Quantitative assessment of the relationship between cellular morphodynamics and signaling events by stochastic analysis of fluorescent images.

Cell motility involves a number of strategies that cells use in order to seek nutrients, escape danger, and fulfill morphogenetic roles. Here we present a methodology to quantify morphological changes and their relationship with signaling events from time-lapse imaging microscopy experiments, in order to characterize physiological and pathological processes. To this aim, the stationary spatial pattern of signaling events is determined through an intracellular fluorescent probe, and it is related with the frequency and entity of morphodynamic events, which are in turn quantified through a stochastic approach: two pseudoimages are obtained from a time series of moving cells that describe the probability that a pixel belongs to the cell, and the probability that a pixel is subject to a dynamic event. The simultaneous construction of these maps permits visualization of hot spots of dynamic events, i.e., zones of formation of membrane protrusions and retractions and their relationship with the signaling events reported by the specific probe employed. The method is tested on spontaneous movement of cells, trasfected with redox-sensitive yellow fluorescent protein, in which the distribution of the hot spots and its change upon expression of constitutively active Rac (V12-Rac), is related to the distribution of oxidized spots.