Tag: molecular biology

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/.

Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response

Liquid–liquid phase-separated biomolecular condensates, liquid droplets play an important role in many biological processes, such as gene expression, protein translation, stress response, and protein degradation, by incorporating a variety of RNA and client proteins into their interior depending on the intracellular context. *

Autophagy is involved in the degradation of several cytoplasmic liquid droplets, including stress granules and P bodies, and defects in this process are thought to cause transition of these droplets to the solid phase, resulting in the development of intractable diseases such as neurodegenerative disorders and cancer. *

Of the droplets that have a unique biological function and are degraded by autophagy, p62 bodies (also called p62 droplets) are liquid droplets formed by liquid–liquid phase separation (LLPS) of p62 and its binding partners, ubiquitinated proteins. *

p62 bodies are involved in the regulation of intracellular proteostasis through their own autophagic degradation, and also contribute to the regulation of the major stress-response mechanism by sequestration of a client protein, kelch-like ECH-associated protein 1 (KEAP1). *

NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid–liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. *

However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear. *

In the article “Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response” Ryo Ikeda, Daisuke Noshiro, Hideaki Morishita, Shuhei Takada, Shun Kageyama, Yuko Fujioka, Tomoko Funakoshi, Satoko Komatsu-Hirota, Ritsuko Arai, Elena Ryzhii, Manabu Abe, Tomoaki Koga, Hozumi Motohashi, Mitsuyoshi Nakao, Kenji Sakimura, Arata Horii, Satoshi Waguri, Yoshinobu Ichimura, Nobuo N Noda and Masaaki Komatsu identify ULK1 as a kinase responsible for the phosphorylation of p62. *

ULK1 colocalizes with p62 bodies, directly interacting with p62. ULK1-dependent phosphorylation of p62 allows KEAP1 to be retained within p62 bodies, thus activating NRF2. p62S351E/+ mice are phosphomimetic knock-in mice in which Ser351, corresponding to human Ser349, is replaced by Glu. *

These mice, but not their phosphodefective p62S351A/S351A counterparts, exhibit NRF2 hyperactivation and growth retardation. This retardation is caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis, a phenotype also observed in systemic Keap1-knockout mice. *

The authors’ results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insights into the role of phase separation in this process. *

To clarify whether the ULK1 kinase itself has an effect on the physical properties and physiological role of p62 bodies, Ryo Ikeda et al. first studied the physical interaction of p62 with ULK1 or its yeast homolog Atg1 using high-speed atomic force microscopy (HS-AFM). *

HS-AFM of p62 (268–440 aa) visualized a homodimeric structure, mediated by the dimerization of the UBA domain, that formed a hammer-shaped structure with IDRs wrapped around each other. *

HS-AFM images were acquired in tapping mode using a sample-scanning HS-AFM instrument. NanoWorld Ultra-Short Cantilevers of the  USC-F1.2-k0.15 AFM probe type were used. ( ~7 μm long, ~2 μm wide, and ~0.08 μm thick with electron beam-deposited (EBD) tips (tip radius < 10 nm). Their resonant frequency and spring constant were 1.2 MHz in air and 0.15 N/m, respectively.*

Figure EV1 from “Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response” by Ryo Ikeda et al.:HS-AFM observation of SNAP-ULK1 and p62 (268–440 aa), and complex of SNAP-Atg1/p62 (268–440 aa) A, B. Successive HS-AFM images of SNAP-ULK1 (A) and p62_268–440 (B). Height scale: 0–4.4 nm (A), 0–3.4 nm (B); scale bar: 20 nm (A, B). C. Successive HS-AFM images of p62_268–440 with SNAP-Atg1. Height scale: 0–3.6 nm; scale bar: 30 nm. D. Schematics showing the molecular characteristics determined by HS-AFM. Gray spheres, globular domains consisting of N-terminal KD and C-terminal MIT of Atg1; pink spheres, globular domains consisting of C-terminal UBA domain of p62; blue thick solid lines, IDRs. NanoWorld Ultra-Short Cantilevers of the USC-F1.2-k0.15 AFM probe type were used.
Figure EV1 from “Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response” by Ryo Ikeda et al.:
HS-AFM observation of SNAP-ULK1 and p62 (268–440 aa), and complex of SNAP-Atg1/p62 (268–440 aa)
A, B. Successive HS-AFM images of SNAP-ULK1 (A) and p62_268–440 (B). Height scale: 0–4.4 nm (A), 0–3.4 nm (B); scale bar: 20 nm (A, B).
C. Successive HS-AFM images of p62_268–440 with SNAP-Atg1. Height scale: 0–3.6 nm; scale bar: 30 nm.
D. Schematics showing the molecular characteristics determined by HS-AFM. Gray spheres, globular domains consisting of N-terminal KD and C-terminal MIT of Atg1; pink spheres, globular domains consisting of C-terminal UBA domain of p62; blue thick solid lines, IDRs.

*Ryo Ikeda, Daisuke Noshiro, Hideaki Morishita, Shuhei Takada, Shun Kageyama, Yuko Fujioka, Tomoko Funakoshi, Satoko Komatsu-Hirota, Ritsuko Arai, Elena Ryzhii, Manabu Abe, Tomoaki Koga, Hozumi Motohashi, Mitsuyoshi Nakao, Kenji Sakimura, Arata Horii, Satoshi Waguri, Yoshinobu Ichimura, Nobuo N Noda and Masaaki Komatsu
Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response
The EMBO Journal (2023)42:e113349
DOI: https://doi.org/10.15252/embj.2022113349

The article “Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response” by Ryo Ikeda, Daisuke Noshiro, Hideaki Morishita, Shuhei Takada, Shun Kageyama, Yuko Fujioka, Tomoko Funakoshi, Satoko Komatsu-Hirota, Ritsuko Arai, Elena Ryzhii, Manabu Abe, Tomoaki Koga, Hozumi Motohashi, Mitsuyoshi Nakao, Kenji Sakimura, Arata Horii, Satoshi Waguri, Yoshinobu Ichimura, Nobuo N Noda and Masaaki Komatsu 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/.