scholarly journals Construction of the endoplasmic reticulum.

1989 ◽  
Vol 109 (5) ◽  
pp. 2045-2055 ◽  
Author(s):  
C Lee ◽  
M Ferguson ◽  
L B Chen
Keyword(s):  
Intermediate Filaments ◽  
Cytochalasin B ◽  
Network Formation ◽  
Living Cells ◽  
Necessary Condition ◽  
Construction Process ◽  
Microtubule Polymerization ◽  
Complete Breakdown ◽  
Complete Disruption ◽  
Drug Free

To study the construction of the ER, we used the microtubule-disrupting drug nocodazole to induce the complete breakdown of ER structure in living cells followed by recovery in drug-free medium, which regenerates the ER network within 15 min. Using the fluorescent dye 3,3'-dihexyloxacarbocyanine iodide to visualize the ER, we have directly observed the network construction process in living cells. In these experiments, the ER network was constructed through an iterative process of extension, branching, and intersection of new ER tubules driven by the ER motility previously described as tubule branching. We have tested the cytoskeletal requirements of this process. We find that newly formed ER tubules are aligned with single microtubules but not actin fibers or vimentin intermediate filaments. Microtubule polymerization preceded the extension of ER tubules and, in experiments with a variety of different drugs, appeared to be a necessary condition for the ER network formation. Furthermore, perturbations of the pattern of microtubule polymerization with microtubule-specific drugs caused exactly correlated perturbations of the pattern of ER construction. Induction of abnormally short, nonintersecting microtubules with 20 microM taxol prevented the ER network formation; ER tubules only extended along the few microtubules contacting the aggregated ER membranes. This requirement for a continuous network of intersecting microtubules indicates that ER network formation takes place through the branching and movement of ER membranes along microtubules. Cytochalasin B had no apparent effect on the construction of the ER network during recovery, despite apparently complete disruption of actin fibers as stained by phalloidin. Blockage of protein synthesis and disorganization of intermediate filaments with cycloheximide pretreatment also failed to perturb ER construction.

Visualization of intermediate filaments in living cells using fluorescently labeled desmin

1989 ◽  
Vol 12 (3) ◽  
pp. 127-138 ◽  
Author(s):  
B. Mittal ◽  
J. M. Sanger ◽  
J. W. Sanger
Keyword(s):  
Intermediate Filaments ◽  
Living Cells

scholarly journals Apparent stiffness of vimentin intermediate filaments in living cells and its relation with other cytoskeletal polymers

2020 ◽  
Vol 1867 (8) ◽  
pp. 118726 ◽  
Author(s):  
Mariano Smoler ◽  
Giovanna Coceano ◽  
Ilaria Testa ◽  
Luciana Bruno ◽  
Valeria Levi
Keyword(s):  
Intermediate Filaments ◽  
Living Cells ◽  
Apparent Stiffness

Altered cell spreading in cytochalasin B: a possible role for intermediate filaments

1983 ◽  
Vol 3 (1) ◽  
pp. 113-125
Author(s):  
A S Menko ◽  
Y Toyama ◽  
D Boettiger ◽  
H Holtzer
Keyword(s):  
Intermediate Filaments ◽  
Rous Sarcoma Virus ◽  
Cytochalasin B ◽  
Dermal Fibroblasts ◽  
Transformed Cells ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Rous Sarcoma ◽  
Microfilament Bundles ◽  
Sarcoma Virus

Trypsinized chicken embryo dermal fibroblasts plated in the presence of cytochalasin B (CB) quickly attached to the substrate and within 24 h obtained an arborized morphology. This morphology is the result of the pushing out of pseudopodial processes along the substrate from the round central cell body. There were no microfilament bundles in the processes of these cells plated in the presence of CB; however, the processes were packed with highly oriented, parallel-aligned intermediate filaments. Only a few scattered microtubules were seen in these processes. These results demonstrated that in CB, cells are capable of a form of movement, i.e., the extension of pseudopodial processes, without the presence of the microfilament structures usually associated with extensions of the cytoplasm and pseudopodial movements. We also found that arborization did not depend on fibronectin since cells plated in CB did not have fibronectin fibers associated with the processes. Chicken fibroblasts transformed with tsLA24A, a Rous sarcoma virus which is temperature sensitive for pp60src, formed arborized cells with properties similar to those of uninfected fibroblasts when plated in the presence of CB at the nonpermissive temperature (41 degrees C). At the permissive temperature for transformation (36 degrees C), the cells attached to the substrate but remained round. These round cells were not only deficient in microfilament bundles but also lacked the highly organized intermediate filaments found in the processes of the arborized cells at 41 degrees C. Although both microfilament bundles and the fibronectin matrix were decreased after transformation with Rous sarcoma virus, neither was involved in the formation of processes in normal cells plated in CB. Therefore, the inability of the transformed cells to form or maintain processes in CB must be the result of another structural alteration in the transformed cells, such as that of the intermediate filaments.

scholarly journals Physiology, structure, and susceptibility to injury of skeletal muscle in mice lacking keratin 19-based and desmin-based intermediate filaments

2011 ◽  
Vol 300 (4) ◽  
pp. C803-C813 ◽  
Author(s):  
Richard M. Lovering ◽  
Andrea O'Neill ◽  
Joaquin M. Muriel ◽  
Benjamin L. Prosser ◽  
John Strong ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Intermediate Filaments ◽  
Striated Muscle ◽  
Contractile Function ◽  
Double Knockout ◽  
Specific Tension ◽  
Keratin 19 ◽  
Keratin Filaments ◽  
Complete Disruption ◽  
Fast Twitch

Intermediate filaments, composed of desmin and of keratins, play important roles in linking contractile elements to each other and to the sarcolemma in striated muscle. Our previous results show that the tibialis anterior (TA) muscles of mice lacking keratin 19 (K19) lose costameres, accumulate mitochondria under the sarcolemma, and generate lower specific tension than controls. Here we compare the physiology and morphology of TA muscles of mice lacking K19 with muscles lacking desmin or both proteins [double knockout (DKO)]. K19−/− mice and DKO mice showed a threefold increase in the levels of creatine kinase (CK) in the serum. The absence of desmin caused a larger change in specific tension (−40%) than the absence of K19 (−19%) and played the predominant role in contractile function (−40%) and decreased tolerance to exercise in the DKO muscle. By contrast, the absence of both proteins was required to obtain a significantly greater loss of contractile torque after injury (−48%) compared with wild type (−39%), as well as near-complete disruption of costameres. The DKO muscle also showed a significantly greater misalignment of myofibrils than either mutant alone. In contrast, large subsarcolemmal gaps and extensive accumulation of mitochondria were only seen in K19-null TA muscles, and the absence of both K19 and desmin yielded milder phenotypes. Our results suggest that keratin filaments containing K19- and desmin-based intermediate filaments can play independent, complementary, or antagonistic roles in the physiology and morphology of fast-twitch skeletal muscle.

scholarly journals Identification of Novel Principles of Keratin Filament Network Turnover in Living Cells

2004 ◽  
Vol 15 (5) ◽  
pp. 2436-2448 ◽  
Author(s):  
Reinhard Windoffer ◽  
Stefan Wöll ◽  
Pavel Strnad ◽  
Rudolf E. Leube
Keyword(s):  
Network Formation ◽  
De Novo ◽  
Peripheral Zone ◽  
Living Cells ◽  
Cell Cortex ◽  
Cell Periphery ◽  
Main Site ◽  
Filament System ◽  
Keratin Filaments ◽  
Keratin Filament

It is generally assumed that turnover of the keratin filament system occurs by exchange of subunits along its entire length throughout the cytoplasm. We now present evidence that a circumscribed submembranous compartment is actually the main site for network replenishment. This conclusion is based on the following observations in living cells synthesizing fluorescent keratin polypeptides: 1) Small keratin granules originate in close proximity to the plasma membrane and move toward the cell center in a continuous motion while elongating into flexible rod-like fragments that fuse with each other and integrate into the peripheral KF network. 2) Recurrence of fluorescence after photobleaching is first seen in the cell periphery where keratin filaments are born that translocate subsequently as part of the network toward the cell center. 3) Partial keratin network reformation after orthovanadate-induced disruption is restricted to a distinct peripheral zone in which either keratin granules or keratin filaments are transiently formed. These findings extend earlier investigations of mitotic cells in which de novo keratin network formation was shown to originate from the cell cortex. Taken together, our results demonstrate that the keratin filament system is not homogenous but is organized into temporally and spatially distinct subdomains. Furthermore, the cortical localization of the regulatory cues for keratin filament turnover provides an ideal way to adjust the epithelial cytoskeleton to dynamic cellular processes.

scholarly journals Analysis of De Novo Golgi Complex Formation after Enzyme-based Inactivation

2007 ◽  
Vol 18 (11) ◽  
pp. 4637-4647 ◽  
Author(s):  
Florence Jollivet ◽  
Graça Raposo ◽  
Ariane Dimitrov ◽  
Rachid Sougrat ◽  
Bruno Goud ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
De Novo ◽  
Living Cells ◽  
Integrative Model ◽  
Interphase Cells ◽  
Golgi Structure ◽  
Dynamics And Function ◽  
And Function ◽  
Drug Free

The Golgi complex is characterized by its unique morphology of closely apposed flattened cisternae that persists despite the large quantity of lipids and proteins that transit bidirectionally. Whether such a structure is maintained through endoplasmic reticulum (ER)-based recycling and auto-organization or whether it depends on a permanent Golgi structure is strongly debated. To further study Golgi maintenance in interphase cells, we developed a method allowing for a drug-free inactivation of Golgi dynamics and function in living cells. After Golgi inactivation, a new Golgi-like structure, containing only certain Golgi markers and newly synthesized cargos, was produced. However, this structure did not acquire a normal Golgi architecture and was unable to ensure a normal trafficking activity. This suggests an integrative model for Golgi maintenance in interphase where the ER is able to autonomously produce Golgi-like structures that need pre-existing Golgi complexes to be organized as morphologically normal and active Golgi elements.

scholarly journals Altered cell spreading in cytochalasin B: a possible role for intermediate filaments.

1983 ◽  
Vol 3 (1) ◽  
pp. 113-125 ◽  
Author(s):  
A S Menko ◽  
Y Toyama ◽  
D Boettiger ◽  
H Holtzer
Keyword(s):  
Intermediate Filaments ◽  
Rous Sarcoma Virus ◽  
Cytochalasin B ◽  
Dermal Fibroblasts ◽  
Transformed Cells ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Rous Sarcoma ◽  
Microfilament Bundles ◽  
Sarcoma Virus

Trypsinized chicken embryo dermal fibroblasts plated in the presence of cytochalasin B (CB) quickly attached to the substrate and within 24 h obtained an arborized morphology. This morphology is the result of the pushing out of pseudopodial processes along the substrate from the round central cell body. There were no microfilament bundles in the processes of these cells plated in the presence of CB; however, the processes were packed with highly oriented, parallel-aligned intermediate filaments. Only a few scattered microtubules were seen in these processes. These results demonstrated that in CB, cells are capable of a form of movement, i.e., the extension of pseudopodial processes, without the presence of the microfilament structures usually associated with extensions of the cytoplasm and pseudopodial movements. We also found that arborization did not depend on fibronectin since cells plated in CB did not have fibronectin fibers associated with the processes. Chicken fibroblasts transformed with tsLA24A, a Rous sarcoma virus which is temperature sensitive for pp60src, formed arborized cells with properties similar to those of uninfected fibroblasts when plated in the presence of CB at the nonpermissive temperature (41 degrees C). At the permissive temperature for transformation (36 degrees C), the cells attached to the substrate but remained round. These round cells were not only deficient in microfilament bundles but also lacked the highly organized intermediate filaments found in the processes of the arborized cells at 41 degrees C. Although both microfilament bundles and the fibronectin matrix were decreased after transformation with Rous sarcoma virus, neither was involved in the formation of processes in normal cells plated in CB. Therefore, the inability of the transformed cells to form or maintain processes in CB must be the result of another structural alteration in the transformed cells, such as that of the intermediate filaments.

Effect of microtubules and intermediate filaments on mitochondrial distribution

1983 ◽  
Vol 61 (1) ◽  
pp. 87-105
Author(s):  
I.C. Summerhayes ◽  
D. Wong ◽  
L.B. Chen
Keyword(s):  
Intermediate Filaments ◽  
Dominant Role ◽  
Single Cells ◽  
Living Cells ◽  
Laser Dye ◽  
Experimental Conditions ◽  
Mitochondrial Distribution ◽  
Experimental Approaches ◽  
Absolute Correlation ◽  
Cytoskeletal Elements

The laser dye rhodamine 123 specifically stains mitochondria in living cells and facilitates the observation of changes in mitochondrial distribution in single cells under a variety of experimental conditions. Visualization of mitochondria in a number of cell lines followed by processing of these cells to study different cytoskeletal elements by indirect immunofluorescence, revealed good but not absolute correlation between mitochondria and microtubules or intermediate filaments. Mitochondria and microfilament distribution within the same cell did not show such a correlation. On the basis of observations made by various experimental approaches, we suggest that mitochondrial distribution is under the strong influence of the two systems, microtubules and intermediate filaments. Neither plays an absolute role but one seems able to play a more dominant role in the absence of the other.

Vector analysis of steerable mechanical tension across nuclear lamina

2018 ◽  
Author(s):  
TingTing Chen ◽  
HuiWen Wu ◽  
YuXuan Wang ◽  
JinJun Shan ◽  
JiaRui Zhang ◽  
...  
Keyword(s):  
Osmotic Pressure ◽  
Intermediate Filaments ◽  
Nuclear Lamina ◽  
Optical Probe ◽  
Living Cells ◽  
Vector Analysis ◽  
Eukaryotic Cells ◽  
Mechanical Tension ◽  
Protein Nanoparticles ◽  
Cytoskeletal Tension

SUMMARYThe nucleus is the most prominent organelle in eukaryotic cells, and its deformation depends on interactions between the nuclear lamina (NL) and cytoskeleton structural tensions. The structural tensions can be quantified at a pico-Newton (pN) level using a genetically encoded optical probe. In living cells, NL tensions countered the 4.26pN resting strain imposed competitively by cytoskeletal tension. The depolymerization of microfilaments or microtubules drove an aberrant increase in outward osmotic pressure through the production of mass protein-nanoparticles. The osmotic pressure also served as a directional converter of inward cytoskeletal force, and contributed to the outward expansion of NL via the passive pull of intermediate filaments (IFs). The NL, but not IFs, can remotely detect extracellular osmosis pressure alterations, which are closely associated with highly polarized microfilament and microtubule structures and their directional force activities. The oxidative-induced increase of NL tension results from intracellular hyper-osmosis, associated closely with protein-nanoparticles production elicited by cofilin and stathmin activation. These data reveal that intracellular steerable forces interact direction-dependently to control NL tension in terms of their magnitude and vectors.

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