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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: h ttp://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)

Real time dynamics of Gating-Related conformational changes in CorA

Magnesium (Mg²⁺) is a key divalent cation in biology. It regulates and maintains numerous, physiological functions such as nucleic acid stability, muscle contraction, heart rate and vascular tone, neurotransmitter release, and serves as cofactor in a myriad of enzymatic reactions. Most importantly, it coordinates with ATP, and is thus crucial for energy production in mitochondria.*

In order to store Mg²⁺ in the mitochondrial lumen it is imported via Mrs2 and Alr2 ion channels that are closely related to CorA, the main Mg2+-importer in bacteria. Although these Mg2+-transport proteins do not show much sequence conservation, they all share two trans-membrane domains (TMDs) with the signature motif Glycine-Methionine-Asparagine (GMN) at the extracellular loop.*

CorA, a divalent-selective channel in the metal ion transport superfamily, is the major Mg²⁺-influx pathway in prokaryotes. CorA structures in closed (Mg²⁺-bound), and open (Mg²⁺-free) states, together with functional data showed that Mg²⁺-influx inhibits further Mg²⁺-uptake completing a regulatory feedback loop. While the closed state structure is a symmetric pentamer, the open state displayed unexpected asymmetric architectures.*

In the article “Real time dynamics of Gating-Related conformational changes in CorA” Martina Rangl, Nicolaus Schmandt, Eduardo Perozo and Simon Scheuring used high-speed atomic force microscopy (HS-AFM), to explore the Mg²⁺-dependent gating transition of single CorA channels: HS-AFM movies during Mg²⁺-depletion experiments revealed the channel’s transition from a stable Mg²⁺-bound state over a highly mobile and dynamic state with fluctuating subunits to asymmetric structures with varying degree of protrusion heights from the membrane.*

Their data shows that at Mg²⁺-concentration below Kd, CorA adopts a dynamic (putatively open) state of multiple conformations that imply structural rearrangements through hinge-bending in TM1. They also discuss how these structural dynamics define the functional behavior of this ligand-dependent channel.*

All Atomic Force Microscopy experiments described in the article were performed using NanoWorld Ultra-Short Cantilevers USC-F1.2-k0.15 for high-speed Atomic Force Microscopy ( HS-AFM ). Videos of CorA membranes were recorded with imaging rates of ~1–2 frames s−1 and at a resolution of 0.5 nm pixel−1.

Figure 1 from “*Real time dynamics of Gating-Related conformational changes in CorA*”:
Sample morphology of CorA reconstitutions for HS-AFM.

(a) HS-AFM overview topograph of densely packed CorA in a POPC/POPG (3:1) lipid bilayer exposing the periplasmic side and a loosely packed protein area with diffusing molecules exposing the intracellular face (full color scale: 20 nm). Left: Height histogram of the HS-AFM image with two peaks representative of the mica and the CorA surface (∆Height (peak-peak): 12 nm (20,500 height values)). The dashed line indicates the position of the cross-section analysis shown in (b). (b) Profile of the membrane shown in a), including a cartoon (top) of the membrane in side view. The height profile (~12 nm) corresponds well to the all-image height analysis (a, left) and the CorA structure (Matthies et al., 2016). (c) High-resolution image (top) and cross-section analysis along dashed line (bottom) of the periplasmic face. The height and dimension of the periplasmic face is in good agreement with the structure (left), and the periodicity (~14 nm, n = 40) corresponds well with the diameter of the intracellular face spacing the molecules on the other side of the membrane (full color scale: 2 nm). (d) HS-AFM image of densely packed CorA embedded in a DOPC/DOPE/DOPS (4:5:1) membrane. This reconstitution resulted in two stacked membrane layers, both exposing the CorA intracellular face. The dashed line indicates the position of the cross-section analysis shown in (e). Left: Height histogram of the HS-AFM image with two peaks at ~12 nm and ~17 nm (32,500 height values), corresponding to the proteins in two stacked membranes (full color scale: 20 nm). (e) Section profile of the membrane shown in d), including a cartoon (top) of the membrane in side view. (f) High-resolution view and cross-section analysis along dashed line (bottom) of the CorA intracellular face revealing the individual subunits of the pentamers (full color scale: 3 nm). Inset: 5-fold symmetrized average of CorA. The dimensions of CorA observed with HS-AFM are in good agreement with the structure (left: PDB 3JCF). The structures in (c) and (f) are shown in ribbon (top) and surface (bottom) representations, respectively.

*Martina Rangl, Nicolaus Schmandt, Eduardo Perozo, and Simon Scheuring
Real time dynamics of Gating-Related conformational changes in CorA
eLife. 2019; 8: e47322
DOI: 10.7554/eLife.47322

Please follow this external link to read the full article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927688/

Open Access: The article “Real time dynamics of Gating-Related conformational changes in CorA” by Martina Rangl, Nicolaus Schmandt, Eduardo Perozo 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 http://creativecommons.org/licenses/by/4.0/.