Limits...
DNA-based digital tension probes reveal integrin forces during early cell adhesion.

Zhang Y, Ge C, Zhu C, Salaita K - Nat Commun (2014)

Bottom Line: Mechanical stimuli profoundly alter cell fate, yet the mechanisms underlying mechanotransduction remain obscure because of a lack of methods for molecular force imaging.Quantitative imaging reveals that integrin tension is highly dynamic and increases with an increasing integrin density during adhesion formation.DNA-based tension probes are among the most sensitive optical force reporters to date, overcoming the force and spatial resolution limitations of traction force microscopy.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, Georgia 30322, USA.

ABSTRACT
Mechanical stimuli profoundly alter cell fate, yet the mechanisms underlying mechanotransduction remain obscure because of a lack of methods for molecular force imaging. Here to address this need, we develop a new class of molecular tension probes that function as a switch to generate a 20- to 30-fold increase in fluorescence upon experiencing a threshold piconewton force. The probes employ immobilized DNA hairpins with tunable force response thresholds, ligands and fluorescence reporters. Quantitative imaging reveals that integrin tension is highly dynamic and increases with an increasing integrin density during adhesion formation. Mixtures of fluorophore-encoded probes show integrin mechanical preference for cyclized RGD over linear RGD peptides. Multiplexed probes with variable guanine-cytosine content within their hairpins reveal integrin preference for the more stable probes at the leading tip of growing adhesions near the cell edge. DNA-based tension probes are among the most sensitive optical force reporters to date, overcoming the force and spatial resolution limitations of traction force microscopy.

Show MeSH

Related in: MedlinePlus

DNA-based digital tension probes(a) Theoretical plot showing the expected increase in fluorescence signal asa function of applied force for the 100% and 22% GC-content hairpin probesand the PEG-based tension probe. DNA-hairpin tension probe response was obtained byfitting into a two-state Boltzmann distribution. The response of the PEG-based tensionprobe was based on experimental parameters obtained from recent work19. (b) Schematic of the integrin tensionsensor, which is comprised of an anchor strand immobilized onto a surface (blue), ahairpin strand that unfolds under sufficient tension (black), and a ligand strandpresenting an adhesive peptide (green). At the apposing termini of the ligand andanchoring strands, a fluorophore and quencher were coupled to report the force-inducedunfolding of the hairpin. (c) Schematic showing the predicted secondarystructure of the folded hairpin (top). Table summarizes the calculated and measuredF1/2 values, GC content, and the calculated free energy of hybridization ofall hairpins used in this study. The quenching efficiency (QE) for Cy3B-BHQ1 and Cy5-QSY21fluorophore-quencher reporters was measured on supported lipid membrane and is alsoincluded in the table. Error represents the standard deviation from three different pairsof samples.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4209443&req=5

Figure 1: DNA-based digital tension probes(a) Theoretical plot showing the expected increase in fluorescence signal asa function of applied force for the 100% and 22% GC-content hairpin probesand the PEG-based tension probe. DNA-hairpin tension probe response was obtained byfitting into a two-state Boltzmann distribution. The response of the PEG-based tensionprobe was based on experimental parameters obtained from recent work19. (b) Schematic of the integrin tensionsensor, which is comprised of an anchor strand immobilized onto a surface (blue), ahairpin strand that unfolds under sufficient tension (black), and a ligand strandpresenting an adhesive peptide (green). At the apposing termini of the ligand andanchoring strands, a fluorophore and quencher were coupled to report the force-inducedunfolding of the hairpin. (c) Schematic showing the predicted secondarystructure of the folded hairpin (top). Table summarizes the calculated and measuredF1/2 values, GC content, and the calculated free energy of hybridization ofall hairpins used in this study. The quenching efficiency (QE) for Cy3B-BHQ1 and Cy5-QSY21fluorophore-quencher reporters was measured on supported lipid membrane and is alsoincluded in the table. Error represents the standard deviation from three different pairsof samples.

Mentions: To this end, we report a new class of molecular tension probes that employs aDNA-hairpin as a “switch” element, thus unfolding at a threshold force andreporting tension in a digital rather than analog fashion (Fig.1a). DNA is ideally-suited for this purpose because an oligonucleotide’snucleobase sequence can be used to rationally tune its force-response function. Probes weredesigned to be highly adaptable, consisting of three oligonucleotides assembled throughhybridization of 21-mer handles (Fig. 1b): a stem-loopDNA hairpin that is unmodified (black), a peptide-displaying ligand strand conjugated to afluorophore (green), and a surface-anchor strand that is tagged with a quencher (blue). Theapposing termini of the ligand and anchor oligonucleotides were modified with afluorophore-quencher pair, such that a sufficient force leads to hairpin unfolding accompaniedby a drastic increase in fluorescence intensity.


DNA-based digital tension probes reveal integrin forces during early cell adhesion.

Zhang Y, Ge C, Zhu C, Salaita K - Nat Commun (2014)

DNA-based digital tension probes(a) Theoretical plot showing the expected increase in fluorescence signal asa function of applied force for the 100% and 22% GC-content hairpin probesand the PEG-based tension probe. DNA-hairpin tension probe response was obtained byfitting into a two-state Boltzmann distribution. The response of the PEG-based tensionprobe was based on experimental parameters obtained from recent work19. (b) Schematic of the integrin tensionsensor, which is comprised of an anchor strand immobilized onto a surface (blue), ahairpin strand that unfolds under sufficient tension (black), and a ligand strandpresenting an adhesive peptide (green). At the apposing termini of the ligand andanchoring strands, a fluorophore and quencher were coupled to report the force-inducedunfolding of the hairpin. (c) Schematic showing the predicted secondarystructure of the folded hairpin (top). Table summarizes the calculated and measuredF1/2 values, GC content, and the calculated free energy of hybridization ofall hairpins used in this study. The quenching efficiency (QE) for Cy3B-BHQ1 and Cy5-QSY21fluorophore-quencher reporters was measured on supported lipid membrane and is alsoincluded in the table. Error represents the standard deviation from three different pairsof samples.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4209443&req=5

Figure 1: DNA-based digital tension probes(a) Theoretical plot showing the expected increase in fluorescence signal asa function of applied force for the 100% and 22% GC-content hairpin probesand the PEG-based tension probe. DNA-hairpin tension probe response was obtained byfitting into a two-state Boltzmann distribution. The response of the PEG-based tensionprobe was based on experimental parameters obtained from recent work19. (b) Schematic of the integrin tensionsensor, which is comprised of an anchor strand immobilized onto a surface (blue), ahairpin strand that unfolds under sufficient tension (black), and a ligand strandpresenting an adhesive peptide (green). At the apposing termini of the ligand andanchoring strands, a fluorophore and quencher were coupled to report the force-inducedunfolding of the hairpin. (c) Schematic showing the predicted secondarystructure of the folded hairpin (top). Table summarizes the calculated and measuredF1/2 values, GC content, and the calculated free energy of hybridization ofall hairpins used in this study. The quenching efficiency (QE) for Cy3B-BHQ1 and Cy5-QSY21fluorophore-quencher reporters was measured on supported lipid membrane and is alsoincluded in the table. Error represents the standard deviation from three different pairsof samples.
Mentions: To this end, we report a new class of molecular tension probes that employs aDNA-hairpin as a “switch” element, thus unfolding at a threshold force andreporting tension in a digital rather than analog fashion (Fig.1a). DNA is ideally-suited for this purpose because an oligonucleotide’snucleobase sequence can be used to rationally tune its force-response function. Probes weredesigned to be highly adaptable, consisting of three oligonucleotides assembled throughhybridization of 21-mer handles (Fig. 1b): a stem-loopDNA hairpin that is unmodified (black), a peptide-displaying ligand strand conjugated to afluorophore (green), and a surface-anchor strand that is tagged with a quencher (blue). Theapposing termini of the ligand and anchor oligonucleotides were modified with afluorophore-quencher pair, such that a sufficient force leads to hairpin unfolding accompaniedby a drastic increase in fluorescence intensity.

Bottom Line: Mechanical stimuli profoundly alter cell fate, yet the mechanisms underlying mechanotransduction remain obscure because of a lack of methods for molecular force imaging.Quantitative imaging reveals that integrin tension is highly dynamic and increases with an increasing integrin density during adhesion formation.DNA-based tension probes are among the most sensitive optical force reporters to date, overcoming the force and spatial resolution limitations of traction force microscopy.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, Georgia 30322, USA.

ABSTRACT
Mechanical stimuli profoundly alter cell fate, yet the mechanisms underlying mechanotransduction remain obscure because of a lack of methods for molecular force imaging. Here to address this need, we develop a new class of molecular tension probes that function as a switch to generate a 20- to 30-fold increase in fluorescence upon experiencing a threshold piconewton force. The probes employ immobilized DNA hairpins with tunable force response thresholds, ligands and fluorescence reporters. Quantitative imaging reveals that integrin tension is highly dynamic and increases with an increasing integrin density during adhesion formation. Mixtures of fluorophore-encoded probes show integrin mechanical preference for cyclized RGD over linear RGD peptides. Multiplexed probes with variable guanine-cytosine content within their hairpins reveal integrin preference for the more stable probes at the leading tip of growing adhesions near the cell edge. DNA-based tension probes are among the most sensitive optical force reporters to date, overcoming the force and spatial resolution limitations of traction force microscopy.

Show MeSH
Related in: MedlinePlus