ABSTRACT
Clathrin-mediated endocytosis (CME) regulates the uptake of cell-surface receptors as well as their downstream signaling activities. We recently reported that signaling can reciprocally regulate CME in cancer cells and that this crosstalk can contribute to cancer progression. To further explore the nature and extent of the crosstalk between signaling and CME in cancer cell biology, we analyzed a panel of oncogenic signaling kinase inhibitors for their effects on CME across a panel of normal and cancerous cells. Inhibition of several kinases selectively affected CME in cancer cells, including inhibition of ERK1/2, which selectively inhibited CME by decreasing the rate of clathrin-coated pit (CCP) initiation. We identified an ERK1/2 substrate, the FCH/F-BAR and SH3 domain-containing protein FCHSD2, as being essential for the ERK1/2-dependent effects on CME and CCP initiation. Our data suggest that ERK1/2 phosphorylation activates FCHSD2 and regulates EGF receptor (EGFR) endocytic trafficking as well as downstream signaling activities. Loss of FCHSD2 activity in nonsmall cell lung cancer (NSCLC) cells leads to increased cell-surface expression and altered signaling downstream of EGFR, resulting in enhanced cell proliferation and migration. The expression level of FCHSD2 is positively correlated with higher NSCLC patient survival rates, suggesting that FCHSD2 can negatively affect cancer progression. These findings provide insight into the mechanisms and consequences of the reciprocal regulation of signaling and CME in cancer cells.
Subject(s)
Carcinoma, Non-Small-Cell Lung/metabolism , Carrier Proteins/biosynthesis , Clathrin/pharmacokinetics , Endocytosis , Gene Expression Regulation, Neoplastic , Lung Neoplasms/metabolism , MAP Kinase Signaling System , Membrane Proteins/biosynthesis , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Neoplasm Proteins/metabolism , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Carrier Proteins/genetics , Cell Line, Tumor , Clathrin/genetics , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Membrane Proteins/genetics , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/genetics , Neoplasm Proteins/geneticsABSTRACT
AIM: With regards to nanoparticles, all biomedical applications require cellular uptake, which to date remains a hurdle to further progress. This study aims to compare both the attractive force of a static magnetic field and the cell penetrating capability of penetratin; two techniques currently employed to enhance cell uptake. MATERIALS & METHODS: Fluorescent magnetic nanoparticles were functionalized with penetratin and cells were challenged with or without the particles in the presence/absence of a static magnetic field (350 mT). Following analysis of the magnetic field applied, cellular uptake and behavior was assessed in terms of fluorescence microscopy, clathrin and caveolin levels, scanning electron microscopy and transmission electron microscopy. RESULTS: Modeling of the field applied demonstrated varying field patterns across the cell culture area, reflected by higher particle uptake at higher field strengths. Both penetratin and the magnetic field increased cell uptake with penetratin proving more efficient. Interestingly, the magnetic field stimulated clathrin-mediated endocytosis and subsequent particle uptake.
Subject(s)
Carrier Proteins/pharmacokinetics , Ferric Compounds/pharmacokinetics , Fibroblasts/metabolism , Magnetic Fields , Metal Nanoparticles/administration & dosage , Carrier Proteins/chemistry , Caveolins/pharmacokinetics , Cell-Penetrating Peptides , Cells, Cultured , Clathrin/pharmacokinetics , Endocytosis/radiation effects , Ferric Compounds/chemistry , Ferric Compounds/radiation effects , Fibroblasts/ultrastructure , Humans , Metal Nanoparticles/chemistry , Metal Nanoparticles/radiation effects , Microscopy, Electron/methods , Particle SizeABSTRACT
Protein transport and sorting in the secretory and endocytic pathways via vesicles is required for organelle biogenesis, constitutive and regulated secretion and constitutive and regulated endocytosis. It is essential for a multicellular organism and the function of its specialised cell types that the multiple transport and sorting events are highly accurate. They determine the protein and lipid composition of specialised compartments, receptor protein function and membrane homeostasis. This review describes the individual events involved in the process of vesicle mediated protein transport and sorting and summarizes the knowledge about the function of proteins and lipids orchestrating the process.
