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Cover  Reversible protein phosphorylation regulates many processes in eukaryotic cells, including cell division, and multiple kinases activated in mitosis have been identified. Although protein phosphatase 1 (PP1) has been implicated as a major counteracting phosphatase, in vivo evidence linking PP1 to specific sites of action within mitotic mammalian cells remains elusive. Fluorescent protein (FP) fusions facilitate studies on the localization of PP1 in live cells. Following FP-PP1 through mitosis in live cells, however, required the establishment of cell lines stably expressing FP-PP1 fusion proteins. The 1 isoform was chosen for these experiments because its upregulation in certain tumor cell lines (Sogawa et al. [1995]. Cancer Lett. 89:1-6; Sogawa et al. [1997]. Cancer Lett. 112:263-268) suggested an important role in cell division. Live cell imaging reveals that the localization of FP-PP1 is highly dynamic throughout cell division. Upon entry into mitosis, the predominant nucleolar localization of FP-PP1 is disrupted as nucleoli disassemble, and PP1 accumulates at kinetochores, which appear at the end of microtubules and are distinct from centromeres. The cover shows a fluorescence micrograph of a 2D additive projection of 3D data collected from a metaphase HeLa cell stably expressing YFP-tagged PP1 (green). The cell was fixed and stained for microtubules with anti--tubulin (red) and for chromosomes with DAPI (blue). Kinetochore localization has also been observed for the yeast PP1 homologue, GFP-Glc7 (Bloecher and Tatchell [2000]. J Cell Biol. 149:125-140). Importantly, it places PP1 at precise sites where and when phosphatase activity is known to be required, based on evidence for a role for PP1 in both the microtubule binding of the kinetochore complex (Sassoon et al. [1999]. Genes Dev. 13:545-555) and in spindle stability (Tournebize et al. [1997] Embo J. 16:5537-5549).Laura Trinkle-Mulcahy