PROJECT SUMMARY Plasma Membrane blebbing and the relevant three-dimensional cell migration are vital biophysical processes, which trigger some critical cellular events and are implicated in a wide range of disease. Despite the preliminary success in identifying a key gene pathway and characterizing the basic cell morphology, the underlying mechanism are still poorly understood, due to the lack of effective research platforms. It remains to be a key question how the blebbing transduce the cell environmental stimuli into the cell and drive the cell migration. We recently developed a microfluidic device for the study of blebbing and cell migration. This device recapitulates the native environment for blebbing-involved migration, while providing some unprecedented advantages for investigating the cellular biophysics. We also established a data-driven mathematical modeling framework to study the complex morphology of bleb-like protrusions. Based on our preliminary data and other previous studies, we hypothesize that it is through the polarization of sensory ion channels that the environmental stimuli induce polarized blebbing, which provides the driving force for the migration. To test the hypothesis, we propose to study the blebbing dynamics, using a novel experimental-theoretical integrated platform, which is based on the previously established microfluidic device and data-driven mathematical model. First, we will define the correlation and cause-effect relationship of blebbing dynamics and the cell motility. Then we will define the correlation of blebbing dynamics to ion channel activities and regulation of pathways. Finally, we will assess the influence of the environmental stimuli on the blebbing-related cellular behavior and molecular regulation. The outcome of this study will significantly enhance our understanding towards the role of PM blebbing in cell migration and, in the long run, promote the bleb-targeting therapy. The developed experimental-theoretical integrated platform will also serve as a valuable technology for other cellular biophysical studies.