Direct Visualization of Platelet Integrins Using ANTI-Transmembrane Domain Peptides Containing a BLUE Fluorescent Amino Acid

Antibodies containing a fluorescent label or recombinant proteins containing a fluorescent reporter are commonly used to visualize proteins in situ. However, antibodies and bulky reporters can perturb protein structure and function. To overcome this problem, we have designed a minimally perturbing blue fluorescent unnatural amino acid that enables the direct imaging of intrinsically-fluorescent proteins with their interaction partners. Specifically, we used a nitrile-derivatized tryptophan, 4-cyanotryptophan (4CNTrp), which differs by only two atoms from native tryptophan. 4CNTrp has unique photophysical properties in the visible blue region: an absorption maximum at ~325 nm, an emission maximum at ~420 nm, a large fluorescence quantum yield (>0.8 in aqueous solution), a long fluorescence lifetime (ca. 13 ns), and good photostability (Hilaire et al. PNAS 2017; 114: 6005-09). Based on these properties, 4CNTrp-labeling was recently used to visualize the binding of a model peptide pHLIP (pH-(Low) Insertion Peptide) to cell membranes via wide-field fluorescence microscopy. 4CNTrp is also a viable FRET donor for acceptor pairs in the green visible region. 4CNTrp can be prepared by a simple, high-yielding cost-effective synthetic route and because it does not inhibit bacterial growth, it may be practical for cellular applications (Zhang et al. Chem. Comm. 2019; 55: 5095-98). Here, we used 4CNTrp-labeled peptides to directly image integrins on cell surfaces. Previously, we described the synthesis of peptides, called CHAMP peptides for Computed Helical Anti-Membrane Protein, that target the transmembrane (TM) domains of integrins in a sequence-specific manner (Yin et al. Science 2007; 315: 1817-22). Thus, the CHAMP peptides anti-αIIb and anti-αv specifically bind to the TM domains of αIIb and αv in platelet membranes, causing separation of the αIIbβ3 and αvβ3 TM domains and αIIbβ3 and αvβ3 activation. Anti-αIIb also caused αIIbβ3 clustering that was visualized using the β3-specific monoclonal antibody SSA6 labeled with Alexa Fluor 488 but only after the platelets were fixed with paraformaldehyde to prevent antibody-induced clustering. To enable the direct visualization of αIIβ3, αvβ3, and α2β1 on cell surfaces, we used solid-phase peptide synthesis to generate CHAMP peptides labeled with 4CNTrp at their N-termini (CN-αIIb, CN-αv, CN-β1). We found that 4CNTrp labeling did not perturb the ability of the CHAMP peptides to bind to integrins and to cause specific integrin activation. Thus, CN-αIIb caused αIIbβ3-dependent platelet aggregation, CN-αv caused the αvβ3-dependent platelet adhesion to osteopontin, and CN-α1 caused α2β1-mediated platelet adhesion to collagen. We then used high resolution wide-field deconvolution microscopy to image 4CNTrp-containing integrins on the surface of platelets, HEL cells, and transfected CHO cells. Compared to 4CNTrp-containing random peptides that uniformly decorate cell surfaces, the CN-labeled CHAMP peptides were present as a limited number of discrete foci on the cell surface. To confirm that these foci represented CN-peptide containing integrins, we co-stained the cells with Alexa Fluor 488-labeled integrin-specific monoclonal antibodies and found that CN-peptide and antibody fluorescence coincided. Thus, these studies demonstrate the specific and direct imaging of integrins embedded in cell membranes in their activated state using 4CNTrp-labeled anti-integrin TM peptides. Because 4CNTrp can readily be incorporated into proteins and peptides with little if any structural perturbation, 4CNTrp-labeling provides a facile way to directly monitor protein behavior and protein-protein interactions in cellular environments.