In situ Nuclear Matrix preparation in Drosophila melanogaster and its use in studying the components of nuclear architecture

The study of Nuclear Matrix (NuMat) over the last 40 years has been limited to either isolated nuclei from tissues or cells grown in culture. Here, we provide a protocol for NuMat preparation in intact Drosophila melanogaster embryos and its use in dissecting the components of nuclear architecture. The protocol does not require isolation of nuclei and therefore maintains the three-dimensional milieu of an intact embryo, which is biologically more relevant compared to cells in culture. One of the advantages of this protocol is that only a small number of embryos are required. The protocol can be extended to larval tissues like salivary glands and imaginal discs with little modification. Taken together, it becomes possible to carry out such studies in parallel to genetic experiments using mutant and transgenic flies. This protocol, therefore, opens the powerful field of fly genetics to cell biology in the study of nuclear architecture.

Single nucleus RNA-sequencing: how it's done, applications and limitations

Single nuclei RNA-sequencing (sNuc-Seq) is a methodology which uses isolated nuclei instead of whole cells to profile gene expression. By using droplet microfluidic technologies, users are able to profile thousands of single transcriptomes at high throughput from their chosen tissue. This article aims to introduce sNuc-Seq as a method and its utility in multiple tissue types. Furthermore, we discuss the risks associated with the use of nuclei, which must be considered before committing to a methodology.

Biochemically distinct cohesin complexes mediate positioned loops between CTCF sites and dynamic loops within chromatin domains

The ring-like cohesin complex mediates sister chromatid cohesion by encircling pairs of sister chromatids. Cohesin also extrudes loops along chromatids. Whether the two activities involve similar mechanisms of DNA engagement is not known. We implemented an experimental approach based on isolated nuclei carrying engineered cleavable RAD21 proteins to precisely control cohesin ring integrity so that its role in chromatin looping could be studied under defined experimental conditions. This approach allowed us to identify cohesin complexes with distinct biochemical, and possibly structural properties, that mediate different sets of chromatin loops. When RAD21 is cleaved and the cohesin ring is opened, cohesin complexes at CTCF sites are released from DNA and loops at these elements are lost. In contrast, cohesin-dependent loops within chromatin domains and that are not anchored at CTCF sites are more resistant to RAD21 cleavage. The results show that the cohesin complex mediates loops in different ways depending on genomic context and suggests that it undergoes structural changes as it dynamically extrudes and encounters CTCF sites.

Analysis of Ultrasound Backscatter from Ensembles of Cells and Isolated Nuclei

Analysis of Ultrasound Backscatter from Ensembles of Cells and Isolated Nuclei

Analysis of Ultrasound Backscatter from Ensembles of Cells and Isolated Nuclei

Analysis of Ultrasound Backscatter from Ensembles of Cells and Isolated Nuclei

An optimised method for intact nuclei isolation from diatoms

Due to their abundance in the oceans, their extraordinary biodiversity and the increasing use for biotech applications, the study of diatom biology is receiving more and more attention in the recent years. One of the limitations in developing molecular tools for diatoms lies in the peculiar nature of their cell wall, that is made of silica and organic molecules and that hinders the application of standard methods for cell lysis required, for example, to extract organelles. In this study we present a protocol for intact nuclei isolation from diatoms that was successfully applied to three different species: two pennates, Pseudo-nitzschia multistriata and Phaeodactylum tricornutum, and one centric diatom species, Chaetoceros diadema. Intact nuclei were extracted by treatment with acidified NH4F solution combined to low intensity sonication pulses and separated from cell debris via FAC-sorting upon incubation with SYBR Green. Microscopy observations confirmed the integrity of isolated nuclei and high sensitivity DNA electrophoresis showed that genomic DNA extracted from isolated nuclei has low degree of fragmentation. This protocol has proved to be a flexible and versatile method to obtain intact nuclei preparations from different diatom species and it has the potential to speed up applications such as epigenetic explorations as well as single cell (“single nuclei”) genomics, transcriptomics and proteomics in different diatom species.

Cardiac endothelial cells maintain open chromatin and expression of cardiomyocyte myofibrillar genes

Endothelial cells (ECs) are widely heterogenous depending on tissue and vascular localization. Jambusaria et al. recently demonstrated that ECs in various tissues surprisingly possess mRNA signatures of their underlying parenchyma. The mechanism underlying this observation remains unexplained, and could include mRNA contamination during cell isolation, in vivo mRNA paracrine transfer from parenchymal cells to ECs, or cell-autonomous expression of these mRNAs in ECs. Here, we use a combination of bulk RNASeq, single-cell RNASeq datasets, in situ mRNA hybridization, and most importantly ATAC-Seq of FACS-isolated nuclei, to show that cardiac ECs actively express cardiomyocyte myofibril (CMF) genes and have open chromatin at CMF gene promoters. These open chromatin sites are enriched for sites targeted by cardiac transcription factors, and closed upon expansion of ECs in culture. Together, these data demonstrate unambiguously that the expression of CMF genes in ECs is cell-autonomous, and not simply a result of technical contamination or paracrine transfers of mRNAs, and indicate that local cues in the heart in vivo unexpectedly maintain fully open chromatin in ECs at genes previously thought limited to cardiomyocytes.

Single-nucleus transcriptomics reveals functional compartmentalization in syncytial skeletal muscle cells

Syncytial skeletal muscle cells contain hundreds of nuclei in a shared cytoplasm. We investigated nuclear heterogeneity and transcriptional dynamics in the uninjured and regenerating muscle using single-nucleus RNA-sequencing (snRNAseq) of isolated nuclei from muscle fibers. This revealed distinct nuclear subtypes unrelated to fiber type diversity, previously unknown subtypes as well as the expected ones at the neuromuscular and myotendinous junctions. In fibers of the Mdx dystrophy mouse model, distinct subtypes emerged, among them nuclei expressing a repair signature that were also abundant in the muscle of dystrophy patients, and a nuclear population associated with necrotic fibers. Finally, modifications of our approach revealed the compartmentalization in the rare and specialized muscle spindle. Our data identifies nuclear compartments of the myofiber and defines a molecular roadmap for their functional analyses; the data can be freely explored on the MyoExplorer server (https://shiny.mdc-berlin.de/MyoExplorer/).

Isolation of nuclei and downstream processing of cell-type-specific nuclei from micro-dissected mouse brain regions – techniques and caveats

The mammalian brain consists of several structurally and functionally distinct regions equipped with an equally complex cell-type system. Due to its relevance in uncovering disease mechanisms, the study of cell-type-specific molecular signatures of different brain regions has increased. The rapid evolution of newer and cheaper sequencing techniques has also boosted the interest in cell-type-specific epigenetic studies. In fact, the nucleus holds most of the cell’s epigenetic information and is quite resistant to tissue dissociation processes as compared to cells. As such, nuclei are continually preferred over cells for epigenetic studies. However, the isolation of nuclei from cells is still a biochemically complex process, with every step affecting downstream results. Therefore, it is necessary to use protocols that fit the experimental design to yield nuclei of high quality and quantity. However, the current protocols are not suitable for nuclei isolation of small volumes of micro-dissected brain regions from individual mouse brains.Additionally, the caveats associated with centrifugation steps of nuclei extraction and the effects of different buffers have not been thoroughly investigated. Therefore, in this study, we describe an iodixanol based density gradient ultracentrifugation protocol suitable for micro-dissected brain regions from individual mice using ArccreERT2 (TG/WT).R26CAG-Sun1-sfGFP-Myc (M/WT or M/M). This mouse model shows sfGFP expression (sfGFP+) in the nuclear membrane of specific stimulus activated cells, thereby providing a good basis for the study - nuclei isolation and separation of cell-type-specific nuclei. The study also introduces new tools for rapid visualization and assessment of quality and quantity of nascent extracted nuclei. These tools were then used to examine critical morphological features of nuclei derived from different centrifugation methods and the use of different buffers to uncover underlying effects. Finally, to obtain cell-type-specific nuclei (sfGFP+ nuclei) from the isolated nuclei pool of high viscosity, an optimized protocol for fluorescence activated nuclei sorting (FANS) was established to speed up sorting. Additionally, we present a 1% PFA protocol for fixation of isolated nuclei for long term microscopic visualization.