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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - OSS (Spatio-temporal control of the Src kinase activation through Optogenetics in Cell invasion)

Teaser

Summary

The OSS project investigated how the same oncogene (a gene that causes cancer) named Src can control different cellular signalling circuitries and consequently different cell behaviors. Our hypothesis is that Src signalling should be regulated in space and time to be able to induce many behaviors, including cell invasion, important for cancer cell dissemination.
Cell invasion is based on adhesive structures named invadosomes that are responsible for microenvironment degradation which is crucial for cancer metastasis. Src controls both assembly and disassembly of invadosomes. It is puzzling to understand how one single protein can affect opposite functions of the same structure. The OSS project is based on the development of a very innovative technique named optogenetics, which allows to turn on and off the molecule Src, by simply enlightening the cell with blue light. Being able to control Src activity in space and time might help for fighting cancer cell dissemination. Furthermore, it would represent a major advance in Src oncogene targeted therapy.

Work performed

Src is a kinase, an enzyme able to add phosphate to proteins which affects their activity. Src can also auto-phosphorylate itself, leading to its own activation. This Src activation forms invadosomes and makes cells become invasive. The number and size of invadosomes in cancer cells are propotional to the level of Src activation. Invadosomes invade the extracellular matrix by degrading it (ECM degradation). The efficiency of cell invasion is measured by the degraded area per invadosome. Our results have shown that the size and number of invadosomes do not result proportionally in an increased extracellular matrix degradation. An invadosome is a circular structure on the cell membrane that dynamically changes its shape and movement. The OSS project discovered, for the very first time in the field, that different dynamics of invadosomes were crucial for efficient ECM degradation. It was revealed that an optimal speed of invadosome movement controls the efficiency of the degradation.
In order to digest the matrix specific enzymes, called MT1-MMP, are secreted in the vicinity of invadosomes . The amount of MT1-MMP was investigated during different stages of ECM degradation to correlate the quantity of MT1-MMP with different dynamics of invadosomes. A stable cell line expressing Src optogenetic system was generated . Preliminary data showed that a specific frequency of blue light is correlated with a specific frequency of Src activation and results in specific dynamics of invadosomes. However additional experiments should be performed in order to confirm the specificity of the frequency and time needed to control different invadosome dynamics. The OSS project identified protein partners of activated Src which were controlled in space and time by the Src optogenetic system. The most relevant identified protein found is an ion channel named TRPV4, which is phosphorylated by Src and is involved in signalling pathways connecting invadosome size and number with ECM degradation. The first phase resulted in the most exploitable results since it has never previously been shown that different Src driven invadosomes lead to different abilites to degrade the ECM.

CONFERENCES:
During the OSS project, the researcher attended a conference of Integrated Mechano-Chemical Signals In Invasion, 16-20 October, Saint Paul De Vence, France which allowed her to be integrated into a cell invasion society, meeting crucial people in the field, discussing with them in person about new advancements in the field and collaboration opportunities.
During the Friday committee thesis in the IAB, she presented her work in front of the institute and all of its departments. During the Department day in October 2017 she was selected to present the lab and give a talk about her work. This was a unique opportunity, because of the valuable feedback obtained from participants. She also participated in the first French Optogenetic Club meeting on the 23rd and 24th June 2016 at the Albert Bonniot Institute in Grenoble where she had the opportunity to discuss her work with prominent scientists in the optogenetic field.

COLLABORATION:
During the OSS project the researcher established a collaboration between the team of Dr.Valverde and the team of Dr. Albiges-Rizo. This work has been published in 2018 in the high impact journal PNAS (DOI: 10.1073/pnas.1718177115). Her contribution allowed two previously unrelatedly investigated fields, ion channels and cell invasion, to meet and merge.

REVIEW:
During the OSS project the researcher had the opportunity to collect data, design and write a scientific review that was published in the journal Matrix biology in 2017 (DOI: 10.1016/j.matbio.2016.06.007).

PAPER IN PREPARATION:
The work done during the OSS project will be submitted to the Nature Cell Biology journal in 2018 which will greatly impact future researchers career as an independent researcher.

BLOG: The researcher opened a blog to help Croatian students to inte

Final results

For the proposed project OSS, the main research training aims and objectives comprised: 1) Increasing researcher scientific background as invadosome adhesion, ECM degradation, complexity of cell invasion 2) Gaining technical expertise combining molecular genetics, biochemistry, cellular biology, imaging and physics. 3) Developing organizational and managing skills: The researcher was responsible for monitoring the projectâ€™s progress and report on it, making adjustments to the work plan when it was necessary, maintaining regular contacts with collaborators, writing scientific papers and grant applications, as well as supervising master students. All of the goals contributed to develop and expand the researcherâ€™s scientific maturity, leadership and organizational skills, which will be crucial for the development of researcher career.
The ability to control Src will have a huge impact for society since Src is crucial for invadosome based cancer metastasis. Controlling Src would allow us to slow down or stop the spread of cancer. Moreover understanding of Src dynamicity would lead to deeper understanding of how a same protein can regulate different processes simultanouesly in space and time. This discovery would greatly impact society.

CONCLUSION
All of these trainings contributed to develop and expand the researcherâ€™s scientific maturity, leadership and organizational skills, which will be crucial for the development of her career as an independent investigator and researcher for the CNRS and will contribute to the scientific excellence of the European Research Area community.