Tag: cellular processes

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

Please follow this external link to read the full article: https://rdcu.be/dnNba

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/.

Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides

Ceramides are central intermediates of sphingolipid metabolism that also function as potent messengers in stress signaling and apoptosis. Progress in understanding how ceramides execute their biological roles is hampered by a lack of methods to manipulate their cellular levels and metabolic fate with appropriate spatiotemporal precision.*

In the article “Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides” Matthijs Kol, Ben Williams, Henry Toombs-Ruane, Henri G Franquelim, Sergei Korneev, Christian Schroeer, Petra Schwille, Dirk Trauner, Joost CM Holthuis and James A Frank report on clickable, azobenzene-containing ceramides, caCers, as photoswitchable metabolic substrates to exert optical control over sphingolipid production in cells.*

They combine atomic force microscopy on model bilayers with metabolic tracing studies in cells, and demonstrate that light-induced alterations in the lateral packing of caCers lead to marked differences in their metabolic conversion by sphingomyelin synthase and glucosylceramide synthase. These changes in metabolic rates are instant and reversible over several cycles of photoswitching. The findings described in the article disclose new opportunities to probe the causal roles of ceramides and their metabolic derivatives in a wide array of sphingolipid-dependent cellular processes with the spatiotemporal precision of light.*

The High-speed AFM in AC mode described in the article was done with NanoWorld Ultra-Short Cantilevers USC-F0.3-k0.3 with a typical stiffness of 0.3 N/m. The AFM cantilever oscillation was tuned to a frequency of 100–150 kHz and the amplitude kept below 10 nm. The scan rate was set to 25–150 Hz. Images were acquired at 256 × 256 pixel resolution. All measurements were performed at room temperature. The force applied on the sample was minimized by continuously adjusting the set point and gain during imaging. Height, error, deflection and phase-shift signals were recorded and images were line-fitted as required.*

Figure 2 b from “ Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides “ by Matthijs Kol et al. :
Photo-isomerization of caCers affects membrane fluidity and lipid domain structure in supported lipid bilayers.
(b) Atomic force microscopy of supported lipid bilayers ( SLBs )prepared as in (a). Isomerization of caCer-3 (top) and caCer-4 (bottom) to cis with UV-A light (365 nm) resulted in a fluidification inside the Lo domains, as indicated by the appearance of small fluid Ld lakes and an increased Ld/Lo area ratio. This effect was reversed on isomerization back to trans with blue light (470 nm), marked by a drop in the Ld/Lo area ratio. Scale bars, 2 μm. (c) Time-course plotting the normalized Lo area over multiple 365/470 nm irradiation cycles for caCer-3 (top) and caCer-4 (bottom).
Please refer to https://doi.org/10.7554/eLife.43230.007 for the full figure.

*Matthijs Kol, Ben Williams, Henry Toombs-Ruane, Henri G Franquelim, Sergei Korneev, Christian Schroeer, Petra Schwille, Dirk Trauner, Joost CM Holthuis, James A Frank
Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides
eLife 2019;8:e43230
DOI: 10.7554/eLife.43230
https://doi.org/10.7554/eLife.43230.001
https://doi.org/10.7554/eLife.43230.007

Please follow this external link to read the full article: https://elifesciences.org/articles/43230

Open Access The article “Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides “ by Matthijs Kol, Ben Williams, Henry Toombs-Ruane, Henri G Franquelim, Sergei Korneev, Christian Schroeer, Petra Schwille, Dirk Trauner, Joost CM Holthuis and  James A Frank 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/.