Molecular analysis of the properties of human Arp2/3 complex family members

Project Information

MolecularArp

Grant agreement ID: 101028239

DOI

10.3030/101028239

Project closed

EC signature date 17 March 2021

Start date 1 April 2021

End date 31 March 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 212 933,76

EU contribution

€ 212 933,76

Coordinated by

THE FRANCIS CRICK INSTITUTE LIMITED
United Kingdom

Periodic Reporting for period 1 - MolecularArp (Molecular analysis of the properties of human Arp2/3 complex family members)

Reporting period: 2021-04-01 to 2023-03-31

Summary of the context and overall objectives of the project

Arp2/3 has been known for over two decades, yet many questions remain regarding its regulation and the disassembly of actin branches generated by the complex. Recent breakthroughs have revealed that Arp2/3 in humans is actually a family of 8 complexes with distinct properties, which has important implications for human development and disease. However, a full understanding of why these complexes differ and whether they have unique cellular functions is still lacking. To address these outstanding questions, the project utilized a combination of in vitro biochemical approaches with defined recombinant Arp2/3 complexes and cell-based assays using CRISPR-Cas9 genome edited cells in combination with advanced live cell imaging.
The project has achieved most of its objectives. During the project, it has been discovered that different type I Nucleation Promoting Factors (NPFs) can regulate Arp2/3 iso-complexes similarly, but type II NPFs regulate them differently, indicating that specific Arp2/3 iso-complexes are involved in different cell signalling pathways. Additionally, the fellow’s study on the dissociation of Arp2/3-mediated branches revealed that actin nucleated by various Arp2/3 iso-complexes has different stability, which contributes to understanding the regulation of actin dynamics through different Arp2/3 iso-complexes. However, due to difficulties in obtaining purified MICAL2, it was not examined how it promotes the rapid debranching of Arp3B-containing complexes.
Although the analysis of the cellular function of individual Arp2/3 family members is still ongoing, the preliminary results suggest that ArpC5-containing Arp2/3 complexes play an important role in cell migration. Further work is needed to fully understand the cellular functions of Arp2/3 iso-complexes.
Overall, this project has achieved most of its objectives. The scientific outputs have contributed significantly to our molecular understanding of Arp2/3 iso-complexes mediated actin nucleation and dissociation. During the project, the researcher was able to share their knowledge of biochemistry and molecular biology with the host lab and gained valuable expertise in cell biology. Moreover, the communication and collaboration established during the project have benefited the wider scientific community.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

This project comprises five working packages.
In WP1, the fellow conducted examinations on the properties of Arp2/3 complexes that were activated by various NPFs, such as WASH, WHAMM, N-WASP, WASP, JMY, and WAVE, using in vitro approaches. It was observed that all iso-complexes of Arp2/3 can be activated by different NPFs.
In WP2, the mechanism of actin filament debranching was studied. Thanks to the collaboration with Dr. Guillaume Romet-Lemonne and Dr. Antoine Jegou at the Jacques Monod Institute, France, the fellow had access to the microfluidics setups which were used quantify the dissociation rate of actin filaments nucleated by different Arp2/3 complexes. Interestingly, it was discovered that actin branches generated by distinct Arp2/3 iso-complexes exhibit varying degrees of stability.
In WP3, the plan was to investigate the specific cellular functions of Arp2/3 iso-complexes. Utilising the B16 melanoma cell line as a model system, a significant finding that ArpC5-containing Arp2/3 complexes play a crucial role in cell migration was made. Furthermore, various Crispr-KO cell lines were generated, which will be used in the future research to gain a deeper understanding of the cellular function of other Arp2/3 iso-complexes.
WP4: Dissemination, exploitation and communication. The scientific results generated in the project were presented in national and international conferences, including 2022 European Cytoskeletal Forum Meeting, Hannover, Germany and the British actin meeting, Bristol. The results regarding the stability of Arp2/3 nucleated branch and linear actin filaments were published in EMBO Journal 2023 (https://doi.org/10.15252/embj.2022113008) ‘Regulation of branches versus linear Arp2/3-generated actin filaments’. The work was also presented at the ERC synergy meeting held among Way lab, Dr. Carolyn Moores lab (Birkbeck University, UK) and Dr. Edgar Gomes lab (Instituto de Medicina Molecular, Portugal).
WP5: Knowledge transfer. During the fellowship, the fellow learnt cell biology approaches from the cell biologists in the Way lab. Meanwhile, the fellow gave support for the protein production in the Way lab using her biochemistry background. To learn how to perform cell migration assay with B16 cell line, the fellow went to Dr. Klemens Rottner lab at Helmholtz Centre for Infection Research, Braunschweig, Germany. The two-week training helped her establishing the cell migration study in the Way lab. Thanks to the unique Postdoc Teaching Programme which is organised by the Crick in collaboration with King’s College London, the fellow gave biochemistry practical courses and Pymol workshop to undergraduate students from the King’s college London. She also participated in the UCL Medical School Student Selected Component (SSC) programme. By giving a lecture and a lab tour, she explained how biologists study cytoskeletal organisation to medical students. Therefore, the fellow gained teaching skills which will enhance her future academic career.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The expected results until the end of the project was to understand the molecular and cellular function of Arp2/3 family members. This project has successfully achieved most of its objectives, while others are still ongoing to fully understand the cellular function of Arp2/3 iso-complexes. The results and outcomes of the project provided a strong foundation for future research that will further elucidate the cellular functions of different Arp2/3 iso-complexes. The project has already yielded insights into how these iso-complexes function at the molecular level, which improved our understanding of diseases and disorders related to Arp2/3 isoform deficiencies.
During the project, the fellow conducted research on the properties of Arp2/3 complexes activated by different NPFs and the mechanism of actin filament debranching. The findings on the stability of Arp2/3 nucleated actin were published in the high-impact, open-access, peer-reviewed EMBO Journal. The results were also presented at the European Cytoskeletal Forum and the British Actin Meeting, and it was scheduled to present the results at the upcoming EMBO workshop this year. Additionally, the fellow used Twitter to boost the exposure of the research, benefiting the scientific community directly.
Thanks to the teaching training program at the Crick Institute, the fellow was able to share her knowledge of biochemistry and structural biology with undergraduate students from King’s College London and give lectures to medical students from University College London. These lectures provided students with an insightful view of how research is conducted at the Institute, contributing to the education of our future generation. Moreover, the advancement of scientific knowledge and understanding of cellular processes resulting from this project could lead to broader societal benefits such as improved technologies for cellular imaging and diagnostics.

micro fluidics de branching assay

Summary of my EMBO J paper