UNICELLINTEGRIN · Unravelling the function of integrins in the unicellular relatives of Metazoa

Unravelling the function of integrins in the unicellular relatives of Metazoa

Together with regulated signaling and cell differentiation, adhesion is one of the key features of multicellularity. Integrins are a family of major cell adhesion receptors whose properties and function have been extensively studied in animals. They are heterodimeric transmembrane proteins consisting of alpha and beta subunits. Their canonical function is adhesion to the extracellular matrix ligands, typically short peptide motifs such as arginine-glycine-aspartate – RGD. Integrin heterodimers bind and interact with several other proteins in the cell forming the integrin mediated adhesion and signaling complex. A few years ago, Dr. Ruiz-Trillo's group made a surprising discovery of the presence of integrins in unicellular organisms related to animals. The main aim of the UNICELLINTEGRIN project was to elucidate the function of integrins and other components of the integrin mediated adhesion and signaling complex in unicellular context. We chose to work on two species closely related to animals: filasterean Capsaspora owczarzaki and ichthiosporean Creolimax fragrantissima. We devised a series of experiments, including transcriptome analyses, gene silencing, and determining the localization of integrins in the cell by immunocytochemistry. The feasibility and outcome of most of these experiments would depend on the development of molecular biology tools and techniques (gene silencing or knock out, transfection, selection, etc.) for our organisms, as well as on the development of custom antibodies for integrins and other proteins of interest. Therefore, a significant part of the project dealt with these matters and a lot of effort was invested in resolving them. Although progress was made on all issues mentioned above, they did prove to be a significant obstacle in achieving the goals of the project. In spite of the extensive testing, morpholinos and RNAi proved inefficient in gene silencing with our organisms. Instead, the group is currently developing CRISPR-Cas system for genome editing in C. owcarzaki. Transfection is now possible in both organisms, but with low efficiency (~ 1%). We are continuing the work to optimize the transfection protocols in order to increase the efficiency. Also, we are performing extensive testing of antibiotics that should enable us to selectively grow the transfected cells. Within the project, we have been working with several specialised companies in developing custom antibodies to perform localization and immunoprecipitation studies. We have produced antibodies against several proteins against proteins involved in integrin-mediated adhesion and signalling (integrins alpha and beta, talin, vinculin, parvin, and paxillin) in both organisms. Most antibodies were delivered only recently and are being tested and optimised. In spite of the challenges with the technical and methodological aspects of the research, we plan to continue the work on integrins in unicellular relatives of animals. Initial results are encouraging and indicate that integrins do have a role in the adhesion of C. owczarzaki cells. Integrin genes and a majority of genes involved in integrin-mediated adhesion and signalling are significantly more highly expressed in the C. owczarzaki adherent stage. In addition, we started related projects. One deals with molecular ecology of of C. owczarzaki and the potential role of integrins in interaction with its host -snail Biomphalaria glabrata. Another one investigates the glycome of the unicellular relatives of animals, with a potential to broaden the research into the role of glycosylation in the origin of multicellularity. The scientist in charge, Dr.Iñaki Ruiz-Trillo, has been awarded an ERC Consolidator Grant in 2014. Furthermore, his Multicellgenome lab (www.multicellgenome.com) receives Spanish national funding. This will provide a solid base for further research on the subjects mentioned above. The researcher, Dr. Matija Harcet, has got a position of a research associate (a five-year contract with possible extension in five-year increments) at Institut Ruder Boskovic in Zagreb, Croatia. In the next year, he will spend most of the time in Dr. Ruiz-Trillo's lab, continuing the work on integrins and developing additional projects mentioned above. Scientist in charge: Dr. Iñaki Ruiz-Trillo Multicellgenome lab Institut de biologia evolutiva (UPF-CSIC) Pssg. de la Barceloneta 37-49. 08003 Barcelona, Spain Phone: (+34) 93 230 96 40 E-mail: inaki.ruiz@ibe.upf-csic.e Web: www.multicellgenome.com Researcher Dr. Matija Harcet Multicellgenome lab Institut de biologia evolutiva (UPF-CSIC) Pssg. de la Barceloneta 37-49. 08003 Barcelona (Spain) or Laboratory for molecular genetics Institut Ruder Boskovic Bijenicka cesta 54, 10 000 Zagreb, Croatia Phone: +34 93 2309500 (Barcelona) or +385 (0)1 4561111 Email: matija.harcet@ibe.upf-csic.es, matija.harcet@irb.hr Web: http://www.irb.hr/eng/People/Matija-Harcet

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