Dynamic Changes In The Monomer-Dimer Equilibrium Of The Intercellular Adhesion Molecule CEACAM1 Revealed By Live Cell TIRF-Based FRET Microscopy
| Autor: | Jonathan V. Rocheleau, Hannah S. W. Lee, Scott D. Gray-Owen, John Oreopoulos, Christopher M. Yip |
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| Rok vydání: | 2009 |
| Předmět: |
Yellow fluorescent protein
Cell signaling Total internal reflection fluorescence microscope Förster resonance energy transfer Membrane protein biology biology.protein Biophysics Protein tyrosine phosphatase Photobleaching hormones hormone substitutes and hormone antagonists Proto-oncogene tyrosine-protein kinase Src Cell biology |
| Zdroj: | Biophysical Journal. 96(3) |
| ISSN: | 0006-3495 |
| DOI: | 10.1016/j.bpj.2008.12.3569 |
| Popis: | The carcinoembryonic-antigen-related cell-adhesion molecule (CEACAM) family of proteins has been implicated in various intercellular-adhesion and intracellular signaling-mediated effects that govern the growth and differentiation of normal and cancerous cells. Recent studies also show that members of the CEACAM family play an important role in modulating the immune responses associated with infection, inflammation and cancer. At the epithelial cell surface, CEACAM1 (also known as BGP, C-CAM and CD66a) is believed to exist as a cis-homodimer. The dimeric state is thought to regulate the ability of the receptor to recruit signaling molecules, including SRC-family kinases and the tyrosine phosphatase SRC homology 2 (SH2)-domain-containing protein tyrosine phosphatase 1 (SHP1). To characterize the dynamics and monomer-dimer equilibrium of CEACAM1, we have applied a live-cell combinatorial microscopy imaging approach. Time-lapse total internal reflection fluorescence (TIRF) imaging of HELA cells overexpressing cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-CEACAM1 constructs revealed a heterogeneous distribution of bright, micron-sized jagged structures that rapidly changed shape and diffused across the cell surface. Homo- and hetero-Forster resonance energy transfer (FRET) imaging techniques (acceptor photobleaching, sensitized emission, and polarization anisotropy) were then used in combination with TIRF microscopy to determine that these jagged structures are likely aggregates of monomeric CEACAM1 proteins while the rest of the membrane contains lower concentrations of dimeric CEACAM1. This combinatorial microscopy strategy not only compliments traditional biochemical assays, but also provides new quantitative insights into spatial-temporal dynamics of this important membrane protein. We are exploiting these insights to further understand the various immune responses associated with the CEACAM family. |
| Databáze: | OpenAIRE |
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