MOTIVATION: Designing mathematical tools that can formally describe the dynamics of complex intracellular processes remains a challenge. Live cell imaging reveals changes in the cellular states, but current simple approaches extract only minimal information of a static snapshot. RESULTS: We implemented a novel approach for analyzing organelle behavior in live cell imaging data based on hidden Markov models (HMMs) and showed that it can determine the number and evolution of distinct cellular states involved in a biological process. We analyzed insulin-mediated exocytosis of single Glut4-vesicles, a process critical for blood glucose homeostasis and impaired in type II diabetes, by using total internal reflection fluorescence microscopy (TIRFM). HMM analyses of movie sequences of living cells reveal that insulin controls spatial and temporal dynamics of exocytosis via the exocyst, a putative tethering protein complex. Our studies have validated the proof-of-principle of HMM for cellular imaging and provided direct evidence for the existence of complex spatial-temporal regulation of exocytosis in non-polarized cells. We independently confirmed insulin-dependent spatial regulation by using static spatial statistics methods. CONCLUSION: We propose that HMM-based approach can be exploited in a wide avenue of cellular processes, especially those where the changes of cellular states in space and time may be highly complex and non-obvious, such as in cell polarization, signaling and developmental processes.

Exocytosis is an essential cellular trafficking process integral to the proper distribution and function of a plethora of molecules, including transporters, receptors, and enzymes. Moreover, incorrect protein targeting can lead to pathological conditions. Recently, the application of evanescent wave microscopy has allowed us to image the final steps of exocytosis. However, spatio-temporal analysis of fusion of constitutive vesicular traffic with the plasma membrane has not been systematically performed. Also, the spatial sites and times of vesicle fusion have not yet been analyzed together. In addition, more formal tests are required in testing biological hypotheses, rather than visual inspection combined with statistical descriptives. Ripley K-functions are used to examine the joint and marginal behavior of locations and fusion times. Semiautomatic detection and mapping of constitutive fusion sites reveals spatial and temporal clustering, but no dependency between the locations and times of fusion events. Our novel approach could be translated to other studies of membrane trafficking in health and diseases such as diabetes.