Digital Protein Biophysics of Aggregation

Project Information

DiProPhys

Grant agreement ID: 101001615

DOI

10.3030/101001615

EC signature date 11 March 2021

Start date 1 July 2021

End date 30 June 2026

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 999 715,00

EU contribution

€ 1 999 715,00

Coordinated by

THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE
United Kingdom

Periodic Reporting for period 2 - DiProPhys (Digital Protein Biophysics of Aggregation)

Reporting period: 2023-01-01 to 2024-06-30

Summary of the context and overall objectives of the project

Problem/Issue Being Addressed: The project focuses on understanding the fundamental principles governing the formation, self-replication, and impact on living systems of aberrant protein aggregates, particularly amyloid aggregates implicated in neurodegenerative disorders. The inherent heterogeneity and complexity of protein aggregation processes present challenges for conventional bulk methods to probe and understand these phenomena effectively.

Importance for Society: Understanding protein aggregation is crucial for addressing a range of increasingly prevalent and currently incurable neurodegenerative disorders. Aberrant protein aggregation is associated with severe consequences for biological systems, making it imperative to decipher its mechanisms to develop potential therapeutic interventions.

Overall Objectives:
1. Understanding Nucleation Events: Investigate the triggering mechanisms of nucleation events in protein aggregation, exploring pre-nucleation clusters, non-classical nucleation pathways, and the role of liquid-liquid phase separation.
2. Characterizing Spatial Propagation: Monitor the spatial propagation of protein aggregation, studying its transmission in space and across soft barriers like cell membranes.
3. Single-Cell Analysis: Analyse the effects of specific types of protein aggregates on biological function at the single-cell level, shedding light on cellular protection mechanisms against aberrant protein aggregation.
4. Developing Digital Biophysics Toolkit: Develop and apply a novel digital biophysics platform combining microfluidics and single-molecule spectroscopy to study protein aggregation at the single aggregate and single-cell levels.

Methodological Approach: The project focuses on the development of a digital biophysics toolkit utilizing microfluidic compartmentalization and single-molecule detection techniques. This toolkit aims to enable the study of protein aggregation processes with unprecedented resolution and accuracy, allowing for real-time monitoring of aggregation dynamics and characterization of fundamental physical properties.
Summary: The project aims to introduce fundamentally novel approaches to studying protein aggregation, bridging the gap between in vitro biophysics and real biological systems. The envisioned platform holds promise for revolutionizing the understanding of protein aggregation dynamics, potentially leading to breakthroughs in both basic science and therapeutic interventions for neurodegenerative disorders.

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

We have been able to make good progress across the work packages. In project A, we have been able to set up the single aggregate characterisation platform as a digital assay. We have been pursuing two main avenues for detection: single molecule confocal spectroscopy and direct imaging of microdroplets using epifluorescence microscopy (WP-A1). We now have proof of concept data showing that both approaches can yield single particle resolution. We have also been able to make good progress in isolating and physically separating the products of aggregation (WP-A2). In particular, we have had good success with electrophoretic separation on chip. We have initiated work on the transport of aggregates in space (WP-A3) and have seen some interesting phenomena with spatial patterns of aggregation triggered in time and space. In this context, we have also looked at the interactions of aggregates with soft barriers, including membranes on chip. We have also made initial steps in establishing the platform for reading out protein aggregate and cluster states from lysed cells (WP-A4). In particular this area is likely to be very promising for understanding phase behaviour of proteins in health and disease in the context of cellular complexity, but while maintaining the level of control characteristic of bottom up biophysical measurements.

Work package B has also progressed well. In particular, we have found that the phenomenon postulated in WP-B1, namely the formation of solid aggregates from liquid precursor states, is very general and exists for many different proteins, including both Abeta and tau, the two main proteins associated with the onset and development of Alzheimer’s disease. This is a very exciting finding and we anticipate this workpackage becoming even more important than initially anticipated in the grant during the second half of the project. We have also been able to make very good progress on detecting low concentrations of oligomers using single molecule spectroscopy coupled to microfluidics and applied this approach to understand the mechanism of secondary nucleation (WP-B2) in vitro, but also very recently in the CSF of patient samples. The findings from these studies are exciting as they reveal evidence for secondary nucleation under both scenarios, suggesting it would have an even more general role than previous anticipated.

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)

This project aims to progress beyond the state of the art both in terms of methodology as well as in terms of understanding of aggregating protein systems. On the methodology front, we have made very good progress in establishing a digital detection toolkit by bringing together advanced optical detection with microfluidics. On the systems side, we are uncovering some of the fundamental molecular mechanisms of protein aggregation including how aberrant protein aggregation may start as a liquid so solid phase transition, and the general role of secondary nucleation.

Example results from the first part of the project as described in the text.