Analysis of EGF Receptor Endocytosis Using Fluorogen Activating Protein Tagged Receptor
BIO-PROTOCOL
Authors: Verdaguer, Mireia Perez; Larsen, Mads B.; Bruchez, Marcel P.; Watkins, Simon C.; Sorkin, Alexander
Abstract
Functional activities of many transmembrane proteins are controlled by their endocytosis. One of the most studied experimental models is the epidermal growth factor (EGF) receptor (EGFR). However, endocytic trafficking of EGFR has been predominantly analyzed using labeled EGF, whereas quantitative analyses of the endocytosis of the receptor itself have been sparse. The fluorescence microscopy methods described here are designed to directly quantify EGFR internalization in living cells without labeled EGFR ligands or antibodies. These methods utilize an engineered EGFR chimera in which the fluorogen activating protein (FAP) is fused to the receptor extracellular domain (FAP-EGFR). Binding of malachite green (MG) based dyes to FAP results in a strong far-red fluorescence of MG, thus efficiently labeling FAP-EGFR. In particular, binding of the cell impermeant MG-Bis-SA dye to FAP produces the pH-sensitive dual-excitation fluorescence, which allows differentiation of the cell-surface and internalized pools of FAP-EGFR. Two modifications of the methodology are described: 1) single cell three-dimensional confocal imaging; and 2) high-throughput assay in multi-well plates. These methodologies can be adopted to study endocytosis of any other transmembrane protein extracellularly tagged with FAP.
A rare case of the recurrent surgery for cribriform-morular variant of papillary thyroid carcinoma
INTERNATIONAL JOURNAL OF SURGERY CASE REPORTS
Authors: Enomoto, Keisuke; Tamagawa, Shunji; Kumashiro, Naoko; Warigaya, Kenji; Takeda, Saori; Gunduz, Mehmet; Murata, Shin-ichi; Hotomi, Muneki
Abstract
INTRODUCTION: The cribriform-morular variant of papillary thyroid carcinoma (CMV-PTC) is an uncommon subtype of PTC, and it is associated with familial adenomatous polyposis (FAP). PRESENTATION OF CASE: We describe a 32-year-old female who presented recurrent CMV-PTC with FAP. Surgery performed after the recurrent disease resulted in left recurrent laryngeal nerve palsy due to formation of strong scar after initial operation. The histopathological examination revealed the recurrent CMV-PTC without thyroid capsular invasion in remnant thyroid tissue of berry ligament. In addition, the immunohistochemical analysis showed that p-Catenin was diffusely positive in both cytoplasm and nucleus in CMV-PTC tissue. CONCLUSION: Total thyroidectomy in CMV-PTC with FAP should be performed at initial surgery due to high recurrence. (C) 2019 Published by Elsevier Ltd on behalf of IJS Publishing Group Ltd.