scholarly journals Vimentin Intermediate Filaments Stabilize Dynamic Microtubules by Direct Interactions

2020 ◽  
Author(s):  
Laura Schaedel ◽  
Charlotta Lorenz ◽  
Anna V. Schepers ◽  
Stefan Klumpp ◽  
Sarah Köster
Keyword(s):  
Intermediate Filaments ◽  
Cell Mechanics ◽  
Physical Nature ◽  
Cellular Functions ◽  
In Vitro System ◽  
Direct Mechanism ◽  
Cytoskeletal Dynamics ◽  
Novel Strategy ◽  
Insight Into

AbstractThe cytoskeleton determines cell mechanics and lies at the heart of important cellular functions. Growing evidence suggests that the manifold tasks of the cytoskeleton rely on the interactions between its filamentous components, known as actin filaments, intermediate filaments and microtubules. However, the nature of these interactions and their impact on cytoskeletal dynamics are largely unknown. Here, we show in a re-constituted in vitro system that vimentin intermediate filaments stabilize microtubules against depolymerization and support microtubule rescue. To understand these stabilizing effects, we directly measure the interaction forces between individual microtubules and vimentin filaments. Combined with numerical simulations, our observations provide detailed insight into the physical nature of the interactions and how they affect microtubule dynamics. Thus, we describe an additional, direct mechanism for cells to establish the fundamental cross-talk of cytoskeletal components alongside linker proteins. Moreover, we suggest a novel strategy to estimate the binding energy of tubulin dimers within the microtubule lattice.

scholarly journals Vimentin intermediate filaments stabilize dynamic microtubules by direct interactions

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Laura Schaedel ◽  
Charlotta Lorenz ◽  
Anna V. Schepers ◽  
Stefan Klumpp ◽  
Sarah Köster
Keyword(s):  
Intermediate Filaments ◽  
Cell Mechanics ◽  
Physical Nature ◽  
Cellular Functions ◽  
Interaction Forces ◽  
In Vitro System ◽  
Direct Mechanism ◽  
Cytoskeletal Dynamics ◽  
Insight Into

AbstractThe cytoskeleton determines cell mechanics and lies at the heart of important cellular functions. Growing evidence suggests that the manifold tasks of the cytoskeleton rely on the interactions between its filamentous components—actin filaments, intermediate filaments, and microtubules. However, the nature of these interactions and their impact on cytoskeletal dynamics are largely unknown. Here, we show in a reconstituted in vitro system that vimentin intermediate filaments stabilize microtubules against depolymerization and support microtubule rescue. To understand these stabilizing effects, we directly measure the interaction forces between individual microtubules and vimentin filaments. Combined with numerical simulations, our observations provide detailed insight into the physical nature of the interactions and how they affect microtubule dynamics. Thus, we describe an additional, direct mechanism by which cells establish the fundamental cross talk of cytoskeletal components alongside linker proteins. Moreover, we suggest a strategy to estimate the binding energy of tubulin dimers within the microtubule lattice.

scholarly journals Myosin-II activity generates a dynamic steady state with continuous actin turnover in a minimal actin cortex

2018 ◽  
Author(s):  
Sonal ◽  
Kristina A. Ganzinger ◽  
Sven K. Vogel ◽  
Jonas Mücksch ◽  
Philipp Blumhardt ◽  
...  
Keyword(s):  
Steady State ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Fine Tuning ◽  
Cellular Functions ◽  
Actin Cortex ◽  
Actin Turnover ◽  
Cytoskeletal Dynamics

ABSTRACTDynamic reorganization of the actomyosin cytoskeleton allows a fine-tuning of cell shape that is vital to many cellular functions. It is well established that myosin-II motors generate the forces required for remodeling the cell surface by imparting contractility to actin networks. An additional, less understood, role of myosin-II in cytoskeletal dynamics is believed to be in the regulation of actin turnover; it has been proposed that myosin activity increases actin turnover in various cellular contexts, presumably by contributing to disassembly. In vitro reconstitution of actomyosin networks has confirmed the role of myosin in actin network disassembly, but factors such as diffusional constraints and the use of stabilized filaments have thus far limited the observation of myosin-assisted actin turnover in these networks. Here, we present the reconstitution of a minimal dynamic actin cortex where actin polymerization is catalyzed on the membrane in the presence of myosin-II activity. We demonstrate that myosin activity leads to disassembly and redistribution in this simplified cortex. Consequently, a new dynamic steady state emerges in which actin filaments undergo constant turnover. Our findings suggest a multi-faceted role of myosin-II in fast remodeling of the eukaryotic actin cortex.

scholarly journals APC binds intermediate filaments and is required for their reorganization during cell migration

2013 ◽  
Vol 200 (3) ◽  
pp. 249-258 ◽  
Author(s):  
Yasuhisa Sakamoto ◽  
Batiste Boëda ◽  
Sandrine Etienne-Manneville
Keyword(s):  
Cell Migration ◽  
Glial Fibrillary Acidic Protein ◽  
Intermediate Filaments ◽  
Carcinoma Cells ◽  
Adenomatous Polyposis ◽  
Polyposis Coli ◽  
Cellular Functions ◽  
Migration Assay ◽  
Armadillo Repeats

Intermediate filaments (IFs) are components of the cytoskeleton involved in most cellular functions, including cell migration. Primary astrocytes mainly express glial fibrillary acidic protein, vimentin, and nestin, which are essential for migration. In a wound-induced migration assay, IFs reorganized to form a polarized network that was coextensive with microtubules in cell protrusions. We found that the tumor suppressor adenomatous polyposis coli (APC) was required for microtubule interaction with IFs and for microtubule-dependent rearrangements of IFs during astrocyte migration. We also show that loss or truncation of APC correlated with the disorganization of the IF network in glioma and carcinoma cells. In migrating astrocytes, vimentin-associated APC colocalized with microtubules. APC directly bound polymerized vimentin via its armadillo repeats. This binding domain promoted vimentin polymerization in vitro and contributed to the elongation of IFs along microtubules. These results point to APC as a crucial regulator of IF organization and confirm its fundamental role in the coordinated regulation of cytoskeletons.

scholarly journals Assessment of the anti-norovirus activity in cell culture using the mouse norovirus: Early mechanistic studies

2021 ◽  
Vol 29 ◽  
pp. 204020662110251
Author(s):  
Jana Van Dycke ◽  
Jasper Rymenants ◽  
Johan Neyts ◽  
Joana Rocha-Pereira
Keyword(s):  
Small Molecules ◽  
Continuous Improvement ◽  
Antiviral Effect ◽  
Viral Gastroenteritis ◽  
Mechanistic Studies ◽  
Preclinical Development ◽  
In Vitro System ◽  
Insight Into

Human norovirus is the main cause of viral gastroenteritis, resulting annually in ∼ 700 million infections and 200,000 deaths, of whom most are children <5 years. Mouse norovirus-infected macrophages are the most widely used in vitro system to screen and characterize the antiviral effect of norovirus-targeting small molecules. We have previously established antiviral assays using this system, identified novel inhibitors and performed additional studies in order to have a first insight into their mechanism of action. After the identification of novel small molecules with anti-norovirus activity (part 1 of this protocol), we here describe the logical next step which entails the generation of early information of their mode of action. This information together with a continuous improvement of the potency of compounds will contribute to the optimization of a compound class towards in vivo efficacy and a successful preclinical development.

scholarly journals Structural Insight into the In Vitro Anti-Intravasative Properties of Flavonoids

2019 ◽  
Vol 87 (3) ◽  
pp. 23 ◽  
Author(s):  
Julia Eichsteininger ◽  
Kerstin Kirisits ◽  
Claudia Smöch ◽  
Christa Stadlbauer ◽  
Chi Huu Nguyen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Three Dimensional ◽  
Positive Control ◽  
Gap Formation ◽  
In Vitro System ◽  
Cancer Spheroids ◽  
Induced Defects ◽  
Insight Into ◽  
Mcf 7

We investigated the effect of 21 flavonoids in a three-dimensional in vitro system for their ability to inhibit gap formation by MCF-7 breast cancer spheroids in monolayers of lymphendothelial cells. Different representatives of the classes of flavones, flavonols, and flavanones were tested in the circular chemorepellent-induced defects (CCID)-assay. Bay11-7082, a known inhibitor of CCID formation served as the positive control. This study provides the first comparison of the potential of flavonoids to suppress features influencing the intravasation of MCF-7 breast cancer cells aggregates through the lymph endothelial barrier. The most significant effects were seen after incubation with the flavones luteolin, chrysin, and apigenin. Additional hydroxylation or methoxylation in positions 6 or 8, as expected, resulted in decreased activity. The tested flavanones remained without or low efficacy.

scholarly journals Assessment of the anti-norovirus activity in cell culture using the mouse norovirus: Identification of active compounds

2021 ◽  
Vol 29 ◽  
pp. 204020662110268
Author(s):  
Jana Van Dycke ◽  
Jasper Rymenants ◽  
Johan Neyts ◽  
Joana Rocha-Pereira
Keyword(s):  
Cell Culture ◽  
Small Molecules ◽  
Mechanism Of Action ◽  
Antiviral Effect ◽  
Viral Gastroenteritis ◽  
Human Norovirus ◽  
In Vitro System ◽  
Insight Into ◽  
Detailed Protocol

Human norovirus is the main cause of viral gastroenteritis, resulting annually in ∼ 700 million infections and 200,000 deaths, of whom most are children <5 years. Mouse norovirus-infected macrophages are the most widely used in vitro system to screen and characterize the antiviral effect of norovirus-targeting small molecules. We have previously established antiviral assays using this system, identified novel inhibitors and performed additional studies in order to have a first insight into their mechanism of action. As potent and safe anti-norovirus small molecules are urgently needed, we here describe the detailed protocol for a set of assays that will allow the identification of novel norovirus inhibitors.

Efficiency and efficacy of using a sophisticated 3D in vitro system of the human epithelial airway barrier to gain insight into the hazard posed by nanomaterials

2013 ◽  
Vol 221 ◽  
pp. S146-S147
Author(s):  
Martin J.D. Clift ◽  
Carola Endes ◽  
Dimitri Vanhecke ◽  
Peter Wick ◽  
Peter Gehr ◽  
...  
Keyword(s):  
In Vitro System ◽  
Insight Into

Correlation Between Cellular Functions and Morphological Changes of Human Trophoblast in Vitro

1975 ◽  
Vol 33 ◽  
pp. 600-601
Author(s):  
John C. Garancis ◽  
Robert O. Hussa ◽  
Michael T. Story ◽  
Donald Yorde ◽  
Roland A. Pattillo
Keyword(s):  
Electron Microscopy ◽  
Cellular Proliferation ◽  
Morphological Changes ◽  
Culture Fluid ◽  
Cellular Functions ◽  
Human Trophoblast ◽  
Transmission Electron ◽  
Marked Activity ◽  
Malignant Trophoblast

Human malignant trophoblast cells in continuous culture were incubated for 3 days in medium containing 1 mM N6-O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) and 1 mM theophylline. The culture fluid was replenished daily. Stimulated cultures secreted many times more chorionic gonadotropin and estrogens than did control cultures in the absence of increased cellular proliferation. Scanning electron microscopy revealed remarkable surface changes of stimulated cells. Control cells (not stimulated) were smooth or provided with varying numbers of microvilli (Fig. 1). The latter, usually, were short and thin. The surface features of stimulated cells were considerably different. There was marked increase of microvilli which appeared elongated and thick. Many cells were covered with confluent polypoid projections (Fig. 2). Transmission electron microscopy demonstrated marked activity of cytoplasmic organelles. Mitochondria were increased in number and size; some giant forms with numerous cristae were observed.

The Interrelationship between Thromboxane Biosynthesis, Aggregation and 5-Hydroxytryptamine Secretion in Human Platelets in Vitro

1980 ◽  
Vol 43 (01) ◽  
pp. 038-040 ◽  
Author(s):  
L C Best ◽  
T K Holland ◽  
P B B Jones ◽  
R G G Russell
Keyword(s):  
Human Platelet ◽  
Human Platelets ◽  
Dense Granule ◽  
Thromboxane B2 ◽  
Essential Requirement ◽  
Direct Mechanism ◽  
Thromboxane Synthetase ◽  
Thromboxane Production ◽  
Higher Doses

SummaryPlatelet aggregation, secretion of 5-hydroxy tryptamine and production of thromboxane B2 were monitored simultaneously in human platelet suspensions in the absence and presence of cyclooxygenase or thromboxane synthetase inhibitors. Aggregation, secretion and thromboxane B2 formation in response to either sodium arachidonate or epinephrine were blocked by aspirin or by 1-N-butyl imidazole suggesting that thromboxane biosynthesis was an essential requirement for platelet activation by these agents. In contrast, thrombin and collagen could apparently induce aggregation and secretion via two pathways: at low doses involving thromboxane production, but at higher doses by a direct mechanism independent of thromboxane biosynthesis. In the case of ADP, inhibition of thromboxane production blocked secretion but had little effect on aggregation, indicating that secretion was probably dependent on thromboxane biosynthesis which probably occurred as a result of aggregation. Thus it appears that although the processes of thromboxane production, release of dense granule constituents and aggregation may often be intimately linked, each process can occur independently of the other, depending upon the stimulus used.

In Vitro Assay of Platelet Adhesiveness with Washed and Tanned Human Red Cells

1968 ◽  
Vol 20 (03/04) ◽  
pp. 384-396 ◽  
Author(s):  
G Zbinden ◽  
S Tomlin
Keyword(s):  
Platelet Adhesion ◽  
Sodium Citrate ◽  
Blood Platelets ◽  
In Vitro Assay ◽  
Red Cells ◽  
Platelet Adhesiveness ◽  
Vitro Assay ◽  
In Vitro System ◽  
Human Red Cells

SummaryAn in vitro system is described in which adhesion of blood platelets to washed and tannic acid-treated red cells was assayed quantitatively by microscopic observation. ADP, epinephrine and TAME produced a reversible increase in platelet adhesiveness which was antagonized by AMP. With Evans blue, polyanetholsulfonate, phthalanilide NSC 38280, thrombin and heparin at concentrations above 1-4 u/ml the increase was irreversible. The ADP-induced increase in adhesiveness was inhibited by sodium citrate, EDTA, AMP, ATP and N-ethylmaleimide. EDTA, AMP and the SH-blocker N-ethylmaleimide also reduced spontaneous platelet adhesion to red cells. No significant effects were observed with adenosine, phenprocoumon, 5-HT, phthalanilide NSC 57155, various estrogens, progestogens and fatty acids, acetylsalicylic acid and similarly acting agents, hydroxylamine, glucose and KCN. The method may be useful for the screening of thrombogenic and antithrombotic properties of drugs.

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