Biochemical purification of native histone core particles
Drosophila S2 cells (100 ml) were grown to mid-log phase. Cells were collected by centrifugation and processed as described previously [11] to obtain purified CenH3 and bulk nucleosomal particles with the following adaptations. Chromatin was released from interphase nuclei in S2 cells by micrococcal nuclease (MNase) digestion, and treated in situ within intact nuclei with 0.05% glutaraldehyde to preserve histone core particle organization. The nuclei were subsequently bound to hydroxylapatite in 1× phosphate buffered saline (PBS) supplemented with 0.35 M NaCl, 0.2 mM ethylenediaminetetraacetic acid (EDTA) and 0.5 mM phenylmethanesulphonyl fluoride (PMSF) for several hours with slow stirring to create a slurry [18]. Hydroxylapatite specifically binds DNA, therefore only DNA-associated histones are enriched during this process. Hydroxylapatite-bound chromatin was collected by low-speed centrifugation and washed twice with 0.35 M NaCl-PBS to remove nuclear proteins that are not bound to DNA, most non-histone chromosomal proteins, and incompletely assembled nucleosomes. The hydroxylapatite-bound chromatin was then incubated with 2 × 20 ml volumes of ice-cold 2 M NaCl-PBS supplemented to 0.5 mM PMSF and 0.2 mM EDTA with slow stirring at 4°C. Under these conditions, intact histone core particles completely dissociate from DNA [18]. The core particles were concentrated to 2 ml using an Amicon pressure cell. The salt was dialyzed down to 0.35 M NaCl-PBS supplemented with 0.5 mM PMSF. Centromeric particles were enriched from this material by immunoprecipitation using a 1:1000 dilution of CenH3 antibody overnight in an end-over-end rotator. The unbound fraction containing mostly canonical histone core particles was saved as the control. Immunoprecipitated samples were washed once in 0.35 M NaCl-PBS, once in 0.15 M NaCl-PBS, and then eluted from the beads by competition with 1 mg ml-1 CenH3-specific peptide for 2–4 hours. Eluted particles were dialyzed as 100 μl droplets on 0.025 μm pore dialysis disks (Millipore) against 50 ml of 1 × PBS buffer for 1 hour at room temperature to remove unbound peptide, diluted between 1:10 and 1:100 and imaged by AFM directly in 1 × PBS within 48 hours of preparation. The dialysis step eliminated a high recognition signal background that was seen without dialysis (Figure 5), which indicates that the recognition signals obtained for samples used in this study did not result from residual peptide sticking to the mica surface. The affinity-purified CenH3 antibody had been raised to a peptide epitope present at end of the 125-aa N-terminal tail of Drosophila CenH3 (Cid [19]). This epitope is not thought to participate in intra-nucleosome histone:histone interactions, and in the absence of DNA is likely to remain accessible to the imaging tip during recognition events.
APTES-mica preparation
A desiccator was purged with argon for 2 minutes and 30 μl of APTES (99% 3-aminopropyl triethoxysilane, Sigma-Aldrich, St. Louis, MO) placed into a small container at the bottom of the desiccator. Ten microliters of N,N-diisopropylethylamine (99%, distilled, Sigma-Aldrich) was placed into another small container, and the desiccator purged with argon for a further 2 minutes. Mica sheets were stripped on one side until smooth and immediately placed into the desiccator. The desiccator was purged for another 3 minutes and then sealed off, leaving the mica exposed to APTES vapor for 1 hour. After this exposure, the APTES was removed, the desiccator purged, and the APTES-mica stored in the sealed desiccator until needed.
Preparing samples for AFM imaging
Two hundred microliters of a 2 μM glutaradehyde (grade I, Sigma-Aldrich) solution in water was added via a pipette onto APTES-mica immediately upon removal from the storage desiccator and incubated for 10 minutes [20]. The surface was rinsed with water from a Nanopure ultrapure water system, and 60 μl core particle solution (about 0.2 μg core particles per milliliter in PBS buffer) was added via a pipette onto the treated surface and allowed to incubate for 30 minutes. The surface was then rinsed again with PBS buffer (100 mM NaCl, 50 mM Na-phosphate, pH 7.5). The prepared sample was mounted into the scanning probing microscopy (SPM) liquid flow cell and imaged immediately.
Functionalizing AFM tips
Silicon-nitride cantilever tips (Microlever, Veeco, Santa Barbara, CA, coated for MacMode AFM by Agilent Technologies, Chandler, AZ) for recognition imaging were used as described [16]. Briefly, anti-CenH3 antibody was reacted with N-Succinimidyl 3-(acetylthio)propionate (SATP, Sigma inc.) and purified in a PD-10 column (Amersham Pharmacia Biotech). The cantilevers were cleaned in a ultraviolet (UV) cleaner, vapor-treated with APTES and reacted with polyethylene glycol (PEG) crosslinker using triethylamine and CHCl3. The SATP-labeled antibodies were then bound to the PEG crosslinkers with NH2OH (Sigma) in NaCl/Phosphate buffer. The tips were then rinsed in PBS buffer and stored at 4°C until use.
Imaging of native core particles
Conventional imaging was performed on a Pico I MacMode AFM (Agilent Technologies, Chandler, AZ) with an amplitude setting between 2.0 and 2.5 V. Recognition imaging was performed on a Pico I AFM with a Picotrec recognition imaging attachment (Agilent Technologies) with an amplitude setting of about 14–16 nm. AFM engagement was performed at 30% amplitude reduction. The images were taken in PBS buffer. Peptide blocking experiments (Figure 2D) were performed by adding 200 μl CenH3 peptide (30 μg ml-1) into the liquid cell (diameter 1.2 cm, height 0.5 cm) during imaging of samples resulting in an effective peptide concentration of 10 μg cm-2.
Data analysis
Recognition imaging was analyzed using custom software [21, 22]. This program compiles histograms of the pixel intensity distribution from background regions containing no features, and compares them with regions containing visible recognition spots. Recognition events give rise to a second peak in the intensity distribution and these were clearly separated from the background by selecting a cut-off of 75% of the background intensity (recognition spots correspond to a decrease in the signal). Particle heights were measured using FemtoScan (Advanced Technologies Center). A maximum height was taken as the peak height relative to the local background. Only particles with an apparent diameter of more than 12 nm were counted. The true diameter of histone core particle ranges from 8 to 10 nm in the absence of DNA [23], which produces features from 15 to 25 nm in diameter in the AFM image owing to the limited resolution of the probe.