Tag: biochemistry

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/.

Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides

Ceramides are central intermediates of sphingolipid metabolism that also function as potent messengers in stress signaling and apoptosis. Progress in understanding how ceramides execute their biological roles is hampered by a lack of methods to manipulate their cellular levels and metabolic fate with appropriate spatiotemporal precision.*

In the article “Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides” Matthijs Kol, Ben Williams, Henry Toombs-Ruane, Henri G Franquelim, Sergei Korneev, Christian Schroeer, Petra Schwille, Dirk Trauner, Joost CM Holthuis and James A Frank report on clickable, azobenzene-containing ceramides, caCers, as photoswitchable metabolic substrates to exert optical control over sphingolipid production in cells.*

They combine atomic force microscopy on model bilayers with metabolic tracing studies in cells, and demonstrate that light-induced alterations in the lateral packing of caCers lead to marked differences in their metabolic conversion by sphingomyelin synthase and glucosylceramide synthase. These changes in metabolic rates are instant and reversible over several cycles of photoswitching. The findings described in the article disclose new opportunities to probe the causal roles of ceramides and their metabolic derivatives in a wide array of sphingolipid-dependent cellular processes with the spatiotemporal precision of light.*

The High-speed AFM in AC mode described in the article was done with NanoWorld Ultra-Short Cantilevers USC-F0.3-k0.3 with a typical stiffness of 0.3 N/m. The AFM cantilever oscillation was tuned to a frequency of 100–150 kHz and the amplitude kept below 10 nm. The scan rate was set to 25–150 Hz. Images were acquired at 256 × 256 pixel resolution. All measurements were performed at room temperature. The force applied on the sample was minimized by continuously adjusting the set point and gain during imaging. Height, error, deflection and phase-shift signals were recorded and images were line-fitted as required.*

Figure 2 b from “ Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides “ by Matthijs Kol et al. :
Photo-isomerization of caCers affects membrane fluidity and lipid domain structure in supported lipid bilayers.
(b) Atomic force microscopy of supported lipid bilayers ( SLBs )prepared as in (a). Isomerization of caCer-3 (top) and caCer-4 (bottom) to cis with UV-A light (365 nm) resulted in a fluidification inside the Lo domains, as indicated by the appearance of small fluid Ld lakes and an increased Ld/Lo area ratio. This effect was reversed on isomerization back to trans with blue light (470 nm), marked by a drop in the Ld/Lo area ratio. Scale bars, 2 μm. (c) Time-course plotting the normalized Lo area over multiple 365/470 nm irradiation cycles for caCer-3 (top) and caCer-4 (bottom).
Please refer to https://doi.org/10.7554/eLife.43230.007 for the full figure.

*Matthijs Kol, Ben Williams, Henry Toombs-Ruane, Henri G Franquelim, Sergei Korneev, Christian Schroeer, Petra Schwille, Dirk Trauner, Joost CM Holthuis, James A Frank
Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides
eLife 2019;8:e43230
DOI: 10.7554/eLife.43230
https://doi.org/10.7554/eLife.43230.001
https://doi.org/10.7554/eLife.43230.007

Please follow this external link to read the full article: https://elifesciences.org/articles/43230

Open Access The article “Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides “ by Matthijs Kol, Ben Williams, Henry Toombs-Ruane, Henri G Franquelim, Sergei Korneev, Christian Schroeer, Petra Schwille, Dirk Trauner, Joost CM Holthuis and  James A Frank 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)