which suppresses early autophagosome formation.

scholarly journals Interorganelle communication and membrane shaping in the early secretory pathway

2021 ◽  
Vol 71 ◽  
pp. 95-102
Author(s):  
Pablo Lujan ◽  
Jessica Angulo-Capel ◽  
Morgan Chabanon ◽  
Felix Campelo
Keyword(s):  
Secretory Pathway ◽  
Early Secretory Pathway

scholarly journals Toxicity Induced by Cytokines, Glucose, and Lipids Increase Apoptosis and Hamper Insulin Secretion in the 1.1E7 Beta Cell-Line

2021 ◽  
Vol 22 (5) ◽  
pp. 2559
Author(s):  
Antonia Diaz-Ganete ◽  
Aranzazu Quiroga-de-Castro ◽  
Rosa M. Mateos ◽  
Francisco Medina ◽  
Carmen Segundo ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Cell Line ◽  
High Glucose ◽  
Secretory Pathway ◽  
Early Stage ◽  
Hybrid Cell ◽  
Basic Research ◽  
Stress Markers ◽  
Beta Cell Line ◽  
Starting Point

Basic research on types 1 and 2 diabetes mellitus require early stage studies using beta cells or cell lines, ideally of human origin and with preserved insulin secretion in response to glucose. The 1.1E7 cells are a hybrid cell line resulting from the electrofusion of dispersed human islets and PANC-1 cells, capable of secreting insulin in response to glucose, but their survival and function under toxic conditions remains untested. This characterization is the purpose of the present study. We treated these cells with a cytokine mix, high glucose, palmitate, and the latter two combined. Under these conditions, we measured cell viability and apoptosis (MTT, Caspase Glo and TUNEL assays, as well as caspase-8 and -9 levels by Western blotting), endoplasmic reticulum stress markers (EIF2AK3, HSPA4, EIF2a, and HSPA5) by real-time PCR, and insulin secretion with a glucose challenge. All of these stimuli (i) induce apoptosis and ER stress markers expression, (ii) reduce mRNA amounts of 2–5 components of genes involved in the insulin secretory pathway, and (iii) abrogate the insulin release capability of 1.1E7 cells in response to glucose. The most pronounced effects were observed with cytokines and with palmitate and high glucose combined. This characterization may well serve as the starting point for those choosing this cell line for future basic research on certain aspects of diabetes.

scholarly journals Geotechnical Approach to Early-Stage Site Characterisation of Shallow Wave Energy Sites

2021 ◽  
Vol 9 (6) ◽  
pp. 605
Author(s):  
Craig Heatherington ◽  
Alistair Grinham ◽  
Irene Penesis ◽  
Scott Hunter ◽  
Remo Cossu
Keyword(s):  
Wave Energy ◽  
Early Stage ◽  
Breaking Waves ◽  
Time Frame ◽  
High Energy ◽  
Specific Information ◽  
Terrain Analysis ◽  
Site Suitability ◽  
Site Characterisation ◽  
A Site

Marine renewable energy is still in its infancy and poses serious challenges due to the harsh marine conditions encountered for wave or tidal installations and the survivability of devices. Geophysical and hydrodynamic initial site surveys need to be able to provide repeatable, reliable, and economical solutions. An oscillating water column wave energy converter is to be installed on the west coast of King Island, Tasmania. The location is in a high-energy nearshore environment to take advantage of sustained shoaling non-breaking waves of the Southern Ocean and required site-specific information for the deployment. We provide insight into scalable geophysical site surveys capable of capturing large amounts of data within a short time frame. This data was incorporated into a site suitability model, utilising seabed slope, sediment depth, and water depth to provide the terrain analysis needed to match deployment-specific characteristics. In addition, short-term hydrology and geotechnical work found a highly energetic seabed (near seafloor water velocities <1 m/s) with sufficient bearing capacity (6 MPa). In a highly energetic environment, care was taken to collect the relevant data needed for an assessment of critical information to an emerging technology companies primary project. This is in addition to the malleable methodology for a site suitability model that can incorporate various weighted parameters to prioritise the location for shallow wave energy sites in general.

scholarly journals Saccharomyces cerevisiae Grx6 and Grx7 Are Monothiol Glutaredoxins Associated with the Early Secretory Pathway

2008 ◽  
Vol 7 (8) ◽  
pp. 1415-1426 ◽  
Author(s):  
Alicia Izquierdo ◽  
Celia Casas ◽  
Ulrich Mühlenhoff ◽  
Christopher Horst Lillig ◽  
Enrique Herrero
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Inhibitory Effect ◽  
Protein Accumulation ◽  
Oxidoreductase Activity ◽  
Early Secretory Pathway ◽  
Glycosylation Inhibitor

ABSTRACT Saccharomyces cerevisiae Grx6 and Grx7 are two monothiol glutaredoxins whose active-site sequences (CSYS and CPYS, respectively) are reminiscent of the CPYC active-site sequence of classical dithiol glutaredoxins. Both proteins contain an N-terminal transmembrane domain which is responsible for their association to membranes of the early secretory pathway vesicles, facing the luminal side. Thus, Grx6 localizes at the endoplasmic reticulum and Golgi compartments, while Grx7 is mostly at the Golgi. Expression of GRX6 is modestly upregulated by several stresses (calcium, sodium, and peroxides) in a manner dependent on the Crz1-calcineurin pathway. Some of these stresses also upregulate GRX7 expression under the control of the Msn2/4 transcription factor. The N glycosylation inhibitor tunicamycin induces the expression of both genes along with protein accumulation. Mutants lacking both glutaredoxins display reduced sensitivity to tunicamycin, although the drug is still able to manifest its inhibitory effect on a reporter glycoprotein. Grx6 and Grx7 have measurable oxidoreductase activity in vivo, which is increased in the presence of tunicamycin. Both glutaredoxins could be responsible for the regulation of the sulfhydryl oxidative state at the oxidant conditions of the early secretory pathway vesicles. However, the differences in location and expression responses against stresses suggest that their functions are not totally overlapping.

Investigation of the role of microtubules in protein secretion from lactating mouse mammary epithelial cells

1992 ◽  
Vol 102 (2) ◽  
pp. 239-247 ◽  
Author(s):  
M.E. Rennison ◽  
S.E. Handel ◽  
C.J. Wilde ◽  
R.D. Burgoyne
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Mammary Epithelial Cells ◽  
Early Stage ◽  
Major Effect ◽  
Vesicle Transport ◽  
Mammary Epithelial ◽  
Mammary Cells

Disruption of microtubules has been shown to reduce protein secretion from lactating mammary epithelial cells. To investigate the involvement of microtubules in the secretory pathway in these cells we have examined the effect of nocodazole on protein secretion from mammary epithelial cells derived from the lactating mouse. Mouse mammary cells have extensive microtubule networks and 85% of their tubulin was in a polymeric form. Treatment with 1 micrograms/ml nocodazole converted most of the tubulin into a soluble form. In a continuous labelling protocol it was found that nocodazole did not interfere with protein synthesis but over a 5 h period secretion was markedly inhibited. To determine whether the inhibition was at the level of early or late stages of the secretory pathway mammary cells were pulse-labelled for 1 h to label protein throughout the secretory pathway before nocodazole treatment. When secretion was subsequently assayed it was found to be slower and only partially inhibited. These findings suggest that the major effect of nocodazole is on an early stage of the secretory pathway and that microtubules normally facilitate vesicle transport to the plasma membrane. An involvement of microtubules in vesicle transport to the plasma membrane is consistent with an observed accumulation of casein vesicles in nocodazole-treated cells. Exocytosis stimulated by the calcium ionophore ionomycin was unaffected by nocodazole treatment. We conclude from these results that the major effect of nocodazole is at an early stage of the secretory pathway, one possible target being casein vesicle biogenesis in the trans-Golgi network.

In progressive nephropathies, overload of tubular cells with filtered proteins translates glomerular permeability dysfunction into cellular signals of interstitial inflammation.

1998 ◽  
Vol 9 (7) ◽  
pp. 1213-1224 ◽  
Author(s):  
M Abbate ◽  
C Zoja ◽  
D Corna ◽  
M Capitanio ◽  
T Bertani ◽  
...  
Keyword(s):  
Time Course ◽  
Early Stage ◽  
Protein Accumulation ◽  
Glomerular Injury ◽  
Cellular Mechanisms ◽  
Proinflammatory Response ◽  
Glomerular Permeability ◽  
Tubular Cells ◽  
Interstitial Inflammation ◽  
Tubulointerstitial Lesions

Progression to end-stage renal failure is the final common pathway of many forms of glomerular disease, independent of the type of initial insult. Progressive glomerulopathies have in common persistently high levels of urinary protein excretion and tubulointerstitial lesions at biopsy. Among the cellular mechanisms that may determine progression regardless of etiology, the traffic of excess proteins filtered from glomerulus in renal tubule may have functional importance by initiating interstitial inflammation in the early phase of parenchymal injury. This study analyzes the time course and sites of protein accumulation and interstitial cellular infiltration in two different models of proteinuric nephropathies. In remnant kidneys after 5/6 renal mass ablation, albumin and IgG accumulation by proximal tubular cells was visualized in the early stage, preceding interstitial infiltration of MHC-II-positive cells and macrophages. By double-staining, infiltrates developed at or near tubules containing intracellular IgG or luminal casts. This relationship persisted thereafter despite more irregular distribution of infiltrate. Similar patterns were found in an immune model (passive Heymann nephritis), indicating that the interstitial inflammatory reaction develops at the sites of protein overload, regardless of the type of glomerular injury. Osteopontin was detectable in cells of proximal tubules congested with protein in both models at sites of interstitial infiltration, and by virtue of its chemoattractive action this is likely mediator of a proximal tubule-dependent inflammatory pathway in response to protein load. Protein overload of tubules is a key candidate process translating glomerular protein leakage into cellular signals of interstitial inflammation. Mechanisms underlying the proinflammatory response of tubular cells to protein challenge in diseased kidney should be explored, as well as ways of limiting protein reabsorption/deposition to prevent consequent inflammation and progressive disease.

scholarly journals A permeabilized cell system identifies the endoplasmic reticulum as a site of protein degradation.

1991 ◽  
Vol 115 (5) ◽  
pp. 1225-1236 ◽  
Author(s):  
F J Stafford ◽  
J S Bonifacino
Keyword(s):  
Protein Degradation ◽  
Secretory Pathway ◽  
Fibroblast Cell ◽  
Cell System ◽  
Current Evidence ◽  
Permeabilized Cell ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
A Site

Analysis of the fate of a variety of newly synthesized proteins in the secretory pathway has provided evidence for the existence of a novel protein degradation system distinct from that of the lysosome. Although current evidence suggests that proteins degraded by this system are localized to a pre-Golgi compartment before degradation, the site of proteolysis has not been determined. A permeabilized cell system was developed to examine whether degradation by this pathway required transport out of the ER, and to define the biochemical characteristics of this process. Studies were performed on fibroblast cell lines expressing proteins known to be sensitive substrates for this degradative process, such as the chimeric integral membrane proteins, Tac-TCR alpha and Tac-TCR beta. By immunofluorescence microscopy, these proteins were found to be localized to the ER. Treatment with cycloheximide resulted in the progressive disappearance of intracellular staining without change in the ER localization of the chimeric proteins. Cells permeabilized with the pore-forming toxin streptolysin O were able to degrade these newly synthesized proteins. The protein degradation seen in permeabilized cells was representative of that seen in intact cells, as judged by the similar speed of degradation, substrate selectivity, temperature dependence, and involvement of free sulfhydryl groups. Degradation of these proteins in permeabilized cells took place in the absence of transport between the ER and the Golgi system. Moreover, degradation occurred in the absence of added ATP or cytosol, and in the presence of apyrase, GTP gamma S, or EDTA; i.e., under conditions which prevent transport of proteins out of the ER. The efficiency and selectivity of degradation of newly synthesized proteins were also conserved in an isolated ER fraction. These data indicate that the machinery responsible for pre-Golgi degradation of newly synthesized proteins exists within the ER itself, and can operate independent of exogenously added ATP and cytosolic factors.

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