Abstract
This work probes the binding kinetics of COOH-terminus of Clostridium perfringens enterotoxin (c-CPE) and claudin expressing MCF-7 cells using force spectroscopy with optical tweezers. c-CPE is of high biomedical interest due to its ability to specifically bind to claudin with high affinity as well as reversibly disrupt tight junctions whilst maintaining cell viability. We observed single-step rupture events between silica particles functionalized with c-CPE and MCF-7 cells. Extensive calibration of the optical tweezers’ trap stiffness and displacement of the particle from trap center extracted a probable bond rupture force of ≈ 18 pN. The probability of rupture events with c-CPE functionalized silica particles increased by 50% compared to unfunctionalized particles. Additionally, rupture events were not observed when probing cells not expressing claudin with c-CPE coated particles. Overall, this work demonstrates that optical tweezers are invaluable tools to probe ligand-receptor interactions and their potential to study dynamic molecular events in drug-binding scenarios.
Highlights
Dynamic force spectroscopy is a technique which measures the distribution of rupture forces between molecular bonds as a function of loading rate (Capitanio and Pavone, 2013)
We investigate the interaction of claudin and COOH-terminus of Clostridium perfringens enterotoxin (c-Clostridium perfringens enterotoxin (CPE)) by performing force spectroscopy measurements using optical tweezers
Based on the work of Litvinov et al (2002) and Shergill et al (2012) we develop a protocol wherein claudin-expressing cells attached to a coverslip are brought into contact with optically trapped particles functionalized with c-CPE and subsequently retracted
Summary
Dynamic force spectroscopy is a technique which measures the distribution of rupture forces between molecular bonds as a function of loading rate (Capitanio and Pavone, 2013). Along with biomembrane force probe, atomic force microscopy and magnetic tweezers, optical tweezers are one of the few known tools that can be implemented to probe the binding strength of molecular bonds such as cell membrane receptors and ligands (Merkel et al, 1999; Neuman and Nagy, 2008). A tightly focused laser beam traps and manipulates microscopic particles with nanometer precision. The optically trapped particle can be functionalized by ligands in order to bind them to receptor proteins present on the membrane of living cells. By monitoring the displacement of the particle from the trap center, the rupture force required to dissociate the bond formed between the ligand and their receptor can be obtained and measured.
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