Real-Time Monitoring of Transferrin-Induced Endocytic Vesicle Formation by Mid-Infrared Surface Plasmon Resonance

Abstract

We report on the application of surface plasmon resonance (SPR), based on Fourier transform infrared spectroscopy in the mid-infrared wavelength range, for real-time and label-free sensing of transferrin-induced endocytic processes in human melanoma cells. The evanescent field of the mid-infrared surface plasmon penetrates deep into the cell, allowing highly sensitive SPR measurements of dynamic processes occurring at significant cellular depths. We monitored in real-time, infrared reflectivity spectra in the SPR regime from living cells exposed to human transferrin (Tfn). We show that although fluorescence microscopy measures primarily Tfn accumulation in recycling endosomes located deep in the cell's cytoplasm, the SPR technique measures mainly Tfn-mediated formation of early endocytic organelles located in close proximity to the plasma membrane. Our SPR and fluorescence data are very well described by a kinetic model of Tfn endocytosis, suggested previously in similar cell systems. Hence, our SPR data provide further support to the rather controversial ability of Tfn to stimulate its own endocytosis. Our analysis also yields what we believe is novel information on the role of membrane cholesterol in modulating the kinetics of endocytic vesicle biogenesis and consumption.

Src-Mediated Phosphorylation of Dynamin and Cortactin Regulates the “Constitutive” Endocytosis of Transferrin

“…The internalization and recycling of the TfR have been closely studied by multiple groups for over 2 decades. While some have observed that internalization of the TfR is linear over time regardless of Tf addition (24), others have observed an increase in the endocytic rate of this receptor in the presence of ligand (19,48,49,60). This concept of regulated internalization has been supported by studies implicating protein kinase C (PKC) (29,47) and casein kinase activation (12), increases in cytoplasmic calcium (46), and, most recently, Src activity (61).…”

Neuroprotective peptides fused to arginine-rich cell penetrating peptides: Neuroprotective mechanism likely mediated by peptide endocytic properties

“…Furthermore, it is important to note that the rapid and transient (lasting up to 4 h with peptide pre-treatment) nature of the neuroprotection induced by poly-arginine peptides (Meloni et al, 2015) corresponds closely to the timeframes of endocytosis and endosomal receptor re-cycling (Gundelfinger et al, 2003;Maxfield & McGraw, 2004;Yashunsky et al, 2009). Importantly, it is known that TAT, penetratin and R9 can induce the internalisation of EGFR and TNFR in HeLa cells (Fotin-Mleczek et al, 2005).…”

Infrared Surface Plasmon Spectroscopy of Living Cells

“…So far we used this technique to study alterations in membrane cholesterol levels in HeLa cells [12], de novo formation of early endocytic transport vesicles in response to transferrin internalization by human melanoma cells [13], and detection of glucose uptake by erythrocytes [11].…”