High-Fidelity Tracking of Endocytosis in Cancer Cells under Cellular Stress Using a Long-Term Anchoring Cell Membrane Fluorescent Probe

Endocytosis plays a critical role in regulating protein dynamics, and cancer cells often exploit this process to enhance their survival capacity. However, the development of reliable tools for high-fidelity visualization of endocytosis under cellular stress remains a significant challenge. In this study, we introduce a fluorescent probe, <b><i>o</i>-IP-IP</b>, based on a dimeric dual-anchoring strategy, to investigate plasma membrane dynamics and endocytosis processes. The probe incorporates two alkyl chains for targeted binding to the plasma membrane while maintaining water solubility through two negatively charged units. This design enables <b><i>o</i>-IP-IP</b> to exhibit target-activated fluorescence enhancement, broad applicability across various cell types, and the ability to distinguish between normal and cancerous cells based on membrane viscosity. The probe's dual anchoring minimizes passive transport-induced internalization, allowing prolonged labeling of the plasma membrane and enabling high-fidelity imaging of both membranes and endocytosis processes under endocytosis inhibition. This feature facilitated real-time observation of endocytosis in living cells under osmotic stress. Furthermore, <b><i>o</i>-IP-IP</b> enabled visualization of endocytosis induced by different extracellular calcium ion concentrations, confirming its utility as a simple and effective tool for high-fidelity endocytosis tracking in cellular stress and cell death studies. The probe was also applied to study dynamic surface protein distribution under specific external stress, highlighting endocytosis as the dominant mechanism for adjusting surface proteins. These findings underscore endocytosis' central role in maintaining cellular homeostasis and provide effective strategies for designing targeted probes and drugs based on membrane-impermeable substances' endocytosis mechanisms.