SPATIALLY RESOLVED FORCE SPECTROSCOPY OF BIOMOLECULAR SYSTEMS

CATALINA VON BILDERLING; MARÍA ANTONIETA DAZA MILLONE; PAULA CECILIA DOS SANTOS CLARO; MARÍA ELENA VELA; LÍA ISABEL PIETRASANTA

Buzios, Brasil

Congreso; VII Iberoamerican Congress of Biophysics 2009 and Latin American Postgraduate Program of Biophysics Course 2009; 2009

During the last decade, atomic force microscopy (AFM) has emerged as a powerful tool for imaging the structure and function of biological specimens in their physiological environment. This instrument uses a sharp stylus at the end of a flexible cantilever to scan over the sample surface. Cantilever deflections measured at a resolution of a few angstroms are used to determine the surface contour of the sample, exploiting a sensitive feedback to minimize the force applied to the stylus. Recent progress in the application of the AFM to imaging and manipulating biomolecules is the result of improved sample preparation methods, image acquisition conditions, and continuous developments in the instrumentation. Additionally, force spectroscopy using the AFM has been used extensively for measuring intermolecular forces between an AFM tip and a wide range of different systems. AFM force spectroscopy has allowed a deeper insight into the energetics of structural phase transitions, conformational changes, and the unfolding of single stretched molecules with piconewton sensitivity and subnanometer accuracy. Recently, single molecule force spectroscopy combined with single molecule imaging has provided unique information on surface structure, protein folding and protein-protein interactions. Mechanical forces play an important role in the organization, growth, maturation, and function of living tissues. At the cellular level, many of the biological responses to external forces originate at two types of specialized structures: focal adhesions and adherent junctions. In order to explore the dynamic regulation of adhesive contacts and of the cytoskeleton architecture of cells, as well as its resultant influence on cellular activities, we will focus on the relationship between local force applied to the cell and the assembly, mechanics and dynamics of focal adhesions. In this work we describe how we are applying AFM and force spectroscopy on different biomolecular systems with the final goal of measuring forces on single focal adhesions in cells. Our first approach on AFM force spectroscopy was to map the spatial arrangement of chemical functional groups and their interactions. Measurements were made with a Multimode AFM, Nanoscope IIIa controller with Quadrex extender (Veeco-Digital Instruments). For all experiments the cantilevers were previously calibrated using the thermal tune method (Rev. Sci. Instrum. 64:1868-1873, 1993), analyzing the free vibration movement data of the cantilever with a homemade Matlab routine. We measured the adhesive and friction forces between molecularly modified AFM probe tips and organic monolayers arranged in a distinct patterned surface. By monitoring the friction between specifically functionalized tip and sample, we obtained friction images that display the expected contrast and correlate with the spatial distribution of functional groups on the sample surface. We also acquired an array of force versus cantilever displacement curves, so-called force volumes, to obtain high-resolution 2-D adhesion maps of the specific interactions between sample and AFM probe tips.