Imaging of formin-mediated actin assembly using interferometric scattering microscopy (iSCAT)
Biochemistry
Final Report Abstract
Interferometric scattering microscopy provides a means of visualising biomolecules label-free in solution and surpasses many of the abilities of other single-molecule methods in terms of spatial and temporal resolution. My project aimed at evaluating the potential of iSCAT to image the process of actin filament polymerisation and to observe its dynamics at the unprecedented level of individual subunit turnover. This ambitious goal drove the technological development of iSCAT such that images of unlabelled individual proteins can now be recorded at high signalto-noise ratio and temporal resolution. Not only did I succeed in directly observing the turnover of subunits at the actin filament tip, but I was also involved in the development in a new application of iSCAT which we call interferometric scattering mass photometry (iSCAMS). The ability to measure the mass of individual biomolecules in a solution environment at high precision and a resolution that allows distinction of small molecular size differences opens up a huge array of new possibilities for experiments in all fields of the biological sciences. We are only beginning to understand the impact that this new technique will have on future research. The Kukura group has started a spin-off company which commercialises this new application of iSCAT and strives to make it available to a wider scientific community. The formation of physiological actin complexes, e.g. with formins and tropomyosins as proposed in this project, remains to be investigated and could be elucidated in even more detail now than anticipated in the original draft of the project. However, other biopolymer systems such as amyloid fibrils which are found in the brains of neurodegenerative disease patients would be extremely interesting targets to study with iSCAT considering the preliminary results I have presented on actin nucleation. In many of these diseases, such as Alzheimer’s, Parkinson’s and Huntington’s, small soluble amyloid oligomers are now considered the most toxic to patients’ neurons and are a premiere target for drugbased intervention. Similar to the case of actin, these nucleating species are particularly difficult to study with the available set of techniques. I believe that iSCAT will become a valuable tool to gaining new insights into these medically important protein systems.
Publications
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(2017) Revealing intricate acto-myosin dynamics at a membrane surface using interferometric scattering microscopy
Koester D, Hundt N, Young G, Fineberg A, Kukura P & Mayor S
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(2017), Direct Single Molecule Observations of the Unique Mechanical State of Human Myosin-6, Biophysical J. 112(3): 265A
Takagi Y, Hundt N, Billington N, Andrecka J, Cole D, Fineberg AJ, Katagiri N, Bird JE, Friedman TB, Kukura P, Sellers JR
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(2018), Label-Free Visualisation of Actin Nucleation and Polymerisation at the Single-Molecule Level using Interferometric Scattering Microscopy, Biophysical J. 114(3): 381A
Hundt N, Tyler A, Young G, Cole D, Fineberg AJ, Andrecka J, Kukura P
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(2018), Quantitative mass imaging of single biological macromolecules. Science. 306(6387): 423-427
Young G, Hundt N, Cole D, Fineberg A, Andrecka J, Tyler A, Olerinyova A, Ansari A, Marklund EG, Collier MP, Chandler SA, Tkachenko O, Allen J, Crispin M, Billington N, Takagi Y, Sellers JR, Eichmann C, Selenko P, Frey L, Riek R, Galpin MR, Struwe WB, Benesch JLP, Kukura P
