Periodic Reporting for period 1 - GTPaseNet (Synthetic and structural biology of Rab GTPase networks)
Periodo di rendicontazione: 2023-01-01 al 2025-06-30
Eukaryotic cells are like tiny, highly organized cities, filled with many different compartments (organelles) that each have specific jobs. These compartments are surrounded by membranes and need to be carefully managed to function properly. A group of proteins called Rab GTPases act as traffic controllers, helping to organize and direct movement within the cell. However, we still don’t fully understand how these proteins create order in such a busy and complex environment.
So far, most research on Rab GTPases has either been performed in living cells or by studying them in very simplified experiments. However, neither approach is sufficient to reveal or explain their complex behavior in the cell. We now know that that Rab GTPases work as part of intricate biochemical networks that can self-organize—like a city that manages itself rather than needing constant outside control. To truly understand how they function, new experimental methods are needed.
In this project, we will take a “bottom-up” approach, rebuilding the Rab GTPase networks from scratch using purified components. This will allow us to see how they naturally organize themselves outside of a cell while still providing experimental access to uncover the underlying molecular interactions. We will also create artificial cell-like environments using microfabrication techniques to understand how these proteins respond to physical and chemical signals, similar to what happens in real cells. Finally, we will use cryo-electron microscopy techniques to look at the molecular structure of Rab GTPases and their partners.
By combining these approaches, this project will give us a better understanding of how Rab GTPases control the biochemical identities of intracellular membranes. This will also help us see how cells maintain their internal order, which is crucial for their survival and function.
So far, most research on Rab GTPases has either been performed in living cells or by studying them in very simplified experiments. However, neither approach is sufficient to reveal or explain their complex behavior in the cell. We now know that that Rab GTPases work as part of intricate biochemical networks that can self-organize—like a city that manages itself rather than needing constant outside control. To truly understand how they function, new experimental methods are needed.
In this project, we will take a “bottom-up” approach, rebuilding the Rab GTPase networks from scratch using purified components. This will allow us to see how they naturally organize themselves outside of a cell while still providing experimental access to uncover the underlying molecular interactions. We will also create artificial cell-like environments using microfabrication techniques to understand how these proteins respond to physical and chemical signals, similar to what happens in real cells. Finally, we will use cryo-electron microscopy techniques to look at the molecular structure of Rab GTPases and their partners.
By combining these approaches, this project will give us a better understanding of how Rab GTPases control the biochemical identities of intracellular membranes. This will also help us see how cells maintain their internal order, which is crucial for their survival and function.
Over the past two years, we first focused on refining protocols to obtain high-quality reproducible data.
We improved protocols to obtain lipid-modified proteins and protein complexes, streamlining the process to ensure high-quality samples with greater speed and reliability. In parallel, we were designing experiments to investigate how various proteins influence the activation of Rab5 on biomimetic membranes, shedding light on the molecular mechanisms that control where and when Rab5 is activated in living cells. We were developing new fluorescence microscopy experiments to follow Rab5 activation in real time under well-controlled biochemical conditions. These experiments allowed us to even analyze transient binding events so for not possible using conventional techniques, which have also been beneficial in our collaborations with other research teams. In parallel, we are also optimizing cryo-electron microscopy experiments to further our structural studies and to examine the conformational states of different protein complexes.
Our experiments have allowed us to develop the principal concepts of membrane-localized biochemical reactions, which has led to a publication in Development Cell. Our objective is to stay on this path until the project's completion while continuing to work toward the goals outlined in the grant proposal.
We improved protocols to obtain lipid-modified proteins and protein complexes, streamlining the process to ensure high-quality samples with greater speed and reliability. In parallel, we were designing experiments to investigate how various proteins influence the activation of Rab5 on biomimetic membranes, shedding light on the molecular mechanisms that control where and when Rab5 is activated in living cells. We were developing new fluorescence microscopy experiments to follow Rab5 activation in real time under well-controlled biochemical conditions. These experiments allowed us to even analyze transient binding events so for not possible using conventional techniques, which have also been beneficial in our collaborations with other research teams. In parallel, we are also optimizing cryo-electron microscopy experiments to further our structural studies and to examine the conformational states of different protein complexes.
Our experiments have allowed us to develop the principal concepts of membrane-localized biochemical reactions, which has led to a publication in Development Cell. Our objective is to stay on this path until the project's completion while continuing to work toward the goals outlined in the grant proposal.
In the last two years, we have developed new techniques to measure and quantify interactions between Rab5 and its regulators with membranes. This has enabled the quantification of even weak interactions down to the single-molecule level, which has previously been impossible using traditional methods. Importantly, such weak interactions can significantly alter the outcome and emergent properties of biochemical networks and therefore play crucial roles for the spatiotemporal organization of the cell. Our work has therefore contributed significantly to a better understanding of how membranes can enable and accelerate protein interactions and therefore influence cellular organization and signaling dynamics. To disseminate these insights to the scientific community, we published a review article detailing how membranes serve as platforms for organizing biochemical processes.
Moving forward, developing new assays to study the self-organized properties of Rab GTPase networks will be critical for achieving our scientific aims. Therefore, we need to further refine protocols to ensure reproducibility and throughput, while also investing in computational to advance quantitative analysis. Overall, our work has provided fundamental insights into how membranes regulate Rab5 activity, revealing the principles of cellular self-organization. While we are still refining some of our approaches, our findings so far represent an important step toward understanding how cells self-organize at the molecular level.
Moving forward, developing new assays to study the self-organized properties of Rab GTPase networks will be critical for achieving our scientific aims. Therefore, we need to further refine protocols to ensure reproducibility and throughput, while also investing in computational to advance quantitative analysis. Overall, our work has provided fundamental insights into how membranes regulate Rab5 activity, revealing the principles of cellular self-organization. While we are still refining some of our approaches, our findings so far represent an important step toward understanding how cells self-organize at the molecular level.