Five challenges to bringing single-molecule force spectroscopy into living cells

Universite catholique de Louvain, Institute of Condensed Matter and Nanosciences, Louvain-la-Neuve, Belgium.
Nature Methods (Impact Factor: 32.07). 02/2011; 8(2):123-7. DOI: 10.1038/nmeth0211-123
Source: PubMed


In recent years, single-molecule force spectroscopy techniques have been used to study how inter- and intramolecular interactions control the assembly and functional state of biomolecular machinery in vitro. Here we discuss the problems and challenges that need to be addressed to bring these technologies into living cells and to learn how cellular machinery is controlled in vivo.

Download full-text


Available from: Daniel J Müller
  • Source
    • "Unauthenticated Download Date | 6/13/15 8:43 PM technique has been used to study how interand intramolecular interactions control the assembly and functional state of biomolecular machinery in vitro [34]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: This review highlights relevant issues about applications and improvements of atomic force microscopy (AFM) toward a better understanding of neurodegenerative changes at the molecular level with the hope of contributing to the development of effective therapeutic strategies for neurodegenerative illnesses. The basic principles of AFM are briefly discussed in terms of evaluation of experimental data, including the newest PeakForce Quantitative Nanomechanical Mapping (QNM) and the evaluation of Young’s modulus as the crucial elasticity parameter. AFM topography, revealed in imaging mode, can be used to monitor changes in live neurons over time, representing a valuable tool for high-resolution detection and monitoring of neuronal morphology. The mechanical properties of living cells can be quantified by force spectroscopy as well as by new AFM. A variety of applications are described, and their relevance for specific research areas discussed. In addition, imaging as well as non-imaging modes can provide specific information, not only about the structural and mechanical properties of neuronal membranes, but also on the cytoplasm, cell nucleus, and particularly cytoskeletal components. Moreover, new AFM is able to provide detailed insight into physical structure and biochemical interactions in both physiological and pathophysiological conditions.
    Full-text · Article · Jun 2015 · Translational Neuroscience
  • Source
    • "In this context, an AFM-based technique, single-molecule force spectroscopy (SMFS), has recently emerged in the field. In this technique, AFM tips interact with biomolecules immobilized on innate substrates or artificial biomembranes (in vitro studies) or present at the surface of living cells so to understand the intramolecular and intermolecular interactions of biomolecular systems (Müller et al., 2009; Dufrêne et al., 2011). SMFS techniques have been widely used in vitro, to monitor, for example, the interaction of cellular adhesion molecules, such as cadherins (Baumgartner et al., 2000) or oligosaccharides (Rief et al., 1997a) or to characterize the anchoring forces of peptides in lipid membranes (Ganchev et al., 2004). "
    [Show abstract] [Hide abstract]
    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. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.
    Full-text · Article · Feb 2015 · Journal of Molecular Recognition
  • Source
    • "Recently, SMFS has been proven to be a useful method to analyze the activities of single molecules in situ on living bacteria [5] and mammalian cells [6]. However, current SMFS experiments are performed on purified biomolecules isolated from cell lines [7] or directly on cell lines [8]. Cell lines cultured in vitro are known to be quite different from cells in the human body, due to the huge difference between the in vitro and in vivo environment . "
    [Show abstract] [Hide abstract]
    ABSTRACT: Rituximab is a monoclonal antibody drug approved for the treatment of patients with lymphomas. Rituximab’s main killing mechanism is antibody-dependent cellular cytotoxicity (ADCC). During ADCC, rituximab’s fragment antigen binding (Fab) region binds to the CD20 antigen on the tumor cell and its fragment crystallizable (Fc) region binds to the Fc receptor (FcR) on the natural killer (NK) cells. In this study, two types of molecular interactions (CD20-rituximab, FcR-rituximab) involved in ADCC were measured simultaneously on cells prepared from biopsy specimens of lymphoma patients by utilizing atomic force microscopy (AFM) with functionalized tips carrying rituximab. NK cells were detected by specific NKp46 fluorescent labeling and tumor cells were detected by specific ROR1 fluorescent labeling. Based on the fluorescence recognition, the binding affinity and distribution of FcRs on NK cells, and CD20 on tumor cells, were quantitatively measured and mapped. The binding affinity and distribution of FcRs (on NK cells) and CD20 (on tumor cells) were associated with rituximab clinical efficacy. The experimental results provide a new approach to simultaneously quantify the multiple types of molecular interactions involved in rituximab ADCC mechanism on patient biopsy cells, which is of potential clinical significance to predict rituximab efficacy for personalized medicine.
    Full-text · Article · Aug 2014 · Cellular Immunology
Show more