Browsing by Subject "Live cell imaging"
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Item Open Access Cells infected with human papilloma pseudovirus display nuclear reorganization and heterogenous infection kinetics(John Wiley & Sons, Ltd, 2022-06-06) Molenberghs, F.; Verschuuren, M.; Bogers, J. J.; Cools, N.; Delputte, P.; Schelhaas, M.; Barbier, Michaël; De Vos, W.Human papillomaviruses (HPV) are small, non-enveloped DNA viruses, which upon chronic infection can provoke cervical and head-and-neck cancers. Although the infectious life cycle of HPV has been studied and a vaccine is available for the most prevalent cancer-causing HPV types, there are no antiviral agents to treat infected patients. Hence, there is a need for novel therapeutic entry points and a means to identify them. In this work, we have used high-content microscopy to quantitatively investigate the early phase of HPV infection. Human cervical cancer cells and immortalized keratinocytes were exposed to pseudoviruses (PsV) of the widespread HPV type 16, in which the viral genome was replaced by a pseudogenome encoding a fluorescent reporter protein. Using the fluorescent signal as readout, we measured differences in infection between cell lines, which directly correlated with host cell proliferation rate. Parallel multiparametric analysis of nuclear organization revealed that HPV PsV infection alters nuclear organization and inflates promyelocytic leukemia protein body content, positioning these events at the early stage of HPV infection, upstream of viral replication. Time-resolved analysis revealed a marked heterogeneity in infection kinetics even between two daughter cells, which we attribute to differences in viral load. Consistent with the requirement for mitotic nuclear envelope breakdown, pharmacological inhibition of the cell cycle dramatically blunted infection efficiency. Thus, by systematic image-based single cell analysis, we revealed phenotypic alterations that accompany HPV PsV infection in individual cells, and which may be relevant for therapeutic drug screens.Item Open Access Reaction-based BODIPY probes for selective bio-imaging(Elsevier B.V., 2018) Kolemen, S.; Akkaya, E. U.Complex intracellular environment of cells, which involves interaction of a large variety of bio-molecules, is a dynamic medium with full of important information that can be recovered as well as many unanswered questions. It is highly critical to image and track biologically relevant molecules in their native media without interfering with the regular cellular processes in order to gather as much data as possible to illuminate intricacies of the biological mechanisms. To that end, small-molecule fluorescent probes have been extensively developed during the last few decades with the help of current advances in imaging technologies. Although conventional probes utilizing non-covalent supramolecular interactions with the analyte of interest are successful, significant effort has been also put into the design of reaction-based probes (chemodosimeters). Chemodosimeters exploit selective reactions of analytes with fluorophores in attempt to improve the selectivity of the probes, address the limitations of former sensors and broaden the palette of useful probes. Various types of fluorophore scaffolds can be used in the design of chemodosimeters for visualization of different analytes. In this review, we highlight the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) based chemodosimeters which have been used to image bio-thiols, reactive oxygen/nitrogen species, and gaseous molecules in living cells.