Fig. 1: Schematic drawing depicting the players in the histone acetylation/deacetylation process.
Eighteen distinct HDACs have been identified to date. They are classified into four groups based on their structural divergence, namely class I, II, III and IV HDACs (see Table 1). The first two classes are considered "classical" HDACs whose activities are inhibited by trichostatin A (TSA), whereas the third class is a family of NAD+-dependent proteins not affected by TSA.
Tab. 1: HDAC overview and localization.
Homologues to these three classes are found in yeast having the names: reduced potassium dependency 3 (Rpd3), which corresponds to Class I; histone deacetylase 1 (hda1), corresponding to Class II; and silent information regulator 2 (Sir2), corresponding to Class III. Class IV (HDAC11) is homologous with class I and class II enzymes. The Class III group is considered an atypical category of its own, which are NAD+-dependent, whereas other groups require Zn2+ as a cofactor. The function and activity of HDACs vary depending on their structure and intracellular localization. The importance of HDACs in the regulation of cellular activities under physiological and diseased conditions makes HDACs critical targets in in the treatment of cancer and inflammatory disorders. Understanding their cellular function, localization and their inhibition is crucial for cancer research and therapeutics.
Immunohistochemistry (IHC) analysis of tissue samples (Figures 2–4) is essential for the detection of the protein in a sample and is often used in diagnostic experiments, but does not result in an image that will allow co-localization experiments.
Studying protein localization within a cell requires precise high-resolution imaging techniques. Confocal microscopy can provide information on subcellular localization, achieving up to 200 nm resolution. Additionally, optical sectioning of multiple image planes on a confocal allows for the study of protein location in a volume. With the introduction of the Leica TCS SP8 STED 3X (Stimulated Emission Depletion) super-resolution microscope, subcellular localization can now be performed in the sub-50 nm range, breaking the diffraction limit. The use of STED super-resolution microscopy can give new insight into the localization and function of individual HDAC family members and other corresponding molecules.
The use of high specificity and high affinity antibodies is critical in any localization experiment to assure the positive outcome of the assay. Quality control is essential in the development and manufacture of such antibodies allowing visual evidence of proper target localization and low background staining. Here we present staining and compare high resolution localization of HDAC1 analysis performed on a confocal microscope to STED microscopy. HDAC1 has been shown to be involved in the suppression of a number of genes implicated in multi-lineage blood cell development and to be required for the transcriptional repression of circadian target genes, such as PER1, mediated by the large PER complex or CRY1 through histone deacetylation. HDAC1 together with MAT2 deacetylates p53 and modulates its effect on cell growth and apoptosis.
Sample tissue was fixated by formalin fixed paraffin embedding. Protein detection was performed using primary antibodies at 5 µg/mL (HDAC5 and HDAC10) or 1:500 (HDAC1) for 1 h at room temperature. Secondary antibody: peroxidase rabbit secondary antibody (Rockland, p/n 611-103-122) at 1:10,000 for 45 min at room temperature. Proteins were visualized as precipitated red or purple signal with hematoxylin purple or blue nuclear counterstain. Primary antibodies shown: HDAC1 (Rockland, p/n 600-401-879), HDAC5 (Rockland, p/n 600-401-J69) and HDAC10 (Rockland, p/n 600-401-J75).
Conditions for STED microscopy
A431 cells (ATCC CRL-1555) were incubated at 37 °C with 5 % CO2 in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen, MO) with 10 % FBS (Rockland, p/n FBS-01-0500) in 25 cm2 cell culture flask. When cells reached 70 to 80 % confluence, they were detached using TrypLE (Invitrogen, MO), and then placed on # 1.5 coverslips in 6 wells plates until reaching ~30 % confluency. Cells were fixed using ice-cold (–20 °C) methanol for 7 minutes. Once fixed, cells were rinsed in PBS at room temperature and then incubated in blocking solution for 15 minutes (PBS/Normal Goat Serum 10 %, Triton-X100 at 0.2 %). This step was followed by incubation with the following primary antibodies for 1 hour at room temperature: rabbit anti-HDAC1 1:50 (Rockland, p/n 600-401-879) and mouse anti-Keratin 1:50 (Rockland, p/n 200-301-390). Cells were rinsed three times in PBS for 10 minutes/rinse, and then incubated in blocking solution for 15 minutes. DyLight 488 anti-Mouse IgG (Rockland, p/n 610-141-002) and ATTO 550 anti-Rabbit IgG (Rockland, p/n 611-154-122) were diluted 1:100 each in blocking solution and incubated for one hour. Cells were rinsed in PBS three times and then in tap water once. Coverslips were then mounted in ProLong Antifade Kit (Life Technologies, p/n P-7481) and allowed to cure in the dark for at least 3 days. It is important to wait at least 3 days, as it takes time for ProLong products to reach the desired refractive index (RI of 1.46) for imaging. The slides were examined using a Leica TCS SP8 STED 3X microscope (Leica Microsystems, Inc.) using a white light laser tuned to 470 nm to excite the DyLight 488 fluorescence and 550 nm to excite the ATTO 550. For STED images, DyLight 488 was used with the 592 nm depletion laser set to 95 % power with a time gating range of 1.5–6.0 ns, and ATTO 550 was used with the 660 nm depletion laser set to 100 % power with a time gating range of 1.5–6.0 ns.
Fig. 5: Confocal vs STED imaging. Epidermoid carcinoma cell line A431 stained for HDAC1-ATTO 550 (red) and Keratin-DyLight 488 (green). The same cell was imaged using standard confocal imaging (left) and STED 3X imaging (right). HDAC1 localizes at the nucleolar periphery.
Stimulated Emission Depletion (STED) microscopy is a confocal super-resolution imaging technique. STED super-resolution is obtained by overlaying an excitation laser and a depletion laser to achieve an optical "sculpting" of the point spread function (PSF), which is then raster scanned over the field of view. The depletion laser beam is introduced in to the light path to create a "donut" shape, which is aligned and overlaid with the excitation beam (Figure 6).
Fig. 6: The excitation beam and STED depletion beam.
The donut depletion laser de-excites the fluorophores through stimulated emission process while leaving the molecules in the donut hole to go through normal fluorescence process. By increasing the depletion laser power the size of the donut hole where normal fluorescence process occurs decreases. Utilization of time gating, which takes advantage of the fluorescence lifetime differences between normal fluorescence and stimulated emission, further enhances the resolution down to 40nm. Two of the major advantages of STED compared to other super-resolution techniques is a purely optical resolution improvement, without post-processing, and the ability to use optical sectioning. The Leica TCS SP8 STED 3X is available with a 592 nm depletion laser and/or a 660 nm depletion laser. The 592 nm depletion beam is best suited for dyes that are in the green spectral range (Figure 7), such as DyLight 488, while the 660 nm depletion laser is recommended for dyes that emit in the yellow to orange range (Figure 7), such as ATTO 550.
Fig. 7: Dye spectra examples from ATTO with the Leica STED lasers at 592 nm and 660 nm.
Here we show staining of HDAC1 in cancer tissue (Figure 2) and epidermoid carcinoma cells (Figure 5). HDAC1 localization in the analyzed samples was confirmed to be nuclear. Cytosolic staining of HDAC1 reported to occur in certain cancer samples was not observed. Using STED microscopy compared to confocal imaging showed a visible increase in resolution (Figure 5). These results clearly show that the use of appropriate validated antibodies and STED microscopy are important tools to study subcellular structures beyond the diffraction limit correcting ill-defined images. This is critical in co-localization studies of proteins inside cells.
It is still to be determined in future experiments if STED microscopy can resolve co-localization of HDAC proteins with acetylated groups on histones or other proteins.
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