Cell Biology

The duplication of DNA is a fundamental process that is required for the transfer of the genetic information from parent to daughter cells. Aberrant DNA replication processes are associated with diverse disease phenotypes, including developmental defects, ageing disorders, blood disorders such as Fanconi Anemia, increased inflammation and cancer. Therefore, the development of tools to study proteins associated with error-free DNA replication processes is of paramount importance. So far, methods to study proteins associated with nascent replication forks relied on conventional immunofluorescence and immunoprecipitation assays of 5′-ethylene-2′-deoxyuridine (EdU) labeled DNA (iPOND). While greatly informative and important, these methods lack specificities for nascent fork interactions (e.g., IF) or assay an average change of millions of cells without single-cell resolution (e.g., iPOND). The assay system described here combines proximity ligation assay (PLA) with EdU coupled click-iT chemistry, which we termed “in situ Protein Interaction with Nascent DNA Replication Forks (SIRF)”. This method enables sensitive and quantitative analysis of protein interactions with nascent DNA replication forks with single-cell resolution, and can further be paired with conventional immunofluorescence marker analysis for added multi-parameter analysis.

The in vitro and in vivo genotoxicity of new metallodrugs either as Small Bioactive Molecules (SBAMs) or Conjugates of Metals with Drugs (CoMeDs) is evaluated by the micronucleus test and the Allium cepa assay, respectively. Fetal lung fibroblast cells (MRC-5), normal human corneal epithelial cells (HCEC) and immortalized human keratinocytes cells (HaCaT) were incubated with solutions of SBAMs or CoMeDs at their IC₅₀ values for 48 h (the concentration of a compound which is required to inhibit the cells growth by 50% in relation to the non-treated cells). The micronucleus abundance percentage towards the corresponding one, of the non-treated cells indicates the in vitro genotoxicity of the formulations. The in vivo Allium cepa test comprises the exposing of the plant Allium cepa roots to an SBAMs or a CoMeDs solution for 48 h. The percentages of the mitotic index, the chromosome aberrations, the nuclear abnormalities and the presence of the micronucleus are calculated indicating the in vivo genotoxicity of the agent.

The protocol described here has been developed to detect RNA at the single cell level. Fluorescent probes hybridize to target RNAs and are detected by flow cytometry after multiple amplification steps. Different types of RNA can be detected such as mRNA, long noncoding RNA, viral RNA or telomere RNA and up to 4 different target probes can be used simultaneously. We used this protocol to specifically measure the expression of two transcription factor mRNAs, MAFB and IRF4, in human monocytes.

The spatio-temporal expression pattern of a gene provides important indications to better understand its biological function. In situ hybridization (ISH) uses a labeled complementary single-stranded RNA or DNA probe to localize gene transcripts in a whole organism, a whole organ or a section of tissue. We adapted the ISH technique to the plant parasite Meloidogyne spp. (root-knot nematode) to visualize RNAs both in free-living preparasitic juveniles and in parasitic stages settled in the plant tissues. We describe each step of the probe synthesis, digoxigenin (DIG) labeling, nematode extraction from plant tissue, and ISH procedure.

To investigate the chromosome dynamics during mitosis, it is convenient to mark the discrete chromosome foci and then analyze their spatial rearrangements during prophase condensation and telophase decondensation. To label the chromosome regions in plant chromosomes, we incorporated the synthetic nucleotide, 5-ethynyl-2’-deoxyuridine (EdU), which can be detected by click-chemistry, into chromatin during replication. Here, we described a protocol of a method based on the application of semi-thin sections of Nigella damascena L. roots embedded in LR White acrylic resin. The thickness of semi-thin (100-250 nm) sections is significantly lower than that of optical sections even if a confocal microscope was used. This approach may also be suitable for work with any tissue fragments or large cells (oocytes, cells with polytene chromosomes, etc.).

Single-molecule RNA fluorescence in situ hybridization (smFISH) is a technique to visualize individual RNA molecules using multiple fluorescently-labeled oligonucleotide probes specific to the target RNA (Raj et al., 2008; Lee et al., 2016a). We adapted this technique to visualize RNAs in the C. elegans whole adult worm or its germline, which enabled simultaneous recording of nascent transcripts at active transcription sites and mature mRNAs in the cytoplasm (Lee et al., 2013 and 2016b). Here we describe each step of the smFISH procedure, reagents, and microscope settings optimized for C. elegans extruded gonads.

During mitosis chromosomes are condensed into dense X-shaped structures that allow for microscopic determination of karyotype as well as inspection of chromosome morphology.

This protocol describes a method to perform immunostaining of formaldehyde-fixed metaphase chromosomes from the avian cell line DT40. It was developed to characterize the localization of YFP-tagged TopBP1 on mitotic chromosomes and specifically determine the percentage of TopBP1 foci that formed on breaks/gaps as well as ends of individual metaphase macrochromosomes (Pedersen et al., 2015). For this purpose immunostaining of YFP was applied. However, the protocol may be optimized for other cell lines or epitopes.

RNA fluorescence in situ hybridization is a method to localize and measure gene expression in individual cell or tissue. Using multiple specific fluorescently labeled oligonucleotides greatly increases signal-to-noise ratio and thus enables detection of single RNA molecule. Around forty different DNA oligonucleotides designed to common RNA target and labeled with single fluorophore at 3´ terminus hybridizes with target RNA in fixed cells. We adapt this method to visualize target RNA in the mammalian oocyte. The ability to detect single transcript in the mammalian oocyte was challenging due to its large cell size. This method consists of four simple steps: fixation, permeabilization, hybridization and imaging. The protocol is adapted to this large nonattached cell to visualize maternal RNAs.

Combination of various fluorophores allows detection of more RNA targets. This method might be used with organelle markers or expanded with immunofluorescence protocol.

One of the major topics in plant and animal biology is sexual reproduction. It is, therefore, of great interest to isolate and study germ cells and accessory cells. The male gametophyte of the flowering plant Arabidopsis thaliana (A. thaliana), pollen, is the product of two post-meiotic mitotic divisions. Each mature pollen grain consists of two sperm cells contained within the vegetative cell, the non-reproductive companion cell. The tough pollen wall and its special nested structure make it difficult to study pollen cells separately. Here, we describe a simple and efficient method to fractionate A. thaliana sperm and vegetative cell nuclei by fluorescence activated cell sorting (FACS). Our protocol is based on differences in fluorescence intensity of sperm and vegetative cell nuclei stained with SYBR Green I. 100 plants yield about 1 x 10⁶ sperm and 350,000 vegetative cell nuclei. This method can be used for purifying pollen nuclei of various A. thaliana wild-type accessions and mutant lines, and can, in principle, be adapted for pollen of other plant species.

This protocol describes an effective method of in situ RT-PCR that was developed to localize specific gene expression directly in thin cross-sections of nematode feeding sites induced by the cyst nematode Heterodera schachtii (H. schachtii) or the root-knot nematode Meloidogyne incognita (M. incognita) in Arabidopsis roots using DIG (Digoxigenin-11dUTP) labeling coupled with AP (alkaline phosphatase) and nitro-blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate-based detection. This method is applicable to any other Arabidopsis root tissue.