Electrostatic-Directed Covalent Labeling of Pan-Membrane Proteins for Ultralong-Term Tracking of Plasma Membrane Dynamics

Abstract

Stable fluorescent labeling holds significant value in elucidating the roles of plasma membrane during various cellular processes. However, current plasma membrane probes primarily rely on noncovalent labeling strategies that often results in poor label stability over long-term observations. To address this limitation, we introduce an innovative electrostatic-directed covalent labeling strategy. Our findings demonstrate that this method enables ultralong-term tracking of plasma membrane dynamics through covalent labeling of pan-membrane proteins in live cells, addressing the growing demand for prolonged monitoring of plasma membranes during cellular processes requiring temporal resolution down to the hourly scale. The optimal probe, Sulfo-Cy5-Br, consists of a Cy5 fluorophore conjugated with two sulfonate groups and a central bromide atom. The reactive bromide group facilitates covalent attachment to proteins via nucleophilic substitution with amino or thiol groups in proteins, which ensures stable binding to plasma membrane proteins. The incorporation of two negatively charged sulfonate groups not only ensures excellent water solubility and cell impermeability during the labeling process but also enables electrostatic interaction between negatively charged probes and partially protonated amino groups on plasma membrane proteins. Moreover, the probe can detect plasma membrane damage by transferring its label, enabling real-time assessment of plasma membrane damage status during long-term cellular events. Notably, the labeling stability of the plasma membrane with Sulfo-Cy5-Br persists for up to 44 h, allowing continuous tracking of plasma membrane morphology and damage during prolonged cellular processes such as pyroptosis and necroptosis, which typically span several-to-tens hours.

Topics & Concepts