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The dynamics of focal adhesions, cell-surface receptors, endocytosis, cell membrane dynamics, and the binding or secretion of neurotransmitters, and many other molecular events on cell surfaces occur at a resolution below the diffraction limit of light microscopy. The development of total internal fluorescence (TIRF) microscopy permits the visualization of biological events at the cell surface at the single-molecule level, with virtually eliminating out-of-focus background noise. The combination of TIRF microscopy and genetically encoded affinity tags have also been applied with great success in cytoskeletal in vitro reconstitution systems and dramatically improved our understanding of cytoskeletal dynamics including the timescale and components required for actin and microtubule assembly, motor protein complex movement and components – basically visualizing 'biochemistry on a coverslip'. While, in vitro reconstitution systems do not contain the hundreds of factors that regulate the actin and microtubule cytoskeletons in cells, such systems can reveal the dynamic interactions between cytoskeletal binding proteins at high spatial resolution and at very rapid temporal scales. Using a new in vitro reconstitution system that permits the simultaneous monitoring of the dynamic properties of both actin and microtubules we described the first molecular mechanism to provide a direct explanation for how microtubule plus-ends direct cellular actin assembly, where three interacting proteins (End Binding-1, Cytoplasmic Linker Protein-170, and Formin) work in concert to promote the nucleation and elongation of actin filaments from the surface of growing microtubule ends. We used this system to explore additional novel actin-microtubule crosstalk mechanisms.

Research Goal

To use a minimal component single-molecule TIRF microscopy system to elucidate actin-microtubule crosstalk mechanisms that can only be fully understood by studying both cytoskeletons simultaneously.

TIRF Microscopy of the Cytoskeleton

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