Lighting Up the Force: Investigating Mechanisms of Mechanotransduction Using Fluorescent Tension Probes.

The ability of cells to sense the physical nature of their surroundings is critical to the survival of multicellular organisms. Cellular response to physical cues from adjacent cells and the extracellular matrix leads to a dynamic cycle in which cells respond by remodeling their local microenvironment, fine-tuning cell stiffness, polarity, and shape. Mechanical regulation is important in cellular development, normal morphogenesis, and wound healing. The mechanisms by which these finely balanced mechanotransduction events occur, however, are not well understood. In large part, this is due to the limited availability of tools to study molecular mechanotransduction events in live cells. Several classes of molecular tension probes have been recently developed which are rapidly transforming the study of mechanotransduction. Molecular tension probes are primarily based on fluorescence resonance energy transfer (FRET) and report on piconewton scale tension events in live cells. In this minireview, we describe the two main classes of tension probes, genetically encoded tension sensors and immobilized tension sensors, and discuss the advantages and limitations of each type. We discuss future opportunities to address major biological questions and outline the challenges facing the next generation of molecular tension probes.

Integrin-generated forces lead to streptavidin-biotin unbinding in cellular adhesions.

The interplay between chemical and mechanical signals plays an important role in cell biology, and integrin receptors are the primary molecules involved in sensing and transducing external mechanical cues. We used integrin-specific probes in molecular tension fluorescence microscopy to investigate the pN forces exerted by integrin receptors in living cells. The molecular tension fluorescence microscopy probe consisted of a cyclic Arg-Gly-Asp-D-Phe-Lys(Cys) (cRGDfK(C)) peptide tethered to the terminus of a polyethylene glycol polymer that was attached to a surface through streptavidin-biotin linkage. A fluorescence resonance energy transfer mechanism was used to visualize tension-driven extension of the polymer. Surprisingly, we found that integrin receptors dissociate streptavidin-biotin tethered ligands in focal adhesions within 60 min of cell seeding. Although streptavidin-biotin binding affinity is described as the strongest noncovalent bond in nature, and is ~10(6) - 10(8) times larger than that of integrin-RGD affinity, our results suggest that individual integrin-ligand complexes undergo a marked enhancement in stability when the receptor assembles in the cell membrane. Based on the observation of streptavidin-biotin unbinding, we also conclude that the magnitude of integrin-ligand tension in focal adhesions can reach values that are at least 10 fold larger than was previously estimated using traction force microscopy-based methods.

Visualizing mechanical tension across membrane receptors with a fluorescent sensor.

We report a fluorescence-based turn-on sensor for mapping the mechanical strain exerted by specific cell-surface proteins in living cells. The sensor generates force maps with high spatial and temporal resolution using conventional fluorescence microscopy. We demonstrate the approach by mapping mechanical forces during the early stages of regulatory endocytosis of the ligand-activated epidermal growth factor receptor (EGFR).

DPN-generated nanostructures as positive resists for preparing lithographic masters or hole arrays.

Experiments that utilize structures generated by dip-pen nanolithography (DPN) as positive resists for fabricating nanohole arrays and lithographic masters are described. The technique takes advantage of the difference in desorption potentials for patterned structures made from 16-mercaptohexadecanoic acid (MHA) and 1-octadecanethiol (ODT), respectively. In this approach, patterns of MHA on gold are generated by DPN, and surrounding areas are passivated by ODT. Electrochemistry is used to selectively remove the MHA nanofeatures made by DPN. The exposed gold can be used as an electrode to plate silver from solution, generating raised features and structures that can be transferred to PDMS to make a lithographic master, or alternatively, they can be etched to make arrays of nanoholes.

Metallomacrocycles that incorporate cofacially aligned diimide units.

Pyromellitic diimide and naphthalene diimide moieties were incorporated into hemilabile phosphanylalkyl thioether ligands. These ligands reacted with [Cu(CH3CN)4]PF6 and [Rh(NBD)Cl]2 (NBD = norbornadiene) by the weak-link approach to form condensed intermediates. Upon reaction of each diimide ligand with these transition-metal precursors, the two diimide units became cofacially aligned within a supramolecular macrocyclic architecture. The introduction of ancillary ligands to each of these condensed intermediates caused the weak thioether-metal bonds to break, thus generating a large macrocycle in which the distance between diimide units is significantly larger than for the condensed intermediates. The two Rh(I) cationic condensed intermediates were characterized by single-crystal X-ray diffraction studies, and the electrochemical activity of these macrocycles was demonstrated with the naphthalene diimide-Cu(I) macrocycles.