Tag: biology AFM probes

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/.

Carbon nanotube porin diffusion in mixed composition supported lipid bilayers

Lipid membranes play a key role in living systems by providing a structural barrier that separates cellular compartments. Bilayer fluidity in the lateral plane is a key property of lipid membranes, that allows the membrane to have sufficient flexibility to accommodate dynamic stresses, shape changes and rearrangements accompanying the cellular lifecycle.*

In the article “Carbon nanotube porin diffusion in mixed composition supported lipid bilayers” Kylee Sullivan, Yuliang Zhang, Joseph Lopez, Mary Lowe and Aleksandr Noy describe how they used high-speed atomic force microscopy (HS-AFM) and all-atom molecular dynamics (MD) simulations to study the behavior of CNTPs in a mixed lipid membrane consisting of DOPC lipid with a variable percentage of DMPC lipid added to it. HS-AFM data reveal that the CNTPs undergo diffusive motion in the bilayer plane.*

Motion trajectories extracted from the HS-AFM movies indicate that CNTPs exhibit diffusion coefficient values broadly similar to values reported for membrane proteins in supported lipid bilayers. The data also indicate that increasing the percentage of DMPC leads to a marked slowing of CNTP diffusion. MD simulations reveal a CNTP-lipid assembly that diffuses in the membrane and show trends that are consistent with the experimental observations. *

The above-mentioned study confirms that CNTPs mimic the major features of the diffusive movement of biological pores in lipid membranes and shows how the increase in bilayer viscosity leads to a corresponding slowdown in protein motion. It should be possible to extend this approach to studies of other membrane protein dynamics in supported lipid bilayers. The authors note that those studies, however, will need to be mindful of the challenge of unambiguous visualization of the membrane components, especially in systems that incorporate smaller proteins, such as antimicrobial peptides. Another challenge that could complicate these studies would be microscopic phase separation of the lipid matrix that could lead to complicated pore dynamics in the membrane. *

NanoWorld Ultra-Short AFM cantilevers with high-density carbon/diamond-like carbon (HDC/DLC) AFM tips of the USC-F1.2-k0.15 type were used for the high-speed atomic force microscopy described in the article. *

Figure 2 from “Carbon nanotube porin diffusion in mixed composition supported lipid bilayers” by Kylee Sullivan et al.: CNTP motion in supported lipid bilayers. (a) Representative frames (with times in seconds indicated on each image) from an HS-AFM movie showing a CNTP diffusing in a supported lipid bilayer with 80:20 DOPC-DMPC ratio (see also Supplementary Movie 2). (b) A representative trajectory for CNTP diffusion in the bilayer. The time step between each datapoint is 0.5 s. NanoWorld Ultra-Short AFM Cantilvers USC-F1.2-k0.15 were used for the HS-AFM imaging
Figure 2 a and b from “Carbon nanotube porin diffusion in mixed composition supported lipid bilayers” by Kylee Sullivan et al.:

CNTP motion in supported lipid bilayers. (a) Representative frames (with times in seconds indicated on each image) from an HS-AFM movie showing a CNTP diffusing in a supported lipid bilayer with 80:20 DOPC-DMPC ratio (see also Supplementary Movie 2). (b) A representative trajectory for CNTP diffusion in the bilayer. The time step between each datapoint is 0.5 s.
Please refer to the full article cited below for the full figure.

*Kylee Sullivan, Yuliang Zhang, Joseph Lopez, Mary Lowe and Aleksandr Noy
Carbon nanotube porin diffusion in mixed composition supported lipid bilayers
Nature Scientific Reports volume 10, Article number: 11908 (2020)
DOI: https://doi.org/10.1038/s41598-020-68059-2

Please follow this external link to read the full article: https://rdcu.be/b69wj

Open Access : The article “Carbon nanotube porin diffusion in mixed composition supported lipid bilayers” by Kylee Sullivan, Yuliang Zhang, Joseph Lopez, Mary Lowe and Aleksandr Noy 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/.

NanoWorld Screencast on Ultra-Short Cantilevers (USC) for High-Speed AFM (HS-AFM) and video rate Atomic Force Microscopy passes 1000 views mark

The screencast on Ultra-Short Cantilevers (USC) for High Speed AFM (HS-AFM)  and video rate Atomic Force Microscopy held by Mathieu Burri  has just reached the 1000 views mark. Congratulations Mathieu!

The Ultra-Short Cantilevers series combines very small cantilevers capable of resonating in the MHz regime and a very sharp and wear resistant tip and is dedicated to High-Speed AFM (HS-AFM). The Ultra-Short Cantilevers series consists of six different types of probes which cover the complete range of high speed scanning applications, from non-contact mode to contact mode, from measurements in air to measurements in liquid.

More information such as high speed videos, an image gallery and a regularly updated reference list of scientific articles on high speed AFM can be found on the dedicated website: https://www.highspeedscanning.com/ . You can also find references to scientific articles mentioning the use of NanoWorld USC AFM probes on the NanoWorld blog.

If you haven’t seen the USC screencast yet have a look at:

NanoWorld ULTRA-SHORT CANTILEVERS for High-Speed AFM

A Chinese Version of this screencast is also available: