iPUT: a minimal-tag probe for imaging putrescine dynamics in cellular homeostasis and stress
Description
Polyamines regulate multiple cellular pathways, yet their cellular distribution remains poorly defined. To overcome limitations of available approaches, we developed iPUT, a clickable putrescine analogue that preserves its native properties and enables bioorthogonal fluorophore conjugation for high-resolution imaging. In MCF-7 cells, unlike fluorophore-conjugated probe BODIPY-PUT, iPUT localized to nuclei, demonstrating the advantage of minimal tagging. In hippocampal co-cultures and acute slices, neurons showed higher iPUT accumulation than astrocytes, challenging the view that glia is the main polyamine reservoir. iPUT accumulation was ATPase transporter-dependent, consistent with enrichment of hippocampal neurons in ATP13A2 carrier. Subcellular distribution differed across models: nuclear enrichment in dividing MCF-7 cells contrasted with extranuclear localization in mature neurons in hippocampal slices. Mimetic hypoxia and excitotoxicity reduced iPUT accumulation in CA1 but not CA2/3 neurons and promoted nuclear redistribution in CA1, suggesting involvement in stress-activated pathways and contribution to higher vulnerability of this region. iPUT is thus a versatile tool for tracking putrescine dynamics, offering new insights into polyamine biology in health and disease.