Tag: membrane biophysics

Correlation of membrane protein conformational and functional dynamics

Membrane proteins (MPs) reside in the plasma membrane and perform various biological processes including ion transport, substrate transport, and signal transduction.*

Function-related conformational changes in membrane proteins occur in times scales ranging from nanoseconds to seconds.*

Obtaining time-resolved dynamic information of MPs in their membrane environment is still a major challenge.*

Although High Speed Atomic Force Microscopy (HS-AFM) images label-free samples such as DNA, soluble proteins, MPs, and intrinsically disordered proteins at ~1n~m lateral, ~0.1 nm vertical and ~100 ms temporal solution in aqueous environment and at ambient temperature and pressure, its temporal resolution is too slow to characterize many dynamic biological processes.*

In order to overcome this limitation Raghavendar Reddy Sanganna Gari, Joel José Montalvo-Acosta, George R. Heath, Yining Jiang, Xiaolong Gao, Crina M. Nimigean, Christophe Chipot and Simon Scheuring in their article Correlation of membrane protein conformational and functional dynamics use High Speed Atomic Force Microscopy Height Spectroscopy ( HS-AFM-HS) to characterize the microsecond timescale conformational changes of an integral-MP model system, i.e., the outer membrane protein G (OmpG) in a membrane environment.*

The positioning of the AFM tip is guided by HS-AFM imaging immediately before HS-AFM-HS-operation.*

NanoWorld Ultra-Short Cantilevers (USC) of the USC-F1.2-k0.15 type were used for the HS-AFM and HS-AFM-HS presented in the article.*

Figure 1 from “Correlation of membrane protein conformational and functional dynamics” by R.R. Sanganna Gari et al: HS-AFM imaging of OmpG in lipid bilayers at pH 7.6 and pH 5.0. a OmpG at pH 7.6 (Supplementary movie 1, left; frame rate: 200 ms per frame). A OmpG dimer is highlighted with dashed outline in all frames. Arrowheads in t = 11.6 s: Loop-6 fluctuating over the lumen. Arrowhead in t = 12.0 s: Fully open state. b Correlation average (n = 2752) of the HS-AFM movie frames (344 frames recorded over 68.8 s, full color scale: 0.0 nm < height < 1.25 nm, where the membrane level was set to 0.0 nm). c Correlation average of OmpG dimers. The topography outline (based on the molecular structure in 1e), serves as a visual guide to locate loop-6 and loop-2 in the topography and is highlighted by the dashed outline (the position of loop-6 is indicated by the asterisk based on its location in the structure (e)). Inner dashed outline show barrel lumen. d Standard deviation (std) map (n = 2752) from the averaging process in (b) (full color scale from blue to red: 0.05 nm < std < 0.19 nm) and topography outlines as in (c). e X-ray structure (PDB 2iwv) of the open OmpG conformation. Loop-6 (arrowhead L6) stands out of the image plane towards the viewer. Loop-2 (L2) forms a beta strand pointing away from the β-barrel, well detected by HS-AFM in the open state (b). f OmpG at pH 5.0 (Supplementary movie 1, right; frame rate: 200 ms per frame). A OmpG dimer is highlighted with dashed outline in all frames. g Correlation average (n = 2472) of the HS-AFM movie frames (309 frames recorded over 61.8 s, full color scale: 0.0 nm < height < 0.7 nm, where the membrane level was set to 0.0 nm). h Correlation average of OmpG dimers. For comparison, the topography outline of the open state (e) is shown (the position of loop-6 is indicated by the asterisk). i Standard deviation (std) map (n = 2472) from the averaging process in (g) (full color scale from blue to red: 0.04 nm < std < 0.07 nm) and topography outlines as in (h). j X-ray structure (PDB 2iww) of the closed OmpG conformation shown in the same orientation as in (e). Loop-6 (L6) folds over the β-barrel lumen in a lid-like manner. Loop-2 (L2) does not form a β-strand in the closed state, in agreement with absence of topography in this region in (h). Black dashed line: outline based on (e) for comparison. NanoWorld USC-F1.2-k0.15 AFM probes were used for the HS-AFM.
Figure 1 from “Correlation of membrane protein conformational and functional dynamics” by R.R. Sanganna Gari et al:
HS-AFM imaging of OmpG in lipid bilayers at pH 7.6 and pH 5.0.
a OmpG at pH 7.6 (Supplementary movie 1, left; frame rate: 200 ms per frame). A OmpG dimer is highlighted with dashed outline in all frames. Arrowheads in t = 11.6 s: Loop-6 fluctuating over the lumen. Arrowhead in t = 12.0 s: Fully open state. b Correlation average (n = 2752) of the HS-AFM movie frames (344 frames recorded over 68.8 s, full color scale: 0.0 nm < height < 1.25 nm, where the membrane level was set to 0.0 nm). c Correlation average of OmpG dimers. The topography outline (based on the molecular structure in 1e), serves as a visual guide to locate loop-6 and loop-2 in the topography and is highlighted by the dashed outline (the position of loop-6 is indicated by the asterisk based on its location in the structure (e)). Inner dashed outline show barrel lumen. d Standard deviation (std) map (n = 2752) from the averaging process in (b) (full color scale from blue to red: 0.05 nm < std < 0.19 nm) and topography outlines as in (c). e X-ray structure (PDB 2iwv) of the open OmpG conformation. Loop-6 (arrowhead L6) stands out of the image plane towards the viewer. Loop-2 (L2) forms a beta strand pointing away from the β-barrel, well detected by HS-AFM in the open state (b). f OmpG at pH 5.0 (Supplementary movie 1, right; frame rate: 200 ms per frame). A OmpG dimer is highlighted with dashed outline in all frames. g Correlation average (n = 2472) of the HS-AFM movie frames (309 frames recorded over 61.8 s, full color scale: 0.0 nm < height < 0.7 nm, where the membrane level was set to 0.0 nm). h Correlation average of OmpG dimers. For comparison, the topography outline of the open state (e) is shown (the position of loop-6 is indicated by the asterisk). i Standard deviation (std) map (n = 2472) from the averaging process in (g) (full color scale from blue to red: 0.04 nm < std < 0.07 nm) and topography outlines as in (h). j X-ray structure (PDB 2iww) of the closed OmpG conformation shown in the same orientation as in (e). Loop-6 (L6) folds over the β-barrel lumen in a lid-like manner. Loop-2 (L2) does not form a β-strand in the closed state, in agreement with absence of topography in this region in (h). Black dashed line: outline based on (e) for comparison.
Figure 2 from “Correlation of membrane protein conformational and functional dynamics” by R.R. Sanganna Gari et al: Single channel electrophysiology and HS-AFM height spectroscopy recordings of OmpG in lipid bilayers. Representative 60-ms segments of OmpG single channel recordings at pH 7.6 (a) and pH 5.0 (b) at +40 mV membrane potential (longer traces in Supplementary Figs. 2 and 3). Cartoon representation of single channel recording experimental setup is shown in inset of (a). OmpG (yellow) in open state (PDB:2IWV) is placed in a lipid bilayer (green) surrounded by buffer (light blue shade) and potassium and chloride ions are shown as red and blue spheres. Red arrow indicates ion flow through OmpG in response to voltage application. Dwell time histograms of open and closed states at pH 7.6 (c) and pH 5.0 (d) from single-channel recordings (see Supplementary Table 1). Representative 60-ms segments of OmpG HS-AFM-HS recordings at pH 7.6 (e) and pH 5.0 (f) (longer traces in Supplementary Fig. 4). Cartoon representation of HS-AFM height spectroscopy experimental setup is shown in inset of (e). An oscillating AFM tip (orange) detects conformational changes of loop motion. Dwell time histograms of open and closed states at pH 7.6 (g) and pH 5.0 (h) from HS-AFM-HS recordings (Supplementary Table 2). In HS-AFM-HS the low state represents the open state, where the HS-AFM tip can descend into the β-barrel, and the high state represents the closed state, where loop-6 covers the beta barrel barring access of the HS-AFM tip to the cavity. All current-time and height-time traces were filtered at 20 kHz during analysis. The state dwell-time histograms are shown using log binning for better visualization of the components49. Red traces in (a) and (b) represent idealized current-time traces using clampfit software. Red traces in (e) and (f) represent idealized height-time traces using the STaSI algorithm (see Methods). NanoWorld USC-F1.2-k0.15 AFM probes were used for the HS-AFM-HS.
Figure 2 from “Correlation of membrane protein conformational and functional dynamics” by R.R. Sanganna Gari et al:
Single channel electrophysiology and HS-AFM height spectroscopy recordings of OmpG in lipid bilayers.
Representative 60-ms segments of OmpG single channel recordings at pH 7.6 (a) and pH 5.0 (b) at +40 mV membrane potential (longer traces in Supplementary Figs. 2 and 3). Cartoon representation of single channel recording experimental setup is shown in inset of (a). OmpG (yellow) in open state (PDB:2IWV) is placed in a lipid bilayer (green) surrounded by buffer (light blue shade) and potassium and chloride ions are shown as red and blue spheres. Red arrow indicates ion flow through OmpG in response to voltage application. Dwell time histograms of open and closed states at pH 7.6 (c) and pH 5.0 (d) from single-channel recordings (see Supplementary Table 1). Representative 60-ms segments of OmpG HS-AFM-HS recordings at pH 7.6 (e) and pH 5.0 (f) (longer traces in Supplementary Fig. 4). Cartoon representation of HS-AFM height spectroscopy experimental setup is shown in inset of (e). An oscillating AFM tip (orange) detects conformational changes of loop motion. Dwell time histograms of open and closed states at pH 7.6 (g) and pH 5.0 (h) from HS-AFM-HS recordings (Supplementary Table 2). In HS-AFM-HS the low state represents the open state, where the HS-AFM tip can descend into the β-barrel, and the high state represents the closed state, where loop-6 covers the beta barrel barring access of the HS-AFM tip to the cavity. All current-time and height-time traces were filtered at 20 kHz during analysis. The state dwell-time histograms are shown using log binning for better visualization of the components49. Red traces in (a) and (b) represent idealized current-time traces using clampfit software. Red traces in (e) and (f) represent idealized height-time traces using the STaSI algorithm (see Methods).

*Raghavendar Reddy Sanganna Gari, Joel José Montalvo-Acosta, George R. Heath, Yining Jiang, Xiaolong Gao, Crina M. Nimigean, Christophe Chipot and Simon Scheuring
Correlation of membrane protein conformational and functional dynamics
Nature Communications volume 12, Article number: 4363 (2021)
DOI: https://doi.org/10.1038/s41467-021-24660-1

Please follow this external link to read the full article: https://rdcu.be/cAE2S

Open Access : The article “Correlation of membrane protein conformational and functional dynamics” by Raghavendar Reddy Sanganna Gari, Joel José Montalvo-Acosta, George R. Heath, Yining Jiang, Xiaolong Gao, Crina M. Nimigean, Christophe Chipot 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/.

Human ESCRT-III polymers assemble on positively curved membranes and induce helical membrane tube formation

The Endosomal Sorting Complex Required for Transport-III (ESCRT-III) is part of a conserved membrane remodeling machine. ESCRT-III employs polymer formation to catalyze inside-out membrane fission processes in a large variety of cellular processes, including budding of endosomal vesicles and enveloped viruses, cytokinesis, nuclear envelope reformation, plasma membrane repair, exosome formation, neuron pruning, dendritic spine maintenance, and preperoxisomal vesicle biogenesis.*

How membrane shape influences ESCRT-III polymerization and how ESCRT-III shapes membranes is yet unclear.*

In the article “Human ESCRT-III polymers assemble on positively curved membranes and induce helical membrane tube formation” Aurélie Bertin, Nicola de Franceschi, Eugenio de la Mora, Sourav Maity, Maryam Alqabandi, Nolwen Miguet, Aurélie di Cicco, Wouter H. Roos, Stéphanie Mangenot, Winfried Weissenhorn and Patricia Bassereau describe how human core ESCRT-III proteins, CHMP4B, CHMP2A, CHMP2B and CHMP3 are used to address this issue in vitro by combining membrane nanotube pulling experiments, cryo-electron tomography and Atomic Force Microscopy.*

The authors show that CHMP4B filaments preferentially bind to flat membranes or to tubes with positive mean curvature.*

The results presented in the article cited above underline the versatile membrane remodeling activity of ESCRT-III that may be a general feature required for cellular membrane remodeling processes.*

The authors provide novel insight on how mechanics and geometry of the membrane and of ESCRT-III assemblies can generate forces to shape a membrane neck.*

NanoWorld Ultra-Short AFM Cantilevers USC-F1.2-k0.15 were used for the High-speed Atomic Force Microscopy ( HS-AFM ) experiments presented in this article.*

Figure 1 from «Human ESCRT-III polymers assemble on positively curved membranes and induce helical membrane tube formation” by Aurélie Bertin et al.: CHMP4-ΔC flattens LUVs and binds preferentially to flat membranes or to membranes with a positive mean curvature. 1a CHMP4B-ΔC spirals observed by HS-AFM on a lipid bilayer. Scale bar: 50 nm. Please refer to the full article for the complete figure: https://rdcu.be/b5rOe
Figure 1 from «Human ESCRT-III polymers assemble on positively curved membranes and induce helical membrane tube formation” by Aurélie Bertin et al.:
CHMP4-ΔC flattens LUVs and binds preferentially to flat membranes or to membranes with a positive mean curvature.
1a CHMP4B-ΔC spirals observed by HS-AFM on a lipid bilayer. Scale bar: 50 nm.
Please refer to the full article for the complete figure: https://rdcu.be/b5rOe

*Aurélie Bertin, Nicola de Franceschi, Eugenio de la Mora, Sourav Maity, Maryam Alqabandi, Nolwen Miguet, Aurélie di Cicco, Wouter H. Roos, Stéphanie Mangenot, Winfried Weissenhorn and Patricia Bassereau
Human ESCRT-III polymers assemble on positively curved membranes and induce helical membrane tube formation
Nature Communications volume 11, Article number: 2663 (2020)
DOI: https://doi.org/10.1038/s41467-020-16368-5

Please follow this external link to read the full article: https://rdcu.be/b5rOe

Open Access The article “ Human ESCRT-III polymers assemble on positively curved membranes and induce helical membrane tube formation “ by Aurélie Bertin, Nicola de Franceschi, Eugenio de la Mora, Sourav Maity, Maryam Alqabandi, Nolwen Miguet, Aurélie di Cicco, Wouter H. Roos, Stéphanie Mangenot, Winfried Weissenhorn and Patricia Bassereau 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/.

Adhesion strategies of Dictyostelium discoideum – a force spectroscopy study

“Motile cells require reversible adhesion to solid surfaces to accomplish force transmission upon locomotion. In contrast to mammalian cells, Dictyostelium discoideum ( a soil dwelling amoeba) cells do not express integrins forming focal adhesions but are believed to rely on more generic interaction forces that guarantee a larger flexibility; even the ability to swim has been described for Dictyostelium discoideum (D.d.).”*

In order to understand the origin of D.d. adhesion, Nadine Kamprad, Hannes Witt, Marcel Schröder, Christian Titus Kreis, Oliver Bäumchen, Andreas Janshoff and Marco Tarantola  describe in their publication “Adhesion strategies of Dictyostelium discoideum – a force spectroscopy study”* how they realized and modified a variety of conditions for the amoeba comprising the absence and presence of the specific adhesion protein Substrate Adhesion A (sadA), glycolytic degradation, ionic strength, surface hydrophobicity and strength of van der Waals interactions by generating tailored model substrates. By employing AFM-based single cell force spectroscopy (using NanoWorld Arrow-TL2 tipless cantilevers) they could show that experimental force curves upon retraction exhibit two regimes described in detail in the article cited above. The study describes a versatile mechanism that allows the cells to adhere to a large variety of natural surfaces under various conditions.

Fig. 2 A from "Adhesion strategies of Dictyostelium discoideum – a force spectroscopy study": Cell parametrization: β, angle between the normal on the cell membrane and the cell axis; R1, contact radius between the cell and substrate; R0, equatorial cell radius; R2, contact radius between the cell and cantilever, ϕ1 contact angle towards the substrate; ϕ2, contact angle between the cell and cantilever, in the background is a section of the confocal image in B. B: morphology of the carA-1-GFP labelled D.d. cell attached to the cantilever subjected to a pulling force of 0.2 nN. NanoWorld Arrow-TL2 tipless cantilevers were used.
Fig. 2 A from “Adhesion strategies of Dictyostelium discoideum – a force spectroscopy study” by N. Kamprad et al.: Cell parametrization: β, angle between the normal on the cell membrane and the cell axis; R1, contact radius between the cell and substrate; R0, equatorial cell radius; R2, contact radius between the cell and cantilever, ϕ1 contact angle towards the substrate; ϕ2, contact angle between the cell and cantilever, in the background is a section of the confocal image in B. B: morphology of the carA-1-GFP labelled D.d. cell attached to the cantilever subjected to a pulling force of 0.2 nN.

*Nadine Kamprad, Hannes Witt, Marcel Schröder, Christian Titus Kreis, Oliver Bäumchen, Andreas Janshoff, Marco Tarantola
Adhesion strategies of Dictyostelium discoideum – a force spectroscopy study
Nanoscale, 2018, 10, 22504-22519
DOI: 10.1039/C8NR07107A

To read the full article follow this external link: https://pubs.rsc.org/en/content/articlehtml/2018/nr/c8nr07107a

Open Access The article “Adhesion strategies of Dictyostelium discoideum – a force spectroscopy study” by Nadine Kamprad, Hannes Witt, Marcel Schröder, Christian Titus Kreis, Oliver Bäumchen, Andreas Janshoff and Marco Tarantola is licensed under a Creative Commons Attribution 3.0 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/3.0/.