Dissociation and re-assembly of the endoplasmic reticulum in live cells

1988 ◽  
Vol 91 (4) ◽  
pp. 511-522 ◽  
Author(s):  
G.L. Koch ◽  
C. Booth ◽  
F.B. Wooding
Keyword(s):  
Endoplasmic Reticulum ◽  
Channel Blocker ◽  
External Medium ◽  
Calcium Ionophore ◽  
Live Cells ◽  
3T3 Cells ◽  
Normal Medium ◽  
Linear Arrays ◽  
Daughter Cells ◽  
Rough Er

The endoplasmic reticulum (ER) of a typical interphase 3T3 fibroblast consists of a compact perinuclear arrangement of cisternae and lamellae which can be observed by immunofluorescence with anti-endoplasmin. During mitosis the reticulum dissociates into small fragments from which it appears to re-assemble in the daughter cells. When interphase 3T3 cells are exposed to calcium ionophores, but not other ionophores, there is a similar dissociation of the ER into small uniform fragments, which are dispersed throughout the cytoplasm. Electron microscopy shows that the fragments consist of small vesicular structures and that essentially all of the rough ER except the nuclear envelope is dissociated. The dissociation of the ER by calcium ionophore is a relatively specific process since other organelles and supramolecular assemblies remain unaffected. When cells with dissociated ER are returned to normal medium, there is a rapid reassembly of the fragments into the continuous reticulum. In a proportion of the cells it is possible to observe linear arrays of the fragments, which probably represent intermediates in the re-assembly process. These observations demonstrate that the ER in interphase 3T3 cells can be dissociated into, and re-assembled from, small fragments. Re-assembly of the ER from the fragments is dependent on the presence of millimolar levels of calcium in the external medium. In the presence of calcium, re-assembly is inhibited by the calcium channel blocker, verapamil. Thus calcium ions appear to play an important role in ER structure and assembly.

In Vitro Induction of Crystals of MT Protein by Vinblastine-Colcemid in Mouse Spermatids

1974 ◽  
Vol 32 ◽  
pp. 132-133
Author(s):  
John J. Wolosewick ◽  
John H. D. Bryan
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Nuclear Ring ◽  
Rough Er ◽  
Distal Direction ◽  
In Vitro Induction

Early in spermiogenesis the manchette is rapidly assembled in a distal direction from the nuclear-ring-densities. The association of vesicles of smooth endoplasmic reticulum (SER) and the manchette microtubules (MTS) has been reported. In the mouse, osmophilic densities at the distal ends of the manchette are the organizing centers (MTOCS), and are associated with the SER. Rapid MT assembly and the lack of rough ER suggests that there is an existing pool of MT protein. Colcemid potentiates the reaction of vinblastine with tubulin and was used in this investigation to detect this protein.

Growth factors and the primary cilium in BALB/c 3T3 cells

1987 ◽  
Vol 45 ◽  
pp. 830-831
Author(s):  
R. W. Tucker ◽  
N. S. More ◽  
S. Jayaraman
Keyword(s):  
Growth Factors ◽  
Primary Cilium ◽  
Cultured Cells ◽  
Morphological Changes ◽  
Calcium Ionophore ◽  
Growth Stimulation ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
3T3 Cells ◽  
Microtubule Depolymerization

The mechanisms by which polypeptide growth factors Induce DNA synthesis in cultured cells is not understood, but morphological changes Induced by growth factors have been used as clues to Intracellular messengers responsible for growth stimulation. One such morphological change has been the transient disappearance of the primary cilium, a “9 + 0” cilium formed by the perinuclear centriole in interphase cells. Since calcium ionophore A23187 also produced both mitogenesis and ciliary changes, microtubule depolymerization might explain ciliary disappearance monitored by indirect immunofluorescence with anti-tubulin antibody. However, complete resorption and subsequent reformation of the primary cilium occurs at mitosis, and might also account for ciliary disappearance induced by growth factors. To settle this issue, we investigated the ultrastructure of the primary cilium using serial thin-section electron microscopy of quiescent BALB/c 3T3 cells before and after stimulation with serum.

scholarly journals Network Organisation and the Dynamics of Tubules in the Endoplasmic Reticulum

2020 ◽  
Author(s):  
H. Perkins ◽  
P. Ducluzaux ◽  
P. Woodman ◽  
V. Allan ◽  
T. Waigh
Keyword(s):  
Endoplasmic Reticulum ◽  
Binary Classification ◽  
Persistence Length ◽  
Gaussian Mixture ◽  
Live Cells ◽  
Hereditary Spastic Paraplegias ◽  
Membrane Contact Sites ◽  
Subcellular Organelle ◽  
Membrane Contact ◽  
Network Organisation

ABSTRACTThe endoplasmic reticulum (ER) is a eukaryotic subcellular organelle composed of tubules and sheet-like areas of membrane connected at junctions. The tubule network is highly dynamic and undergoes rapid and continual rearrangement. There are currently few tools to evaluate network organisation and dynamics. We quantified ER network organisation in Vero and MRC5 cells, and developed a classification system for ER dynamics in live cells. The persistence length, tubule length, junction coordination number and angles of the network were quantified. Hallmarks of imbalances in ER tension, indications of interactions with microtubules and other subcellular organelles, and active reorganisation and dynamics were observed. Live cell ER tubule dynamics were classified using a Gaussian mixture model, defining tubule motion as active or thermal and conformational phase space analysis allowed this classification to be refined by tubule curvature states.STATEMENT OF SIGNIFICANCEThe endoplasmic reticulum (ER), a subcellular organelle, is an underexplored real-world example of active matter. Many processes essential to cell survival are performed by the ER, the efficacy of which may depend on its organisation and dynamics. Abnormal ER morphology is linked to diseases such as hereditary spastic paraplegias and it is possible that the dynamics are also implicated. Therefore, analysing the ER network in normal cells is important for the understanding of disease-related alterations. In this work, we outline the first thorough quantification methods for determining ER organisation and dynamics, deducing that tubule motion has a binary classification as active or thermal. Active reorganisation and dynamics along with indications of tension imbalances and membrane contact sites were observed.

scholarly journals The ER Stress Surveillance (ERSU) pathway regulates daughter cell ER protein aggregate inheritance

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Francisco J Piña ◽  
Maho Niwa
Keyword(s):  
Cell Cycle ◽  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Mother Cell ◽  
Daughter Cell ◽  
Protein Aggregates ◽  
Cytoplasmic Protein ◽  
Protein Aggregate ◽  
Daughter Cells ◽  
Simultaneous Visualization

Stress induced by cytoplasmic protein aggregates can have deleterious consequences for the cell, contributing to neurodegeneration and other diseases. Protein aggregates are also formed within the endoplasmic reticulum (ER), although the fate of ER protein aggregates, specifically during cell division, is not well understood. By simultaneous visualization of both the ER itself and ER protein aggregates, we found that ER protein aggregates that induce ER stress are retained in the mother cell by activation of the ER Stress Surveillance (ERSU) pathway, which prevents inheritance of stressed ER. In contrast, under conditions of normal ER inheritance, ER protein aggregates can enter the daughter cell. Thus, whereas cytoplasmic protein aggregates are retained in the mother cell to protect the functional capacity of daughter cells, the fate of ER protein aggregates is determined by whether or not they activate the ERSU pathway to impede transmission of the cortical ER during the cell cycle.

A novel mesoionic carbene based highly fluorescent Pd(ii) complex as an endoplasmic reticulum tracker in live cells

2018 ◽  
Vol 47 (44) ◽  
pp. 15646-15650 ◽  
Author(s):  
Sanjay K. Verma ◽  
Pratibha Kumari ◽  
Shagufi Naz Ansari ◽  
Mohd Ovais Ansari ◽  
Dondinath Deori ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluorescence Recovery After Photobleaching ◽  
Fluorescence Recovery ◽  
Live Cells

Synthesis of new organometallic MIC based mononuclear Pd(ii) complex 1, specifically target ER of live cells and have fluorescence recovery after photobleaching (FRAP) property.

Inhibition by extracellular Na+ replacement of GRF-induced GH secretion from rat pituitary cells

1988 ◽  
Vol 254 (4) ◽  
pp. E476-E481 ◽  
Author(s):  
M. Kato ◽  
M. A. Hattori ◽  
M. Suzuki
Keyword(s):  
Anterior Pituitary ◽  
Channel Blocker ◽  
Na Channel ◽  
Pituitary Cells ◽  
Ionic Mechanism ◽  
Camp Production ◽  
Normal Medium ◽  
Gh Secretion ◽  
Rat Pituitary ◽  
Rat Pituitary Cells

To further clarify the ionic mechanism of the action of growth hormone (GH)-releasing factor (hGRF) on GH secretion, the involvement of extracellular Na+ was studied in perifused dispersed rat anterior pituitary cells. Replacing extracellular Na+ with mannitol or tris(hydroxymethyl)aminomethane (Tris+) suppressed hGRF- and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP)-induced GH secretion. The peak responses to a 2-min application of 1 nM hGRF were 165.0 +/- 5.6 ng/ml (normal medium, mean +/- SE), 21.2 +/- 1.4 ng/ml (Na+-free, mannitol medium), and 18.0 +/- 1.7 ng/ml (Na+-free, Tris+ medium). GH secretion induced by DBcAMP was also suppressed by Na+ replacement to less than 50% of that in normal medium. However, either 15 or 30 mM KCl-stimulated GH secretion was not markedly affected by replacement of Na+ with either compound. Tetrodotoxin, a voltage-sensitive Na+ channel blocker, had no effect on either hGRF- or excess K+-induced GH secretion. cAMP production by hGRF was not greatly affected by replacing extracellular Na+. Thus extracellular Na+ plays an important role in hGRF-induced GH secretion, especially in the process after cAMP production. The involvement of cAMP-sensitive Na+ channels in hGRF-stimulated GH secretion is discussed.

scholarly journals Endoplasmic Reticulum Stress Is a Determinant of Retrovirus-Induced Spongiform Neurodegeneration

2003 ◽  
Vol 77 (23) ◽  
pp. 12617-12629 ◽  
Author(s):  
Derek E. Dimcheff ◽  
Srdjan Askovic ◽  
Audrey H. Baker ◽  
Cedar Johnson-Fowler ◽  
John L. Portis
Keyword(s):  
Endoplasmic Reticulum ◽  
Transmissible Spongiform Encephalopathy ◽  
Viral Envelope ◽  
Spongiform Encephalopathy ◽  
Envelope Gene ◽  
Viral Envelope Protein ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT FrCasE is a mouse retrovirus that causes a fatal noninflammatory spongiform neurodegenerative disease with pathological features strikingly similar to those induced by transmissible spongiform encephalopathy (TSE) agents. Neurovirulence is determined by the sequence of the viral envelope protein, though the specific role of this protein in disease pathogenesis is not known. In the present study, we compared host gene expression in the brain stems of mice infected with either FrCasE or the avirulent virus F43, differing from FrCasE in the sequence of the envelope gene. Four of the 12 disease-specific transcripts up-regulated during the preclinical period represent responses linked to the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Among these genes was CHOP/GADD153, which is induced in response to conditions that perturb endoplasmic reticulum function. In vitro studies with NIH 3T3 cells revealed up-regulation of CHOP as well as BiP, calreticulin, and Grp58/ERp57 in cells infected with FrCasE but not with F43. Immunoblot analysis of infected NIH 3T3 cells demonstrated the accumulation of uncleaved envelope precursor protein in FrCasE- but not F43-infected cells, consistent with ER retention. These results suggest that retrovirus-induced spongiform neurodegeneration represents a protein-folding disease and thus may provide a useful tool for exploring the causal link between protein misfolding and the cytopathology that it causes.

Heterologous expression of polycystin-1 inhibits endoplasmic reticulum calcium leak in stably transfected MDCK cells

2008 ◽  
Vol 294 (6) ◽  
pp. F1279-F1286 ◽  
Author(s):  
Kimberly H. Weber ◽  
Eun Kyung Lee ◽  
Uma Basavanna ◽  
Sabina Lindley ◽  
Roy C. Ziegelstein ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Calcium Release ◽  
Mdck Cells ◽  
Calcium Ionophore ◽  
Calcium Flux ◽  
Calcium Stores ◽  
Endoplasmic Reticulum Calcium ◽  
Calcium Indicator ◽  
Luminal Calcium ◽  
Er Membrane

We previously found that polycystin-1 accelerated the decay of ligand-activated cytoplasmic calcium transients through enhanced reuptake of calcium into the endoplasmic reticulum (ER; Hooper KM, Boletta A, Germino GG, Hu Q, Ziegelstein RC, Sutters M. Am J Physiol Renal Physiol 289: F521–F530, 2005). Calcium flux across the ER membrane is determined by the balance of active uptake and passive leak. In the present study, we show that polycystin-1 inhibited calcium leak across the ER membrane, an effect that would explain the capacity of this protein to accelerate clearance of calcium from the cytoplasm following a calcium release response. Calcium leak was detected by measurement of the accumulation of calcium in the cytoplasm following treatment with thapsigargin. Heterologous polycystin-1, stably expressed in Madin-Darby canine kidney cells, attenuated the thapsigargin-induced calcium peak with no effect on basal calcium stores, mitochondrial calcium uptake, or extrusion of calcium across the plasma membrane. The capacity of polycystin-1 to limit the rate of decay of ER luminal calcium following inhibition of the pump was shown indirectly using the calcium ionophore ionomycin, and directly by loading the ER with a low-affinity calcium indicator. We conclude that disruption of ER luminal calcium homeostasis may contribute to the cyst phenotype in autosomal dominant polycystic kidney disease.

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