Using Fluorescence Recovery After Photobleaching (FRAP) to study dynamics of the Structural Maintenance of Chromosome (SMC) complex in vivo

The SMC complex, MukBEF, is important for chromosome organization and segregation in Escherichia coli. Fluorescently tagged MukBEF forms distinct spots (or 'foci') in the cell, where it is thought to carry out most of its chromosome associated activities. This chapter outlines the technique of Fluorescence Recovery After Photobleaching (FRAP) as a method to study the properties of YFP-tagged MukB in fluorescent foci. This method can provide important insight into the dynamics of MukB on DNA and be used to study its biochemical properties in vivo

DNA motor-protein hybrids for molecular transport and self-organisation

Kinesin is a molecular motor which walks on microtubule tracks in the eukaryotic cytoskeleton. It transports cargo but is also involved in cytoskeletal organisation. This thesis demonstrates fusing kinesin and DNA to construct a molecular transport system using self-organised tracks and to study the mechanics of the minimal motor unit of kinesin. The programmability of DNA allows for the formation of nanostructures with controllable interactions. Kinesin is conjugated to various DNA nanostructures to accomplish different tasks. Instructions encoded into DNA sequences are used to direct the assembly of a polar array of microtubules, to control the loading, active concentration and unloading of cargo on this track network and to trigger the disassembly of the network. Fluorescence microscopy was used to observe these microtubule arrays and the movement of cargo. It was found that the DNA signals used to control the unloading of cargo and the disassembly of the network had to be actively transported, rather than relying on diffusion, for effective delivery of the signal. This work lead to a first author publication, Wollman et al. (2013). DNA was also used to study kinesin by linking defined numbers of minimal functional motor units, single kinesin heads, into teams of 4-12 heads and observing their movement along microtubules via fluorescent labelling. A minimum of 5 heads were required for sustained movement, in agreement with the predictions of Hancock and Howard (1998). The velocity of teams increased with more heads, up to 8, and then a decrease was observed in teams with more heads.</p