Imaging of formin-mediated actin assembly using interferometric scattering microscopy (iSCAT)

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The actin cytoskeleton is essential for many fundamental cellular functions, such as cell migration, cell division, establishment of cell-cell contacts, endo- and exocytosis and vesicle transport. However, the molecular details underlying actin filament dynamics are not yet fully elucidated. One reason for this lack of understanding is the fact that standard fluorescence microscopy techniques do not reach the required combination of dynamic range and spatio-temporal resolution to track individual actin subunits entering and leaving the filament. The proposed project aims to set up a new imaging approach for actin filament assembly using interferometric scattering microscopy (iSCAT). This technique is capable of detecting, imaging and localising single molecules based on their mass without the necessity of any labels. The method promises to shed light onto the precise mechanisms of actin filament assembly, as it will be able to distinguish single subunit addition from filament annealing and enable the measurement of the associated kinetics for the first time. Furthermore, I will combine iSCAT with single molecule fluorescence microscopy to investigate the impact of the formins mDia1 and mDia2 on actin assembly. My imaging approach promises to directly reveal the individual steps of formins at the tip of actin filament ends by fluorescence microscopy and correlate them with the incorporation of actin subunits into filaments. Once the imaging system for formin-mediated actin assembly is established, the ultimate goal is to investigate, whether and how formins coordinate tropomyosin recruitment to actin filaments.