Endocytic pathways: combined scanning ion conductance and surface confocal microscopy study

We introduce a novel high resolution scanning surface confocal microscopy technique that enables imaging of endocytic pits in apical membranes of live cells for the first time. The improved topographical resolution of the microscope together with simultaneous fluorescence confocal detection produces pairs of images of cell surfaces sufficient to identify single endocytic pits. Whilst the precise position and size of the pit is detected by the ion conductance microscope, the molecular nature of the pit, e.g. clathrin coated or caveolae, is determined by the corresponding green fluorescent protein fluorescence. Also, for the first time, we showed that flotillin 1 and 2 can be found co-localising with ~200-nm indentations in the cell membrane that supports involvement of this protein in endocytosis

Imaging Single Virus Particles on the Surface of Cell Membranes by High-Resolution Scanning Surface Confocal Microscopy

We have developed a high-resolution scanning surface confocal microscopy technique capable of imaging single virus-like particles (VLPs) on the surfaces of cells topographically and by fluorescence. The technique combines recently published single-molecule-resolution ion-conductance microscopy that acquires topographical data with confocal microscopy providing simultaneous fluorescent imaging. In our experiments we have demonstrated that the cell membrane exhibits numerous submicrometer-sized surface structures that could be topographically confused with virus particles. However, simultaneous acquisition of confocal images allows the positions of fluorescently tagged particles to be identified. Using this technique, we have, for the first time, visualized single polyoma VLPs adsorbed onto the cell membrane. Observed VLPs had a mean width of 108 ± 16 nm. The particles were randomly distributed across the cell membrane, and no specific interactions were seen with cell membrane structures such as microvilli. These experiments demonstrate the utility of this new microscope for imaging the interactions of nanoparticles with the cell surface to provide novel insights into the earliest interactions of viruses and other nanoparticles such as gene therapy vectors with the cell

Role for Centromeric Heterochromatin and PML Nuclear Bodies in the Cellular Response to Foreign DNA

Nuclear spatial positioning plays an important role in the epigenetic regulation of eukaryotic gene expression. Here we show a role for nuclear spatial positioning in regulating episomal transgenes that are delivered by virus-like particles (VLPs). VLPs mediate the delivery of plasmid DNA (pDNA) to cell nuclei but lack viral factors involved in initiating and regulating transcription. By tracking single fluorescently labeled VLPs, coupled with luciferase reporter gene assays, we found that VLPs transported pDNA to cell nuclei efficiently but transgenes were immediately silenced by the cell. An investigation of the nuclear location of fluorescent VLPs revealed that the pDNAs were positioned next to centromeric heterochromatin. The activation of transcription by providing viral factors or inhibiting histone deacetylase activity resulted in the localization to euchromatin regions. Further, the activation of transcription induced the recruitment of PML nuclear bodies (PML-NBs) to the VLPs. This association did not play a role in regulating transgene expression, but PML protein was necessary for the inhibition of transgene expression with alpha interferon (IFN-α). These results support a model whereby cells can prevent foreign gene expression at two levels: by positioning transgenes next to centromeric heterochromatin or, if that is overcome, via the type I IFN response facilitated by PML-NB recruitment