“Different endocytosis of insulin receptor A and B developed by fluorescence microscopy and quantum dots”.

JIMENA GIUDICE; FEDERICO COLUCCIO LESKOW; DONNA J. ARNDT-JOVIN; THOMAS M. JOVIN; ELIZABETH JARES-ERIJMAN

Buzios. Brasil

Simposio; IV INTERNATIONAL SYMPOSIUM ON MYOSIN V - 21 Years of History. EMBO.; 2009

European Molecular Biology Organization

<!-- /* Font Definitions */ @font-face {font-family:SimSun; panose-1:2 1 6 0 3 1 1 1 1 1; mso-font-alt:宋体; mso-font-charset:134; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:3 135135232 16 0 262145 0;} @font-face {font-family:"@SimSun"; panose-1:2 1 6 0 3 1 1 1 1 1; mso-font-charset:134; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:3 135135232 16 0 262145 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:SimSun;} p.Default, li.Default, div.Default {mso-style-name:Default; mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; mso-layout-grid-align:none; text-autospace:none; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:SimSun; color:black;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Insulin signalling comprises a complex cascade of events with a plethora of consequences, playing a key role in the regulation of glucose metabolism and being also involved in cellular growth. It has been shown that failures in its function lead to diabetes regulating as well pathways involved in cancer (1-5). The insulin receptor (IR) is a tetrameric tyrosine kinase receptor. Two splice variants exist in mammalian cells: IR-A lacking exon 11, and the full length IR-B. Our study focuses on the spatio-temporal dynamics of the activation of the different IR isoforms and their differential downstream signaling. We applied a combination of labeling techniques designed to study insulin receptor dynamics using high resolution microscopy in living cells and taking advantage of 3 powerful tools: quantum dots (QDs) conjugated with streptavidin (SA) and then with biotinylated ligands; visible fluorescent proteins (VFPs); and the covalent specific enzymatic modification of cell surface proteins that makes use of the post-translational modification of acyl carrier protein (ACP) by a phosphopantetheinyl transferase, ACPwt-S (6-9). Biotinylated insulin bound with fluorescent streptavidin or QDs conjugated with SA was applied in order to imaged ligand binding and endocytosis of the ligand receptor complex by confocal microscopy and Programmable Array Microscopy (PAM). Here we show that in transfected HeLa cells biotin amido caproyl bovine insulin was able to bind IR and IR fussed to green fluorescent protein at its C-terminal (IR-GFP), to activate them and to induce their internalization which could be imaged in fixed and in living cells by confocal microscopy and PAM. This strategy provided an interesting tool to study the interactions between insulin and its receptor, the conformational changes of it after activation, and binding, endocytosis and recycling processes. In addition it allowed a cell by cell analysis showing differential internalization of both isoform. The analysis leaded a smaller internalization after 20 minutes for IR-B-GFP (47 ± 13%) than for IR-A-GFP (65 ± 11%), and the difference was statistically significant (p ≤ 0.001).