Synchronized Temporal-spatial Analysis via Microscopy and Phoshoproteomics (STAMP) of Quiescence.

Tightly coordinated cell cycle regulation is essential for homeostasis. G , or quiescence, is especially crucial for cells to respond to extracellular stimuli. Little is known about the mechanisms that establish the G program, though the primary cilium (a key signaling hub formed only in G ) is the most widely recognized marker. The study of ciliogenesis is challenging due to its small size, relative to the cell body. To address this gap in our understanding, we developed STAMP (Spatio-Temporal Analysis via Microscopy and Proteomics) to temporally map the changes in cellular landscape occurring in G and ciliogenesis. Using synchronized RPE cells, we used fixed and live cell imaging combined with phosphoproteomics to uncover new signals and order them in these processes, which also allows further, more targeted, analyses (e.g., using genetic and pharmacological perturbations). We propose that STAMP is broadly applicable for studying temporal-spatial signaling processes and the underlying mechanisms in various biological contexts and cell types.