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Force is a signal that cells cannot ignore.

Yusko EC, Asbury CL - Mol. Biol. Cell (2014)

Bottom Line: Cells sense biochemical, electrical, and mechanical cues in their environment that affect their differentiation and behavior.The molecular details underlying how cells respond to force are only beginning to be understood.Here we review tools for probing force-sensitive proteins and highlight several examples in which forces are transmitted, routed, and sensed by proteins in cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195-7290.

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Methods for applying and measuring precise forces to single molecules and molecular complexes. (A–C) Instruments often used to apply precise forces to individual macromolecules or complexes. (A) In laser trapping, a focused laser beam behaves roughly like a Hookean spring, pulling a submicrometer bead toward its center with a force proportional to the stiffness of the laser trap, k, multiplied with the displacement of the bead from the trap center, Δx; beads are often decorated with a protein or receptor of interest and can be controlled by manipulating the position of the laser beam relative to the microscope slide. (B) Atomic force microscopes employ a micrometer-width cantilever, at the tip of which is a nanometer-sized pointer that can be decorated with proteins or receptors; once these proteins bind their receptors on the surface of a glass slide, the cantilever is retracted causing it to deflect. (C) Magnetic tweezers employ magnetic beads with a magnetic moment, μ; when subjected to a magnetic field, the force on the beads is proportional to the magnetic field strength multiplied by μ. Up to several hundred magnetic beads can be pulled at the same time. (D–F) Techniques for measuring forces precisely between and within molecules. (D) Pillars with diameters and lengths on the nanometer to submicrometer scale can be formed from elastic polymers and decorated with extracellular matrix proteins, such that cultured cells adhere and form focal adhesions; the deflection of each nanopillar from its resting position reveals the contractile forces exerted at the corresponding focal adhesion. (E) Hybridized dsDNA molecules for which one strand is tethered to a surface and the complementary strand is tethered to a protein or receptor can act as a “tension-gauge-tether” by which the number of base pairs within the dsDNA that support the load dictates a well-defined force at which the dsDNA will unzip or melt; unzipping of the dsDNA can be observed using fluorescent tags on the DNA molecules or by cell phenotypes, allowing estimation of the range of forces to which a protein–ligand interaction might be subjected during a cellular event such as early stages of cell adhesion. (F) An intramolecular strain sensor based on FRET can be used to determine the forces exerted through a protein by engineering the probe into the protein structure and monitoring the level of FRET.
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Figure 1: Methods for applying and measuring precise forces to single molecules and molecular complexes. (A–C) Instruments often used to apply precise forces to individual macromolecules or complexes. (A) In laser trapping, a focused laser beam behaves roughly like a Hookean spring, pulling a submicrometer bead toward its center with a force proportional to the stiffness of the laser trap, k, multiplied with the displacement of the bead from the trap center, Δx; beads are often decorated with a protein or receptor of interest and can be controlled by manipulating the position of the laser beam relative to the microscope slide. (B) Atomic force microscopes employ a micrometer-width cantilever, at the tip of which is a nanometer-sized pointer that can be decorated with proteins or receptors; once these proteins bind their receptors on the surface of a glass slide, the cantilever is retracted causing it to deflect. (C) Magnetic tweezers employ magnetic beads with a magnetic moment, μ; when subjected to a magnetic field, the force on the beads is proportional to the magnetic field strength multiplied by μ. Up to several hundred magnetic beads can be pulled at the same time. (D–F) Techniques for measuring forces precisely between and within molecules. (D) Pillars with diameters and lengths on the nanometer to submicrometer scale can be formed from elastic polymers and decorated with extracellular matrix proteins, such that cultured cells adhere and form focal adhesions; the deflection of each nanopillar from its resting position reveals the contractile forces exerted at the corresponding focal adhesion. (E) Hybridized dsDNA molecules for which one strand is tethered to a surface and the complementary strand is tethered to a protein or receptor can act as a “tension-gauge-tether” by which the number of base pairs within the dsDNA that support the load dictates a well-defined force at which the dsDNA will unzip or melt; unzipping of the dsDNA can be observed using fluorescent tags on the DNA molecules or by cell phenotypes, allowing estimation of the range of forces to which a protein–ligand interaction might be subjected during a cellular event such as early stages of cell adhesion. (F) An intramolecular strain sensor based on FRET can be used to determine the forces exerted through a protein by engineering the probe into the protein structure and monitoring the level of FRET.

Mentions: Table 1 shows that tools for direct observation of individual protein interactions must operate with nanometer precision and on the piconewton force scale. Biophysical tools capable of this precision can be grouped into two categories: those that actively control position and therefore apply force (Figure 1, A–C) and those that passively measure force (similar to a strain gauge; Figure 1, D–F). To apply force actively to a protein interaction of interest, the interaction is typically recapitulated in vitro with one protein tethered to a glass coverslip and its binding partner tethered to a polystyrene bead for laser trapping (Ashkin and Dziedzic, 1987; Neuman and Block, 2004; Matthews, 2009), to a magnetic bead for magnetic tweezers (Smith et al., 1992; Strick et al., 1996; Guttenberg et al., 2000), or to the tip of a submicrometer cantilever for atomic force microscopy (AFM; Binnig et al., 1986). Another method, not illustrated in Figure 1, called the biomembrane force probe, can also be used to apply forces to protein–protein interactions; however, most investigators have moved toward the laser trapping, AFM, or magnetic beads platforms (Evans et al, 1995; Gourier et al., 2008; Neuman and Nagy, 2008). Proteins are typically tethered using well-established conjugation chemistries (Hermanson, 2013; Kim and Herr, 2013), and in general tethering is not a major limitation. The physical principles underlying these instruments are outside the scope of this Perspective (see review by Neuman and Nagy, 2008). However, all of them share the ability to manipulate the position of a tethered protein or complex with nanometer precision while observing the deflection of a sensor element (i.e., bead or AFM tip) from its resting position. This deflection is usually proportional to the force applied on the protein–protein interaction with sub-piconewton sensitivity.


Force is a signal that cells cannot ignore.

Yusko EC, Asbury CL - Mol. Biol. Cell (2014)

Methods for applying and measuring precise forces to single molecules and molecular complexes. (A–C) Instruments often used to apply precise forces to individual macromolecules or complexes. (A) In laser trapping, a focused laser beam behaves roughly like a Hookean spring, pulling a submicrometer bead toward its center with a force proportional to the stiffness of the laser trap, k, multiplied with the displacement of the bead from the trap center, Δx; beads are often decorated with a protein or receptor of interest and can be controlled by manipulating the position of the laser beam relative to the microscope slide. (B) Atomic force microscopes employ a micrometer-width cantilever, at the tip of which is a nanometer-sized pointer that can be decorated with proteins or receptors; once these proteins bind their receptors on the surface of a glass slide, the cantilever is retracted causing it to deflect. (C) Magnetic tweezers employ magnetic beads with a magnetic moment, μ; when subjected to a magnetic field, the force on the beads is proportional to the magnetic field strength multiplied by μ. Up to several hundred magnetic beads can be pulled at the same time. (D–F) Techniques for measuring forces precisely between and within molecules. (D) Pillars with diameters and lengths on the nanometer to submicrometer scale can be formed from elastic polymers and decorated with extracellular matrix proteins, such that cultured cells adhere and form focal adhesions; the deflection of each nanopillar from its resting position reveals the contractile forces exerted at the corresponding focal adhesion. (E) Hybridized dsDNA molecules for which one strand is tethered to a surface and the complementary strand is tethered to a protein or receptor can act as a “tension-gauge-tether” by which the number of base pairs within the dsDNA that support the load dictates a well-defined force at which the dsDNA will unzip or melt; unzipping of the dsDNA can be observed using fluorescent tags on the DNA molecules or by cell phenotypes, allowing estimation of the range of forces to which a protein–ligand interaction might be subjected during a cellular event such as early stages of cell adhesion. (F) An intramolecular strain sensor based on FRET can be used to determine the forces exerted through a protein by engineering the probe into the protein structure and monitoring the level of FRET.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230779&req=5

Figure 1: Methods for applying and measuring precise forces to single molecules and molecular complexes. (A–C) Instruments often used to apply precise forces to individual macromolecules or complexes. (A) In laser trapping, a focused laser beam behaves roughly like a Hookean spring, pulling a submicrometer bead toward its center with a force proportional to the stiffness of the laser trap, k, multiplied with the displacement of the bead from the trap center, Δx; beads are often decorated with a protein or receptor of interest and can be controlled by manipulating the position of the laser beam relative to the microscope slide. (B) Atomic force microscopes employ a micrometer-width cantilever, at the tip of which is a nanometer-sized pointer that can be decorated with proteins or receptors; once these proteins bind their receptors on the surface of a glass slide, the cantilever is retracted causing it to deflect. (C) Magnetic tweezers employ magnetic beads with a magnetic moment, μ; when subjected to a magnetic field, the force on the beads is proportional to the magnetic field strength multiplied by μ. Up to several hundred magnetic beads can be pulled at the same time. (D–F) Techniques for measuring forces precisely between and within molecules. (D) Pillars with diameters and lengths on the nanometer to submicrometer scale can be formed from elastic polymers and decorated with extracellular matrix proteins, such that cultured cells adhere and form focal adhesions; the deflection of each nanopillar from its resting position reveals the contractile forces exerted at the corresponding focal adhesion. (E) Hybridized dsDNA molecules for which one strand is tethered to a surface and the complementary strand is tethered to a protein or receptor can act as a “tension-gauge-tether” by which the number of base pairs within the dsDNA that support the load dictates a well-defined force at which the dsDNA will unzip or melt; unzipping of the dsDNA can be observed using fluorescent tags on the DNA molecules or by cell phenotypes, allowing estimation of the range of forces to which a protein–ligand interaction might be subjected during a cellular event such as early stages of cell adhesion. (F) An intramolecular strain sensor based on FRET can be used to determine the forces exerted through a protein by engineering the probe into the protein structure and monitoring the level of FRET.
Mentions: Table 1 shows that tools for direct observation of individual protein interactions must operate with nanometer precision and on the piconewton force scale. Biophysical tools capable of this precision can be grouped into two categories: those that actively control position and therefore apply force (Figure 1, A–C) and those that passively measure force (similar to a strain gauge; Figure 1, D–F). To apply force actively to a protein interaction of interest, the interaction is typically recapitulated in vitro with one protein tethered to a glass coverslip and its binding partner tethered to a polystyrene bead for laser trapping (Ashkin and Dziedzic, 1987; Neuman and Block, 2004; Matthews, 2009), to a magnetic bead for magnetic tweezers (Smith et al., 1992; Strick et al., 1996; Guttenberg et al., 2000), or to the tip of a submicrometer cantilever for atomic force microscopy (AFM; Binnig et al., 1986). Another method, not illustrated in Figure 1, called the biomembrane force probe, can also be used to apply forces to protein–protein interactions; however, most investigators have moved toward the laser trapping, AFM, or magnetic beads platforms (Evans et al, 1995; Gourier et al., 2008; Neuman and Nagy, 2008). Proteins are typically tethered using well-established conjugation chemistries (Hermanson, 2013; Kim and Herr, 2013), and in general tethering is not a major limitation. The physical principles underlying these instruments are outside the scope of this Perspective (see review by Neuman and Nagy, 2008). However, all of them share the ability to manipulate the position of a tethered protein or complex with nanometer precision while observing the deflection of a sensor element (i.e., bead or AFM tip) from its resting position. This deflection is usually proportional to the force applied on the protein–protein interaction with sub-piconewton sensitivity.

Bottom Line: Cells sense biochemical, electrical, and mechanical cues in their environment that affect their differentiation and behavior.The molecular details underlying how cells respond to force are only beginning to be understood.Here we review tools for probing force-sensitive proteins and highlight several examples in which forces are transmitted, routed, and sensed by proteins in cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195-7290.

Show MeSH
Related in: MedlinePlus