microscopy of in situ dna and rna-containing structures · 1.2 in situ localization of...

Microscopy of in situ DNA and RNA-containing structures

M.L. Segura-Valdez1, R. Chávez-Rosales1, L.T. Agredano-Moreno1, E. Ubaldo1, E.F. del Toro-Rangel1, R. Lara-Martínez1, C.E. Villegas-Mercado1, G. Zavala2, P.F. Islas-Morales1 and L.F. Jiménez-García1,* 1 Cell Nanobiology Laboratory, Department of Cell Biology, Faculty of Sciences, National Autonomous University of

Mexico (Universidad Nacional Autónoma de México-UNAM), Circuito Exterior, C.U. 04510 Coyoacán, México D.F., Mexico

2 Institute of Biotechnology, National Autonomous University of Mexico (Universidad Nacional Autónoma de México-UNAM), Cuernavaca, Morelos, Mexico

In situ analysis of gene expression requires visualizing the localization of nucleic acids and proteins within the cell. Localization of DNA and RNA by microscopy involves the use of several techniques for light and electron microscopy. Here we will expose several of those classical cytochemical techniques and illustrate examples of applications. We include images of Feulgen and DAPI staining for light microscopy, EDTA regressive method for RNP and osmium-amine for electron microscopy, among others. We also illustrate the visualization of chromatin by atomic force microscopy and mention current perspectives.

Keywords: DNA; in situ localization; nucleus; nucleolus; RNA; RNP

1. In situ localization of DNA and RNA-containing structures

Localization of nucleic acids within the cell may contribute to know the distribution of DNA and RNA in space and time. Therefore, it is now possible to study gene expression in situ. The approach of studying the chemical and molecular composition in tissues and cell originally allowed the development of classic histochemistry and cytochemistry. More specific techniques were then invented to detect nucleic acids as DNA. Feulgen reaction for DNA is a technique which specifically stains DNA in red. More recent advances use fluorochromes to stain DNA specifically, DAPI (4',6-diamidino-2-phenylindole) staining for DNA, produces a blue color where DNA is associated to DAPI. However, the design of molecules detecting specific sequences of aminoacids in proteins by antibodies and specific sequences of nucleotides in nucleic acids by molecular probes, made cytochemistry more specific even to localize sequences, so cytochemistry turned to a molecular level. For DNA and RNA, staining and other procedures as in situ hybridization have been used since 1924, when Feulgen and Rossenbeck proposed the so called Feulgen reaction [1]. Although several techniques were also developed, the Feulgen reaction is widely used as it produces very accurate results. In situ hybridization was invented in 1969 [2], becoming a powerful tool for detection of DNA and RNA. Protocols have been used for light and transmission electron microscopy. Novel approaches also include the analysis of chromatin in situ by atomic force microscopy and ultraresolution imaging in vivo light microscopy [3-6].

1.1 In situ localization of DNA-containing structures

1.1.1 Light microscopy

Feulgen reaction and DAPI staining produce red and blue color by bright field microscopy and epifluorescence, respectively. In Fig. 1 Vicia faba root tip cells were treated by Feulgen staining and sperm were treated with DAPI. Fluorescence protocols can be combined with immunolocalization to simultaneously observe spatial relationship between proteins and nucleic acids. Fig. 2 illustrates the combination of both. DNA was localized with DAPI while two nuclear proteins were observed by a double immunolocalization procedure. In addition, the staining of DNA by DAPI and the simultaneous visualization of cytoskeleton is also seen.

1.1.2 Electron microscopy

For electron microscopy, similar techniques include the use of contrast procedures with phosphotungstic acid (PTA) [7], the NAMA-Ur technique [8] or the osmium amine method [9]. Labelling of DNA with specific antibodies provides localization of DNA with secondary antibodies coupled to 10 nm gold particles at high resolution [10]. Fig. 3 shows contrast by the PTA method in HeLa cells, osmium amine in Aloe vera cells, NAMA-Ur in rat liver cells and immunoelectron microscopy for DNA in Giardia lamblia [10]. The PTA technique is preferential for DNA while the others are specific techniques.

Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.)

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1.1.3 Atomic force microscopy

Atomic force microscopy has been used in several cell types to visualize compact chromatin in three dimensions [3-6]. Fig. 4 shows reticulated chromatin in onion root tip cells.

Fig. 1 Localization of DNA by light microscopy. (a) bright field after Feulgen reaction in Vicia faba root tip cells. While chromosomes are stained in mitotic cells (large arrows). In interphase the nucleus (N) is positive while cytoplasm (C) and nucleolus (n) are negative. (b) epifluorescence microscopy combined with phase contrast of DAPI stained human sperm cells. Heads (arrows) of sperm cells are blue.

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1.2 In situ localization of RNA-containing structures

1.2.1 Light microscopy

Toluidine blue prepared as an acidic and alcoholic solution stains RNA at light microscopy [11]. It preferentially stains rRNA. It is a suitable and easy protocol. Figure 5a illustrates HeLa cells stained by this technique. In addition, RNA may be localized by a protocol of fluorescent in situ hybridization (FISH) using a total biotinylated DNA probe [12-13]. Figure 5b shows localization of total viral RNA in HeLa cells infected by adenovirus 2.

Fig. 2 Localization of DNA combined with localization of proteins. (a) epifluorescence of triple labeled HeLa cell. DNA stained with DAPI is blue (large arrow), nucleolar protein fibrillarin stains the nucleolus (n) in green and nuclear splicing factor is red by immunofluorescence. (b) double labelling of DNA with DAPI stains the nuclei (blue, arrows) and cytoskeleton protein α-actin (C) in green in human lung fibroblasts.

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1.2.2 Electron microscopy

RNA-containing intranuclear structures as nucleolus, perichromatin granules, nuclear bodies and cytoplasmic ribonucleoproteins [14-23] may be visualized by the EDTA regressive technique for ribonucleoproteins (RNPs) [14-15] or by ultrastructural high resolution in situ hybridization [10, 24-26].

Fig. 3 Localization of DNA by electron microscopy using PTA in HeLa cells (a), osmium, amine in Aloe vera cells (b), NAMA-Ur in liver cells (c), and immunoelectron microscopy in Giardia lamblia trophozoites (d). In (a), (b), and (c), contrast is seen in compact chromatin (arrows) within the nuclei (N). Cytoplasm (Cy) and nucleoli (n) are negative. .In G. lamblia (d), labelling is present in the nucleoplasm within the nucleus (N) and is absent in the nucleolus (n) and cytoplasm (Cy). In (d) small arrows point to cross sections of flagella. Large arrow indicates the ventral disc of the parasite. Aloe vera sample was prepared by Yolanda. Lozada.

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Fig. 4 Atomic force microscopy of DNA-containing structures as reticulated compact chromatin (arrows) and RNA-containing structures as the nucleolus (n) in onion root tip cells. Cw, cell wall; v, vacuole; N, nucleus.

Fig. 6 shows a cell nucleus after the EDTA regressive technique for RNPs. Fine structure of RNA-containing particles as nucleolus and RNP fibers and granules such as perichromatin fibers and granules as well as interchromatin granule clusters, are seen. Fig. 7 shows a high magnification of the interphase between compact chromatin and transcription zone where perichromatin fibers are observed in a region extending about 200 nm. EDTA technique is performed in acrylic or epoxy sections mounted in copper grids. First, grids containing thin sections (40-60 nm thickness), are stained with uranyl citrate which stain deoxyribonucleoproteins and ribonucleoproteins (RNPs). After staining, grids are rinsed with deionized water and air dried. In the second step grids are treated with ethylenediaminetetraacetic acid (EDTA), as chelating agent to remove uranyl from deoxyribonucleoproteins. After rinse, grids are stained with lead citrate which binds to uranyl ions still present in RNPs to strength the staining on these structures. Finally the grids are rinsed and air dried. This technique must be adapted to different biological samples embedded in hydrophobic and hydrophilic resins. In immunoelectron microscopy protocols, a pre-embedding method can be used. The antigen–antibody reaction is performed before (pre) plastic embedding and subsequent ultrathin sectioning. For pre-embedding immunogold labeling, samples are exposed to both the primary and secondary antibody (conjugated with 5, 10 or 15 nm gold particles), in situ. Once the samples have been immunolabeled, the specimen is fixed and processed for standard electron microscopy and embedded in plastic blocks of epoxy resin. Ultra-thin sections (40-60 nm), are cut from these blocks, using an ultramicrotome, and the resulting sections are stained with uranyl acetate and lead citrate and examined in the transmission electron microscope. In post-embedding methods, the antigen–antibody reaction is performed after (post) plastic embedding. For immunolabeling of acrylic sections, cells and tissues are fixed with formaldehyde/glutaraldehyde mixture and then embedded in water-soluble plastic embedding materials such as LR White and Lowicryl at cold temperatures (-20°C). Labeling is done on ultrathin plastic sections (60-90 nm), using the first antibody and after that a secondary antibody

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Fig. 5 RNA localization by light microscopy. (a) toluidine blue for RNA in HeLa cells. The nucleoli (arrows) and cytoplasm (Cy) are stained. (b) Epifluorescence microscopy of adenovirus infected HeLa cells after in situ hybridization for total RNA. Labeling is present in the nucleus (N) and cytoplasm (Cy). The nucleolus (n) is negative.

conjugated with 5, 10 or 15 nm gold particles. Finally, plastic sections are stained with uranyl acetate and lead citrate and examined in the transmission electron microscope. Post-embedding in situ hybridization techniques are performed on thin sections of cell and tissues fixed in formaldehyde and embedded in acrylic resins. The first step of in situ hybridization is selection and labeling of the probe to be detected (DNA or RNA sequence), with Biotin by the nick translation procedure. The next step is the application of these probes to tissue sections to allow DNA or RNA to be localized within tissue regions and cell types. Finally, labeled probes are detected by avidin or streptavidin molecules that are covalently linked to heavy metals as colloidal gold.

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Fig. 6 Localization of RNA-containing structures by electron microscopy after the EDTA regressive staining for ribonucleoproteins. Within the cell nucleus (N), compact chromatin (ch) is bleached, while RNA containing structures are positive including the nucleolus (n), interchromatin granule clusters (IGC), perichromatin fibrils (thin arrows) and perichromatin granules (thick arrows). Courtesy of Dr. G.H. Vázquez-Nin and O.M. Echeverría, UNAM, México.

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Fig. 7 Localization of RNA-containing structures by electron microscopy after the EDTA regressive staining in the plant Lacandonia schismatica tegument cells. High magnification in the interphase (thin arrow) between DNA-containing structures (bleached , ch) and RNA-containing structures (thick arrow) shows a fibro-granular component.

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Fig. 8 Localization of RNA-containing structures after in situ hybridization using a genomic DNA probe in Ginkgo biloba cell nucleus (N) by transmission electron microscopy. Labeling is present in the nucleoplasm (arrows) among compact chromatin strands (ch).

Fig. 8 shows a high resolution in situ hybridization for total RNA in Ginkgo biloba cell nucleus using a total genomic DNA probe.

1.2.3 Final comments

In summary, several techniques, including those not shown here [27], are used to preferentially or specifically localize DNA or RNA within the cell, associated to cell structures as to analyze those involved in gene expression in situ. These techniques may be combined to study space and time relationships between nucleic acids and proteins. Currently, cytochemical approaches include the combination of molecular and microscopy tools [28-29]. In vivo imaging of chromatin, chromosome territories, loci and single molecules is an exciting perspective since it is now possible to perform in vivo and dynamic analysis even with nanometer resolution [30-35]. Recognition of loci and repetitive elements in living cells has been possible by a CRISPR-cas9 dependent fluorescence method [36]. Single molecule ultraresolution in vivo demands optical fluorescent microscopy beyond the limit of diffraction. Resolution up to tens of nanometers is possible for single molecules using photo-active localization microscopy (PALM) and

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stochastic optical reconstruction (STORM) [37-39]. Photobleaching approaches of nucleolar behavior have been very successful [40]. More recently, excitation of common fluorophores by diffusible acceptor molecules (FRET) and ulterior stochastic integration (FRET-dSOFI) has proven to reach ultraresolution without the need of photobleaching or photoactive illumination systems and complex image analysis [41]. As these techniques become more accessible, crucial advantages on structure and dynamics of DNA, DNA-protein, RNA, and RNA-protein interactions in the cell are expected.

Acknowledgements The support by CONACyT 180835 and DGAPA UNAM PAPIIT IN220713 is gratefully acknowledged. Adenovirus image was taken during a postdoctoral training of LFJ-G in 1992 at Cold Spring Harbor Laboratory, New York (under Dr. D. Spector). Authors thanks Martha Montaño and Carina Becerril from INER-México, for supplying lung fibroblast cells. F. del Toro Rangel and C.E. Villegas-Mercado are graduate students of the program Posgrado en Ciencias Médicas, Odontológicas y de la Salud-UNAM under support of CONACyT. P.F. Islas-Morales was a visiting student at Stanford University while this manuscript was under writing, supported by Secretaría de Educación Pública through Subsecretaría de Educación Superior and by Dirección General de Cooperación e Internacionalización (DGCI)-UNAM, México. Prof. Dr. R. Dirzo is greatly acknowledged for support and encouragement to P.F. Islas-Morales at Stanford.

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