Charcot-Marie-Tooth and Centronuclear myopathy induced mechanistic impairment in endocytosis Tayyibah Ali1, Andrew Shevchuk1 1Nanomedicine Laboratory, Department of Medicine, Imperial college London, Hammersmith Hospital Introduction Aims 0.1 mm 1.5 mm F-F0 COS-7 with R465W-DNM2-GFP (246.5 sec/frame) DNM2-R465W- GFP Topography 3697 sec B. Clc-GFP A. Human skin fibroblast with R465W-DNM2 (17.36 second/frame) 1302 sec - indicates pit flattening Dynamin 2 (DNM2) is involved in the scission of nascent vesicles during endocytosis and is also implicated in a wide range of cellular processes (1). Mutations in the DNM2 gene have been associated with Charcot-Marie-Tooth (CMT) and Centronuclear Myopathy (CNM) in a tissue specific manner. To determine the role of DNM2 mutations in endocytic vesicle formation and internalisation. Scanning Ion Conductance Microscopy / Figure 3: Principle of SICM. As the pipette reaches the surface of the cell the ion current drops which is detected via the feedback loop, recording the position and moving to the next point thereby building a topographical view of the cell surface. The laser beam can be aligned with the pipette to produce confocal images simultaneously. Figure 1: Diagram depicting the mutations studied. R465W and R522H are both missense mutations in the Dynamin 2 gene (2). Figure 5: CNM patient skin fibroblasts with R465W-DNM2 mutation and modelling of R465W-DNM2 in COS-7 show increase in pit life time compared with control (Shevchuk et al. unpublished) Current research has characterised the biochemical properties of DNM2 mutant however none have yet related these properties to functioning in the cell (3). Figure 6: Mean lifetime of clathrin mediated endocytotic events observed with SICM. Bars represents standard error of the mean. Figure 2: Model of action for dynamin at the neck of clathrin-coated pits (CCP) (4). The development of correlative Scanning Ion Conductance Microscopy (SICM) combined with laser confocal microscopy have allowed live cell topographical imaging directly correlated with fluorescently labelled cargo/proteins (5). We applied this technique to characterise the effect of DNM2 mutations on clathrin mediated endocytosis. Conclusion Results The Cos-7 model permits monitoring of mutant dynamin recruitment due to fluorescent labelling, however this model has potential difficulties related to over-expression and cytotoxicity of dynamin, whilst human skin fibroblasts allow investigation of how endogenous levels of mutant dynamin affect CCP kinetics. Both R465W and 522H mutations in either model adversely affect the distribution of CCPs in fibroblasts. The current investigation does not address cargo uptake, therefore future work will focus on weather cargo uptake is affected by the mutations in DNM2. 0.1 µm F-F0 Wt DNM2 Human skin fibroblasts (17 sec/frame) CLC-GFP Topography DNM2-WT-GFP 0.2 mm 1.5 mm 102 sec Wt DNM2 COS-7 (11.3 sec/frame) 357 seconds A. B. Materials and Methods Human skin fibroblasts from healthy subjects and from CNM patients with DNM2 mutations were kindly gifted by Dr Marc Bitoun (University Pierre et Marie Curie, France). CNM patients have DNM2 mutations; R645W or R522H. Cells were cultured in Dulbecco's Modified Eagle Medium. We applied correlative scanning ion conductance microscopy (SICM) and fluorescence confocal (FC) microscopy to characterise the temporal and spatial recruitment of mutant dynamins and their effect on the formation and internalisation of individual endocytic pits. - indicate pit closure Figure 4: SCIM and confocal localisation of transfected human skin fibroblasts with Clathrin light chain (CLC)-GFP and COS-7 cell line with wild type DNM2-GFP (Shevchuk et al. unpublished). References G. Praefcke, H. McMahon, The dynamin superfamily: universal membrane tubulation and fission molecules? Nat. Rev. Mol. Cell. Biol. 5, 133-147(2004). A. M. González-Jamett et al., Dynamin-2 function and dysfunction along the secretory pathway. Front. Endocrinol. 4, 1-9 (2013). A. Durieux, B. Prudhon, P. Guicheney, M. Bitoun, Dynamin 2 and Human diseases. J. Mol. Med. 88, 339-350 (2010). K. Faelber et al., Structural insights into dynamin-mediated membrane fission. Structure. 20, 1621-1628 (2012). A. I. Shevchuk et al., An alternative mechanism of clathrin-coated pit closure revealed by ion conductance microscopy. JCB. 197, 499-508 (2012).