高速AFMカンチレバー

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/.

Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis

Cells communicate with their environments via the plasma membrane and various membrane proteins. Clathrin-mediated endocytosis (CME) plays a central role in such communication and proceeds with a series of multiprotein assembly, deformation of the plasma membrane, and production of a membrane vesicle that delivers extracellular signaling molecules into the cytoplasm.*

In the article “Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis”, Aiko Yoshida, Nobuaki Sakai, Yoshitsugu Uekusa, Yuka Imaoka, Yoshitsuna Itagaki, Yuki Suzuki and Shige H. Yoshimura describe how they utilized their home-built correlative imaging system comprising high-speed atomic force microscopy (HS-AFM) and confocal fluorescence microscopy to simultaneously image morphological changes of the plasma membrane and protein localization during CME in a living cell.*

Overlaying AFM and fluorescence images revealed the dynamics of protein assembly and concomitant morphological changes of the plasma membrane with high spatial resolution. In particular, the authors elucidate the role of actin in the closing step of CME.*

The results revealed a tight correlation between the size of the pit and the amount of clathrin assembled. Actin dynamics play multiple roles in the assembly, maturation, and closing phases of the process, and affects membrane morphology, suggesting a close relationship between endocytosis and dynamic events at the cell cortex. Knock down of dynamin also affected the closing motion of the pit and showed functional correlation with actin.*

An AFM tip-scan–type HS-AFM unit combined with an inverted fluorescence/optical microscope equipped with a phase contrast system and a confocal unit was used for this study.*

The modulation method was set to phase modulation mode to detect AFM tip–sample interactions. A customized NanoWorld Ultra-Short AFM cantilever with an electron beam–deposited sharp AFM tip with a spring constant of 0.1 N m−1 (USC-F0.8-k0.1-T12) was used. *

All observations were performed at 28 °C. The AFM tip was aligned with confocal views as described in the Results section of the article. The images from the confocal microscope and AFM were simultaneously acquired at a scanning rate of 10 s/frame. The captured sequential images were overlaid by using AviUTL (http://spring-fragrance.mints.ne.jp/aviutl/) based on the AFM tip position.
The fluorescence intensity was quantified by Image J software (http://rsbweb.nih.gov/ij/). *

Fig 1 from Aiko Yoshida et al 2018 “Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis” :Aligning the confocal image and the AFM image. (A) Schematic illustration of the sample stage. A cross-shaped movable XY-stage (orange) is mounted on the base plate (light green) of the inverted optical microscope (IX83) via a stage guide (gray) equipped at each of the 4 ends of the cross. A 3-point support plate (purple) for mounting the AFM scanner unit is fixed on the base plate with a configuration that does not hinder the sliding of the XY-stage along the x-axis and y-axis. These setups allow the sample stage to move independently of the AFM unit and the optical axis. (B) Side view of the HS-AFM unit mounted on the stage illustrated in panel A. (C) Overlaying a confocal image and an AFM image. COS-7 cells expressing EGFP-CLCa were fixed with 5% paraformaldehyde and subjected to AFM (left) and CLSM (middle) imaging. The x-y position of the probe tip was determined as described in S1 Fig. Two images were overlaid (right) based on the x-y center position. Scale bar: 1 μm. Autofluorescence of the probe was much weaker than clathrin spot and could not be detected during the fast scanning. (D) AFM images of CCP on the cytoplasmic surface of the plasma membrane. COS-7 cells were “unroofed” by mild sonication as described in Materials and methods and then fixed with glutaraldehyde. Scale bar: 0.1 μm. AFM, atomic force microscopy; CCP, clathrin-coated pit; CLSM, confocal laser scanning microscopy; COS-7, CV-1 in origin with SV40 gene line 7; EGFP, enhanced green fluorescent protein; EGFP-CLCa, EGFP-fused clathrin light chain a; HS-AFM, high-speed AFM. https://doi.org/10.1371/journal.pbio.2004786.g001 customized NanoWorld Ultra-Short AFM cantilevers with an electron beam–deposited sharp AFM tip with a spring constant of 0.1 N m−1 (USC-F0.8-k0.1-T12) were used — Fig 1 from Aiko Yoshida et al 2018 “Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis” :
Aligning the confocal image and the AFM image.
(A) Schematic illustration of the sample stage. A cross-shaped movable XY-stage (orange) is mounted on the base plate (light green) of the inverted optical microscope (IX83) via a stage guide (gray) equipped at each of the 4 ends of the cross. A 3-point support plate (purple) for mounting the AFM scanner unit is fixed on the base plate with a configuration that does not hinder the sliding of the XY-stage along the x-axis and y-axis. These setups allow the sample stage to move independently of the AFM unit and the optical axis. (B) Side view of the HS-AFM unit mounted on the stage illustrated in panel A. (C) Overlaying a confocal image and an AFM image. COS-7 cells expressing EGFP-CLCa were fixed with 5% paraformaldehyde and subjected to AFM (left) and CLSM (middle) imaging. The x-y position of the probe tip was determined as described in S1 Fig. Two images were overlaid (right) based on the x-y center position. Scale bar: 1 μm. Autofluorescence of the probe was much weaker than clathrin spot and could not be detected during the fast scanning. (D) AFM images of CCP on the cytoplasmic surface of the plasma membrane. COS-7 cells were “unroofed” by mild sonication as described in Materials and methods and then fixed with glutaraldehyde. Scale bar: 0.1 μm. AFM, atomic force microscopy; CCP, clathrin-coated pit; CLSM, confocal laser scanning microscopy; COS-7, CV-1 in origin with SV40 gene line 7; EGFP, enhanced green fluorescent protein; EGFP-CLCa, EGFP-fused clathrin light chain a; HS-AFM, high-speed AFM.
https://doi.org/10.1371/journal.pbio.2004786.g001

Supporting figure 1 from Aiko Yoshida et al 2018 “Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis”:1 Fig. Aligning confocal and AFM images. (A) Scanning electron microscopy (SEM) images of a cantilever equipped with an EBD tip with tilt angle of 12°. Scale bar, 5 μm. Note that the cantilever is held on the AFM head unit with a tilt angle of 102° (from the x-y plane) so that the relative tip–sample angle (θ) is 90°. This setup makes it possible to precisely determine the position of the AFM tip. Scale bar, 2 μm. (B) Determining the position of the AFM probe in a fluorescence image. While the AFM probe was attached on the glass surface without scanning, the autofluorescence signal of the probe was imaged by the confocal scanning unit. The observed fluorescence spot (arrowhead in the middle panel) is defined as an origin of the fluorescence image plane (x = 0, y = 0) and used to define the optical axis (left panel). The position of a fluorescence spot derived from EGFP-CLCa was determined on this axis. On the other hand, the scanning area of the AFM scanner covers the area of (−3, 2.25) (left top), (3, 2.25) (right top), (3, −2.25) (right bottom), and (−3, −2.25) (left bottom) (all right panel). By aligning the axis from both images, the x, y position of the AFM image and that of the confocal fluorescence image could be merged. AFM, atomic force microscopy; EBD, electron beam–deposited; EGFP, enhanced green fluorescent protein; EGFP-CLCa, EGFP-fused clathrin light chain a. https://doi.org/10.1371/journal.pbio.2004786.s001 customized NanoWorld Ultra-Short AFM cantilevers with an electron beam–deposited sharp AFM tip with a spring constant of 0.1 N m−1 (USC-F0.8-k0.1-T12) were used — Supporting figure 1 from Aiko Yoshida et al 2018 “Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis”:
1 Fig. Aligning confocal and AFM images.
(A) Scanning electron microscopy (SEM) images of a cantilever equipped with an EBD tip with tilt angle of 12°. Scale bar, 5 μm. Note that the cantilever is held on the AFM head unit with a tilt angle of 102° (from the x-y plane) so that the relative tip–sample angle (θ) is 90°. This setup makes it possible to precisely determine the position of the AFM tip. Scale bar, 2 μm. (B) Determining the position of the AFM probe in a fluorescence image. While the AFM probe was attached on the glass surface without scanning, the autofluorescence signal of the probe was imaged by the confocal scanning unit. The observed fluorescence spot (arrowhead in the middle panel) is defined as an origin of the fluorescence image plane (x = 0, y = 0) and used to define the optical axis (left panel). The position of a fluorescence spot derived from EGFP-CLCa was determined on this axis. On the other hand, the scanning area of the AFM scanner covers the area of (−3, 2.25) (left top), (3, 2.25) (right top), (3, −2.25) (right bottom), and (−3, −2.25) (left bottom) (all right panel). By aligning the axis from both images, the x, y position of the AFM image and that of the confocal fluorescence image could be merged. AFM, atomic force microscopy; EBD, electron beam–deposited; EGFP, enhanced green fluorescent protein; EGFP-CLCa, EGFP-fused clathrin light chain a.
https://doi.org/10.1371/journal.pbio.2004786.s001

*Aiko Yoshida, Nobuaki Sakai, Yoshitsugu Uekusa, Yuka Imaoka, Yoshitsuna Itagaki, Yuki Suzuki and Shige H. Yoshimura
Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis
PLoS Biol 16(5) (2018): e2004786
DOI: https://doi.org/10.1371/journal.pbio.2004786

The article “Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis” by Aiko Yoshida, Nobuaki Sakai, Yoshitsugu Uekusa, Yuka Imaoka, Yoshitsuna Itagaki, Yuki Suzuki and Shige H. Yoshimura 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/.