Visualizing Spatiotemporal Dynamics of Multicellular Cell-Cycle Progression

Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198, Japan.
Cell (Impact Factor: 32.24). 03/2008; 132(3):487-98. DOI: 10.1016/j.cell.2007.12.033
Source: PubMed

ABSTRACT The cell-cycle transition from G1 to S phase has been difficult to visualize. We have harnessed antiphase oscillating proteins that mark cell-cycle transitions in order to develop genetically encoded fluorescent probes for this purpose. These probes effectively label individual G1 phase nuclei red and those in S/G2/M phases green. We were able to generate cultured cells and transgenic mice constitutively expressing the cell-cycle probes, in which every cell nucleus exhibits either red or green fluorescence. We performed time-lapse imaging to explore the spatiotemporal patterns of cell-cycle dynamics during the epithelial-mesenchymal transition of cultured cells, the migration and differentiation of neural progenitors in brain slices, and the development of tumors across blood vessels in live mice. These mice and cell lines will serve as model systems permitting unprecedented spatial and temporal resolution to help us better understand how the cell cycle is coordinated with various biological events.

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Available from: Asako Sakaue-Sawano, Sep 29, 2015
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    • "These microspheres allowed the encapsulation of single cells, eliminating the influence of cell–cell interactions. Using a fluorescent ubiquitinationbased cell cycle indicator (Fucci) [53], it was found that the cell cycle progression of single HeLa cells was delayed within hydrogel microspheres with a higher stiffness. In contrast, a significant delay in the cell cycle was not observed within hydrogel microspheres with a lower stiffness . "
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    ABSTRACT: Given the importance of regulating living cell functions in three-dimensional (3D) environments, we have designed a cytocompatible and spontaneously forming polymer hydrogel matrix. Water-soluble phospholipid polymers bearing a phenylboronic acid unit, poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-vinylphenylboronic acid) (PMBV), and poly(vinyl alcohol) (PVA), are candidate systems for preparing the hydrogel matrices. Aqueous solutions of PMBV and PVA can be used to form hydrogels based on reversible complexation between the phenylboronic acid groups in PMBV and the diol groups in PVA, even under cell culture conditions. Uniform cell encapsulation is easily achieved with hydrogel formation, and cells survived well in the hydrogel. By applying a spinning-assisted layer-by-layer (LbL) process, multilayered PMBV/PVA hydrogels containing living cells can be assembled. This multilayered hydrogel, which mimics the stratified structure of in vivo tissues, allows the layer-specific encapsulation of cells and temporary storage of bioactive molecules. Distance-dependent cell-cell interactions are investigated using the multilayered hydrogel where two cell-laden layers are separated by a finely controlled multilayered hydrogel. In addition, dual-crosslinked multilayered hydrogels are also assembled by alternative depositions of PMBV and photoreactive-PVA solutions, followed by photoirradiation. The dual-crosslinked hydrogel has a lower diffusivity of bioactive molecules than that of single-crosslinked hydrogel and therefore acts as a diffusion-controlling barrier. We demonstrate the utility of this dual-crosslinked hydrogel by examining ways to regulate the diffusion of bioactive molecules in the hydrogel and investigating the diffusion-dependent effects on cell behavior. In conclusion, these hydrogel matrices can provide insights into the regulation of cell behavior in 3D matrices. In turn, our results may contribute to the future design of 3D cell culture systems and tissue regenerated medicine based on cell engineering.
    European Polymer Journal 04/2015; DOI:10.1016/j.eurpolymj.2015.03.030 · 3.01 Impact Factor
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    • "So far, several strategies for cell lineage analyses trace the development and cell progeny of NPCs. Different approaches and transgenic mice have been used to analyze either the cell division pattern or the progenitor cycle progression as FUCCI reporters (Sakaue-Sawano et al., 2008). Moreover, newly generated cells can be labeled by birthdating methods, such as retrovirus infections, incorporation of thymidine analogs and transgenic mice (Imayoshi et al., 2011). "
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    ABSTRACT: Genetic lineage tracing with electroporation is one of the most powerful techniques to target neural progenitor cells and their progeny. However, the spatiotemporal relationship between neural progenitors and their final phenotype remain poorly understood. One critical factor to analyze the cell fate of progeny is reporter integration into the genome of transfected cells. To address this issue, we performed postnatal and in utero co-electroporations of different fluorescent reporters to label, in both cerebral cortex and olfactory bulb, the progeny of subventricular zone neural progenitors. By comparing fluorescent reporter expression in the adult cell progeny, we show a differential expression pattern within the same cell lineage, depending on electroporation stage and cell identity. Further, while neuronal lineages arise from many progenitors in proliferative zones after few divisions, glial lineages come from fewer progenitors that accomplish many cell divisions. Together, these data provide a useful guide to select a strategy to track the cell fate of a specific cell population and to address whether a different proliferative origin might be correlated with functional heterogeneity.
    Frontiers in Neuroscience 03/2015; 1(87). DOI:10.3389/fnins.2015.00087 · 3.66 Impact Factor
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    • "tGnRH-3 and tGnRH-5 Inhibit Cell Cycle Progression by Arresting the G1-S Phase Transition Cell proliferation is regulated by cell cycle progression. To investigate how tGnRH-3 and tGnRH-5 affect the cell cycle, we utilized the fluorescent cell cycle indicator Fucci (Sakaue-Sawano et al., 2008). In the Fucci system, cells at the G1 phase are labeled with red fluorescence derived from the G1 Fucci probe, while cells at the proliferative phases (S, G2, and M phases) are labeled with green fluorescence derived from the S/G2/M Fucci probe. "
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    ABSTRACT: Background: Gonadotropin-releasing hormones (GnRHs) are neuropeptides that play central roles in the reproduction of vertebrates. In the ascidian Ciona intestinalis, GnRHs and their receptors are expressed in the nervous systems at the larval stage, when animals are not yet capable of reproduction, suggesting that the hormones have non-reproductive roles. Results: We showed that GnRHs in Ciona are involved in the animal's metamorphosis by regulating tail absorption and adult organ growth. Absorption of the larval tail and growth of the adult organs are two major events in the metamorphosis of ascidians. When larvae were treated with GnRHs, they completed tail absorption more frequently than control larvae. cAMP was suggested to be a second messenger for the induction of tail absorption by GnRHs. tGnRH-3 and tGnRH-5 (the "t" indicates "tunicate") inhibited the growth of adult organs by arresting cell cycle progression in parallel with the promotion of tail absorption. Conclusions: This study provides new insights into the molecular mechanisms of ascidian metamorphosis conducted by non-reproductive GnRHs.
    Developmental Dynamics 12/2014; 243(12). DOI:10.1002/dvdy.24176 · 2.38 Impact Factor
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