Tag: afm tips

Millisecond dynamics of an unlabeled amino acid transporter

Excitatory amino acid transporters (EAATs) are important in many physiological processes and crucial for the removal of excitatory amino acids from the synaptic cleft.*

In the article “Millisecond dynamics of an unlabeled amino acid transporter “ Tina R. Matin, George R. Heath, Gerard H. M. Huysmans, Olga Boudker and Simon Scheuring develop and apply high-speed atomic force microscopy line-scanning (HS-AFM-LS) combined with automated state assignment and transition analysis for the determination of transport dynamics of unlabeled membrane-reconstituted GltPh, a prokaryotic EAAT homologue, with millisecond temporal resolution.*

Among the bulk and single-molecule techniques, high-speed atomic force microscopy ( HS-AFM ) stands out with its ability to provide real-time structural and dynamical information of single molecules. HS-AFM images label-free molecules under close-to-physiological conditions with ~0.1 nm vertical and ~1 nm lateral imaging resolution. Furthermore, HS-AFM has typically ~100 ms temporal resolution, giving access to structure–dynamics relationship of proteins, though the achievable imaging speed depends on sample characteristics like scan size and surface corrugation.

Recently in a quest to achieve higher temporal resolutions, the authors of the cited article used HS-AFM line scanning (HS-AFM-LS) for the analysis of single-protein dynamics. *

Line scanning, using a conventional AFM, has been used to study protein–protein interactions earlier. In HS-AFM-LS, the slow-scan axis (y-direction) is disabled. Therefore, instead of imaging an x/y-area, the scientists scan over one horizontal x-line several hundreds to thousands of times per second, thus reaching millisecond temporal resolution. The topographical readouts of this line are stacked one after another, resulting in kymographs of the dynamical behavior of the molecules. Therefore, HS-AFM-LS has between 2 and 3 orders of magnitude higher temporal resolution than HS-AFM imaging and should allow the detection of fast transporter dynamics and possible intermediate states that have so far escaped kinetic characterization. *

All AFM images presented in this study were taken using a HS-AFM operated in amplitude modulation mode (with typical free and setpoint amplitudes, Afree = 1.0 nm and Aset = 0.9 nm, respectively using optimized scan and feedback parameters. NanoWorld Ultra-Short Cantilevers ( NanoWorld’s AFM probe series especially dedicated for High Speed Scanning) of the USC-F1.2-k0.15 type were used. In the presented experiments, four different buffer conditions were used. *

As the authors state in their article they find that GltPh transporters can operate much faster than previously reported, with state dwell-times in the 50 ms range, and report the kinetics of an intermediate transport state with height between the outward- and inward-facing states. Transport domains stochastically probe transmembrane motion, and reversible unsuccessful excursions to the intermediate state occur. The presented approach and analysis methodology are generally applicable to study transporter kinetics at system-relevant temporal resolution.*

Figure 2 from “Millisecond dynamics of an unlabeled amino acid transporter” by Tina R. Matin et al. HS-AFM line scanning (HS-AFM-LS): millisecond temporal resolution of unlabeled transporter dynamics.: a HS-AFM image of a membrane packed with GltPh exposing the extracellular face before HS-AFM-LS (apo condition: 20 mM Tris-HCl, pH7.5, 150 mM KCl). Dashed lines indicate the position of the central scan line where subsequent HS-AFM-LS is performed. b Six seconds of a HS-AFM-LS kymograph with 3.3 ms line acquisition speed. Each transporter domain appears as a vertical line. c Projection (top) and height profile (bottom) of b. d HS-AFM image after HS-AFM-LS. The lateral position of recognizable features in a–d are indicated by arrowheads. e One second high-magnification views of dashed regions 1, 2, and 3 in b. Transport domain excursions to the inward-facing state appear as dark dwells along the vertical time axis. f Projection (top) and height profile (bottom) of e. Arrowheads indicate the position of the seven protomers in the kymograph (red: active protomer #5). g Height/time traces (gray) and state fits (red) of the active domain (protomer #5) in e. This figure is representative of the experimental sequence for the >50 replicates analyzed in this work. NanoWorld Ultra-Short Cantilevers ( NanoWorld's AFM probe series especially dedicated for High Speed Scanning) of the USC-F1.2-k0.15 type (8 μm length, nominal spring constant of 0.15 N/m, nominal resonance frequency of ∼650 kHz and quality factor of ∼1.5 in buffer) were used.
Figure 2 from “Millisecond dynamics of an unlabeled amino acid transporter” by Tina R. Matin et al.
HS-AFM line scanning (HS-AFM-LS): millisecond temporal resolution of unlabeled transporter dynamics.:
a HS-AFM image of a membrane packed with GltPh exposing the extracellular face before HS-AFM-LS (apo condition: 20 mM Tris-HCl, pH7.5, 150 mM KCl). Dashed lines indicate the position of the central scan line where subsequent HS-AFM-LS is performed. b Six seconds of a HS-AFM-LS kymograph with 3.3 ms line acquisition speed. Each transporter domain appears as a vertical line. c Projection (top) and height profile (bottom) of b. d HS-AFM image after HS-AFM-LS. The lateral position of recognizable features in a–d are indicated by arrowheads. e One second high-magnification views of dashed regions 1, 2, and 3 in b. Transport domain excursions to the inward-facing state appear as dark dwells along the vertical time axis. f Projection (top) and height profile (bottom) of e. Arrowheads indicate the position of the seven protomers in the kymograph (red: active protomer #5). g Height/time traces (gray) and state fits (red) of the active domain (protomer #5) in e. This figure is representative of the experimental sequence for the >50 replicates analyzed in this work.

*Tina R. Matin, George R. Heath, Gerard H. M. Huysmans, Olga Boudker and Simon Scheuring
Millisecond dynamics of an unlabeled amino acid transporter
Nature Communications volume 11, Article number: 5016 (2020)
DOI: https://doi.org/10.1038/s41467-020-18811-z

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

Open Access : The article “Millisecond dynamics of an unlabeled amino acid transporter” by Tina R. Matin, George R. Heath, Gerard H. M. Huysmans, Olga Boudker and Simon Scheuring 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/.

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/.