We are interested in the structure and function of the contractile apparatus of muscle; the myofibril, the structure and functions of the myosin superfamily, the molecular motors kinesin and dynein, and microtubule associated proteins. We use a wide range of techniques (wet biochemistry (protein isolation, expression and characterisation), biophysical methods (eg analytical ultracentrifugation, atomic force microscopy), light microscopy (confocal, time-lapse fluorescence, deconvolution and super-resolution microscopies) electron microscopy, including time-resolved cryo-EM, molecular genetic tools and cell biology (tissue culture, live cell imaging) to investigate the structure and function of the muscle proteins myosin, titin and actin.
We also investigate cell behaviour, and the dynamic behaviour of molecular motors within cells, using time-lapse phase and fluorescence microscopy, our new instant structured illumination microscope, and dSTORM and PALM. Our research forms part of the major effort in structural biology at Leeds.
New PhD Opportunity in dSTORM and PALM super-resolution microscopy: Applying Machine Learning to 3D single molecule localisation analysis in super-resolution microscopy https://www.findaphd.com/search/ProjectDetails.aspx?PJID=97128 Supervisors: Prof Michelle Peckham (Leeds), Dr Joanna Leng (Leeds). Closing Date is 14 May!
We have begun using Affimers - small non-antibody binding proteins, in 'super-resolution microscopy'. Their small size (~2nm - 10 fold smaller than antibodies) places an unique dye molecule close to the target of interest, resulting in a smaller linkage error (see green plot, in panel D), and enables them to penetrate dense cytoskeleton to label more efficiently than antibodies (see magenta staining - for Affimer in cytokinetic furrow) (for more see Tiede et al., ELife, 2017).)
As part of a group working on the primary cilium, we have used dSTORM (direct Stochastic Optical Reconstruction Microscopy) on a home-built 3D set up at Leeds to image proteins in the transition zone. This example shows the protein RPGRIP1L. For more information see our recent paper Lambacher et al., (2016) TMEM107 recruits ciliopathy proteins to subdomains of the ciliary transition zone and causes Joubert syndrome. Nature Cell Biology, 18 p122-131 link to article
Myosin 10 is required to form filopodia, thin actin rich protrusions and it accumulates at the tip of these filopodia. It uses a combination of diffusion and active translocation to find its way to the right place in the cell. Tracking fluorescent myosin 10 molecules, and plotting their positions shows the tracks that this myosin uses in getting to the tip (as shown above). This image is taken from our recent paper in Journal of Biological Chemistry (Baboolal et al., (2016) J. Biol. Chem. 291, 22373-22385 link to article, and is a collaboration with Justin Molloy at the Crick Institute.
watch this cool animation of myosin 10 moving to the tip, as an animated kymograph.
Our recent paper in Cell Reports (Makowska et al.,(2015) Cell Reports. 13, 2118-2125: link to article) shows that some myosins (myo1b, Myo9b, Myo10 and Myo18a) are overexpressed in metastatic prostate cancer. Specifically knocking down expression of each of these myosins result in the re-organisation of the actin cytoskeleton, and the type of reorganisation seen depends on which myosin has been depleted. Importantly, mis-regulation of myosin expression in these cells, helps to create the metastatic phenotype. (See also Michelle's recent review in Biochem. Soc. Trans (2016) on myosins in cancer http://www.biochemsoctrans.org/content/44/4/1026.long)