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Proceedings Paper

Ultra-fast optical manipulation of single proteins binding to the actin cytoskeleton
Author(s): Marco Capitanio; Lucia Gardini; Francesco Saverio Pavone
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Paper Abstract

In the last decade, forces and mechanical stresses acting on biological systems are emerging as regulatory factors essential for cell life. Emerging evidences indicate that factors such as applied forces or the rigidity of the extracellular matrix (ECM) determine the shape and function of cells and organisms1. Classically, the regulation of biological systems is described through a series of biochemical signals and enzymatic reactions, which direct the processes and cell fate. However, mechanotransduction, i.e. the conversion of mechanical forces into biochemical and biomolecular signals, is at the basis of many biological processes fundamental for the development and differentiation of cells, for their correct function and for the development of pathologies. We recently developed an in vitro system that allows the investigation of force-dependence of the interaction of proteins binding the actin cytoskeleton, at the single molecule level. Our system displays a delay of only ~10 μs between formation of the molecular bond and application of the force and is capable of detecting interactions as short as 100 μs. Our assay allows direct measurements of load-dependence of lifetimes of single molecular bonds and conformational changes of single proteins and molecular motors. We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to now, opening new perspectives for the investigation of the effects of forces on biological processes.

Paper Details

Date Published: 26 February 2014
PDF: 7 pages
Proc. SPIE 8946, Optical Elastography and Tissue Biomechanics, 89460K (26 February 2014); doi: 10.1117/12.2039219
Show Author Affiliations
Marco Capitanio, European Lab. for Non-linear Spectroscopy (Italy)
Univ. degli Studi di Firenze (Italy)
Lucia Gardini, European Lab. for Non-linear Spectroscopy (Italy)
Francesco Saverio Pavone, European Lab. for Non-linear Spectroscopy (Italy)
Univ. degli Studi di Firenze (Italy)
Istituto Nazionale di Ottica, CNR (Italy), International Ctr. of Computational Neurophotonics (Italy)

Published in SPIE Proceedings Vol. 8946:
Optical Elastography and Tissue Biomechanics
Kirill V. Larin; David D. Sampson, Editor(s)

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