Tag: molecular biology

Molecular and nanoscale evaluation of N-cadherin expression in invasive bladder cancer cells under control conditions or GW501516 exposure

N-cadherin is a transmembrane glycoprotein expressed by mesenchymal origin cells and is located at the adherens junctions. It regulates also cell motility and contributes to cell signaling.*

A pharmacological approach to inhibit N-cadherin expression or to block its function could be relevant to prevent disease progression and metastasis development.*

In the article “Molecular and nanoscale evaluation of N-cadherin expression in invasive bladder cancer cells under control conditions or GW501516 exposure” Céline Elie-Caille, Isabelle Lascombe, Adeline Péchery, Hugues Bittard and Sylvie Fauconnet, describe how they aimed at exploring the expression level of N-cadherin in invasive bladder cancer cells upon GW501516 exposure by both molecular biology techniques such as RTqPCR and Western blotting and atomic force microscopy (AFM) using an AFM tip functionalized with a monoclonal antibody directed against this adhesion molecule. *

The Atomic Force Microscope is a mighty nanoanalytical tool for studying biological samples under liquid, in pathological or physiological conditions, and at the scale of a single cell. It allows to characterize cells and their modification upon drug exposure or function alteration, in terms of cell surface topography or cell adhesion. *

The authors demonstrated for the first time, that the PPARβ/δ activator from a concentration of 15 µM decreased the full length N-cadherin at the mRNA and protein level and significantly reduced its cell surface coverage through the measurements of the interaction forces involving this adhesion molecule. *

Using atomic force microscopy the authors carried out a morphological and topographical analysis on bladder cancer cells of different histologic grade. *

AFM imaging was carried out in contact mode on fixed cells (with an applied force of 0.1 V), the QI mode was used for alive cell imaging, all in liquid. *

Force spectroscopy in force mapping was used for cadherin/anti-cadherin antibody measurement interactions and cadherin mapping on cells. *

NanoWorld Pyrex-Nitride PNP-TR triangular shaped silicon nitride cantilevers ( CB2 with a typical spring constant of 0.08 N/m ) were used.

For force mapping the AFM cantilevers were calibrated. The AFM probes, made of silicon nitride, were functionalized by 1% APTES (3-(Aminopropyl)triethoxysilane) in toluene during 2 h, washed extensively with toluene, and then with ethanol.
The second step consisted in an incubation in 0.2% glutaraldehyde solution during 10 min, followed by extensive washing with water. A naked AFM tip was used as a negative control.
The modified AFM tips were then incubated in 50 µg/mL primary antibody solution (N-cadherin GC-4 clone directed against the extracellular domain, N-cadherin 3B9 clone directed against the intracellular domain, E-cadherin HECD-1 clone directed against the extracellular domain) during 30 min, then washed with PBS 1X.
Finally, the functionalized AFM tip was saturated by incubation in 2 mg/mL RSA (rat serum albumin) solution during 30 min. *

Quantitative imaging AFM mode enabled to register more than hundred force spectroscopy curves per condition. The curves registered on cells were overlayed in order to highlight a specific pattern and the interaction peak areas were measured. *

Figure 1 from “Molecular and nanoscale evaluation of N-cadherin expression in invasive bladder cancer cells under control conditions or GW501516 exposure” by Céline Elie-Caille et al.: T24 and RT4 bladder cancer cell morphology and topography. a Images from control confluent cells by phase contrast microscopy. Scale bars: 200 µm. b, c AFM images obtained on control confluent cells, after glutaraldehyde fixation, in contact mode in liquid. b AFM height images. c AFM deflection images. Scale bars: 10 µm NanoWorld Pyrex-Nitride triangular PNP-TR silicon nitride AFM probes were used for the atomic force microscopy.
Figure 1 from “Molecular and nanoscale evaluation of N-cadherin expression in invasive bladder cancer cells under control conditions or GW501516 exposure” by Céline Elie-Caille et al.:
T24 and RT4 bladder cancer cell morphology and topography. a Images from control confluent cells by phase contrast microscopy. Scale bars: 200 µm. b, c AFM images obtained on control confluent cells, after glutaraldehyde fixation, in contact mode in liquid. b AFM height images. c AFM deflection images. Scale bars: 10 µm

* Céline Elie-Caille, Isabelle Lascombe, Adeline Péchery, Hugues Bittard amd Sylvie Fauconnet
Molecular and nanoscale evaluation of N-cadherin expression in invasive bladder cancer cells under control conditions or GW501516 exposure
Molecular and Cellular Biochemistry (2020) 471:113–127
DOI: https://doi.org/10.1007/s11010-020-03771-1

Please follow this external link to read the full article: https://link.springer.com/article/10.1007/s11010-020-03771-1

Open Access : The article “Molecular and nanoscale evaluation of N-cadherin expression in invasive bladder cancer cells under control conditions or GW501516 exposure” by Céline Elie-Caille, Isabelle Lascombe, Adeline Péchery, Hugues Bittard and Sylvie Fauconnet is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/.

The free energy landscape of retroviral integration

Retroviral integration, the process of covalently inserting viral DNA into the host genome, is a point of no return in the replication cycle. Yet, strand transfer is intrinsically iso-energetic and it is not clear how efficient integration can be achieved.*

In the article “The free energy landscape of retroviral integration” published in Nature Communications Willem Vanderlinden, Tine Brouns, Philipp U. Walker, Pauline J. Kolbeck, Lukas F. Milles, Wolfgang Ott, Philipp C. Nickels, Zeger Debyser and Jan Lipfert use biochemical assays, atomic force microscopy (AFM), and multiplexed single-molecule magnetic tweezers (MT) to study tetrameric prototype foamy virus (PFV) strand-transfer dynamics.*

Their finding that PFV intasomes employ auxiliary-binding sites for modulating the barriers to integration raises the question how the topology of higher-order intasomes governs integration of pathogenic retroviruses, most notably HIV. The single-molecule assays developed in this work are expected to be particularly useful to further unravel the complexity of this important class of molecular machines.*

The AFM images were recorded in amplitude modulation mode under ambient conditions and by using NanoWorld high resolution SuperSharpSilicon™ SSS-NCH cantilevers ( resonance frequency ≈300 kHz; typical end-radius 2 nm; half-cone angle <10 deg). Typical scans were recorded at 1–3 Hz line frequency, with optimized feedback parameters and at 512 × 512 pixels.*

Figure 2 e, f and g from “The free energy landscape of retroviral integration” by Willem Vanderlinden et al. (please refer to the full article for the complete figure 2 https://rdcu.be/b0R63 ) : e Atomic Force Microscopy image of intasomes incubated briefly (2 min) with supercoiled plasmid DNA, depicting a branched complex as found in ~50% of early complexes. f Atomic Force Microscopy image of a bridging complex that dominates (~80%) the population of complexes at longer (>45 min) incubation. g Atomic Force Microscopy image of a gel-purified STC
Figure 2 e, f and g from “The free energy landscape of retroviral integration” by Willem Vanderlinden et al.
(please refer to the full article for the complete figure 2 https://rdcu.be/b0R63 ) :
 e AFM image of intasomes incubated briefly (2 min) with supercoiled plasmid DNA, depicting a branched complex as found in ~50% of early complexes.
 f AFM image of a bridging complex that dominates (~80%) the population of complexes at longer (>45 min) incubation.
g AFM image of a gel-purified STC

*Willem Vanderlinden, Tine Brouns, Philipp U. Walker, Pauline J. Kolbeck, Lukas F. Milles, Wolfgang Ott, Philipp C. Nickels, Zeger Debyser, Jan Lipfert
The free energy landscape of retroviral integration
Nature Communications volume 10, Article number: 4738 (2019)
DOI: https://doi.org/10.1038/s41467-019-12649-w

Please follow this external link to read the full article: https://rdcu.be/b0R63

Open Access The article “The free energy landscape of retroviral integration“ by Willem Vanderlinden, Tine Brouns, Philipp U. Walker, Pauline J. Kolbeck, Lukas F. Milles, Wolfgang Ott, Philipp C. Nickels, Zeger Debyser and Jan Lipfert is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

More papers on High Speed Atomic Force Microscopy – list of references updated

We have updated our list of articles in the field of High-Speed AFM (HS-AFM) on the www.highspeedscanning.com website. If you would like to see what has been going on recently in the field of High-Speed AFM (HS-AFM) then you are welcome to have a look at: http://www.highspeedscanning.com/hs-afm-references.html

We are aware that this list is far from complete so if you have used one of our Ultra-Short Cantilevers (USC) for high speed atomic force microscopy in the research for your publication and your article isn’t listed yet then please let us know. We will be happy to add it to the list.

NanoWorld Ultra-Short Cantilevers (USC) for High-Speed AFM (HS-AFM)
NanoWorld Ultra-Short Cantilevers (USC) for High-Speed AFM (HS-AFM)