Mapping HA‐tagged protein at the surface of living cells by atomic force microscopy

Abstract

Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2-adrenergic receptor (β2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process.

A New Function for Amyloid-Like Interactions: Cross-Beta Aggregates of Adhesins form Cell-to-Cell Bonds

“…That outcome may be desirable in biofilms where amyloids can become part of the matrix, as in bacterial biofilms [ 7 , 24 , 25 , 61 , 62 , 63 , 64 ]. In support of this idea, other wall proteins also include cross-β core sequences [ 32 , 65 , 66 , 67 ]. Among these, the glycosyl transferases Gas1, Gas3, Gas 5, Ygp1, and Bgl2 can form intracellular β-aggregates, and form amyloids in E. coli when overexpressed in the Csg-curlin system [ 22 , 66 , 67 , 68 , 69 ].…”

Cell biology of microbes and pharmacology of antimicrobial drugs explored by Atomic Force Microscopy

“…This mode presents the advantages of avoiding lateral forces and thus damaging of the sample, while simultaneously collecting information on its topography and biophysical properties. Moreover, tips used can be functionalized, either with biomolecules or with single living cells, which opens up the way to study specific molecular interactions at the cellular interface [9,10], or between two different or similar cellular interfaces [11][12][13]. However, the time needed to record a high-resolution image in this mode does not match the dynamics of biological processes.…”

Integration of Biochemical, Biophysical and Transcriptomics Data for Investigating the Structural and Nanomechanical Properties of the Yeast Cell Wall

“…In addition, the single molecule force spectroscopy (SMFS), technique that uses a chemically modified AFM-tip allows to directly monitor specific interaction at the cell surface. As a relevant example is the presence of adhesive patches forming nanodomains at the yeast cell surface as identified by AFM-tip functionalized with concanavalin (Alsteens et al, 2010 ; Schiavone et al, 2015 ) Likewise, mapping of cell wall proteins at the cell surface can be monitored using corresponding antibodies covalently fixed on the AFM-tips (Formosa et al, 2015a ).…”