Kiyoko Fukami

Visualizing Spatiotemporal Dynamics of Multicellular Cell-Cycle Progression

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.

Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain

Optical methods for viewing neuronal populations and projections in the intact mammalian brain are needed, but light scattering prevents imaging deep into brain structures. We imaged fixed brain tissue using Scale, an aqueous reagent that renders biological samples optically transparent but completely preserves fluorescent signals in the clarified structures. In Scale-treated mouse brain, neurons labeled with genetically encoded fluorescent proteins were visualized at an unprecedented depth in millimeter-scale networks and at subcellular resolution. The improved depth and scale of imaging permitted comprehensive three-dimensional reconstructions of cortical, callosal and hippocampal projections whose extent was limited only by the working distance of the objective lenses. In the intact neurogenic niche of the dentate gyrus, Scale allowed the quantitation of distances of neural stem cells to blood vessels. Our findings suggest that the Scale method will be useful for light microscopy–based connectomics of cellular networks in brain and other tissues.

Cosignaling of NCAM via lipid rafts and the FGF receptor is required for neuritogenesis

The neural cell adhesion molecule (NCAM) has been reported to stimulate neuritogenesis either via nonreceptor tyrosine kinases or fibroblast growth factor (FGF) receptor. Here we show that lipid raft association of NCAM is crucial for activation of the nonreceptor tyrosine kinase pathway and induction of neurite outgrowth. Transfection of hippocampal neurons of NCAM-deficient mice revealed that of the three major NCAM isoforms only NCAM140 can act as a homophilic receptor that induces neurite outgrowth. Disruption of NCAM140 raft association either by mutation of NCAM140 palmitoylation sites or by lipid raft destruction attenuates activation of the tyrosine focal adhesion kinase and extracellular signal–regulated kinase 1/2, completely blocking neurite outgrowth. Likewise, NCAM-triggered neurite outgrowth is also completely blocked by a specific FGF receptor inhibitor, indicating that cosignaling via raft-associated kinases and FGF receptor is essential for neuritogenesis.

Illuminating cell-cycle progression in the developing zebrafish embryo

By exploiting the cell-cycle-dependent proteolysis of two ubiquitination oscillators, human Cdt1 and geminin, which are the direct substrates of SCFSkp2 and APCCdh1 complexes, respectively, Fucci technique labels mammalian cell nuclei in G1 and S/G2/M phases with different colors. Transgenic mice expressing these G1 and S/G2/M markers offer a powerful means to investigate the coordination of the cell cycle with morphogenetic processes. We attempted to introduce these markers into zebrafish embryos to take advantage of their favorable optical properties. However, although the fundamental mechanisms for cell-cycle control appear to be well conserved among species, the G1 marker based on the SCFSkp2-mediated degradation of human Cdt1 did not work in fish cells, probably because the marker was not ubiquitinated properly by a fish E3 ligase complex. We describe here the generation of a Fucci derivative using zebrafish homologs of Cdt1 and geminin, which provides sweeping views of cell proliferation in whole fish embryos. Remarkably, we discovered two anterior-to-posterior waves of cell-cycle transitions, G1/S and M/G1, in the differentiating notochord. Our study demonstrates the effectiveness of using the Cul4Ddb1-mediated Cdt1 degradation pathway common to all metazoans for the development of a G1 marker that works in the nonmammalian animal model.

Phospholipase Cδ4 is required for Ca2+ mobilization essential for acrosome reaction in sperm

Zona pellucida (ZP)–induced acrosome reaction in sperm is a required step for mammalian fertilization. However, the precise mechanism of the acrosome reaction remains unclear. We previously reported that PLCδ4 is involved in the ZP-induced acrosome reaction in mouse sperm. Here we have monitored Ca2+ responses in single sperm, and we report that the [Ca2+]i increase in response to ZP, which is essential for driving the acrosome reaction in vivo, is absent in PLCδ4−/− sperm. Progesterone, another physiological inducer of the acrosome reaction, failed to induce sustained [Ca2+]i increases in PLCδ4−/− sperm, and consequently the acrosome reaction was partially inhibited. In addition, we observed oscillatory [Ca2+]i increases in wild-type sperm in response to these acrosome inducers. Calcium imaging studies revealed that the [Ca2+]i increases induced by exposure to ZP and progesterone started at different sites within the sperm head, indicating that these agonists induce the acrosome reaction via different Ca2+ mechanisms. Furthermore, store-operated channel (SOC) activity was severely impaired in PLCδ4−/− sperm. These results indicate that PLCδ4 is an important enzyme for intracellular [Ca2+]i mobilization in the ZP-induced acrosome reaction and for sustained [Ca2+]i increases through SOC induced by ZP and progesterone in sperm.

IDENTIFICATION OF A PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE-BINDING SITE IN CHICKEN SKELETAL MUSCLE ALPHA -ACTININ

We previously reported that phosphatidylinositol 4,5-bisphosphate (PIP2) dramatically increases the gelating activity of smooth muscle α-actinin (Fukami, K., Furuhashi, K., Inagaki, M., Endo, T., Hatano, S., and Takenawa, T.(1992) Nature 359, 150-152) and that the hydrolysis of PIP2 on α-actinin by tyrosine kinase activation may be important in cytoskeletal reorganization (Fukami, K., Endo, T., Imamura, M., and Takenawa, T.(1994) J. Biol. Chem. 269, 1518-1522). Here we report that a proteolytic fragment with lysylendopeptidase comprising amino acids 168-184 (TAPYRNVNIQNFHLSWK) from striated muscle α-actinin contains a PIP2-binding site. A synthetic peptide composed of the 17 amino acids remarkably inhibited the activities of phospholipase C (PLC)-γ1 and -δ1. Furthermore, we detected an interaction between PIP2 and a bacterially expressed α-actinin fragment (amino acids 137-259) by PLC inhibition assay. Point mutants in which arginine 172 or lysine 184 of α-actinin were replaced by isoleucine reduced the inhibitory effect on PLC activity by nearly half. Direct interactions between PIP2 and the peptide (amino acids 168-184) or the bacterially expressed protein (amino acids 137-259) were confirmed by enzyme-linked immunosorvent assay. We also found this region homologous to the sequence of the PIP2-binding site in spectrin and the pleckstrin homology domains of PLC-δ1 and Grb7. Synthetic peptides from the homologous regions in spectrin and PLC-δ1 inhibited PLC activities. These results indicate that residues 168-184 comprise a binding site for PIP2 in α-actinin and that similar sequences found in spectrin and PLC-δ1 may be involved in the interaction with PIP2.

Lipid Rafts and Caveolin-1 Are Required for Invadopodia Formation and Extracellular Matrix Degradation by Human Breast Cancer Cells

Invadopodia are ventral membrane protrusions through which invasive cancer cells degrade the extracellular matrix. They are thought to function in the migration of cancer cells through tissue barriers, which is necessary for cancer invasion and metastasis. Although many protein components of invadopodia have been identified, the organization and the role of membrane lipids in invadopodia are not well understood. In this study, the role of lipid rafts, which are cholesterol-enriched membrane microdomains, in the assembly and function of invadopodia in human breast cancer cells was investigated. Lipid rafts are enriched, internalized, and dynamically trafficked at invadopodia sites. Perturbation of lipid raft formation due to depleting or sequestering membrane cholesterol blocked the invadopodia-mediated degradation of the gelatin matrix. Caveolin-1 (Cav-1), a resident protein of lipid rafts and caveolae, accumulates at invadopodia and colocalizes with the internalized lipid raft membranes. Membrane type 1 matrix metalloproteinase (MT1-MMP), a matrix proteinase associated with invadopodia, is localized at lipid raft-enriched membrane fractions and cotrafficked and colocalized with Cav-1 at invadopodia. The small interfering RNA-mediated silencing of Cav-1 inhibited the invadopodia-mediated and MT1-MMP-dependent degradation of the gelatin matrix. Furthermore, Cav-1 and MT1-MMP are coexpressed in invasive human breast cancer cell lines that have an ability to form invadopodia. These results indicate that invadopodia are the sites where enrichment and trafficking of lipid rafts occur and that Cav-1 is an essential regulator of MT1-MMP function and invadopodia-mediated breast cancer cell invasion.

Phospholipase C is a key enzyme regulating intracellular calcium and modulating the phosphoinositide balance.

Spatial and temporal activation of phosphoinositide turnover enables eukaryotic cells to perform various functions such as cell proliferation/differentiation, fertilization, neuronal functions, and cell motility. In this system, phospholipase C (PLC) is a key enzyme, which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) into two second messengers, inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and diacylglycerol (DAG). Ins(1,4,5)P(3) triggers the release of calcium from intracellular stores, and DAG mediates the activation of protein kinase C (PKC). In parallel, PI(4,5)P(2) also directly regulates a variety of cellular functions, including cytoskeletal remodeling, cytokinesis, phagocytosis, membrane dynamics, and channel activity, in addition to its role as a substrate for PLC and phosphatidylinositol 3-kinase (PI3K), which generates PI(3,4,5)P(3). An imbalance of these phosphoinositides contributes to the pathogeneses of various human diseases. Therefore, strict regulation of the levels of PI(4,5)P(2) and PI(3,4,5)P(3) by PLC or other interconverting enzymes is necessary for cellular functions. In this review, we focus on the roles of PLC as a calcium-regulating enzyme and as a modulator of the phosphoinositide balance.

Sperm Factor Induces Intracellular Free Calcium Oscillations by Stimulating the Phosphoinositide Pathway

Abstract Injection of a porcine cytosolic sperm factor (SF) or of a porcine testicular extract into mammalian eggs triggers oscillations of intracellular free calcium ([Ca2+]i) similar to those initiated by fertilization. To elucidate whether SF activates the phosphoinositide (PI) pathway, mouse eggs or SF were incubated with U73122, an inhibitor of events leading to phospholipase C (PLC) activation and/or of PLC itself. In both cases, U73122 blocked the ability of SF to induce [Ca2+]i oscillations, although it did not inhibit Ca2+ release caused by injection of inositol 1,4,5-triphosphate (IP3). The inactive analogue, U73343, had no effect on SF-induced Ca2+ responses. To determine at the single cell level whether SF triggers IP3 production concomitantly with a [Ca2+]i rise, SF was injected into Xenopus oocytes and IP3 concentration was determined using a biological detector cell combined with capillary electrophoresis. Injection of SF induced a significant increase in [Ca2+]i and IP3 production in these oocytes. Using ammonium sulfate precipitation, chromatographic fractionation, and Western blotting, we determined whether PLCγ1, PLCγ2, or PLCδ4 and/or its splice variants, which are present in sperm and testis, are responsible for the Ca2+ activity in the extracts. Our results revealed that active fractions do not contain PLCγ1, PLCγ2, or PLCδ4 and/or its splice variants, which were present in inactive fractions. We also tested whether IP3 could be the sensitizing stimulus of the Ca2+-induced Ca2+ release mechanism, which is an important feature of fertilized and SF-injected eggs. Eggs injected with adenophostin A, an IP3 receptor agonist, showed enhanced Ca2+ responses to CaCl2 injections. Thus, SF, and probably sperm, induces [Ca2+]i rises by persistently stimulating IP3 production, which in turn results in long-lasting sensitization of Ca2+-induced Ca2+ release. Whether SF is itself a PLC or whether it acts upstream of the eggs PLCs remains to be elucidated.

The Role of EF-hand Domains and C2 Domain in Regulation of Enzymatic Activity of Phospholipase Cζ

Sperm-specific phospholipase C-ζ (PLCζ) induces Ca2+ oscillations and egg activation when injected into mouse eggs. PLCζ has such a high Ca2+ sensitivity of PLC activity that the enzyme can be active in resting cells at ∼100 nm Ca2+, suitable for a putative sperm factor to be introduced into the egg at fertilization (Kouchi, Z., Fukami, K., Shikano, T., Oda, S., Nakamura, Y., Takenawa, T., and Miyazaki, S. (2004) J. Biol. Chem. 279, 10408–10412). In the present structure-function analysis, deletion of EF1 and EF2 of the N-terminal four EF-hand domains caused marked reduction of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-hydrolyzing activity in vitro and loss of Ca2+ oscillation-inducing activity in mouse eggs after injection of RNA encoding the mutant. However, deletion of EF1 and EF2 or mutation of EF1 or EF2 at the x and z positions of the putative Ca2+-binding loop little affected the Ca2+ sensitivity of the PLC activity, whereas deletion of EF1 to EF3 caused 12-fold elevation of the EC50 of Ca2+ concentration. Thus, EF1 and EF2 are important for the PLCζ activity, and EF3 is responsible for its high Ca2+ sensitivity. Deletion of four EF-hand domains or the C-terminal C2 domain caused complete loss of PLC activity, indicating that both regions are prerequisites for PLCζ activity. Screening of interactions between the C2 domain and phosphoinositides revealed that C2 has substantial affinity to PI(3)P and, to the lesser extent, to PI(5)P but not to PI(4,5)P2 or acidic phospholipids. PI(3)P and PI(5)P reduced PLCζ activity in vitro, suggesting that the interaction could play a role for negative regulation of PLCζ.