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Biology

Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands

Published: August 21, 2021 doi: 10.3791/62408
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1, 1, 1
1Instituto de Biotecnología, Departamento de Genética del Desarrollo y Fisiología Molecular, Universidad Nacional Autónoma de México

Summary

This protocol aims to visualize heterochromatin aggregates in Drosophila polytene cells.

Abstract

Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila melanogaster. Therefore, it is an excellent model to study heterochromatin formation and maintenance. Polytenized cells, such as the ones found in salivary glands of third instar D. melanogaster larvae, provide an excellent tool to observe the chromatin amplified nearly a thousand times and have allowed researchers to study changes in the distribution of heterochromatin in the nucleus. Although the observation of heterochromatin components can be carried out directly in polytene chromosome preparations, the localization of some proteins can be altered by the severity of the treatment. Therefore, the direct visualization of heterochromatin in cells complements this type of study. In this protocol, we describe the immunostaining techniques used for this tissue, the use of secondary fluorescent antibodies, and confocal microscopy to observe these heterochromatin aggregates with greater precision and detail.

Introduction

Since the early studies of Emil Heitz1, heterochromatin has been considered an important regulator of cellular processes such as gene expression, meiotic and mitotic separation of chromosomes, and the maintenance of genome stability2,3,4.

Heterochromatin is mainly divided into two types: constitutive heterochromatin that characteristically defines repetitive sequences, and transposable elements that are present at specific chromosome sites such as the telomeres and centromeres. This type of heterochromatin is mainly defined epigenetically by specific histone marks such as the di or tri-methylation of lysine 9 of histone H3 (H3K9me3) and the binding of the Heterochromatin protein 1a (HP1a)5,6. On the other hand, facultative heterochromatin localizes through the chromosome's arms and consists mainly of developmentally silenced genes7,8. Immunostaining of heterochromatin blocks in metaphase cells, or the observation of heterochromatin aggregates in interphase cells, has unveiled much light in the understanding of the formation and function of heterochromatic regions9.

The use of Drosophila as a model system has allowed the development of essential tools to study heterochromatin without the use of electron microscopy10. Since the description of position effect variegation and the discovery of heterochromatin-associated proteins, such as HP1a, and histone post-translational modifications, many groups have developed several immunohistochemical techniques that allow visualization of these heterochromatic regions10,11.

These techniques are based on the use of specific antibodies that recognize heterochromatin-associated proteins or histone marks. For every cell type and antibody, the fixation and permeabilization conditions must be determined empirically. Also, conditions may vary if additional mechanical processes such as squashing techniques are used. In this protocol, we describe the use of Drosophila salivary glands to study heterochromatic foci. Salivary glands have polytenized cells that contain more than 1,000 copies of the genome, thus providing an amplified view of most of the chromatin features, with the exception of satellite DNA and some heterochromatic regions which are under replicated. Nevertheless, heterochromatin regions are easily visualized in polytene chromosome preparations, but the squashing techniques may sometimes disrupt characteristic chromatin-bound complexes or the chromatin architecture. Therefore, immunolocalization of proteins in whole salivary gland tissue can surpass these undesired effects. We have used this protocol to detect several chromatin bound proteins, and we have demonstrated that this protocol combined with mutant Drosophila stocks can be used to study heterochromatin disruption12.

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Protocol

1. Third instar larvae culture

  1. Prepare 1 liter of standard media by adding 100 g of yeast, 100 g of unrefined whole cane sugar, 16 g of agar, 10 mL of propionic acid and 14 g of gelatin. Dissolve all ingredients except the yeast in 800 mL of tap water and then dissolve the yeast. Autoclave immediately for 30 minutes.
    1. Afterward, let the media cool down to 60 °C and add propionic acid to a final concentration 0.01%. Let the bottle stand until the gelatin is formed.
  2. To optimize the 3ᵒ instar larvae culture, first collect 5-to-10-day old adults and place 50 (25 males and 25 females) in a broad neck bottle of standard media.
  3. Place the bottle with the flies in a controlled temperature incubator at 25 °C until the number of eggs laid is 50 (approximately 12 hours for the wild-type strain).
  4. After the incubation time is over, remove the adults and transfer them to a new bottle to repeat the procedure. Let the embryos grow at 18 °C for 72 hours
    ​NOTE: For more about Drosophila stock maintenance conditions, see Tennessen & Thymmel13.

2. Larvae collection

  1. For larvae collection choose the wandering larvae that do not have everted spiracles. After the eversion of the spiracles, the larva enters the prepupal stage, while retaining excellent polytene chromosomes suitable for analysis. Only after 12 hours do the cells of the salivary gland begin to prepare for programmed cell death14,15.
  2. Take fifteen 3° instar larvae and put them in a watch glass to wash them. Then transfer them to an ice-cold saline solution or PBS (1x PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, adjust pH to 7.4).
  3. Dissect 15 to 30 pairs of salivary glands (or as many as possible in 30 minutes) in cold PBS with protease inhibitors under the stereoscopic microscope. Transfer the salivary glands to a 1.5 mL tube with ice-cold PBS.
  4. Wash once with 1 mL of PBS plus protease inhibitors. Wait for the tissue to reach the bottom of the tube.
  5. After the wash, remove the PBS with a 1000 µL pipette. Take care not to touch the tissue.
    1. Alternatively, dissect the salivary glands in 5 mL of PBS to eliminate the need for this washing step and proceed to step 3 by transferring the salivary glands to 0.5 mL of the Ruvkun fixing buffer described below.

3. Salivary gland tissue fixation

  1. After removing the PBS from the last step, directly add 0.5 mL of 1x Ruvkun fixing buffer, with 50% methanol (add 0.5 mL of methanol) and 2% formaldehyde.
    ​NOTE: 2x Ruvkun solution is 160 mM KCl, 40 mM NaCl, 20 mM EGTA, 30 mM PIPES at pH 7.4.
  2. Incubate for 2 hours at 4 °C with mild rotation.

4. Salivary gland tissue wash

  1. Carry out one 5-minute rotation wash with 1 mL of Tris/Triton buffer (100 mM Tris pH 7.4, 1% Triton X-100 and 1 mM EDTA).
    ​NOTE: Wait for the tissue to reach the bottom of the tube.

5. Permeabilization

  1. Incubate the salivary glands in 1 mL of Tris/Triton X-100 (the same as above). For some proteins it might be necessary to add 1% β-mercaptoethanol.
  2. Incubate for 2 hours at 37 ° C with mild shaking (300 rpm).

6. Preservation step (optional)

NOTE: If not proceeding immediately to the incubation with the antibody, preserve the tissue as follows.

  1. Wash with 1 mL of BO3 buffer (0.01 M H3BO3 pH 9.2 + 0.01 M NaOH) and then incubate in BO3/10 mM DTT at 37 ° C with mild shaking (300 rpm) for 15 minutes.
  2. At the end of the incubation period, perform a wash with 1 mL of BO3 buffer alone.
    NOTE: Wait for the tissue to reach the bottom of the tube.
  3. Add 1 mL of PBS. Preserve the tissue in this solution at 4 °C for up to 72 hours and then proceed with the next step. This step is particularly helpful when working with different mutant strains that may present a delayed life cycle, so the immunodetection can be performed at the same time along with the controls.

7. Tissue blocking

  1. Incubate the salivary glands in 1 mL of Buffer B (PBS + 0.1% BSA + 0.5% Triton X-100 + 1 mM EDTA) for 2 hours at room temperature with rotation.

8. Immunostaining

  1. Remove all buffer B and add buffer A (PBS + 0.1% BSA) plus antibody of interest.
    ​NOTE: We use the HP1a C1A9c (concentrated antibody) from Hybridoma Bank up to 1:3000. When using the C1A9s (supernatant) we have tried from 1:100 to a 1:500 dilution and any dilution between this rank works well) overnight at 4 ᵒC with rotation. At this point it is important that the shaking does not raise bubbles which might damage the antibody.

9. Immunostaining washing

  1. Perform 3 x 15-minute washes with buffer B under stirring at room temperature using 1 mL each time.
  2. Transfer the glands to buffer B together with the secondary antibody coupled to a fluorophore for 2 hours under rotation at 4 ᵒC (secondary antibody Alexa fluor 568 Invitrogen were used 1:3000).
    1. Cover the tube with aluminum paper foil to protect the secondary antibody from the light.
  3. Carry out 2 x 15-minute washes at room temperature while rotation with 1 mL of Buffer B.
  4. Incubate with a DNA marker such as Sytox (take 2 µL of 5 mM stock and dissolve in 1 mL of Buffer B) or Hoechst (take 1 µL of 10 mg/mL stock and dissolve in 1 mL of Buffer B) for 10 minutes at room temperature with rotation.
  5. Carry out one wash with Buffer B and once with PBS, each wash lasting 10 minutes while rotating at room temperature.
    ​NOTE: Remember to protect it from the light.

10. Imaging

  1. Mount the salivary glands on a slide, making a pool with a coverslip.
  2. Put the salivary glands in the middle of the pool and cover with AF1 citifluor to avoid the formation of bubbles extending the viscous liquid all over the place. Then seal all the sides with clear nail polish.
  3. Observe under a fluorescence or confocal microscope. If the sample is not going to be observed on the same day, store away from light at 4 °C.
  4. Use GraphPad Prism 6 to generate all graphs and statistical analyses.
  5. Analyze the data from HP1a distribution in salivary glands using the Kruskal-Wallis test. Statistical significance was set at (p < 0.05*, < 0.01 **, < 0.001***, < 0.0001****).

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Representative Results

Representative results of HP1a immunostaining in Drosophila salivary glands are shown in Figure 1. A positive result is to observe one focal point (Figure 1a) (heterochromatic aggregate or condensate). A negative result is no signal or a dispersed signal. Sometimes a double signal can be observed, that is, with a double point (Figure 1c), but it usually occurs in smaller quantities.

Data analysis can be represented as bar graphs, comparing the distribution of HP1a within different mutant backgrounds. For example, in Figure 2 we can see that 98% of the wild type nuclei present a distribution of one focal point and 2% of the nuclei present two foci, whereas in the mutant, the proportion changes, and the presence of two foci increases to 40%.

Figure 3 shows representative H3k9me3 immunostaining results in Drosophila salivary glands. We can observe one focal point (Figure 3b) that resembles the HP1a immunostaining, (heterochromatic aggregate or condensate). A double or triple signal (Figure 3c) can be seen on rare occasions in the wild type strains.

Figure 1
Figure 1. Representative confocal microscopy image from salivary gland immunostaining with HP1a antibody from wild type (wt). a) DNA (cyan signal), HP1a (magenta signal), and merge scale bar 100 µm. In immunostaining for HP1a, a nucleus with a focal point is marked with a white arrow and a nucleus with two foci with a dotted line box. The right column shows a magnified image of a single nucleus with a scale bar of 5 µm. b) focal distribution. c) two foci distribution. Both nuclei are marked with a white dashed line. Please click here to view a larger version of this figure.

Figure 2
Figure 2. Examples result from counting nuclei foci distribution ofHP1a immunostaining. The first bar represents the counting of the wild-type nuclei (wt), as in Figure 1. The second bar represents a mutant strain that affects this distribution. Please click here to view a larger version of this figure.

Figure 3
Figure 3. Representative confocal microscopy image from salivary gland immunostaining with H3K9me3 antibody from wild type (wt). a) DNA (cyan signal), H3K9me3 (magenta signal) and merge scale bar 100 µm. In immunostaining for H3K9me3.The right column shows a magnified image of a single nucleus with a scale bar of 5 µm. b) a nucleus with a focal distribution. c) three foci distribution. Both nuclei are marked with a white dashed line. Please click here to view a larger version of this figure.

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Discussion

The cellular function of eukaryotic organisms can define the 3D structure within the nucleus, which is supported by interactions between different proteins with chromatin and various molecules including RNA. In the last three years, the biological condensates that have had relevance, including heterochromatin, have taken a fundamental role in the determination of the phase separation promoting the distinct nuclear spatial organization of active and repressive chromatin 16,17,18.

Heterochromatin is essential to preserve cell functions and identity. Previously it was thought that these dense areas were not transcribed. However, now that we have more powerful technologies, we can see that the heterochromatin is not only transcribed but also a fundamental process to maintain the scaffold of the nucleus and is sensitive to developmental or pathological processes12,19. Besides, certain genes embedded in pericentric heterochromatin need a heterochromatic environment to function properly. HP1a mutations reduce the expression of the light and rolled genes, which were the first to be discovered19. These genes are essential for the organism's survival and are found in heterochromatin blocks. As a result, despite its ability to induce silencing, this peculiar genome component has the potential to be very dynamic20. In a complex balance between chromatin-bound and diffuse types that can be controlled by various biological contexts, heterochromatin-associated proteins such as HP1a also exist. It was also recently suggested that phase-separation properties are shown by the assembly of heterochromatin condensates21,22.

There are a number of papers in which the authors carried out whole-mount immunostaining of Drosophila salivary gland nuclei using different and sometimes simpler protocols23,24. In this case we adapted a protocol first described in C. elegans25, and subsequently used in Drosophila salivary glands by several groups26,27,28,29 and combined it with the use of confocal microscopy and mutant organisms. This protocol also allows visualization of different types of proteins, including transcription factors such as XPD, XPB and TBP27, but also heterochromatin bound proteins such as HP1a and histone marks such as H3K9me3, which positions it as a protocol for broad use in this tissue. It also has the advantage that the tissue can be stored at an intermediate step without affecting polytene chromosome banding.

This protocol is reliable and cost-effective due to the use of a specific antibody to view the HP1a protein. The critical step in this protocol is to avoid losing the glands during washes and waiting for the tissue to bottom out. The advantage of using salivary glands is that a 3D view of the nucleus and its conformation can be obtained easily, in contrast to the polytene chromosome technique that requires a mechanical disruption of the cell and can damage the chromatin. While performing this protocol, special care should be taken during the washing steps. If not carefully performed, the tissue will break, and it would not be possible to obtain high quality images.

To evaluate the importance of the lack of binding of RNA to the regions or proteins that are being observed, it is necessary to add a wash with Buffer C (Buffer B without EDTA) and add 100 µM of RNase. This wash should be carried out for 1 h at 37 °C as previously described. Washing should be done before the step where molecules are added to observe the DNA (between steps 9.3 and 9.4).

Confocal microscopy may not seem like a very new methodology to address questions of heterochromatin condensates25,30, but it has been extremely useful to identify delocalization of the HP1a protein in Drosophila nuclei, which suggests severe problems in chromatin structure that can be evaluated with other techniques more thoroughly. Despite its limitation, it can be used in combination with high-resolution microscopy as a first approach to apply novel techniques to clarify the biological activity that modulates heterochromatin condensate assembly, control, and functions31. Some of these new methodologies that focus on the molecular and biophysical interactions between heterochromatin, RNA, and heterochromatin-associated proteins are gathered from this set of methods to test heterochromatin condensates.

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Disclosures

The authors declare that they have no competing interests.

Acknowledgments

We thank Marco Antonio Rosales Vega and Abel Segura for taking some of the confocal images, Carmen Muñoz for media preparation and Dr. Arturo Pimentel, M.C. Andrés Saralegui, and Dr. Chris Wood from the LMNA for advice on the use of the microscopes.

FUNDING:
This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACyT) (A1-S-8239 to VV-G) and Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (204915 and 200118 to VV-G)

Materials

Name Company Catalog Number Comments
1.5 mL microcentrifuge tubes Axygen MCT-150-C 11351904 brand not critical
16% formaldehyde Thermo Scientific 28908
AF1 Citifluor Ted pella 19470 25 mL
BSA, Molecular Biology Grade Roche 10735078001 brand not critical
Complete, protease inhibitors Ultra EDTA-free
protease inhibitors		 Merck 5892953001
Coverslip Corning CLS285022-200EA 22x22, brand not critical
DTT Sigma d9779 brand not critical
EDTA Sigma E5134 brand not critical
EGTA brand not critical
Glass slide Gold seal 3011 brand not critical
H3BO3 Baker 0084-01 brand not critical
H3K9me3 Abcam 8889
HP1a Hybridoma Bank C1A9 Product Form Concentrate 0.1 mL
KCl Baker 3040-01 brand not critical
Methanol Baker 9070-03 brand not critical
NaCl Sigma 71376 brand not critical
NaOH brand not critical
PIPES brand not critical
Rotator Thermo Scientific 13-687-12Q  Labquake Tube Shaker
Thermo Mixer C Eppendorf 13527550 SmartBlock 1.5 mL
Tris Milipore 648311 brand not critical
Triton X-100 Sigma T8787 100 mL, brand not critical
β-mercaptoethanol Bio-Rad 1610710 25 mL, brand not critical

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References

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  2. Irick, H. A new function for heterochromatin. Chromosoma. 103 (1), 1-3 (1994).
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  5. Eissenberg, J. C., Elgin, S. C. R. HP1a: A structural chromosomal protein regulating transcription. Trends in Genetics. 30 (3), 103-110 (2014).
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  18. Keenen, M. M., Larson, A. G., Narlikar, G. J. Visualization and Quantitation of Phase-Separated Droplet Formation by Human HP1α. Methods in Enzymology. , (2018).
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Tags

Immunofluorescent Staining Visualization Heterochromatin Associated Proteins Drosophila Salivary Glands Polytene Cells Third Instar Larvae Telomeres Chromocenter Heterochromatic Aggregates Genetic Mutants Larvae Culture Temperature Incubator Larvae Collection Salivary Glands PBS
Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in <em>Drosophila</em> Salivary Glands
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Meyer-Nava, S., Zurita, M.,… More

Meyer-Nava, S., Zurita, M., Valadez-Graham, V. Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands. J. Vis. Exp. (174), e62408, doi:10.3791/62408 (2021).

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