biology AFM probes

Cholesterol-regulated cellular stiffness may enhance evasion of NK cell-mediated cytotoxicity in gastric cancer stem cells

Gastric cancer has a high rate of recurrence, and as such, immunotherapy strategies are being investigated as a potential therapeutic strategy. *

Although the involvement of immune checkpoints in immunotherapy is well studied, biomechanical cues, such as target cell stiffness, have not yet been subject to the same level of investigation. *

Changes in the cholesterol content of the cell membrane directly influence tumor cell stiffness. *

In the article “Cholesterol-regulated cellular stiffness may enhance evasion of NK cell-mediated cytotoxicity in gastric cancer stem cells” Lijuan Zhu and Hongjin Wang investigate the effect of cholesterol on NK cell-mediated killing of gastric cancer stem-like cells. *

They report that surviving tumor cells with stem-like properties elevated cholesterol metabolism to evade NK cell cytotoxicity. *

Inhibition of cholesterol metabolism enhances NK cell-mediated killing of gastric cancer stem-like cells, highlighting a potential avenue for improving immunotherapy efficacy. *

This study suggests a possible effect of cancer cell stiffness on immune evasion and offers insights into enhancing immunotherapeutic strategies against tumors. *

Measurement of cell stiffness by AFM:

A customized commercially available atomic force microscope (AFM) and NanoWorld Pyrex-Nitride PNP-TR AFM probes were used for the measurement of cell stiffness by AFM. *

Atomic force microscopy cell stiffness was measured according to standard methodology. *

AFM force curves were captured with a customized AFM placed atop an inverted optical microscope that had a heating stage for live-cell imaging and a ×20 objective. Using an XY stage, the materials were moved until the desired cell, which could be observed under an optical microscope, was positioned beneath the AFM tip. Using a NanoWorld PNP-TR-B AFM cantilever (NanoWorld), the force curves on the cell were collected at a rate of ~ 5 μm·s−1 in the relative trigger mode (15 nm trigger threshold). *

By utilizing a thermal tuning and the deflection sensitivity of 170 nm·V−1, the AFM cantilever spring constant was determined to be 0.08 N·m−1. *

Single cells were measured both before and after treatment at 37 °C. The force curves were processed using the AFM’s analysis software, which also computed the Young’s modulus of the sample. This was accomplished by fitting the approach curve to an indentation of less than 500 nm (to account for stiffness) and assuming a cortical Poisson’s ratio of 0.3.*

NanoWorld Pyrex-Nitride PNP AFM probe - silicon nitride AFM cantilever and silicon nitride AFM tip — NanoWorld Pyrex-Nitride AFM probe series – AFM tip and AFM cantilever made of silicon nitride

*Lijuan Zhu and Hongjin Wang
Cholesterol-regulated cellular stiffness may enhance evasion of NK cell-mediated cytotoxicity in gastric cancer stem cells
FEBS Open Bio, Volume 14, Issue 5, May 2024, Pages 855-866
DOI: https://doi.org/10.1002/2211-5463.13793

Open Access The article “Cholesterol-regulated cellular stiffness may enhance evasion of NK cell-mediated cytotoxicity in gastric cancer stem cells” by Lijuan Zhu and Hongjin Wang 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/.

Mechanism for Vipp 1 spiral formation, ring biogenesis, and membrane repair

The ESCRT-III-like protein Vipp1 couples filament polymerization with membrane remodeling. It assembles planar sheets as well as 3D rings and helical polymers, all implicated in mitigating plastid-associated membrane stress. The architecture of Vipp1 planar sheets and helical polymers remains unknown, as do the geometric changes required to transition between polymeric forms. *

In the article “Mechanism for Vipp1 spiral formation, ring biogenesis, and membrane repair” Souvik Naskar, Andrea Merino, Javier Espadas, Jayanti Singh, Aurelien Roux, Adai Colom and Harry H. Low show how cyanobacterial Vipp1 assembles into morphologically-related sheets and spirals on membranes in vitro.*

The spirals converge to form a central ring similar to those described in membrane budding. Cryo-EM structures of helical filaments reveal a close geometric relationship between Vipp1 helical and planar lattices. Moreover, the helical structures reveal how filaments twist—a process required for Vipp1, and likely other ESCRT-III filaments, to transition between planar and 3D architectures. *

Overall, the authors’ results provide a molecular model for Vipp1 ring biogenesis and a mechanism for Vipp1 membrane stabilization and repair, with implications for other ESCRT-III systems. *

NanoWorld Ultra-Short Cantilevers USC-F0.3-k0.3 for High-Speed AFM (HS-AFM) with a typical spring constant of 0.3 N nm−1 and a typical resonance frequency of about 300 kHz were used for image acquisition with fast scanning atomic force microscopy.*

Fig. 2 from Souvik Naskar et al. 2024 “Mechanism for Vipp1 spiral formation, ring biogenesis, and membrane repair”:Vipp1 assembles dynamic networks of spirals, rings and sheets on membrane a, F-AFM phase timecourse showing Vipp1 recruitment to the highly curved edge of membrane patches. Scan rate, 70 Hz; 256 × 256 pixels. The area in the dashed box is enlarged in b. b, Spiral and ring formation localized to the membrane edge. Scan rate, 70 Hz; 256 × 256 pixels. c, Left, phase timecourse showcasing a dense network of sheets, spirals, and rings that ultimately cover the entire membrane plane. Right, average of six F-AFM height images. Scan rate, 120 Hz; 256 × 256 pixels. d, Average F-AFM height image showing Vipp1 sheet, spiral, and ring detail. Red arrows mark the sheet branching into filaments ~13 nm wide. Scan rate, 20 Hz; 256 × 256 pixels. e, Vipp1 sheet and spiral filament height offset from the membrane. f–i, Quantification of Vipp1 filament and spiral characteristics. n = 124, 13, 278, and 278 independent measurements for panels f, g, h, and i, respectively. Error bars show one s.d. of the mean. NanoWorld Ultra-Short Cantilevers USC-F0.3-k0.3-10 with a typical spring constant of 0.3 N nm−1 and a typical resonance frequency of about 300 kHz were used for image acquisition. — Fig. 2 from Souvik Naskar et al. 2024 “Mechanism for Vipp1 spiral formation, ring biogenesis, and membrane repair”:
Vipp1 assembles dynamic networks of spirals, rings and sheets on membrane
a, F-AFM phase timecourse showing Vipp1 recruitment to the highly curved edge of membrane patches. Scan rate, 70 Hz; 256 × 256 pixels. The area in the dashed box is enlarged in b. b, Spiral and ring formation localized to the membrane edge. Scan rate, 70 Hz; 256 × 256 pixels. c, Left, phase timecourse showcasing a dense network of sheets, spirals, and rings that ultimately cover the entire membrane plane. Right, average of six F-AFM height images. Scan rate, 120 Hz; 256 × 256 pixels. d, Average F-AFM height image showing Vipp1 sheet, spiral, and ring detail. Red arrows mark the sheet branching into filaments ~13 nm wide. Scan rate, 20 Hz; 256 × 256 pixels. e, Vipp1 sheet and spiral filament height offset from the membrane. f–i, Quantification of Vipp1 filament and spiral characteristics. n = 124, 13, 278, and 278 independent measurements for panels f, g, h, and i, respectively. Error bars show one s.d. of the mean.

*Souvik Naskar, Andrea Merino, Javier Espadas, Jayanti Singh, Aurelien Roux, Adai Colom and Harry H. Low
Mechanism for Vipp1 spiral formation, ring biogenesis, and membrane repair
Nature Structural & Molecular Biology (2024)
DOI: https://doi.org/10.1038/s41594-024-01401-8

Open Access The article “Mechanism for Vipp1 spiral formation, ring biogenesis, and membrane repair” by Souvik Naskar, Andrea Merino, Javier Espadas, Jayanti Singh, Aurelien Roux, Adai Colom and Harry H. Low 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/.

Real-time multistep asymmetrical disassembly of nucleosomes and chromatosomes visualized by high-speed atomic force microscopy

During replication, expression, and repair of the eukaryotic genome, cellular machinery must access the DNA wrapped around histone proteins forming nucleosomes. These octameric protein·DNA complexes are modular, dynamic, and flexible and unwrap or disassemble either spontaneously or by the action of molecular motors. Thus, the mechanism of formation and regulation of subnucleosomal intermediates has gained attention genome-wide because it controls DNA accessibility.*

In the article “Real-Time Multistep Asymmetrical Disassembly of Nucleosomes and Chromatosomes Visualized by High-Speed Atomic Force Microscopy” Bibiana Onoa, César Díaz-Celis, Cristhian Cañari-Chumpitaz, Antony Lee and Carlos Bustamante describe how they imaged nucleosomes and their more compacted structure with the linker histone H1 (chromatosomes) using high-speed atomic force microscopy to visualize simultaneously the changes in the DNA and the histone core during their disassembly when deposited on mica.*

Furthermore, Bibiana Onoa et al. trained a neural network and developed an automatic algorithm to track molecular structural changes in real time. *

The authors’ results show that nucleosome disassembly is a sequential process involving asymmetrical stepwise dimer ejection events. The presence of H1 restricts DNA unwrapping, significantly increases the nucleosomal lifetime, and affects the pathway in which heterodimer asymmetrical dissociation occurs. *

Bibiana Onoa et al. observe that tetrasomes are resilient to disassembly and that the tetramer core (H3·H4)2 can diffuse along the nucleosome positioning sequence. Tetrasome mobility might be critical to the proper assembly of nucleosomes and can be relevant during nucleosomal transcription, as tetrasomes survive RNA polymerase passage. These findings are relevant to understanding nucleosome intrinsic dynamics and their modification by DNA-processing enzymes. *

To characterize the nucleosomes dynamics in 2D, individual molecules were observed in buffer using an Ando-type high speed atomic force microscope together with NanoWorld Ultra-Short Cantilevers for HS-AFM of the USC-F1.2-K0.15 AFM probe type ( typical spring constant 0.15 N/m, typical resonance frequency in air 1200 kHz, resonance frequency 500–600 kHz in liquid). *

The AFM data presented in the article allow the authors to directly visualize the dynamics of DNA and histones during nucleosome and chromatosome disassembly, providing a simultaneous observation of DNA unwrapping and histone dissociation. *

The experimental and analytical strategy presented shows that real-time HS-AFM is a robust and powerful tool for studying single nucleosomes and chromatin dynamics. *

graphical abstract from Bibiana Onoa et al 2024 "Real-Time Multistep Asymmetrical Disassembly of Nucleosomes and Chromatosomes Visualized by High-Speed Atomic Force Microscopy" - NanoWorld Ultra-Short Cantilevers of the USC-F1.2-k0.15 AFM probe type were used for the high-speed atomic force microscopy — graphical abstract from Bibiana Onoa et al 2024 “Real-Time Multistep Asymmetrical Disassembly of Nucleosomes and Chromatosomes Visualized by High-Speed Atomic Force Microscopy”

*Bibiana Onoa, César Díaz-Celis, Cristhian Cañari-Chumpitaz, Antony Lee and Carlos Bustamante
Real-Time Multistep Asymmetrical Disassembly of Nucleosomes and Chromatosomes Visualized by High-Speed Atomic Force Microscopy
ACS Central Science 2024, 10, 1, 122–137
DOI: https://doi.org/10.1021/acscentsci.3c00735

Open Access The article “Real-Time Multistep Asymmetrical Disassembly of Nucleosomes and Chromatosomes Visualized by High-Speed Atomic Force Microscopy” by Bibiana Onoa, César Díaz-Celis, Cristhian Cañari-Chumpitaz, Antony Lee and Carlos Bustamante 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/.