Periodic Reporting for period 1 - EndoPos (Endosome positioning in tumour-stroma interactions)
Okres sprawozdawczy: 2019-05-01 do 2021-04-30
More than 150,000 women die each year from ovarian cancer. Rab11 GTPase family (Rab11a/b/c) is implicated in determining the aggressiveness of ovarian cancer by delivering signalling molecules and receptors for extracellular matrix by recycling endosomes where cancer cells need them. The mechanism how is poorly understood and there is very limited option how to study the specific and redundant functions of individual members of Rab11 family. Furthermore, manipulating endosomal trafficking and signalling pathways with subcellular specificity is a challenge to cell biology.
In the project EndoPos we use state-of-the-art innovative approaches to determine recycling endosomes positioning in ovarian cancer cells. First by BioID-based proteomics (a proximity labelling approach), second by developing a magnetogenetic approach to reposition endosomes in live ovarian cancer cells invading the extracellular matrix using external magnetic gradient. Finally, we use state of the art microscopy techniques for imaging Rab11s by exploiting CRISPR-Cas9 mediated knock-ins of ovarian cancer cells.
In the project EndoPos we use state-of-the-art innovative approaches to determine recycling endosomes positioning in ovarian cancer cells. First by BioID-based proteomics (a proximity labelling approach), second by developing a magnetogenetic approach to reposition endosomes in live ovarian cancer cells invading the extracellular matrix using external magnetic gradient. Finally, we use state of the art microscopy techniques for imaging Rab11s by exploiting CRISPR-Cas9 mediated knock-ins of ovarian cancer cells.
The Endopos project started on May 2019 and the main results so far are:
1) BioID based proteomic screen identifying Rab11a, Rab11c (=Rab25) and Rab4 protein complexes followed by verification using an innovative Knocksideways technique and quantitative analysis.
2) We developed and optimized a novel approach allowing one-step CRISPR-Cas9 mediated targeted knock-ins resulting in conditional reversible gene knockouts/rescues ideal for fluorescent microscopy with the option to select cells by FACS sorting independent of traditional clonal methods. The rescue protein level is tunable and possible to control systematically (drug-based) or spatiotemporally (optogenetically) and is not limited to only active genes. Therefore, we were able to provide novel high-resolution colocalization data from simultaneous observation of endogenously fluorophore-tagged Rab11a/b with Rab25 positive vesicles inside live ovarian A2780 cancer cells where Rab25 is otherwise endogenously silenced. In addition, it allowed us to compare endogenous Rab11a, Rab11b and Rab25 expression kinetics (DNA accessibility for transcription), stability and dynamics of all protein coding isoforms at once and their direct effect on cell migration, proliferation and invasion.
3) In collaboration with Prof. Piehler’s lab (University of Osnabrück in Germany) we established magnetogenetic manipulation of endogenously tagged Rab25 (Rab11ab) endosomes by using GFP functionalized nanoparticles and by building home-made magnetic manipulator set-up including microinjection, perfusion system and extended heating chamber within the Bioimaging facility in Manchester. This system uses a state-of-the-art spinning disc microscope so we can image ovarian cancer cells at high spatial-temporal resolution and determine the effects of magnetogenetic manipulation of Rab11s on trafficking, signalling, actin dynamics and migration in living ovarian cancer cells. Our proof-of-concept experiments show how direct control of Rab25 positive endosomes can promote protrusions on demand and how these magnetically controlled endosomes allow ovarian cancer cell migrating in 3D environment to feel and respond to the magnetic field gradient.
1) BioID based proteomic screen identifying Rab11a, Rab11c (=Rab25) and Rab4 protein complexes followed by verification using an innovative Knocksideways technique and quantitative analysis.
2) We developed and optimized a novel approach allowing one-step CRISPR-Cas9 mediated targeted knock-ins resulting in conditional reversible gene knockouts/rescues ideal for fluorescent microscopy with the option to select cells by FACS sorting independent of traditional clonal methods. The rescue protein level is tunable and possible to control systematically (drug-based) or spatiotemporally (optogenetically) and is not limited to only active genes. Therefore, we were able to provide novel high-resolution colocalization data from simultaneous observation of endogenously fluorophore-tagged Rab11a/b with Rab25 positive vesicles inside live ovarian A2780 cancer cells where Rab25 is otherwise endogenously silenced. In addition, it allowed us to compare endogenous Rab11a, Rab11b and Rab25 expression kinetics (DNA accessibility for transcription), stability and dynamics of all protein coding isoforms at once and their direct effect on cell migration, proliferation and invasion.
3) In collaboration with Prof. Piehler’s lab (University of Osnabrück in Germany) we established magnetogenetic manipulation of endogenously tagged Rab25 (Rab11ab) endosomes by using GFP functionalized nanoparticles and by building home-made magnetic manipulator set-up including microinjection, perfusion system and extended heating chamber within the Bioimaging facility in Manchester. This system uses a state-of-the-art spinning disc microscope so we can image ovarian cancer cells at high spatial-temporal resolution and determine the effects of magnetogenetic manipulation of Rab11s on trafficking, signalling, actin dynamics and migration in living ovarian cancer cells. Our proof-of-concept experiments show how direct control of Rab25 positive endosomes can promote protrusions on demand and how these magnetically controlled endosomes allow ovarian cancer cell migrating in 3D environment to feel and respond to the magnetic field gradient.
Deciphering the role of individual Rab11 GTPases can help with understanding of the changes in metastatic ovarian cancer cells that promote invasion and acquire drug resistance. Understanding the mechanisms that drive these routes leading to patient mortality will provide line-of-sight to novel targeted therapies that prevent the spread of ovarian cancer.
Our novel innovative CRISPR-Cas9 mediated gene control allowing conditional reversible gene knockouts/rescues is a very powerful technique as it can be applied to the majority of protein coding genes. By one CRISPR based knock in you can get all you need at once for your research, where it is even possible to choose and adjust later the expression levels of re-activated genes (bright for microscopy vs physiological levels). We expect that our approach once published will bring substantial benefit to the wider scientific community.
Magnetogenetic manipulation belongs to cutting-edge technology that promises high-impact publications which will help to attract additional funds and new collaborations. Remote active spatio-temporal control of cellular functions/components by using magnetic force is not limited by sample thickness, offers unique unprecedented opportunities in fundamental research and allow us to answer fundamental and long standing questions that cannot be addressed with conventional methods. To see direct consequences of our actions. We expect that by the end of the project we will provide a step change in our understanding of the role of vesicle trafficking in tumour progression and the mechanisms that determine how cells decode their surrounding environment.
Our novel innovative CRISPR-Cas9 mediated gene control allowing conditional reversible gene knockouts/rescues is a very powerful technique as it can be applied to the majority of protein coding genes. By one CRISPR based knock in you can get all you need at once for your research, where it is even possible to choose and adjust later the expression levels of re-activated genes (bright for microscopy vs physiological levels). We expect that our approach once published will bring substantial benefit to the wider scientific community.
Magnetogenetic manipulation belongs to cutting-edge technology that promises high-impact publications which will help to attract additional funds and new collaborations. Remote active spatio-temporal control of cellular functions/components by using magnetic force is not limited by sample thickness, offers unique unprecedented opportunities in fundamental research and allow us to answer fundamental and long standing questions that cannot be addressed with conventional methods. To see direct consequences of our actions. We expect that by the end of the project we will provide a step change in our understanding of the role of vesicle trafficking in tumour progression and the mechanisms that determine how cells decode their surrounding environment.