Tag Archives: biophysics

Nanoscale dipole dynamics of protein membranes studied by broadband dielectric microscopy

The dielectric permittivity of membranes is important for many fundamental electrophysiological functions like selective transport in ion channels, action potential propagation and energy generation.*

In their article “Nanoscale dipole dynamics of protein membranes studied by broadband dielectric microscopy” George Gramse, Andreas Schönhals and Ferry Kienberger investigate the nearfield dipole mobility of protein membranes in a wide frequency range from 3 kHz to 10 GHz.*

They achieved their results by adding the frequency as a second fundamental dimension to quantitative dielectric microscopy thereby demonstrating the possibilities of broadband dielectric microscopy for the investigation of dynamic processes in cell bioelectricity at the individual molecular level. Furthermore, the technique may also shed light on local dynamic processes in related materials science applications like semiconductor research or nano-electronics.*

All AFM measurements were carried out at 25 °C using a NANOSENSORS Platinum Silicide AFM probe ( PtSi-FM ).

Fig. 2 from “Nanoscale dipole dynamics of protein membranes studied by broadband dielectric microscopy” by Gramse et al.: image a) shows the AFM topography and image b) shows the corresponding C′′(z)/C′′dry(z) image obtained in lift mode at z = 10 nm above the last scan line and at a frequency of ω = 10 kHz (inset at 1 MHz). The corresponding topography and C′′(z)/C′′dry(z) profile lines are shown in  image c). Solid lines correspond to profile lines at 10 kHz and the dashed line to 1 MHz. Image d) shows the normalized dielectric spectra on the substrate and protein membrane at constant height z′ = 15 nm and lift mode z = 15 nm. Black solid lines represent fitting with eqn (1) and (2). image e) shows the resulting complex dielectric functions ε′r(f) and ε′′r(f)2 (using the relation ε′′r(f) = −(π/2∂)ε′r/∂ln(2πf)38). All measurements are carried out at 25 °C using conductive and wear-resistant Platinum Silicide AFM probes  (PtSi-FM ) from NANOSENSORS (Germany). Humidity was changed and left to stabilize for 2–3 hours. Imaging conditions were adjusted to maintain the lift distance for the dielectric images identical.

Fig. 2 from “Nanoscale dipole dynamics of protein membranes studied by broadband dielectric microscopy” by Gramse et al.: (a) AFM topography and (b) corresponding C′′(z)/C′′dry(z) image obtained in lift mode at z = 10 nm above the last scan line and at a frequency of ω = 10 kHz (inset at 1 MHz). The corresponding topography and C′′(z)/C′′dry(z) profile lines are shown in (c). Solid lines correspond to profile lines at 10 kHz and the dashed line to 1 MHz. (d) Normalized dielectric spectra on the substrate and protein membrane at constant height z′ = 15 nm and lift mode z = 15 nm. Black solid lines represent fitting with eqn (1) and (2). (e). Resulting complex dielectric functions ε′r(f) and ε′′r(f)2 (using the relation ε′′r(f) = −(π/2∂)ε′r/∂ln(2πf)38).
All measurements are carried out at 25 °C using PtSi-FM tips from NANOSENSORS (Germany). Humidity was changed and left to stabilize for 2–3 hours. Imaging conditions were adjusted to maintain the lift distance for the dielectric images identical.

*Georg Gramse, Andreas Schönhals, Ferry Kienberger
Nanoscale dipole dynamics of protein membranes studied by broadband dielectric microscopy
Nanoscale, 2019, 11, 4303-4309
DOI: 10.1039/C8NR05880F

Please follow this external link for the full article: https://pubs.rsc.org/en/content/articlehtml/2019/nr/c8nr05880f

Open Access The article “Nanoscale dipole dynamics of protein membranes studied by broadband dielectric microscopy” by George Gramse, Andreas Schönhals and Ferry Kienberger 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. To view a copy of this license, visit https://creativecommons.org/licenses/by/3.0/

Morphological Evaluation of Meta-stable Oligomers of α-Synuclein with Small-Angle Neutron Scattering

Amyloidogenesis of α-synuclein (αS) is considered to be a pathological phenomenon related to Parkinson’s disease (PD).

In the publication “Morphological Evaluation of Meta-stable Oligomers of α-Synuclein with Small-Angle Neutron Scattering”  Ghibom Bhak, Soonkoo Lee, Tae-Hwan Kim, Ji-Hye Lee, Jee Eun Yang, Keehyoung Joo, Jooyoung Lee, Kookheon Char and Seung R. Paik have studied one particular type of meta-stable αS oligomers (Meta-αS-Os) since they act as a growing unit to exhibit the accelerated amyloid fibril formation in the presence of external stimuli such as shear force10, temperature change11, pH12, and organic solvents13 which are suspected to alter the structure of Meta-αS-Os into a self-associative state.*

Figure 2 from Ghibom Bhak et al., “Morphological Evaluation of Meta-stable Oligomers of α-Synuclein with Small-Angle Neutron Scattering” Unit-assembly of Meta-αS-Os. (a) 3-D AFM images of the scattered Meta-αS-Os species and an extended fibrillar structure on the mica surface. Black arrow represents the heat treatment at 80 °C for 30 min. (b) Thio-T binding fluorescence of αS after the centrifugal membrane filtration in the absence or presence of NaCl at various concentrations of 0, 0.015, 0.15, 1.5, 15, and 150 mM. (c) TEM images of the αS oligomers before (i) and after the membrane filtration at various concentrations of 0 (ii), 0.015 (iii), 0.15 (iv), 1.5 (v), 15 (vi) or 150 mM (vii). (d) AFM image showing coexistence of the oligomers and the amyloid fibrils at 1.5 mM NaCl following the membrane filtration. (e) AFM height profile of the oligomeric and fibrillar species. The oligomeric and fibrillar species of αS on the mica were analyzed with AFM in a tapping mode with a Super Sharp AFM probe (SSS-NCHR, NANOSENSORS, Switzerland).
Figure 2 from Ghibom Bhak et al., “Morphological Evaluation of Meta-stable Oligomers of α-Synuclein with Small-Angle Neutron Scattering”:
Unit-assembly of Meta-αS-Os. (a) 3-D AFM images of the scattered Meta-αS-Os species and an extended fibrillar structure on the mica surface. Black arrow represents the heat treatment at 80 °C for 30 min. (b) Thio-T binding fluorescence of αS after the centrifugal membrane filtration in the absence or presence of NaCl at various concentrations of 0, 0.015, 0.15, 1.5, 15, and 150 mM. (c) TEM images of the αS oligomers before (i) and after the membrane filtration at various concentrations of 0 (ii), 0.015 (iii), 0.15 (iv), 1.5 (v), 15 (vi) or 150 mM (vii). (d) AFM image showing coexistence of the oligomers and the amyloid fibrils at 1.5 mM NaCl following the membrane filtration. (e) AFM height profile of the oligomeric and fibrillar species.

“For the assessment of heat-induced oligomeric unit assembly, an aliquot (10 μl) containing αS oligomers was placed on a cleaved mica surface. Following 30-min incubation at room temperature, the mica was immersed in fresh 20 mM Mes (pH 6.5) and incubated at 80 °C for another 30 min. After the heat-treated mica was cleaned with excessive distillated water, the oligomeric and fibrillar species of αS on the mica were analyzed with AFM in a tapping mode with a Super Sharp AFM tip (SSS-NCHR, NANOSENSORS, Switzerland). To reveal coexistence of the oligomers and fibrils, the αS species (10 μl) was adsorbed on the mica coated with poly-l-lysine for 5 min at room temperature. After the mica was washed with distilled water 5 times and dried in vacuum chamber, AFM analysis was done in a tapping mode.”*

“Meta-αS-Os have played a growing unit for the unit-assembly-based facilitated amyloid fibril formation. Their unit-assembly was examined on the surface of mica immersed in 20 mM Mes (pH 6.5) at 80 °C for 30 min. In the 3-D AFM images, the scattered Meta-αS-Os on the mica surface became aligned with each other to form the extended fibrils following the heat treatment (Fig. 2a). This phenomenon reflects general two-step process of protein-surface interaction showing initial reversible binding of the oligomers to the surface followed by their irreversible stabilization into the amyloid fibrils via the transition of Meta-αS-Os into type-Bon oligomers upon the heat treatment. […] AFM analysis revealed that the particulates and the short fibrils of αS obtained with 1.5 mM NaCl had almost identical height profile with the maximum height of 4 nm (Fig. 2d,e).”*

[…]

“AFM analysis revealed that the particulates and the short fibrils of αS obtained with 1.5 mM NaCl had almost identical height profile with the maximum height of 4 nm (Fig. 2d,e).”*

*Ghibom Bhak, Soonkoo Lee, Tae-Hwan Kim, Ji-Hye Lee, Jee Eun Yang, Keehyoung Joo, Jooyoung Lee, Kookheon Char and Seung R. Paik
Morphological Evaluation of Meta-stable Oligomers of α-Synuclein with Small-Angle Neutron Scattering
Nature Scientific Reports, volume 8, Article number: 14295 (2018)
DOI: https://doi.org/10.1038/s41598-018-32655-0

Open Access: The article “Morphological Evaluation of Meta-stable Oligomers of α-Synuclein with Small-Angle Neutron Scattering” by  Ghibom Bhak et al. 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/.

Substrate properties modulate cell membrane roughness by way of actin filaments

“Cell membrane roughness has been proposed as a sensitive feature to reflect cellular physiological conditions”*
In the article “Substrate properties modulate cell membrane roughness by way of actin filaments” Chao-Hung Chang, Hsiao-Hui Lee, and Chau-Hwang Lee employed the non-interferometric wide-field optical profilometry (NIWOP) technique to measure the membrane roughness of living mouse embryonic fibroblasts with different conditions of the culture substrate to find out whether membrane roughness is associated with the substrate properties. By controlling the surface density of fibronectin (FN) coated on the substrate, they found that cells exhibited higher membrane roughness as the FN density increased in company with larger focal adhesion (FA) sizes.

The examination of membrane roughness was also confirmed with atomic force microscopy.
The long cantilever of NANOSENSORS uniqprobe qp-SCONT AFM probes ( 125-μm long, spring constant 0.01 N/m.) was used to observe the membrane topography on living MEFs.

If you would like to learn more about the NANOSENSORS uniqprobe AFM probes series which offers soft, drift-reduced AFM probes with unsurpassed small variation in spring constant and resonance frequency mainly for biology and life science applications but also for other aplications such as high speed scanning then please have a look at our recently updated Uniqprobe brochure: https://www.nanosensors.com/pdf/NANOSENSORS-uniqprobe-brochure.pdf .

Supplementary Figure S1 from Chao-Hung Chang et al. “Substrate properties modulate cell membrane roughness by way of actin filaments”: Images of membrane topography determined by atomic force microscopy (AFM). MEFs were seeded on the polymer coverslip-bottom μ-dishes coated with 0 or 10 μg/ml FN for 6 hours for the measurement of membrane roughness by AFM. The regions marked by the white squares in the bright-field images are displayed in the membrane topography. Scale bar, 10 μm. NANOSENSORS uniqprobe qp-SCONT AFM probes(long cantilever length 125 um, spring constant 0.01 N/m) were used.
Supplementary Figure S1 from Chao-Hung Chang et al. “Substrate properties modulate cell membrane roughness by way of actin filaments”: Images of membrane topography determined by atomic force microscopy (AFM). MEFs were seeded on the polymer coverslip-bottom μ-dishes coated with 0 or 10 μg/ml FN for 6 hours for the measurement of membrane roughness by AFM. The regions marked by the white squares in the bright-field images are displayed in the membrane topography. Scale bar, 10 μm.

*Chao-Hung Chang, Hsiao-Hui Lee, Chau-Hwang Lee
Substrate properties modulate cell membrane roughness by way of actin filaments
Nature Scientific Reports, volume 7, Article number: 9068 (2017)
DOI: https://doi.org/10.1038/s41598-017-09618-y

Please follow this external link for the full article: https://rdcu.be/bdZm9

Open Access The article “Substrate properties modulate cell membrane roughness by way of actin filaments” by  Chao-Hung Chang et al. 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/.