Targeting Quantum Dots to Surface Proteins in Living Cells with Biotin Ligase

M. Howarth, K. Takao, Y. Hayashi, A.Y. Ting, Proc Natl Acad Sci U S A Nature Methods 102, 7583 (2005).

Can Quantum Dots Be Used to Track Proteins in Live Cells?

Proteins can be specifically labeled with quantum dots through a three-step method, wherein the protein of interest is bound by a primary antibody, the primary is complexed with a biotinylated secondary antibody, and the complex is visualized with a quantum dot–streptavidin conjugate. However, the large size of the resulting label complex can interfere with normal protein function, making it difficult to draw physiologically relevant conclusions in certain types of studies. By first tagging the AMPA receptor—a glutamate-activated ion channel involved in synaptic activity—with a 15-amino acid acceptor peptide (AP), the authors have demonstrated a simple method for directly biotinylating cell surface proteins via the activity of a bacterial biotin ligase enzyme (BirA), thereby allowing direct detection of the biotinylated receptor protein with a quantum dot–streptavidin conjugate.

The authors used this method to examine trafficking of the AMPA receptor in live hippocampal neurons. Labeling was detected after only one minute of biotinylation with BirA; control experiments demonstrated that labeling was restricted to AP-tagged proteins on the cell surface. Pulse–chase experiments using an Alexa Fluor 488–streptavidin conjugate and the quantum dot–streptavidin conjugate were used to demonstrate differential trafficking dynamics of AMPA receptors labeled with AP-GluR1 subunits versus AP-GluR2 subunits in response to treatment with glycine.

Niche-Dependent Translineage Commitment of Endothelial Progenitor Cells, Not Cell Fusion in General, Into Myocardial Lineage Cells

Murasawa S, Kawamoto A, Horii M, Nakamori S, Asahara T. Arterioscler Thromb Vasc Biol. 2005 Jul;25(7):1388-94. Epub 2005 Apr 28.


Objective—
Previous studies from our laboratory have shown therapeutic potential of ex vivo expanded endothelial progenitor cells (EPCs) for myocardial ischemia. Our purpose was to investigate the mechanisms regulating EPC contribution to myocardial regeneration.

Methods and Results—
To evaluate niche-dependent expression profiles of EPCs in vitro, we performed coculture using cultured EPCs derived from human peripheral blood and rat cardiac myoblast cell line (H9C2). Reverse-transcription polymerase chain reaction (PCR) disclosed the expression of human-specific cardiac markers as well as human-specific smooth muscle markers. Cytoimmunochemistry presented several cocultured cells stained with human specific cardiac antibody. To prove this translineage differentiation in vivo, human cultured EPCs were injected into nude rat myocardial infarction model. Reverse-transcription PCR as well as immunohistochemistry of rat myocardial samples demonstrated the expression of human specific cardiac, vascular smooth muscle, and endothelial markers. We observed the distribution of colors (Qtracker; Quantum Dot Corp) in coculture to detect the fused cells, and the frequency of cell fusion was <1%.

Conclusions— EPCs can contribute to not only vasculogenesis but also myogenesis in the ischemic myocardium in vivo. Transdifferentiation, not cell fusion, is dominant for EPCs commitment to myocardial lineage cells. Ex vivo expanded EPCs transplantation might have enhanced therapeutic potential for myocardial regeneration.   Ex vivo expanded EPC transplantation has shown therapeutic potential for myocardial ischemia.

We observed the expression of human-specific cardiac, vascular smooth muscle, and endothelial markers in niche-dependent expression profiles of EPCs via mainly transdifferentiation. EPCs could contribute to not only vasculogenesis but also myogenesis in the ischemic myocardium.