Jürgen Reymann

High-precision structural analysis of subnuclear complexes in fixed and live cells via spatially modulated illumination (SMI) microscopy

Spatially modulated illumination (SMI) microscopy is a method of wide field fluorescence microscopy featuring interferometric illumination, which delivers structural information about nanoscale architecture in fluorescently labelled cells. The first prototype of the SMI microscope proved its applicability to a wide range of biological questions. For the SMI live cell imaging this system was enhanced in terms of the development of a completely new upright configuration. This so called Vertico-SMI transfers the advantages of SMI nanoscaling to vital biological systems, and is shown to work consistently at different temperatures using both oil- and water-immersion objective lenses. Furthermore, we increased the speed of data acquisition to minimize errors in the detection signal resulting from cellular or object movement. By performing accurate characterization, the present Vertico-SMI now offers a fully-fledged microscope enabling a complete three-dimensional (3D) SMI data stack to be acquired in less than 2 seconds. We have performed live cell measurements of a tet-operator repeat insert in U2OS cells, which provided the first in vivo signatures of subnuclear complexes. Furthermore, we have successfully implemented an optional optical configuration allowing the generation of high-resolution localization microscopy images of a nuclear pore complex distribution.

The microtubule affinity regulating kinase MARK4 promotes axoneme extension during early ciliogenesis

A functional screen identified MARK4 as a positive regulator of axonemal extension and ciliogenesis via its interaction with the mother centriolar protein ODF2.

Hypothalamic miR-103 Protects from Hyperphagic Obesity in Mice

The role of neuronal noncoding RNAs in energy control of the body is not fully understood. The arcuate nucleus (ARC) of the hypothalamus comprises neurons regulating food intake and body weight. Here we show that Dicer-dependent loss of microRNAs in these neurons of adult (DicerCKO) mice causes chronic overactivation of the signaling pathways involving phosphatidylinositol-3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) and an imbalance in the levels of neuropeptides, resulting in severe hyperphagic obesity. Similarly, the activation of PI3K-Akt-mTOR pathway due to Pten deletion in the adult forebrain leads to comparable weight increase. Conversely, the mTORC1 inhibitor rapamycin normalizes obesity in mice with an inactivated Dicer1 or Pten gene. Importantly, the continuous delivery of oligonucleotides mimicking microRNAs, which are predicted to target PI3K-Akt-mTOR pathway components, to the hypothalamus attenuates adiposity in DicerCKO mice. Furthermore, loss of miR-103 causes strong upregulation of the PI3K-Akt-mTOR pathway in vitro and its application into the ARC of the Dicer-deficient mice both reverses upregulation of Pik3cg, the mRNA encoding the catalytic subunit p110γ of the PI3K complex, and attenuates the hyperphagic obesity. Our data demonstrate in vivo the crucial role of neuronal microRNAs in the control of energy homeostasis.

From experimental setup to bioinformatics: An RNAi screening platform to identify host factors involved in HIV-1 replication

RNA interference (RNAi) has emerged as a powerful technique for studying loss‐of‐function phenotypes by specific down‐regulation of gene expression, allowing the investigation of virus‐host interactions by large‐scale high‐throughput RNAi screens. Here we present a robust and sensitive small interfering RNA screening platform consisting of an experimental setup, single‐cell image and statistical analysis as well as bioinformatics. The workflow has been established to elucidate host gene functions exploited by viruses, monitoring both suppression and enhancement of viral replication simultaneously by fluorescence microscopy. The platform comprises a two‐stage procedure in which potential host factors are first identified in a primary screen and afterwards re‐tested in a validation screen to confirm true positive hits. Subsequent bioinformatics allows the identification of cellular genes participating in metabolic pathways and cellular networks utilised by viruses for efficient infection. Our workflow has been used to investigate host factor usage by the human immunodeficiency virus‐1 (HIV‐1), but can also be adapted to other viruses. Importantly, we expect that the description of the platform will guide further screening approaches for virus‐host interactions. The ViroQuant‐CellNetworks RNAi Screening core facility is an integral part of the recently founded BioQuant centre for systems biology at the University of Heidelberg and will provide service to external users in the near future.

Correlative light microscopy for high-content screening.

High-throughput microscopy is an effective tool for rapidly collecting data on a large scale. However, high throughput comes at the cost of low spatial resolution. Here we introduce correlative light microscopy by combining fast automated widefield imaging, confocal microscopy and super-resolution microscopy. We demonstrate the potential of this approach for scalable experiments. The workflow consists of a robust approach for selecting cells of interest on a wide-field screening microscope at low resolution and subsequently re-localizing those cells with micrometer precision for confocal and super-resolution imaging. As a case study, we visualized and quantified cis- and trans-Golgi markers at increasing resolution.

Integrated and correlative high-throughput and super-resolution microscopy

AbstractWe have developed a method to perform microscopic temporal and spacial multi-scale experiments by imaging cellular phenotypes of interest on complementary fluorescence microscopy systems. In a low-resolution fast data acquisition screen for phenotypic cellular responses induced by small interfering RNA (siRNA), cells in spots of siRNA cell arrays showing characteristic alterations have been selected automatically by feature space analysis. These objects were imaged on a second super-resolution dSTORM microscope (direct stochastic optical reconstruction microscopy). The coordinate transfer was based on fixed cells as reference points without the use of additional fiducial markers. This procedure is suitable to combine any kind of fluorescence microscopy technique, in order to gain further insights on the observed specimen at multiple temporal or special scales.

A three-dimensional colocalization RNA interference screening platform to elucidate the alternative lengthening of telomeres pathway

A high‐content colocalization RNA interference screen based on automatic three‐color confocal fluorescence microscopy was developed to analyze the alternative lengthening of telomeres (ALT) pathway. Via this pathway telomerase‐negative cancer cells can maintain their telomeres and with it their unlimited proliferative potential. A hallmark of ALT cells is the colocalization of promyelocytic leukemia (PML) nuclear bodies with telomeres to form ALT‐associated PML nuclear bodies (APBs). In our screen, the presence of APBs was used as a marker to identify proteins required for the ALT mechanism. A cell‐based assay and an automatic confocal image acquisition procedure were established. Using automatic image analysis based on 3D parametric intensity models to identify APBs, we conducted an unbiased and quantitative analysis of nine different candidate genes. A comparison with the literature and manual analysis of the gene knockdown demonstrates the reliability of our approach. It extends the available repertoire of high‐content screening to studies of cellular colocalizations and allows the identification of candidate genes for the ALT mechanism that represent possible targets for cancer therapy.

Next-generation 9216-microwell cell arrays for high-content screening microscopy

Reverse transfection on cell arrays is a high-throughput method for the parallel transfection of mammalian cells for use in high-content screening light microscopy. Here, we present novel 9216-microwell cell arrays which combine the advantages of multiwell plates (physically separated samples) and cell microarrays (high sample density and long-term storage).

The catalytically inactive tyrosine phosphatase HD-PTP/PTPN23 is a novel regulator of SMN complex localization.

This first systematic and comprehensive screen of human phosphatases for a regulatory role in the survival motor neuron (SMN) complex identifies the catalytically inactive, non–receptor-type tyrosine phosphatase PTPN23/HD-PTP as a novel SMN complex regulator. PTPN23 maintains a highly phosphorylated state of SMN, which is important for its function in snRNP assembly.

Cell Arrays and High-Content Screening

Endocytosis is one of the most essential cellular processes, which enables cells to internalise diverse -material. It is crucial for regulation of receptor activity and signalling, cell polarisation, attachment and motility, and a great number of other cellular functions. A number of diverse endocytosis pathways are described by now; however, their specificity for different cellular cargoes is poorly resolved. Only few of endocytosis regulators are well-characterised and even less are attributed to the specific cargo. That is very true for the integrin endocytosis pathway, which is a key process in cell migration, adhesion, and signalling. The recent advent of quantitative fluorescent microscopy and cell arrays opened an exciting possibility to systematically characterise molecules playing a role in this crucially important process. Here, we describe a fluorescent screening microscopy-based assay to identify regulators of integrin α2 internalisation. The experimental procedure is the best suited for a highly parallel screening format, such as cell arrays, albeit can be used in single experiments. We provide protocols for sample preparation, fabrication of cell arrays and quantification of integrin α2 internalisation. The approach can be modified to quantify endocytosis of other cargo, and can be used under the conditions of knock-down and knock-in as well as for chemical screening.