Tag: 高速AFM

Structural and optical variation of pseudoisocyanine aggregates nucleated on DNA substrates

The ability to maximize the range of exciton transport while minimizing energy loss has significant implications for the design of future nanoscale light harvesting, optoelectronic, and sensing applications. *

One method of achieving this would be to densely pack dyes into strongly coupled aggregates such that excitations can be coherently delocalized through the partial or full length of the aggregate. *

Coherently coupled aggregates enable exciton migration over discreet spatial distances with near unitary quantum efficiency. As a result, controlled dye aggregation has long been studied by chemists as a method of tuning the photonic and physical properties of the dyes and pigments in light harvesting devices. An example of this coherent coupling phenomenon can be observed in the cyanine dye family and specifically the prototypical example, pseudoisocyanine (PIC) dye. *

In the article «Structural and optical variation of pseudoisocyanine aggregates nucleated on DNA substrates” Matthew Chiriboga, Christopher M Green, Divita Mathur, David A Hastman, Joseph S Melinger, Remi Veneziano, Igor L Medintz and Sebastián A Díaz show that DNA-nucleated PIC aggregates have properties which correlate to different molecular structures and are directed by changing the DNA scaffold. *

To achieve this, they formed PIC aggregates through heterogeneous nucleation by mixing dissolved PIC dye with various DNA nanostructures ranging from a rigid DX-tile to more flexible DNA duplex (dsDNA) or single strand DNA oligonucleotide (ssDNA). *

Although the aggregates Matthew Chiriboga et al formed required elevated excess of PIC dye relative to previously reported J-bits, they exhibited sharper and brighter fluorescence peaks as well as longer Ncoh. *

Therefore, the authors refer to aggregates formed by this approach as super aggregate (SA) in their article, though they note SA formed with different DNA substrates result in unique properties. *

Complementary circular dichroism (CD) and atomic force microscopy (AFM) characterizations were used to analyze the SA and both indicated distinctions in the way each substrate and subsequent dye aggregate incorporates the individual PIC molecules. *

To achieve high resolution imaging of nucleic acid nanostructures, the DNA is often deposited onto a mica substrate, where mica electrostatically binds the DNA. Once deposited onto the mica, the imaging can be done in a hydrated environment as there is no additional required dehydration or staining of the DNA, a particularly convenient advantage of AFM. *

The AFM imaging was performed under AC fast imaging mode (liquid) with NanoWorld  Ultra-Short Cantilevers (USC) for Fast/High-Speed AFM of the USC-F0.3-k0.3 AFM probe type.*

On a segment of freshly cleaved mica mounted to a magnetic puck, 15 μl of PIC-DANN solution was deposited immediately before measurement. A 25 μl droplet of imaging buffer was deposited on the AFM tip, then the AFM tip mount was lowered into the sample buffer to create a liquid ‘chamber’ for imaging. *

When introducing various DNA scaffolds for SA formation and subsequent AFM imaging, Matthew Chiriboga et al. observed significant changes in the aggregates structure. *

The AFM imaging highlighted the stark differences in aggregate formation resulting from the DNA substrates. *

To the author’s knowledge this is the first visualization of DNA-based PIC aggregates. Results from the field have been pointing towards fiber-like or nanotube-like networks of polymerized PIC as a structural model for aggregates suspended in solution. *

On the other hand, other AFM studies demonstrate that PIC aggregates formed on mica substrates adopt a leafy island morphology. *

Interestingly, Matthew Chiriboga et al. observe evidence of both PIC fibers as well as leafy islands that exhibit distinct growth patterns, again depending on the DNA substrate. *

Although this work contributes to the growing body of evidence that solution-based PIC aggregates form fibrous networks structures, the AFM measurements presented in the article highlight the multiplicity of PIC aggregation modes when introduced to DNA scaffolds. *

The results presented in the research article suggest modification of the DNA substrate results in significant changes to how the DNA and companion dye molecules are integrated into larger form PIC aggregates. *

Bearing in mind that the broader motivation for studying DNA based PIC aggregates is to integrate strongly coupled dyes onto modular DNA structural units, PIC SAs should be given due consideration as a versatile option. *

In fact, similar work is being done with other cyanine dyes where DNA template modification is used to switch between quenching and energy transfer. *

Ultimately this could be a path for the PIC SA and one which possibly leads towards applications in optical microcavities for quantum electrodynamical devices and optical switching, molecular plasmonics, biosensors, and light-harvesting arrays. *

Figure 6 from Matthew Chiriboga et al. “Structural and optical variation of pseudoisocyanine aggregates nucleated on DNA substrates”:Atomic Force Microscopy visualisation of pseudoisocyanine aggregates – super aggregates (SA) nucleated on DNA substrates. AFM visualizations of pseudoisocyanine (PIC) aggregates formed in the (A) AT, (B) dsDNA, and (C) ssDNA nanostructures. Each of the samples was formed immediately before measurement by mixing 160 μM PIC dye with 500 nM DNA normalized to the dye-labeled strand concentration (i.e. 320-fold excess). When the SA was formed using an AT DX-tile template (figures 6(A) the authors observed the formation of large and long rod-like aggregates with a relatively isotropic growth axis. This supports the hypothesis proposed by Yoa et al which suggested aggregation along preferential axis due to a preferred interaction between the PIC and the mica NanoWorld USC-F0.3-k0.3 AFM probes were used for the under AC fast imaging mode in liquid.
Figure 6 from Matthew Chiriboga et al. “Structural and optical variation of pseudoisocyanine aggregates nucleated on DNA substrates”:
AFM visualization of SA formations. AFM visualizations of PIC aggregates formed in the (A) AT, (B) dsDNA, and (C) ssDNA nanostructures. Each of the samples was formed immediately before measurement by mixing 160 μM PIC dye with 500 nM DNA normalized to the dye-labeled strand concentration (i.e. 320-fold excess).

*Matthew Chiriboga, Christopher M Green, Divita Mathur, David A Hastman, Joseph S Melinger, Remi Veneziano, Igor L Medintz and Sebastián A Díaz
Structural and optical variation of pseudoisocyanine aggregates nucleated on DNA substrates
Methods and Applications in Fluorescence (2023) 11 014003
DOI: https://doi.org/10.1088/2050-6120/acb2b4

The article “Active self-assembly of piezoelectric biomolecular films via synergistic nanoconfinement and in-situ poling” by Matthew Chiriboga, Christopher M Green, Divita Mathur, David A Hastman, Joseph S Melinger, Remi Veneziano, Igor L Medintz and Sebastián A Díaz 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/.

Intrinsically disordered regions in TRPV2 mediate protein-protein interactions

Transient receptor potential (TRP) ion channels are gated by diverse intra- and extracellular stimuli leading to cation inflow (Na+, Ca2+) regulating many cellular processes and initiating organismic somatosensation. *

Structures of most TRP channels have been solved. However, structural and sequence analysis showed that ~30% of the TRP channel sequences, mainly the N- and C-termini, are intrinsically disordered regions (IDRs). Unfortunately, very little is known about IDR ‘structure’, dynamics and function, though it has been shown that they are essential for native channel function. *

In the article “Intrinsically disordered regions in TRPV2 mediate protein-protein interactions”, Raghavendar R. Sanganna Gari, Grigory Tagiltsev, Ruth A. Pumroy, Yining Jiang, Martin Blackledge, Vera Y. Moiseenkova-Bell and Simon Scheuring imaged TRPV2 channels in membranes using high-speed atomic force microscopy (HS-AFM). *

The dynamic single molecule imaging capability of HS-AFM allowed the authors to visualize IDRs and revealed that N-terminal IDRs were involved in intermolecular interactions. Their work provides evidence about the ‘structure’ of the TRPV2 IDRs, and that the IDRs may mediate protein-protein interactions. *

In total, 1.5 µl of the TRPV2 reconstituted vesicles were deposited on a 1.5-mm2 freshly cleaved mica surface, which was glued with epoxy to the quartz sample stage. After 20–30 min incubation, the sample was gently rinsed with imaging buffer (20 mM Hepes, pH 8.0, 150 mM NaCl) and mounted in the HS-AFM fluid cell. All images in this study were taken using a HS-AFM operated in amplitude modulation mode using optimized scan and feedback parameters and lab-built amplitude detectors and free amplitude stabilizers. *

Short (8 µm) cantilevers (NanoWorld Ultra-Short Cantilevers for High-Speed AFM USC-F1.2-k0.15) with nominal spring constant of 0.15 N/m, resonance frequency of 0.6 MHz, and a quality factor of ∼1.5 in liquid were used. AFM probes were sharpened using oxygen plasma etching to obtain better resolution. *

Fig. 1 from “Intrinsically disordered regions in TRPV2 mediate protein-protein interactions” by Raghavendar R. Sanganna Gari et al. :TRPV2 reconstitution for HS-AFM analysis. b Overview HS-AFM images (Supplementary Movie 1) of TRPV2 (windmill-shaped molecules) in soy polar lipid membranes on mica (dark background areas). False color scale: 0–9 nm. The white oversaturated areas have a height of ~26 nm and represent likely non-ruptured small vesicles. NanoWorld-USC-F1.2-k0.15 AFM probes were used for the HS-AFM
Fig. 1 from “Intrinsically disordered regions in TRPV2 mediate protein-protein interactions” by Raghavendar R. Sanganna Gari et al. :
TRPV2 reconstitution for HS-AFM analysis.
b Overview HS-AFM images (Supplementary Movie 1) of TRPV2 (windmill-shaped molecules) in soy polar lipid membranes on mica (dark background areas). False color scale: 0–9 nm. The white oversaturated areas have a height of ~26 nm and represent likely non-ruptured small vesicles.
Fig. 1 from “Intrinsically disordered regions in TRPV2 mediate protein-protein interactions” by Raghavendar R. Sanganna Gari et al. : TRPV2 reconstitution for HS-AFM analysis. a Negative-stain EM of TRPV2 reconstituted into soy polar lipids at a lipid-to-protein ratio of 0.7. Protruding features (arrow) at the vesicle periphery and the strong contrast of the proteins in the vesicle in the negative-stain EM are indicative of inside-out reconstitution of the TRPV2 channels with the large cytoplasmic domains exposed to the outside of the vesicle. b Overview HS-AFM images (Supplementary Movie 1) of TRPV2 (windmill-shaped molecules) in soy polar lipid membranes on mica (dark background areas). False color scale: 0–9 nm. The white oversaturated areas have a height of ~26 nm and represent likely non-ruptured small vesicles. c Height distribution of TRPV2 above mica from (b). TRPV2 has a full height of 9.5 ± 0.1 nm above mica, in good agreement with the TRPV2 cryo-EM structure. Inset: Cryo-EM structure PDB 6U84 shown with the intracellular side up (as imaged by HS-AFM), membrane indicated in light gray. Short (8 µm) cantilevers (NanoWorld Ultra-Short Cantilevers for High-Speed AFM USC-F1.2-k0.15,) with nominal spring constant of 0.15 N/m, resonance frequency of 0.6 MHz, and a quality factor of ∼1.5 in liquid were used. AFM probes were sharpened using oxygen plasma etching to obtain better resolution. *
Fig. 1 from “Intrinsically disordered regions in TRPV2 mediate protein-protein interactions” by Raghavendar R. Sanganna Gari et al. :
TRPV2 reconstitution for HS-AFM analysis.
a Negative-stain EM of TRPV2 reconstituted into soy polar lipids at a lipid-to-protein ratio of 0.7. Protruding features (arrow) at the vesicle periphery and the strong contrast of the proteins in the vesicle in the negative-stain EM are indicative of inside-out reconstitution of the TRPV2 channels with the large cytoplasmic domains exposed to the outside of the vesicle. b Overview HS-AFM images (Supplementary Movie 1) of TRPV2 (windmill-shaped molecules) in soy polar lipid membranes on mica (dark background areas). False color scale: 0–9 nm. The white oversaturated areas have a height of ~26 nm and represent likely non-ruptured small vesicles. c Height distribution of TRPV2 above mica from (b). TRPV2 has a full height of 9.5 ± 0.1 nm above mica, in good agreement with the TRPV2 cryo-EM structure. Inset: Cryo-EM structure PDB 6U84 shown with the intracellular side up (as imaged by HS-AFM), membrane indicated in light gray.

 

*Raghavendar R. Sanganna Gari, Grigory Tagiltsev, Ruth A. Pumroy, Yining Jiang, Martin Blackledge, Vera Y. Moiseenkova-Bell and Simon Scheuring
Intrinsically disordered regions in TRPV2 mediate protein-protein interactions
Communications Biology volume 6, Article number: 966 (2023)
DOI: https://doi.org/10.1038/s42003-023-05343-7

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The article “Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response” by Raghavendar R. Sanganna Gari, Grigory Tagiltsev, Ruth A. Pumroy, Yining Jiang, Martin Blackledge, Vera Y. Moiseenkova-Bell 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/.

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/.