Manuel Gunkel

Functional Nuclear Organization of Transcription and DNA Replication A Topographical Marriage between Chromatin Domains and the Interchromatin Compartment

We studied the nuclear topography of RNA transcription and DNA replication in mammalian cell types with super-resolution fluorescence microscopy, which offers a resolution beyond the classical Abbe/Raleigh limit. Three-dimensional structured illumination microscopy (3D-SIM) demonstrated a network of channels and wider lacunas, called the interchromatin compartment (IC). The IC starts at nuclear pores and expands throughout the nuclear space. It is demarcated from the compact interior of higher-order chromatin domains (CDs) by a 100-200-nm thick layer of decondensed chromatin, termed the perichromatin region (PR). Nascent DNA, nascent RNA, RNA polymerase II (RNA Pol II), as well as histone modifications for transcriptionally competent/active chromatin, are highly enriched in the PR, whereas splicing speckles are observed in the interior of the IC. In line with previous electron microscopic evidence, spectral precision distance/position determination microscopy (SPDM) confirmed the presence of RNA Pol II clusters indicative of transcription factories. Still, a substantial part of transcription apparently takes place outside of such factories. Previous electron microscopic evidence has suggested that the functional nuclear organization of DNA replication depends on brownian movements of chromatin between the CD interior and the PR. As an incentive for future studies, we hypothesize that such movements also take place during transcription, i.e., only the actually transcribed part of a gene may be located within the PR, whereas its major part, including previously or later transcribed sequences, is embedded in a higher-order chromatin configuration in the interior of the CD.

Dual color localization microscopy of cellular nanostructures

The dual color localization microscopy (2CLM) presented here is based on the principles of spectral precision distance microscopy (SPDM) with conventional autofluorescent proteins under special physical conditions. This technique allows us to measure the spatial distribution of single fluorescently labeled molecules in entire cells with an effective optical resolution comparable to macromolecular dimensions. Here, we describe the application of the 2CLM approach to the simultaneous nanoimaging of cellular structures using two fluorochrome types distinguished by different fluorescence emission wavelengths. The capabilities of 2CLM for studying the spatial organization of the genome in the mammalian cell nucleus are demonstrated for the relative distributions of two chromosomal proteins labeled with autofluorescent GFP and mRFP1 domains. The 2CLM images revealed quantitative information on their spatial relationships down to length‐scales of 30 nm.

Using conventional fluorescent markers for far-field fluorescence localization nanoscopy allows resolution in the 10-nm range

We present a novel technique of far‐field localization nanoscopy combining spectral precision distance microscopy with widely used fluorochromes like the Green Fluorescent Protein (GFP) derivatives eGFP, EmGFP, Yellow Fluorescent Protein (YFP) and eYFP, synthetic dyes like Alexa 488 and Alexa 568, as well as fluoresceine derivates. Spectral precision distance microscopy allows the surpassing of conventional resolution limits in fluorescence far‐field microscopy by precise object localization after the optical isolation of single signals in time. Based on the principles of this technique, our novel nanoscopic method was realized for laser optical precision localization and image reconstruction with highly enhanced optical resolution in intact cells. This allows for spatial assignment of individual fluorescent molecules with nanometre precision. The technique is based on excitation intensity dependent reversible photobleaching of the molecules used combined with fast time sequential imaging under appropriate focusing conditions. A meaningful advantage of the technique is the simple applicability as a universal tool for imaging and investigations to the major part of already available preparations according to standard protocols. Using the above mentioned fluorophores, the positions of single molecules within cellular structures were determined by visible light with an estimated localization precision down to 3 nm; hence distances in the range of 10–30 nm were resolved between individual fluorescent molecules allowing to apply different quantitative structure analysis tools.

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.

Localization Microscopy Reveals Expression-Dependent Parameters of Chromatin Nanostructure

A combined approach of 2D high-resolution localization light microscopy and statistical methods is presented to infer structural features and density fluctuations at the nuclear nanoscale. Hallmarks of nuclear nanostructure are found on the scale below 100 nm for both human fibroblast and HeLa cells. Mechanical measures were extracted as a quantitative tool from the histone density fluctuations inside the cell to obtain structural fluctuations on the scale of several micrometers. Results show that different mechanisms of expression of the same nuclear protein type lead to significantly different patterns on the nanoscale and to pronounced differences in the detected compressibility of chromatin. The observed fluctuations, including the experimental evidence for dynamic looping, are consistent with a recently proposed chromatin model.

Superresolution imaging of biological nanostructures by spectral precision distance microscopy

For the improved understanding of biological systems on the nanoscale, it is necessary to enhance the resolution of light microscopy in the visible wavelength range beyond the limits of conventional epifluorescence microscopy (optical resolution of about 200 nm laterally, 600 nm axially). Recently, various far‐field methods have been developed allowing a substantial increase of resolution (“superresolution microscopy”, or “lightoptical nanoscopy”). This opens an avenue to ‘nano‐image’ intact and even living cells, as well as other biostructures like viruses, down to the molecular detail. Thus, it is possible to combine light optical spatial nanoscale information with ultrastructure analyses and the molecular interaction information provided by molecular cell biology. In this review, we describe the principles of spectrally assigned localization microscopy (SALM) of biological nanostructures, focusing on a special SALM approach, spectral precision distance/position determination microscopy (SPDM) with physically modified fluorochromes (SPDMPhymod. Generally, this SPDM method is based on high‐precision localization of fluorescent molecules, which can be discriminated using reversibly bleached states of the fluorophores for their optical isolation. A variety of application examples is presented, ranging from superresolution microscopy of membrane and cytoplasmic protein distribution to dual‐color SPDM of nuclear proteins. At present, we can achieve an optical resolution of cellular structures down to the 20‐nm range, with best values around 5 nm (∼1/100 of the exciting wavelength).

SPDM: single molecule superresolution of cellular nanostructures

Novel methods of visible light microscopy have overcome the limits of resolution hitherto thought to be insurmountable. The localization microscopy technique presented here based on the principles of Spectral Precision Distance Microscopy (SPDM) with conventional fluorophores under special physical conditions allows to measure the spatial distribution of single fluorescence labeled molecules in entire cells with macromolecular precision which is comparable to a macromolecular effective optical resolution. Based on detection of single molecules, in a novel combination of SPDM and Spatially Modulated Illumination (SMI) microscopy, a lateral (2D) effective optical resolution of cellular nanostructures around 10 - 20 nm (about 1/50th of the exciting wavelength) and a three dimensional (3D) effective optical resolution in the range of 40 - 50 nm are achieved.

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.

The long non-coding RNA LINC00152 is essential for cell cycle progression through mitosis in HeLa cells

In recent years, long non-coding RNA (lncRNA) research has identified essential roles of these transcripts in virtually all physiological cellular processes including tumorigenesis, but their functions and molecular mechanisms are poorly understood. In this study, we performed a high-throughput siRNA screen targeting 638 lncRNAs deregulated in cancer entities to analyse their impact on cell division by using time-lapse microscopy. We identified 26 lncRNAs affecting cell morphology and cell cycle including LINC00152. This transcript was ubiquitously expressed in many human cell lines and its RNA levels were significantly upregulated in lung, liver and breast cancer tissues. A comprehensive sequence analysis of LINC00152 revealed a highly similar paralog annotated as MIR4435-2HG and several splice variants of both transcripts. The shortest and most abundant isoform preferentially localized to the cytoplasm. Cells depleted of LINC00152 arrested in prometaphase of mitosis and showed reduced cell viability. In RNA affinity purification (RAP) studies, LINC00152 interacted with a network of proteins that were associated with M phase of the cell cycle. In summary, we provide new insights into the properties and biological function of LINC00152 suggesting that this transcript is crucial for cell cycle progression through mitosis and thus, could act as a non-coding oncogene.