Jcb_200904137 1..10

DNA in eukaryotes is wrapped around a histone octamer containing H2A, H2B, H3, and H4, forming a nucleosome, which is the fundamental unit of chromatin. Posttranslational modifications of these histones play critical roles in genome function, including the regulation of transcription and maintenance of genome integrity (Jenuwein and Allis, 2001; Kouzarides, 2007). However, little is known about how these modifications change with time in single cells, largely because we lack the appropriate monitoring systems. Although resonance energy transfer between fluorescently tagged proteins has been used for this purpose (Kanno et al., 2004; Lin and Ting, 2004), this approach usually monitors the activity of modifying enzymes rather than the modification of endogenous proteins, and extensive probe improvements are required to obtain higher signal to noise ratios. In this study, we detect endogenous modifications in living cells by introducing specific antibodies (Fab) directed against phosphorylated histone H3. In all organisms investigated so far (Hendzel et al., 1997; Wei et al., 1999; Johansen and Johansen, 2006), H3 is extensively phosphorylated at Ser10 (H3S10) during chromosome condensation and segregation by evolutionarily conserved aurora family kinases. In higher eukaryotes, aurora B is responsible for mitotic H3S10 phosphorylation and is essential for chromosome segregation (Ruchaud et al., 2007; Vader and Lens, 2008).