Defining the role of Arp2/3 complex diversity at multiple scales of biology

Informations projet

ArpComplexity

N° de convention de subvention: 810207

DOI

10.3030/810207

Projet clôturé

Date de signature de la CE 14 Janvier 2019

Date de début 1 Juillet 2019

Date de fin 30 Juin 2026

Financé au titre de

• EXCELLENT SCIENCE - European Research Council (ERC)

Coût total

€ 10 715 153,00

Contribution de l’UE

€ 10 715 153,00

Coordonné par

THE FRANCIS CRICK INSTITUTE LIMITED
United Kingdom

Periodic Reporting for period 4 - ArpComplexity (Defining the role of Arp2/3 complex diversity at multiple scales of biology)

Période du rapport: 2024-01-01 au 2025-06-30

Résumé du contexte et des objectifs généraux du projet

Our skeleton provides our bodies with support and strength. In the same way, albeit on a much smaller scale the cells that make up our body also have an internal skeleton. This, so called cytoskeleton is involved in many essential aspects of life and is part of the machinery that drives cell movement during development and in tissue repair throughout our lives. This project focuses on a part of the cytoskeleton called actin, which forms a complex, dynamic networks of filaments that help drive cell motility and maintain tissue organization. Disruption of actin cytoskeleton function is associated with numerous diseases, including cancer, immunodeficiency and muscle disorders. Studying the actin cytoskeleton is important so we can understand how healthy cells operate and how its misfunction causes disease.

The Arp2/3 complex is a critical component of the actin cytoskeleton that initiates and stabilises the formation of branched actin filament networks. The Way group discovered that humans and other mammals make eight distinct types of Arp2/3 complex, and we are investigating their activities and functions. Using the different expertise of our research groups, we will be exploring their: 1) molecular structures and activities; 2) regulation and function within cells; 3) physiological roles in tissues, - focusing on muscle - to understand their contributions to homeostasias and health. Overall, using biochemistry, structural, cell and developmental biology, and physiology analysis, we will determine how Arp2/3 diversity contributes to biological complexity at multiple scales.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

COVID-19 pandemic caused major limitations in laboratories access, team recruitment and equipment purchase. Nevertheless, the ArpComplexity team have achieved the following:

- We were able to hold a kick-off meeting in London in November 2019 for two days to begin team-building, share expertise and start to build a vision for synergy discovery;
- We have subsequently held a number of whole-team scientific exchanges and journal clubs online.
- Since 2022 we are now having retreats every 6 months and zoom meeting every 2-3 weeks where the three teams present, discuss their ongoing work and collaborations.

- The Way team have created transgenic mice lacking distinct Arp2/3 isoforms both whole-body and in specific tissues including skeletal muscle.
- The Way team have generated, and shared recombinant Arp2/3 complexes and associated proteins with the Moores lab for their structural analysis using cryo-EM – medium-resolution results have already been published (PMID: 32661131).
- The Way team have uncovered that ARP3B containing Arp2/3 complexes promote the disassembly of branched actin networks as the result of the oxidation of Arp3B Met293 by the methionine monooxygenase MICAL2 (PMID: 34106209).
- The Way team have begun to establish biochemical assays to investigate the actin nucleating properties of different Arp2/3 complexes and in branched network disassembly.
- The Way teams has found that mice lacking ARPC5 are embryonic lethal at ~ E9.5 due to defects in heart development (PMID: 37382373).
- The Way team in collaboration with clinicians in Germany has found that ARPC5 deficiency in humans leads to severe early-onset systemic inflammation and mortality (PMID: 37382373).
- The Way teams has found that CK666 and CK869 do not inhibit all Arp2/3 iso-complexes.
- The Way teams has generated a nano body library against Arp2/3 iso-complexes.
- The Way teams has found that mice lacking ARPC5L develop normally but have progressive ataxia from 10 weeks due to Purkinje cell degeneration.

- The Moores team have worked with the Way team to perform cryo-EM structure determination of Arp2/3 complexes and have optimized sample preparation with the goal of determining high resolution structures of these complexes.
- The Moores team have worked with the Way team to perform cryo-EM structure determination on Arp2/3-mediated actin branches and visualize the branch stabilization factor cortactin.
- The Moores team have worked with the Way and Gomes teams to develop cell sample preparation for cryo-ET visualization of the branched actin network.
- The Moores team are developing sample preparation protocols with the Gomes team to study muscle ultrastructure using TEM tomography and SEM tissue imaging to define the roles of specific Arp2/3 complexes in muscle development.

- The Gomes team have initiated the characterization of the dynamics of T-tubules during triad formation by time-lapse microscopy, and the role of different Arp 2/3 isoforms on skeletal muscle development and nuclear positioning.
- The Gomes team have collaborated with the Moores team to optimise muscle sample preparation for their EM studies.
- The Gomes team are also working with several transgenic mouse lines, including from the Way team, to enable them to study the role of Arp2/3 isoforms in skeletal muscle development.
- The Gomes team found that ARPC4 knockout mice exhibit noticeable muscle irregularities and an increase in nuclear volume and area compared to wild-type counterparts.
- The Gomes lab found that ARPC5, but not ARPC5L is associated the process of T-tubule disentanglement.
- The Gomes lab found that membrane tension is involved in T-tubule formation and ARPC5 is involved in this mechanism.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

Our synergistic goal is to define the role of Arp2/3 complex diversity at three hierarchies of biology:

1. Molecular basis of Arp2/3 diversification
With purified isoform-specific complexes we will perform cryo-electron microscopy and single molecule fluorescence microscopy to reveal the structural variations and influence of Arp2/3 diversity on actin networks in vitro.

2. Cellular function of different Arp2/3 complexes
With cells expressing specific Arp2/3 isoforms, we will use quantitative live cell imaging and cryo-electron tomography to reveal the dependence of cellular actin networks on Arp2/3 diversity and its functional consequences.

3. Developmental and physiological role of individual Arp2/3 complexes
With genetically modified cultured myofibers and transgenic mice, we will use an array of imaging approaches to reveal the contribution of different Arp2/3 family members to muscle development, structure and physiology.

Our interdisciplinary plan takes advantage of unique reagents and powerful model systems, and will allow us to determine how Arp2/3 diversity contributes to biological complexity at multiple scales.

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