Cellular Networks Go Spatially and Dynamically in Live Cells

Jung Huang^1*, Chien Y. Lin¹

¹Photonics, Chiao Tung University, Hsinchu, Taiwan

* Presenter:Jung Huang, email:jyhuang@faculty.nctu.edu.tw

In the past decade, scientists have gradually accepted the concept that cellular functions are generated through a variety of coupled networks of interacting proteins. Live cells tend to develop a heterogeneous ensemble to adapt to an ever-changing environment and those cellular stochastic dynamics are often averaged out in an ensemble measurement. Probing the distribution and mobility of single-molecule proteins in live cellular environments is thus crucial for understanding cellular functions and regulatory mechanisms. Single-molecule imaging and tracking can directly probe the properties of molecular assemblies and the kinetics of the interaction in live cells. Mounting evidence suggests that nanocluster and conformational states of a protein may be invoked to resist stochastic fluctuations and thereby significantly improve cellular functions and signaling reliability. However, biological media are spatially inhomogeneous, which causes the information to be poorly conveyed by measuring just a few, sparse single-molecule trajectories. We developed a 2D analysis of single-molecule trajectories with normalized variance (NV) vs mean squared displacement (MSD) to offer high-quality statistics while preserve single-molecule sensitivity. The MSDs were used to quantify the diffusion of a protein and the NV can disclose the nature of those interactions. Thus, the plot can be more informative than MSD alone to reflect the stochastic dynamics of a protein in cellular environments. We applied this 2D analysis technique to the dimerization processes of EGFRs in live cells and found that unliganded EGFR species appear to remain outside the cholesterol-enriched lipid domains. After ligand binding, EGFR molecules can relocate to lipid raft domains. Selectively tagging EGFR species with fluorophores allowed us to further monitor the correlative motions of EGFR species. Liganded receptors diffusing in proximity on the plasma membrane can interact with each other to move correlatively, which can be attributed to from correlated fluctuations in the lipid environment. The correlative motions are also mediated by the membrane cholesterol. Based on this understanding, the following questions can be raised: How a specific signaling protein be recruited to the action site to perform its function? What is the force involved for this targeted long range movement? What is the underlying dynamics and relevant forces involved? To address these important questions, we combine 3D light-sheet optical microscopy with optical flow algorithm to investigate the cascading Raf pathway of EGFR. We found that EGF binding can produce rapid and small movements of Raf near plasma membrane in first 5 minutes. After 10 minutes, those patterns are replaced with larger movements from plasma membrane to the inner region of the cells. Eigen-patterns were deduced. We attribute those Raf movement patterns to be caused by Ras clustering at the tip of cortical actin filaments on plasma membrane. which creates a potential well to promote the rapid movements of Raf in cytosol toward the Ras nanoclusters.

This research was funded by the Ministry of Science and Technology of the Republic of China (grant numbers MOST 103-2112-M-009-012-MY3, and MOST 106-2112-M-009-019-MY3).

Keywords: single-molecule tracking, cellular network, cellular signaling, plasma membrane, EGFR