Covalent Quantum dot Receptor Linkage via the acyl Carrier Protein for Single-Molecule Tracking, Internalization, and Trafficking Studies

Abstract

Here we describe a labeling technique for the covalent linkage of quantum dots to transmembrane receptors for single-molecule tracking. Our method combines the acyl carrier protein (ACP) technique with coenzyme A (CoA)-functionalized quantum dots to covalently attach quantum dots to ACP fusions of receptor proteins. The advantages of this approach include: (i) the use of a smaller attachment linker than in many other quantum dot-labeling systems; (ii) the ability to achieve a reliable 1:1 fluorophore-to-receptor labeling stoichiometry; (iii) the specificity of the method; and (iv) the covalent nature of the quantum dot linkage. We demonstrate the general suitability of this technique in single-molecule tracking, internalization, and trafficking studies by imaging two different transmembrane receptors in living cells.

Nonendocytic Delivery of Functional Engineered Nanoparticles into the Cytoplasm of Live Cells Using a Novel, High-Throughput Microfluidic Device

“…Recent work has addressed the issue of specific conjugation through methods such as antibody targeting, streptavidin-biotin labeling schemes, HaloTag-chloroalkane and acyl carrier protein-acetyl CoA labeling. 6,22 In this report, we address the cytosolic delivery challenge by using a microfluidic device to deliver quantum dots to the cytosol and we confirm their delivery by observing their interaction with the cytosolic environment.…”

Green fluorescent protein fused to peptide agonists of two dissimilar G protein‐coupled receptors: novel ligands of the bradykinin B2 (rhodopsin family) receptor and parathyroid hormone PTH1 (secretin family) receptor

“…While both the activated PTH 1 R and B 2 R were previously shown to transport large receptor-bound cargoes, such as Qdot nanomaterials (Zelman-Femiak et al 2010 ; Bawolak et al 2011 ), the constructs PTH 1-34 -EGFP and EGFP-MK represent bona fide agonists that support the feasibility of a ligand-centered strategy to transport protein cargoes inside cells using GPCRs. Indeed, the typical B 2 R agonist BK, a 1-kDa nonapeptide, and PTH 1-34 (4 kDa) are much smaller than the novel agonist fusion proteins that we have produced and characterized.…”

“…However, the same could not be shown in the PTH 1-34 -EGFP/PTH 1 R system (data not shown), probably due to the low concentration of the agonist in this case (concentrated PTH forms are active in this respect in our hands, data not shown). While both PTH 1 R and B 2 R are essentially recycled back to the plasma membrane after agonist-induced endocytosis (Leeb-Lundberg et al 2005 ; Zelman-Femiak et al 2010 ), the progressive colocalization with Rab7 suggests that both EGFP fusion proteins progress toward lysosomes. In the maximal period of observation (6 h), the associated green fluorescence was not released or appear in the cytosol.…”

“…PTH 1 R is also subjected to agonist-dependent endocytosis behavior via interaction with β-arrestins (Gesty-Palmer and Luttrell 2011 ). PTH 1 R trafficking has notably been documented using synthetic agonist peptides conjugated to a chemical fluorophore (Ferrandon et al 2009 ) or covalent linkage of a quantum dot fluorophore to the receptors (Zelman-Femiak et al 2010 ). Furthermore, recent molecular pharmacologic studies showed that several hormones that bind secretin family GPCR hormones can be elongated at their C-terminus with a linker and transmembrane tether and still act as autocrine full agonists when coexpressed with a cognate receptor (Fortin et al 2009 , 2011 ) (this notably applied to PTH 1-34 ; schematic representation, Fig.…”

The phosphopantetheinyl transferases: catalysis of a post-translational modification crucial for life

“…320–324 Offering a high signal intensity as well as excellent photostability, quantum dots were evaluated as replacement of fluorescent dyes for imaging of cell surface and transmembrane proteins. 325, 326 …”