Time-lapse Imaging Reveals Dynamic Relocalization of PP1γ throughout the Mammalian Cell Cycle

Abstract

Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase that regulates many cellular processes, including cell division. When transiently expressed as fluorescent protein (FP) fusions, the three PP1 isoforms, ␣, ␤/␦, and ␥1, are active phosphatases with distinct localization patterns. We report here the establishment and characterization of HeLa cell lines stably expressing either FP-PP1␥ or FP alone. Time-lapse imaging reveals dynamic targeting of FP-PP1␥ to specific sites throughout the cell cycle, contrasting with the diffuse pattern observed for FP alone. FP-PP1␥ shows a nucleolar accumulation during interphase. On entry into mitosis, it localizes initially at kinetochores, where it exchanges rapidly with the diffuse cytoplasmic pool. A dramatic relocalization of PP1 to the chromosome-containing regions occurs at the transition from early to late anaphase, and by telophase FP-PP1␥ also accumulates at the cleavage furrow and midbody. The changing spatio-temporal distribution of PP1␥ revealed using the stable PP1 cell lines implicates it in multiple processes, including nucleolar function, the regulation of chromosome segregation and cytokinesis.

Ki67 Antigen Contributes to the Timely Accumulation of Protein Phosphatase 1γ on Anaphase Chromosomes

“…Although a good population of YFP-PP1␥ is found in the cytoplasm throughout mitosis, a certain population localizes at kinetochores in metaphase (not seen clearly in the resolution obtained here), and an accumulation to the chromosomal regions occurs at the transition from early to late anaphase ( Fig. 2A) (26). Upon depletion of Ki67 with two different siRNAs (si31 or si32), the characteristic accumulation of YFP-PP1␥ on chromosomes in late anaphase was weakened in common ( Fig.…”

“…Protein phosphatase 1 (PP1), 5 along with protein phosphatase 2A (23,24), is one of the major serine/ threonine-specific phosphatase families involved in mitotic exit by counteracting mitotic kinases (25). Among three PP1 isoforms in mammals (PP1␣, ␤/␦, and ␥), PP1␥ is considered to play an important roles for chromatin decondensation and/or reactions taking place on chromatin, such as nuclear envelope assembly, as a subset of PP1␥ targets on chromosomes in early anaphase (26). PP1␥ has many interaction partners, all of which are regulatory subunits important for targeting PP1␥ to diverse subcellular structures and for restricting its activity and/or specificity (27).…”

“…Ki67 Is Involved in Ensuring the Appropriate Behavior of PP1␥ in Anaphase-To explore the physiological significance of the interaction between Ki67 and PP1␥, we first examined the possible contribution of Ki67 on the behavior of PP1␥ by utilizing HeLa cells stably expressing YFP-PP1␥ ((YG cells) (26). Although a good population of YFP-PP1␥ is found in the cytoplasm throughout mitosis, a certain population localizes at kinetochores in metaphase (not seen clearly in the resolution obtained here), and an accumulation to the chromosomal regions occurs at the transition from early to late anaphase ( Fig.…”

Pre-anaphase chromosome oscillations are regulated by the antagonistic activities of Cdk1 and PP1 on Kif18A

“…However, this seems unlikely because the phosphorylation levels of Hec1-which are highly sensitive to Aurora-B and PP1 activities at KTs 26,27 -were not affected by replacing endogenous Kif18A with GFP-Kif18A PP1D . Furthermore, fluorescence recovery after photobleaching experiments revealed that the dynamic localization of PP1 to KTs was not altered by nocodazole-induced MT depolymerization 40 , a condition that prevents Kif18A from localizing to KTs. Thus, Kif18A does not significantly contribute to the KT localization of PP1 which as shown previously is primarily mediated by KNL1 (ref.…”

Imaging of metabolites by using a fusion protein between a periplasmic binding protein and GFP derivatives: From a chimera to a view of reality

“…This jellyfish protein has three key properties: the chromophore is formed by folding of the protein itself and this occurs readily even in heterologous hosts, it absorbs and fluoresces strongly, and it has been possible to engineer a series of modified forms with altered spectral properties (9). FRET has been widely applied to monitor proteinprotein interactions (10), analyze conformational changes in molecular motors (11), monitor protein kinase activities (12) and the subcellular targeting and regulation of protein phosphatases by regulatory subunits (13), and to monitor changes in cellular free Ca 2ϩ (14,15), pH (16), protein kinase activator (17) cGMP (18), and diacylglycerol and phosphoinositides (19).…”