Morphodynamic Profiling of Cell Protrusion Based on Spatiotemporal Spectrum Decomposition and Unsupervised Clustering

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Cellular morphogenesis is governed and regulated by complex interactions between the cytoskeleton and molecular pathways. Due to their central roles in the differentiation, embryogenesis and homeostasis process, cell morphology and morphodynamics are often used for qualitative and quantitative assessments of cell state. Here, we propose a framework to quantitatively profile cellular morphodynamics based on an adaptive spectrum decomposition method. This method decomposes the motion of cell boundary into analytical instantaneous frequency spectrum through Hilbert-Huang transform (HHT). Subsequently, we computed for each decomposed signal the distribution of instantaneous frequency and amplitude, from which we compiled the HHT spectrum. For spontaneously protrusive Cos7 cells, the instantaneous magnitudes of protrusion were found to greatly vary across different cells with nearly identical genetic and molecular backgrounds, whereas the frequency spectra were remarkably consistent. Meanwhile we examined Cos7 cells in which the Vav2 Guanine Exchange Factor (GEF) activity was acutely perturbed by optogenetic tools, and found significant shifts in the frequency distribution for specific signals. We thus concluded that edge motion velocity is an arbitrary parameter of cell morphogenesis whereas the frequency spectrum encodes the molecular state of regulation. The features extracted from the HHT spectrum could be used as a classifier to group subcellular regions into numerous characteristic sectors with specific molecular states through Statistical Region Merging (SRM) based clustering. Finally, we monitored the Rac1 activity downstream of Vav2 expressed in Cos7 cells and found that those clustering sectors distinguished by morphodynamic parameters indeed reflect regions with different local signaling. Thus, cell morphodynamic behavior can be used to detect different modes of operations or perturbations in cellular signaling.