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The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and 'counts' individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-A\(^{Cnp1}\) with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-A\(^{Cnp1}\) levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-A(Cnp1) is deposited solely during the G2 phase of the cell cycle.
Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304\(\pm\)0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of \(\pm\)0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.