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A novel fluorescent dye enables visualization of Kinesin-1 motor activity in living cells

A recent collaboration between scientists in organic chemistry and biochemistry within the NCCR Chemical Biology at the University of Geneva has led to the discovery of the first fluorescent dye that can be used to visualize native kinesin-1 transport activity in cellulo. We interviewed first author Simona Angerani, PhD student in the lab of Nicolas Winssinger (professor in Organic Chemistry, UNIGE) and Charlotte Aumeier (professor in Biochemistry, UNIGE), to hear the story behind.

 

Microtubules (MTs) are highly dynamic polymers that form part of the cell cytoskeleton, serving several important functions within the cell. One of these is the transport of intracellular cargoes towards the cell periphery, a process that requires movement of the motor protein Kinesin-1 on MTs to carry material. Clearly, visualizing and monitoring the movement of Kinesin-1 along MTs is crucial to understanding the many functions associated with MTs. Yet live visualization of Kinesin-1 motion has never been reported, while existing methods rely on fixing and staining cells.

 

The best discoveries often come from accidental findings

The project that led to this discovery began in the Winssinger lab while pursuing an entirely different aim. Simona Angerani and colleagues had designed a quinazolinone-based probe named QPD-OTf to sense oxidative stress in cells. QPD is a well-known precipitating dye that has been employed to stain a variety of cellular targets in response to enzymatic activity. By functionalizing the phenolic moiety of QPD, caged fluorophores are generated that, in response to specific stimuli, yield a green fluorescent precipitate that remains localized at the site of cleavage. But in this case, QPD-OTf was found not to be responsive to O2.− in vitro, with no precipitation observed. Instead, treatment of zymosan stimulated RAW264.7 cells caused dotted fluorescent precipitate after 10 min that evolved into complex filamentous crystals within 1 h.

It was certainly a surprise!” exclaimed Simona. She then explained how the project took an unexpected turn when she observed that QPD-OTf formed beautiful crystals in cells instead of precipitating in response to O2. While QPD is known to generate fluorescent precipitates, the formation of filamentous crystals growing over time had never been observed before. This peculiar behaviour led the Winssinger group to hypothesise that the crystal had to be related to a specific cellular event, suggesting that QPD-OTf could be able to record the native activity of an enzyme. “This opportunity really sounded exciting and motivated us to investigate further”, added Simona.

From the biochemistry perspective, Charlotte Aumeier confirmed that although the original QPD-OTf was developed to visualize reactive oxygen species (ROS) in cells, yet the small molecule never gave any positive results (fluorescent precipitation) during the reaction but instead formed crystals. At that point, “Nicolas Winssinger contacted me to discuss and assess together whether this crystal formation could be cytoskeletal related. This was the beginning of our collaboration” she shares.

 

A novel fluorescent dye is born of a joint and interdisciplinary effort

At this point, organic chemistry and biochemistry joined forces to unravel what was happening with QPD-OTf. “It was a bit like playing Sherlock Holmes with a molecule”, Simona Angerani laughed as she told us that the collaboration began with very little knowledge of what was really happening, besides the fact that QPD-OTf was generating crystals in living cells.

In fact, Simona Angerani and Charlotte Aumeier had to backtrack the crystal localization and elucidate what triggered crystal formation. The filamentous nature of the crystals pointed towards a correlation with cytoskeletal elements, with tubulin being their first suspect. However, purified microtubules were not sufficient to generate crystals. An important hint was given by observing the centre of the crystals, which colocalizes with the Golgi apparatus. At this point, it was clear to all that the observed crystal formation had to be linked to motor proteins, which are found on both MTs and colocalize with the Golgi apparatus.

From then on, the Winssinger and Aumeier labs focused on elucidating this link. Only after a series of experiments combining in cellulo, in vitro and in silico approaches, could they finally identify the target, Kinesin-1.

 

A promising future for better understanding Kinesin-1 motor activity

Charlotte Aumeier and Simona Angerani then shared their thoughts about where the project may be heading, especially how some unanswered questions in the field of microtubules could be investigated using QPD-OTf. “This fluorogenic dye is a promising tool”, they said, “mainly because it allows us to record native kinesin-1 activity in live cells without any modifications or the need for fixation. Until now, tracking microtubule dynamics has been a major limitation in the field, because it has relied primarily on using Kinesin-1-GFP, which results in high fluorescent background of inactive Kinesin-1-GFP, or fixing cells, which cannot be used to uncover the dynamics of most processes”.

The discovery of the QPD-OTf thus paves the way to study microtubules functions in live cells.

I plan to perform experiments at faster timeframes and lower doses of QPD-OTf to understand if we could visualize any dynamics of the transport process” concluded Prof. Aumeier, as “the discovery is very promising but still further work is necessary to define the ideal experimental conditions for better understanding the trafficking on MTs”.

 

More info

Angerani, S., Lindberg, E., Klena, N. et al.,Kinesin-1 activity recorded in living cells with a precipitating dye“, Nat Commun 12, 1463 (2021).

 

Simona Angerani carried out her undergraduate studies in Milan, Itlay, where she obtained her Master’s degree in chemistry under the supervision of Prof. Cesare Gennari, working on small molecule-drug conjugates. In 2016 she moved to the University of Geneva for her PhD in the group of Prof. Nicolas Winssinger at the Organic Chemistry Department, focusing on the development of supramolecular networks able to perform in biological systems. After the PhD, Simona will continue working in Geneva, starting a Postdoc in the group of Prof. Oliver Hartley at the Centre Médical Universitaire in July 2021.

 


Thanks very to Lluc Farrera Soler & Ilaria Di Meglio for this article.

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