Extracting Diffusive States of Rho GTPase in Live Cells: Towards In Vivo Biochemistry

Resolving distinct biochemical interaction states when analyzing the trajectories of diffusing proteins in live cells on an individual basis remains challenging because of the limited statistics provided by the relatively short trajectories available experimentally. Here, we introduce a novel, machine-learning based classification methodology, which we call perturbation expectation-maximization (pEM), that simultaneously analyzes a population of protein trajectories to uncover the system of diffusive behaviors which collectively result from distinct biochemical interactions. We validate the performance of pEM in silico and demonstrate that pEM is capable of uncovering the proper number of underlying diffusive states with an accurate characterization of their diffusion properties. We then apply pEM to experimental protein trajectories of Rho GTPases, an integral regulator of cytoskeletal dynamics and cellular homeostasis, in vivo via single particle tracking photo-activated localization microcopy. Remarkably, pEM uncovers 6 distinct diffusive states conserved across various Rho GTPase family members. The variability across family members in the propensities for each diffusive state reveals non-redundant roles in the activation states of RhoA and RhoC. In a resting cell, our results support a model where RhoA is constantly cycling between activation states, with an imbalance of rates favoring an inactive state. RhoC, on the other hand, remains predominantly inactive.Author Summary: Single particle tracking is a powerful tool that captures the diffusive dynamics of proteins as they undergo various interactions in living cells. Uncovering different biochemical interactions by analyzing the diffusive behaviors of individual protein trajectories, however, is challenging due to the limited statistics provided by short trajectories and experimental noise sources which are intimately coupled into each protein’s localization. Here, we introduce a novel, unsupervised, machine-learning based classification methodology, which we call perturbation expectation-maximization (pEM), that simultaneously analyzes a population of protein trajectories to uncover the system of diffusive behaviors which collectively result from distinct biochemical interactions. We validate the performance of pEM in silico and in vivo on the biological system of Rho GTPase, a signal transduction protein responsible for regulating cytoskeletal dynamics. We envision that the presented methodology will be applicable to a wide range of single protein tracking data where different biochemical interactions result in distinct diffusive behaviors. More generally, this study brings us an important step closer to the possibility of monitoring the endogenous biochemistry of diffusing proteins within live cells with single molecule resolution.