Arata, Yukinobu et al. (2020) MicroPubl Biol

Sako, Yasushi, Kimura, Hiroshi, Arata, Yukinobu, Ikeda, Yusaku, Oshima, Taku

[MicroPubl Biol, 2020]

OP50 is an Escherichia coli strain conventionally used as a bacterial food in the laboratory maintenance of Caenorhabditis elegans on agar plates. It has also been used to feed C. elegans in longitudinal cultures within microfluidic devices (MFDs) (Hulme et al., 2010; Li et al., 2015), where it has been subject to killing by ultraviolet irradiation or pasteurization performed to suppress clogging due to biofilm formation and aggregation (Li et al., 2015; Zhuo et al., 2017). However, the killed bacterial food can change C. elegans aging dynamics, likely due to influences on C. elegans physiology (Saul et al., 2009; Gruber et al.;, 2007; Garigan et al., 2002). Further development of longitudinal culturing systems for C. elegans in MFDs requires elucidation of the mechanisms that underlie food bacteria clogging and delineation of culture conditions in which living bacterial food can be incorporated without clogging. Bacteria switch from planktonic growth to aggregated growth under conditions of environmental stress, in the presence of toxins (e.g. antibiotics), and when there is a lack of nutrients (Trunk et al., 2018). Biofilms, such as dental plaque, are bacterial communities that are organized in a film-like form in which they are embedded in a self-produced polymeric matrix on biotic or abiotic surfaces; pellicles are floating biofilms that form at liquid-air interfaces. Meanwhile, autoaggregations are aggregated communities of bacteria suspended in solution, such as bacterial flocs formed in activated sludge. Biofilms and autoaggregations are formed by both shared and independent genetic and physico-chemical mechanisms (Trunk et al., 2018; Berne et al., 2018; Berne et al., 2015). In this study, we examined OP50 biofilm formation.Biofilm formation is mediated by flagellin proteins (e.g. FliC), which form flagella, and the adhesion protein FimH, which is located at the tips of type I pili (Berne et al., 2018, Jones et al., 1995; Pratt and Kolter, 1998; Friedlander et al., 2013). We compared the biofilm formation ability of OP50 with that of the biofilm-forming (Wood et al., 2006) wild-type BW251113 E. coli strain as well as that of two BW251113-derived knockouts produced with a kanamycin (Km) cassette characterized as biofilm formation defective mutants: JW4283: BW25113 fimH::Km (a fimH knockout) and JW1908: BW25113 fliC::Km (a fliC knockout) (Baba et al., 2006). Compared to the original BW251113 strain, BW251113 fliC::Km had a significantly reduced ability to form biofilm on glass and polystyrene (Fig. 1A and 1B, p < 0.05) and BW25113 fimH::Km had a significantly reduced ability to form biofilm on glass (Fig. 1A, p < 0.05; biofilm formation on polystyrene showed a near-significant reduction trend Fig. 1B, p = 0.0574). Compared with the original BW251113 strain, we found that OP50 had a significantly reduced biofilm formation ability on polystyrene (Fig. 1B, p < 0.05; biofilm formation on glass showed a near-significant reduction trend, Fig. 1A, p = 0.0507). The biofilm formation ability of OP50 was as low as that seen with the BW251113 biofilm formation defective mutants, and similar to that of OP50 fliC::Km and OP50 fimH::Km mutants (Fig. 1A and 1B), which were constructed by transferring fliC::Km and fimH::Km alleles to OP50 by P1 transduction (Fig. 1C and 1D). Therefore, we conclude that the original OP50 strain is itself a biofilm formation defective mutant.

Arata, Yukinobu et al. (2009) International Worm Meeting "Directional control of PAR-dependent polarization in C. elegans."

Arata, Yukinobu, Sawa, Hitoshi, Kobayashi, Tetsuya

[International Worm Meeting, 2009]

Cell polarity is properly oriented in many developmental processes such as cell migration, axonal projection, and asymmetric cell division. It has been shown that evolutionarily conserved par genes play a central role in these polarity-linked processes. In C. elegans embryo, the fertilized egg (P0) and germline lineage P cells (P1, P2, P3) undergo a series of asymmetric cell divisions to give rise to somatic cells and germline P cells. In the P0 and P1 divisions, daughter P cells are generated on the posterior side, while in the P2 and P3 divisions, daughter P cells are generated on the anterior side. However, how the polarity in P2 and P3 is established and how the direction of the polarity is determined remain unknown. First, we tested the P2 division in the selected embryos with hypomorphic par mutations where P0 and P1 divide normally. Among the embryos of par-2, par-4 and par-6 mutants, size asymmetry between the daughter cells was significantly disrupted. In addition, localizations of PAR-2 and PAR-6 were asymmetric in the isolated wild-type P2 cell using the in vitro cell isolation technique. Therefore, we conclude that asymmetric division of P2 is regulated by a PAR-dependent mechanism. Next, we examined how the direction of P2 polarity is determined. We found that the P2 polarity was always oriented toward the contact site with the adjacent somatic daughter cell, even when the contact sites were randomized in vitro. In addition, we found that this directional control was dependent on the mes-1 gene encoding a transmembrane protein by blastomere recombination, and that mes-1 is required to localize PAR-2 at the contact site. Therefore, PAR-2 localization is oriented by extracellular signal MES-1. Finally, to determine how the extracellular signal orients PAR-2 localization, we compared mobility of cortical PAR-2 protein in the presence or absence of the signal-sending cell by FRAP. The mobility was higher in the presence of the signal-sending cell, suggesting that the extracellular signal entrains asymmetric PAR-2 localization to the contact site by increasing the relative anterograde movement. The directional control of PAR-2 localization appears to be accomplished by modifying cell-autonomous polarization process.

Arata, Yukinobu et al. (2021) International Worm Meeting "Insulin signaling underlies a heavy-tailed temporal organization in C. elegans episodic swimming"

Jurica, Peter, Kimura, Hiroshi, Arata, Yukinobu, Ikeda, Yusaku, Kiyono, Ken, Sako, Yasushi, Shiga, Itsuki

[International Worm Meeting, 2021]

Episodic bouts of animal behaviors are not periodic, but temporally organized in a scale-free manner with long memory in many animal species including human. It is known that depression in human is associated with defects in the scale-free temporal organization of behaviors and impacts on social fitness, and accordingly exacerbates disease state. C. elegans crawls in on agar plate and swim in a solution while switching between actively-moving state and inactive state, which is referred to as episodic behavior. We have shown that the residence time in each state in episodic swimming has a scale-free or heavy-tailed property with long memory. Here, we studied how the residence times between two states are cross-correlated by using detrending moving-average cross-correlation analysis (DMCA) to activity time series obtained from a long-term culture of C. elegans. We found that residence times in the active and inactive states are correlated with each other over the time scale from minutes to hours, or at the ultradian time scale. This temporal cross-correlation disappeared when cultured in M9 buffer without food bacteria, and restored when cultured M9 buffer only with glucose. Additionally, the temporal cross-correlation disappeared in the mutant animals of daf-2 gene, which encodes the membrane receptor for insulin growth factor signal and in the mutant of daf-16, which encodes a downstream transcriptional repressor FOXO. Therefore, we conclude that the temporal cross-correlation is achieved by the insulin signaling. Glucose metabolism disorders and behavioral disorders are known to be closely related in human depression, raising the possibility that the temporal organization by insulin signaling found in C. elegans is conserved in humans.

Arata, Yukinobu et al. (2013) International Worm Meeting "C. elegans meets single-molecule detection technologies; The embryonic cell polarity system is driven by state transition of PAR-2 protein molecules."

Hiroshima, Michio, Pack, Chan-gi, Arata, Yukinobu, Sako, Yasushi, Kobayashi, Tetsuya, Shibata, Tatsuo

[International Worm Meeting, 2013]

Genetics analyses have shown that asymmetric localization of PAR proteins is achieved by mutual inhibition and feedback controls. But how are the physical properties of the polarity proteins controlled in vivo? Recently, we succeeded in detecting single PAR-2 molecules in living embryos using total internal reflection fluorescence microscopy and fluorescence correlation spectroscopy. PAR-2 formed oligomers, and PAR-2's high-degree oligomers accumulated asymmetrically in the posterior side of the embryo. The cortical PAR-2 localization is regulated by PKC-3-dependent phosphorylation. By comparing the dissociation rate constants from the cortex of low- and high-degree oligomers in hyper- and hypo-phosphorylated PAR-2 proteins, we found that PKC-3-dependent phosphorylation promotes, but PAR-2 oligomerization negatively regulates PAR-2's dissociation. This dual regulation broadens the range of dissociation rate constants along the a-p axis. Unexpectedly, the association rate constant of PAR-2 proteins onto the cortex was larger in the posterior region, and thereby asymmetric along the a-p axis. This asymmetry was weakened in phospho-mimic PAR-2 proteins expressed in wild-type embryos normally polarized by endogenous proteins. This suggests that the association rate asymmetry requires dephosphorylation in the cytoplasm. A mathematical model, in which all parameters were determined by experimental measurements reproduced the asymmetric localization as a bi-stable system. The physico-chemical state transitions driven by phospho-dephosphorylation cycle and oligomerization produce asymmetric patterning of PAR-2 protein particles. Thus, quantitative measurements of protein dynamics using single-molecule detection technologies and mathematical modeling provide a bridge between molecular reactions and cellular pattern formation.

Yukinobu ARATA et al. (2002) Japanese Worm Meeting "psa-3, ceh-20, and nob-1 regulate asymmetric cell division."

Yukinobu ARATA, Hiroko Kouike, Hitoshi Sawa, Hideyuki Okano

[Japanese Worm Meeting, 2002]

Asymmetric cell division is a fundamental mechanism to generate cellular diversity. The T cells, which are the blast cells on the tail of C. elegans , asymmetrically divide into the two daughter cells with distinct fates; the anterior daughter generates neural cells, while the posterior daughter generates hypodermal cells. It has been shown that the T cell division is regulated by LIN-44 (Wnt) and LIN-17 (Frizzled). Here we report other three genes, psa-3 , psa-11 , and psa-9 , which regulate the asymmetric cell division of the T cell. First, psa-3 is homologous to transcriptional factors, Meis and Homothorax (HTH). We found a 150 bps-deletion in the promoter of the psa-3 gene in the psa-3 mutant (os8) . This region contains a binding sequence of POP-1 (TCF) (ref, 1). POP-1 is a transcriptional factor involved in Wnt signaling cascade. This presumably indicates that Wnt signaling regulates PSA-3 expression. Second, psa-11 turned out to be ceh-20 , a homologue of transcriptional factor PBX and Extradenticle (EXD) that have an atypical homeobox, TALE-homeobox (ref, 2). Lastly, psa-9 is a HOX gene, nob-1 , which has an essential function in the formation of the posterior structures. It has been shown that Meis (HTH), PBX (EXD) and HOX proteins form a ternary complex and are involved in morphogenesis and tumorigenesis in metazoans. In budding yeast, MAT-alpha2, which has TALE-homeobox, and MAT-a1, which has conventional homeobox, are the cell-fate determinants required after asymmetric cell division. We presume that PSA-3, CEH-20, and NOB-1 form a ternary complex and function as the determinant in the one of the daughters of the T cell. Recently, we have reported that SWI/SNF, chromatin-remodeling complex is involved in the asymmetric cell division in C. elegans as well as in budding yeast (ref, 3). Asymmetric cell division in C. elegans may be regulated by the similar mechanism to that in budding yeast. References 1) Korswagen, H.C. et al. (2000) Nature 406, 527-532. 2) Burglin TR (1997) Nucleic Acids Res. 25, 4173-4180 3) Sawa, H. et al. (2000) Mol. Cell 6, 617-624.

Yukinobu ARATA et al. (2005) International Worm Meeting "Quantitative observations of division timing in C. elegans embryo"

Hitoshi Sawa, Yukinobu ARATA

[International Worm Meeting, 2005]

In early embryonic development, size of blastomeres and gap of cell-division timings are critical for correct cell positioning that is consequently important for cell-cell interactions. Since we are interested in developmental regularities at the cellular level, we precisely measured cell volume and cell cycle duration time. We found that cell cycle duration time was in inverse proportion to approximately the 0.28th power of cell size in at least all the founder cells (except the P4 cell) and AB and MS descendants. This result suggests that cell size is an important factor to determine cell cycle duration time. Therefore, production of daughters with different cell sizes though asymmetric cell division may contribute to make gaps of cell-division timings. In contrast, E cell daughters and P cells (P1, P2, P3, P4) were remarkably out of the regularity. Especially, the E cell daughters underwent gastrulation during the prolonged cell cycle time. Therefore, we are interested in another regularity that determines cell-division timing in E cell daughters and P cells. For this purpose, we carefully observed order of cell divisions. We found that most cell divisions occurred nearly synchronously throughout embryo. However, in each synchronized cell divisions, anteriorly-positioned cells tended to divide slightly earlier than posteriorly-positioned cells. This phenomenon seems to correspond to mitotic wave that has been reported in Drosophila, Xenopus and Zebrafish development. Interestingly, E cell daughters and P cells also followed the rule of the division order, despite the fact that they ignore the regularity of cell size. Therefore, cell-division timing of E cell daughters and P cells are likely to be, if not all, under the control of the mitotic wave rather than that of cell size. In this presentation, we would like to discuss the possibility that precise cell cycle duration time is determined by cooperation between cell autonomous mechanism and regulatory mechanism though cell-cell interaction.

Yukinobu Arata et al. (2007) International Worm Meeting "Gut attracts Germ by orienting an asymmetric cell division in C. elegans."

Yukinobu ARATA, Bob Goldstein, Hitoshi Sawa

[International Worm Meeting, 2007]

In C. elegans</I>, the final step in the segregation of the somatic and germ lines is asymmetric cell division of the P3 cell in the 16 cell-stage embryo. The P3 division produces mesodermal precursor (D) and the primordial germ cell (P4), and P4 is always produced on the side of the endodermal precursor (E.p). Since the endoderm functions as nurse cells of the germ cells in C. elegans</I>, the direction of the P3 division is critical for germ line development. Because PAR-2 protein is localized asymmetrically during the P3 division, the orientation of PAR-2 localization seems to be critical to direct the P3 division. We found that when isolated P3 cells were cultured alone, PAR-2 was asymmetrically segregated into the P4 cell, suggesting that P3 can be polarized in cell autonomous fashion. Nevertheless, the PAR-2 protein was always localized at the boundary between these two cells and the P3 division was oriented toward the E.p cells, even when the positions of contacts between P3 and E.p was randomized in vitro</I>. These results indicate that the PAR-2 localization in the P3 division is directed by an extracellular signal from the endodermal precursor. To identify the extracellular signal, we conducted a cell recombination experiment, in which the P3 cell isolated from wild-type is attached to the E.p cell from a mutant embryo. We found that the P3 divisions were not directed by the E.p cells isolated from mes-1</I> mutants. MES-1, a transmembrane protein containing a tyrosine kinase-like domain, is localized at the cell boundary between P3 and E.p. This suggests that MES-1 provides a positional cue to direct the PAR-2 localization. Now we are studying molecular mechanism how MES-1 determines PAR-2 localization. In our presentation, we will also discuss the role of endodermal tissues on germ line development in other organisms.

Yukinobu ARATA et al. (2008) Development & Evolution Meeting "PAR-dependent polarization is oriented by an extracellular signal MES-1 in C. elegans"

Hitoshi Sawa, Bob Goldstein, Yukinobu ARATA, Tetsuya Kobayashi

[Development & Evolution Meeting, 2008]

Cell polarity needs to be oriented in many developmental processes; cell migration, axonal projection, and asymmetric cell division. At the 16-cell stage in the C. elegans embryo, the P3 cell divides asymmetrically to produce the large C cell and the small P4 cell, which is a primordial germ cell. This division is oriented toward the endodermal precursor (E) such that the P4 cell forms proximal to the E cell. To study mechanisms of the asymmetric division of the P3 cell, we used an in vitro cell-isolation system. When the P3 cell was isolated and cultured alone, the P3 cell divided asymmetrically. Notably, when the isolated P3 cell was re-attached to the E cell, the P3 division was oriented toward the E cell. To identify the extracellular signals that orient the P3 division, we attached the wild-type P3 cell with the E cell of mes-1mutants. mes-1 encodes a transmembrane protein, which is localized at the boundary between the P3 cell and the E cell. In the recombined embryos, the P3 division failed to be directed. These results indicate that the asymmetric division of the P3 cell is polarized spontaneously, but nevertheless is directed by the extracellular signal, MES-1 from the E cell. We further demonstrated that MES-1 and SRC-1 tyrosine kinase regulate the localization of PAR-2, the polarity regulator of zygote (P0), to the boundary between the P3 cell and the E cell. Moreover, we found that par-2 is required for asymmetric division of the P3 cell. Therefore, we conclude that MES-1/SRC-1 signaling provides a positional cue to direct the PAR-dependent cell polarization in the P3 division. We also introduce a novel approach using a program for quantitative data acquisition and mathematical modeling to understand the process of orienting cell polarity as a dynamical system.

Yukinobu Arata et al. (2007) International Worm Meeting "Quantitative measurement of the timing of cell division in C. elegans embryo."

Hitoshi Sawa, Yukinobu ARATA

[International Worm Meeting, 2007]

During animal development, cell cycle progression seems to be regulated tightly. In this study, we quantitatively measured cell volume and cell cycle duration. We found that cell cycle duration was in inverse proportion to the 0.26 (~1/4) th power of cell volume in the AB and MS lineages and the 0.34 (~1/3) th power of cell volume in the C lineage, but was independent of cell volume in the E and D lineages. These findings indicate that cells divide in size-dependent and -independent manners during the development, and suggest that cells possess unidentified mechanisms to regulate cell cycle progression, depending on cell volume. In addition, we also observed a relationship between cell division timing and cell position in the embryo, and showed that most cell divisions occur semi-synchronously throughout the embryo; anteriorly-positioned cells tended to divide slightly earlier than those in the posterior. This phenomenon seems to correspond to the mitotic wave that has been reported in Drosophila</I>, Xenopus</I> and zebrafish development. Is cell division timing affected by cell division in adjacent cells? We changed the relative positions of cells in embryos cultured in vitro</I>. Although most cells divide in the same order as in the embryo in egg shell, the division order of E cell daughters was earlier. This result suggests that cell division timing is affected by the embryonic microenvironment. Given these findings, we propose that cell cycle progression is regulated by size- and position-dependent mechanisms in the C. elegans</I> embryo.

Yukinobu ARATA et al. (2004) East Asia Worm Meeting "Transcriptional integration of cell-polarity signal and positional-identity cue specifies asymmetric cell division."

Hiroko Kouike, Hitoshi Sawa, Yukinobu ARATA, Hideyuki Okano

[East Asia Worm Meeting, 2004]

In C. elegans development, most of cell division is known to be asymmetric. Almost all of the asymmetric cell divisions are supposed to be regulated by a universal cell polarity system that includes at least pop-1 /TCF, lit-1 /NLK, wrm-1 /beta-catenins that are components of the Wnt signaling pathway. Our question is how the universal cell-polarity system generates the specific sets of asymmetric daughter cells in each asymmetric cell division. We identified three genes that are involved in asymmetric cell division of the T cell that are psa-3 /Meis, ceh-20 /Pbx and nob-1 /Hox. Interestingly, expression of PSA-3 was spatially restricted; not only specific in the T cell among the seam cells, but also asymmetric between the T cell daughters. So far, we have demonstrated that psa-3 /Meis is a direct target gene of POP-1/TCF by genetic and molecular approach. This indicates that psa-3 /Meis is a target of the cell polarity signal. In addition, we found that the expression of psa-3 /Meis was remarkably decreased in nob-1 /Hox and ceh-20 /Pbx mutants. By using deletion series of psa-3::gfp , we found that the ~100 bps on the intron 4 that is weakly conserved between C. elegans and C. briggsae , and has two putative Hox-Pbx binding sites, is important for psa-3::gfp expression. By gel shift assay, we demonstrated that NOB-1/Hox directly binds at least to the one of the both sites. Furthermore, by protein binding assay, we demonstrated that NOB-1/Hox directly interacts with CEH-20/Pbx in vitro . These data suggest that the complex of NOB-1/Hox and CEH-20/Pbx that is responsible for positional identity of the T cell, is directly involved in psa-3 /Meis transactivation. Taken together, we discovered that spatially restricted expression pattern of psa-3 /Meis is caused by transcriptional integration of the cell polarity signal and the positional identity cue, whose mechanism plays an essential role to carry out the position-specific asymmetric cell division.