Quantitative single-molecule microscopy reveals that CENP-A ^Cnp1 deposition occurs during G2 in fission yeast

Abstract

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-ACnp1 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-ACnp1 levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-ACnp1 is deposited solely during the G2 phase of the cell cycle.

Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

“…Single molecule counting-PALM is used to determine protein stoichiometry when single step photobleaching is not feasible (such as high density of proteins) [11][12][13][14] . Fluorescent proteins are photoactivated one at a time, detected and subsequently photobleached (Fig.…”

“…These probes can be activated in sparse numbers over time such that their images are optically resolvable and can be precisely localized 4 . These techniques have been exploited to image sub-cellular structures [5][6][7] , visualize protein (co)-organization [8][9][10] and quantify protein stoichiometry [11][12][13][14][15] .…”

Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory

“…Several recent studies made progress in quantifying single-molecule superresolution microscopy data by correcting for dye blinking (25)(26)(27)(28). Other approaches calculated averaged spatial distributions of an ensemble of signaling complexes (33,38) or determined high mEos2 molecule numbers using an average number of blinking events (39). One persistent problem is the strong environmental dependence of the photophysical parameters required for blink correction and the fraction of unobserved fluorophores.…”

Plasticity and Epigenetic Inheritance of Centromere-specific Histone H3 (CENP-A)-containing Nucleosome Positioning in the Fission Yeast

“…Two independent investigations have led to somewhat different conclusions for CENP-A/ Cnp1 deposition in fission yeast. One suggests that two redundant pathways operate in S or G 2 phase, respectively (24), whereas a recent study concludes that Cnp1 is replenished exclusively in G 2 phase (25). Overall, a common feature seen in various species is that CENP-A replenishment in many cases may occur without a tight junction with DNA replication (26).…”

“…Multiple lines of investigation, using quantitative fluorescence microscopy, have shown that on average, over 50% of total nucleosomes in centromeres contain Cnp1 (25,34), although a separate study showed a much higher number of Cnp1 molecules residing in centromeres, exceeding the full capacity of centromeric chromatin (35) (see Fig. 2 for details).…”