Exploring focal adhesion data: dynamic parameter extraction from FRAP and FLAP experiments using chemical master equation

Author(s)

de Oliveira, Luciana Renata ; Fernandes, Matheus Gimenez ; Patane, Jose Salvatore Leister ; Schwartz, Jean-Marc ; Krieger, Jose Eduardo ; Ballestrem, Christoph ; Miyakawa, Ayumi Aurea

• Document type: Journal article
• Source: FRONTIERS IN MOLECULAR BIOSCIENCES; v. 12, p. 15-pg., 2025-05-06.

Abstract

The dynamic behavior of proteins within cellular structures can be studied using fluorescence recovery after photobleaching (FRAP) and fluorescence loss after photobleaching (FLAP) experiments. These techniques provide insights into molecular mobility by estimating parameters such as turnover rates ( k T ) and diffusion coefficients (D). However, traditional deterministic models often rely on simplifying assumptions that may not fully capture the stochastic nature of molecular interactions. In this study, we developed a novel stochastic model based on the analytical solution of the chemical master equation to extract dynamic parameters from FRAP and FLAP experiments in the focal adhesion (FA) network. Our approach extends beyond standard FRAP/FLAP analysis by inferring additional parameters, such as protein-specific entry ( k I n ) and exit ( k Out ) rates, allowing a deeper understanding of protein turnover and interactions. To validate our model, we analyzed previously published experimental data from NIH3T3 fibroblasts expressing GFP-tagged FA proteins, including tensin 1, talin, vinculin, alpha -actinin, ILK, alpha -parvin, kindlin-2, paxillin, p130Cas, VASP, FAK, and zyxin. These proteins participate in mechanotransduction, cytoskeletal organization, and adhesion regulation, exhibiting distinct dynamic behaviors within FA structures. Furthermore, we constructed an interaction network to quantify how vinculin and actin influence talin dynamics, leveraging our model to uncover their regulatory roles in FA turnover. Using an analytical solution of the chemical master equation, our framework provides a generalizable approach for studying protein dynamics in any system where FRAP and FLAP data are available. It can be applied to new experimental datasets and reanalyzed from existing data, revealing previously inaccessible molecular interactions and enhancing our understanding of FA dynamics and broader cellular processes. (AU)

• FAPESP's process: 23/03079-9 - Crp3 is a key element for vascular smooth muscle cells mechanotransduction and functional cell phenotype
• Grantee: Ayumi Aurea Miyakawa Yamaguchi
• Support Opportunities: Regular Research Grants
• FAPESP's process: 14/50889-7 - National Institute of Science and Technology Medicine Assisted by Scientific Computing (INCT-MACC)
• Grantee: José Eduardo Krieger
• Support Opportunities: Research Projects - Thematic Grants