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/.

More papers on High Speed Atomic Force Microscopy – list of references updated

We have updated our list of articles in the field of High-Speed AFM (HS-AFM) on the www.highspeedscanning.com website. If you would like to see what has been going on recently in the field of High-Speed AFM (HS-AFM) then you are welcome to have a look at: http://www.highspeedscanning.com/hs-afm-references.html

We are aware that this list is far from complete so if you have used one of our Ultra-Short Cantilevers (USC) for high speed atomic force microscopy in the research for your publication and your article isn’t listed yet then please let us know. We will be happy to add it to the list.

NanoWorld Ultra-Short Cantilevers (USC) for High-Speed AFM (HS-AFM)
NanoWorld Ultra-Short Cantilevers (USC) for High-Speed AFM (HS-AFM)

Analysis of long dsRNA produced in vitro and in vivo using atomic force microscopy in conjunction with ion-pair reverse-phase HPLC

Long double-stranded (ds) RNA is emerging as a novel alternative to chemical and genetically-modified insect and fungal management strategies. The ability to produce large quantities of dsRNA in either bacterial systems, by in vitro transcription, in cell-free systems or in planta for RNA interference applications has generated significant demand for the development and application of analytical tools for analysis of dsRNA.*

In their article “Analysis of long dsRNA produced in vitro and in vivo using atomic force microscopy in conjunction with ion-pair reverse-phase HPLC” Alison O. Nwokeoji, Sandip Kumar, Peter M. Kilby, David E. Portwood, Jamie K. Hobbs and Mark J. Dickman have utilised atomic force microscopy (AFM) in conjunction with ion-pair reverse-phase high performance liquid chromatography (IP-RP-HPLC) to provide novel insight into dsRNA for RNAi applications.*

The AFM analysis enabled direct structural characterisation of the A-form duplex dsRNA and accurate determination of the dsRNA duplex length.*

The work presented in this study demonstrates the ability of AFM in conjunction with IP RP HPLC to rapidly assess sample heterogeneity and provide important structural information regarding dsRNA.*

For the high resolution images presented in Fig. 1(A, B) and 2(B) in the article NanoWorld Ultra-Short Cantilevers USC-F1.2-k0.15 with a High Density Carbon tip (nominal values: tip radius 10 nm, cantilever length 7 μm, stiffness 0.15 N m−1, resonant frequency 1200 kHz in air) were tuned to 600–650 kHz, oscillated at a free amplitude of <30 mV and scanned at a rate of 0.4–1.0 μm s−1,to visualize the dsRNA and dsDNA grooves.*


Fig. 1 A and B from “Analysis of long dsRNA produced in vitro and in vivo using atomic force microscopy in conjunction with ion-pair reverse-phase HPLC” by Alison O. Nwokeoji et al. :
Analysis of dsRNA monomers, multimers and higher order assemblies under non-denaturing conditions. Non-denaturing gel electrophoretograms (A) in vivo synthesised dsRNA (521 bp and 698 bp) (B) in vitro synthesised dsRNA (504 bp). Each dsRNA sample was run in duplicate. The proposed dsRNA multimers or higher order assemblies with reduced electrophoretic mobility are highlighted above the corresponding dsRNA main band.

*Alison O. Nwokeoji, Sandip Kumar,Peter M. Kilby, David E. Portwood, Jamie K. Hobbs and Mark J. Dickman
Analysis of long dsRNA produced in vitro and in vivo using atomic force microscopy in conjunction with ion-pair reverse-phase HPLC
Analyst, 2019,144, 4985
DOI: 10.1039/c9an00954j

Please follow this external link for the full article: https://pubs.rsc.org/en/content/articlelanding/2019/an/c9an00954j

Open Access: The article « Analysis of long dsRNA produced in vitro and in vivo using atomic force microscopy in conjunction with ion-pair reverse-phase HPLC  by Alison O. Nwokeoji, Sandip Kumar,Peter M. Kilby, David E. Portwood, Jamie K. Hobbs and Mark J. Dickman is licensed under a Creative Commons Attribution 3.0 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/3.0/.