Optical Control of G-Actin with a Photoswitchable Latrunculin

Background of the preprint

Actin is an important structural protein in eukaryotic cells. Due to its biological importance, its homeostasis is tightly regulated by multiple biological mechanisms. This provides many opportunities by which actin can be modulated—several small molecule modulators of actin have been discovered. However, the complexity of these processes also makes the study of actin very challenging.

As a result of highly collaborative efforts led by the Trauner group and the Arndt group, a series of photoswitchable versions of the F-actin stabiliser jasplakinolide, called optojasps, were created that stabilise F-actin precisely and reversibly.^{1, 2} In this work, Vepřek and co-workers (from the Trauner group) develop a series of photoswitchable G-actin stabilisers based on the latrunculins. Consisting of a diazene linker, these probes are usually in the inactive trans- position; when exposed to light irradiation, they then convert to the active cis- position (Figure 1). Due to their ability to photoswitch, these probes can bind to G-actin monomers reversibly when light is shone on them. The authors could thus use these probes to control G-actin dynamics in a space- and time-dependent manner. First, they performed a series of assays to identify the most useful probe, which they named OptoLat. They then tested OptoLat in various cell lines and in more complex tissues.

 

 

Figure 1. Overview of the work reported by Vepřek and co-workers in their preprint.

 

 

Key findings of this preprint

Vepřek and co-workers first set about designing and synthesising their latrunculin photochemical probes based on the structure-activity relationship of known latrunculin binders. Broadly, these latrunculin analogues either bore macrocyclic lactones or ester linkages to the azobenzene and diazocine groups. The authors also made two more structural changes to their latrunculin probes. First, they modified the vector around the aromatic diazene connection to modulate the steric clash between their probes and the protein following photoisomerisation of the diazene bond. Second, they replaced the ester linkages with aryl ethers to promote metabolic stability and to maximise steric effects when the probes bind to the protein pocket.

Among these probes, the authors identified one that exhibited the best photophysical properties, i.e. the most red-shifted absorption spectrum and the fastest thermal relaxation, which they named OptoLat. In further tests, they also confirmed that photoswitching was fully reversible (i.e. could undergo complete deactivation and reactivation) and persisted for at least 30 cycles. In light-dependent cell proliferation assays, OptoLat was activated by irradiation with blue light, with an EC₅₀ in the micromolar range.

Next, Vepřek and co-workers tested OptoLat in cell-based assays to probe actin dynamics and their effects on cellular structure and function. In oligodendrocytes, the authors proved that OptoLat’s antiproliferative effect was caused by its ability to destabilise actin networks upon irradiation with blue light.

With this in hand, the authors investigated OptoLat’s effects in various biological systems. Actin plays important roles in yeast cellular division and proliferation and cell migration and the authors found that exposing OptoLat-treated yeast to blue light led to a significant decrease in actin cables within 5 minutes. Similarly, OptoLat reduced the migration of invasive cancer cells in a light-dependent fashion, though the concentration of OptoLat used under these conditions was not cytotoxic. Further experiments by the authors showed that OptoLat activation in live cells destroyed actin networks and that these changes were fully reversible.

Finally, the authors tested OptoLat in a more complex tissue—the brain. In these experiments, the authors monitored the cell shape and motility of microglia following OptoLat treatment and activation. They found that OptoLat activation did not affect cell shape, but significantly reduced motility; changes in motility were reversed after the OptoLat-treated cells were no longer exposed to light.

 

 

What I like about this preprint

Photoswitchable probes have many uses in chemical biology: they are often used to investigate cellular organisation and dynamics. Here, Vepřek and co-workers outline their development of a light-activated actin de-stabiliser, OptoLat. I think this preprint is interesting for two reasons.

First, the biological applications will be broad. Actin is a highly-conserved structural protein, so probes that target actin will be applicable in almost all organisms. However, selectivity will be essential for specific control. This problem is partly resolved by OptoLat, because it is only activated in the presence of blue light. Vepřek and co-workers could thus control when OptoLat was activated, the duration of the activation, and the regions by which it was activated.

Second, I find the chemistry of photoswitchable probes rather fascinating. Specifically, to be able to interconvert the cis- and trans- isomers of OptoLat readily, reversibly, and in a repeated manner without degradation of the probe is quite challenging because these light-sensitive probes can degrade each time they are excited by light. Moreover, the authors describe a simple route to synthesise OptoLat of just about 10 steps in their preprint. Synthetic simplicity is important in these projects because it directly affects synthetic chemists’ throughput in making a variety of structures to understand the structure-activity relationship of these probes and identify the best one. While one of the synthetic steps in this route produces a pair of diastereomers, they can be separately purified and might anyway be useful intermediates that could lead to the further exploration of diastereomeric analogues.

 

 

Future directions

Future work around the development of OptoLat and its analogues will probably involve looking into their potential biological uses. Open questions prompting future research for instance include: are these diazene structures orthogonal to other functional handles? That is, could we install different functional groups onto these probes, and selectively activate them by shining different wavelengths of light? For example, diazirines are useful photochemical probes in protein labelling. In theory, it would thus be possible to develop probes in which shining light of a certain wavelength would change its shape, and shining light of a different wavelength would allow it to perform another function, such as labelling. These developments would conceivably broaden the applications of these probes.

 

Acknowledgements

Images created using Microsoft Powerpoint, ChemDraw, and BioRender.

 

Questions for authors

1. Did thermal relaxation times differ among the probes and were these differences significant?

 

 

References

1. M. Borowiak, F. Küllmer, F. Gegenfurtner, S. Peil, V. Nasufovic, S. Zahler, O. Thorn-Seshold, D. Trauner and H.-D. Arndt, Journal of the American Chemical Society, 2020, 142, 9240-9249.
2. F. Küllmer, N. A. Vepřek, M. Borowiak, V. Nasufović, S. Barutzki, O. Thorn‐Seshold, H. D. Arndt and D. Trauner, Angewandte Chemie International Edition, 2022, 61.

 

Tags: bioorthogonal chemistry, photoswitching

doi: https://doi.org/10.1242/prelights.35576

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