Magnetoplasmonic nanoparticles can manipulate cell surface receptors with single-molecule precision to clarify the effects of force application and receptor clustering.
Mechanosensitive proteins have a variety of roles, ranging from the perception of inputs to developmental processes. To study the function of such proteins and the signaling pathways they participate in, one can apply mechanical force with tweezer-manipulated microbeads or with the tip of an atomic force microscope. However, “these microprobes are really big compared to the target proteins,” says Young-wook Jun from the University of California, San Francisco. In collaboration with Zev Gartner's team at the University of California, San Francisco, Jun and his team set out to develop tools that are comparable in size to proteins.
The researchers followed up on their earlier work on monovalent quantum dots, which are fluorescent particles that can be targeted to single proteins. The nanoparticles in their latest study not only can be targeted to single proteins as well as imaged, but also can exert force on the target proteins. The particles have a size of about 50 nanometers and consist of a magnetic core and a plasmonic gold shell for manipulating and imaging the particles, respectively. The nanoparticles are coated with an oligonucleotide that is functionalized with a small molecule or protein that targets the nanoparticle to the mechanosensitive protein of interest. Because of its small size and monovalent nature, each nanoparticle attaches to a single target protein.
Jun and his colleagues manipulate the cell-attached nanoparticles with micromagnetic tweezers. Depending on the applied force and the distance of the tip from the cell, the researchers can cluster the nanoparticle-bound proteins or pull on single proteins. Jun explains that the beauty of his nanoparticle-based technology is that it makes it possible to distinguish spatial segregation of target proteins from mechanical activation. “That is not possible with the traditional microbead technology,” says Jun. Owing to the larger size and multivalent nature of microbeads, many receptors are typically recruited, and therefore the mechanical activation of the receptors cannot be separated from the effect of clustering these proteins.
Jun's team has applied their technology to clarify the roles of mechanical activation and oligomerization in Notch activation. Although the importance of mechanical activation has been recognized, it was unclear whether Notch oligomerization or ligand binding was necessary as well. The researchers discovered that mechanical activation alone was sufficient to initiate Notch proteolytic cleavage and downstream signaling processes. Furthermore, the team examined the differential effects of clustered E-cadherin or E-cadherin under mechanical stress and observed differences in the underlying actin cytoskeleton.
Jun thinks that his technology will be extremely useful for studying how mechanoreceptor signaling is regulated by spatial segregation and mechanical activation in a variety of contexts. In addition to Notch and E-cadherin signaling pathways and their roles in developmental processes, he is investigating the mechanism of mechanosensory ion channel signaling.
Jun is actively working on improving and expanding the technology. He explains that because of the small size of the nanoparticles, the magnetic field has to be approached very closely, which is difficult to control. In addition, the forces that can be applied through the nanoparticles are quite weak. By increasing the size of the magnetic core while decreasing the size of the shell, he aims to develop nanoparticles that are capable of generating greater forces.
Seo, D. et al. A mechanogenetic toolkit for interrogating cell signaling in space and time. Cell 165, 1507–1518 (2016).
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Vogt, N. Miniature magnetic force probes. Nat Methods 13, 544 (2016). https://doi.org/10.1038/nmeth.3915