Optical superresolution microscopy of molecular mechanisms of disease

The self-assembly of proteins into ordered macromolecular units is fundamental to a variety of diseases. For example, in Alzheimer’s Disease (AD) and Parkinson’s Disease (PD), proteins that are usually harmless are found to adopt aberrant shapes; one says they ‘misfold’.  In the misfolded state the proteins are prone to aggregate into highly ordered, toxic structures, called protein amyloids and these make up the insoluble deposits found in the brains of patients suffering from these devastating disorders.  Virus particle assembly is another example of a protein self-assembly process associated with disease.  A key requirement to gain insights into molecular mechanisms of disease and to progress in the search for therapeutic intervention is a capability to image the protein assembly process in situ i.e. in cellular models of disease. 
In this talk I will give an overview of research to gain insight on the aggregation state neurotoxic proteins in vitro (1), in cells (2, 3) and in live model organisms (4). In particular we wish to understand how these and similar proteins nucleate to form toxic structures and to correlate such information with phenotypes of disease (3). I will show how direct stochastic optical reconstruction microscopy, dSTORM, and multiparametric imaging techniques, such as spectral and lifetime imaging, are capable of tracking amyloidogenesis in vitro, and in vivo, and how we can correlate the appearance of certain aggregate species with toxic phenotypes of relevance to PD and AD (5).
Finally I will show how superresolution gives insights into the self-assembly process of viral particles at a resolution that rivals electron microscopy techniques (6).

(1) Pinotsi D, Büll AK, Galvagnion C, Dobson CM, Kaminski-Schierle GS, Kaminski CF, "Direct Observation of Heterogeneous Amyloid Fibril Growth Kinetics via Two-Color Super-Resolution Microscopy," Nano Letters (2013), 14 (1), 339–345
(2) Kaminski Schierle GS, van de Linde S, Erdelyi M, Esbjörner EK, Klein T, Rees E, Bertoncini CW, Dobson CM, Sauer M, and Kaminski CF, "In Situ Measurements of the Formation and Morphology of Intracellular ß-Amyloid Fibrils by Super-Resolution Fluorescence Imaging", J. Am. Chem. Soc., 133 (33), pp 12902–12905 (2011)
(3) Esbjörner, E.K., Chan, F., Rees, E., Erdelyi, M., Luheshi, L.M., Bertoncini, C.W., Kaminski, C.F., Dobson, C.M., and Kaminski Schierle, G.S., “Direct Observations of Amyloid β Self-Assembly in Live Cells Provide Insights into Differences in the Kinetics of Aβ(1–40) and Aβ(1–42) Aggregation,” Chemistry & Biology (2014).
(4) Kaminski Schierle GS, Bertoncini CW, Chan FTS, van der Goot AT, Schwedler S, Skepper J, Schlachter S, van Ham T, Esposito A, Kumita JR, Nollen EAA, Dobson CM, Kaminski CF, "A FRET sensor for non-invasive imaging of amyloid formation in vivo", ChemPhysChem, 12(3), 673–680, (2011)
(5) Michel CH, Kumar S, Pinotsi D, Tunnacliffe A, St George-Hyslop P, Mandelkow E, Mandelkow E-M, Kaminski CF, Kaminski Schierle GS, "Extracellular Monomeric Tau is Sufficient to Initiate the Spread of Tau Pathology", J. Biol. Chem. (2014), 289: 956-967.
(6) Pinotsi D, Michel CH, Buell AK, Laine RF, Mahou P, Dobson CM, Kaminski CF, Kaminski Schierle GS, "Nanoscopic insights into seeding mechanisms and toxicity of α-synuclein species in neurons" PNAS (2016), 3815–3819, doi: 10.1073/pnas.1516546113
(7) Laine R F. Albecka A, van de Linde S, Rees E J, Crump C M, Kaminski CF, "Structural analysis of herpes simplex virus by optical super-resolution imaging." Nature Communications (2015), 6:5980

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Dr Nick Ktistakis
The Brian Heap Seminar Room