Singh D et al. (2014) Commun Integr Biol "A function for the midbody remnant in embryonic patterning."

Singh D, Pohl C

[Commun Integr Biol, 2014]

Asymmetric cell divisions combine cell division with fate specification and one general model of how this is achieved was proposed already decades ago(1,2): During interphase, the cell polarity axis is specified, followed by orientation of the spindle along the polarity axis and segregation of fate determinants along the polarity axis during mitosis. In most cells, the polarity axis and the spindle will usually align with the long axis that the cell had before division, also called Hertwig's rule(3-6). In the C. elegans embryo, the first polarity axis also forms along the long axis of the embryo by enrichment of myosin in the anterior(7) and formation of mutually exclusive anterior and posterior cortical polarity domains, mediated through directional cortical contractile flow(8-10). The directionality of this flow is determined by an extrinsic cue, the entry of the sperm, which inhibits Rho-dependent myosin activation at the future posterior pole by bringing with it the Rho GTPase activating protein CYK-4(11,12). Moreover, since there is no previous division 'history' before the first cleavage, mechanisms have to ensure that the nucleus-centrosome complex undergoes a 90 degree rotation so that the spindle can subsequently elongate along the long axis(13-15). Additional mechanisms ensure that the site of cleavage is perpendicular to the long axis(16,17). Hence, tight coupling of an extrinsic cue to intrinsic polarity formation and spindle elongation enables alignment of the division orientation with the long axis of the organism and successful segregation of fate determinants.

Singh D et al. (2023) Org Biomol Chem "Imaging of mitochondria/lysosomes in live cells an C. elegans."

Singh D, Soppina P, Regar R, Kanvah S, Soppina V

[Org Biomol Chem, 2023]

Two rhodamine-phenothiazine conjugates, RP1 and RP2, were synthesized, and their photophysical properties, subcellular localization, and photocytotoxicity were investigated. We observed robust localization of RP1 in mitochondria and dual localization in mitochondria and lysosomes with RP2 in live cells. Live cell imaging with these probes allowed us to track the dynamics of mitochondria and lysosomes during ROS-induced mitochondrial damage and the subsequent lysosomal digestion of the damaged mitochondria. The fluorophores also demonstrated preferential accumulation in cancer cells compared to normal cells and had strong photo-cytotoxicity. However, no cytotoxicity was observed in the dark. The mitochondrial staining and light-induced ROS production were not limited to mammalian cell lines, but were also observed in the animal model C. elegans. The study demonstrated the potential applications of these probes in visualizing the mitochondria-lysosome cross-talk after ROS production and for photodynamic therapy.

Singh D et al. (2019) J Cell Sci "Mechanical stress induces a scalable switch in cortical flow polarization during cytokinesis."

Dutta P, Pohl C, Odedra D, Singh D

[J Cell Sci, 2019]

During animal development, cells need to sense and adapt to mechanical forces from their environment. Ultimately, these forces are transduced through the actomyosin cortex. How the cortex simultaneously responds to and creates forces during cytokinesis is not well understood. Here we show that under mechanical stress, cortical actomyosin flow switches polarization during cytokinesis in the <i>C. elegans</i> embryo. In unstressed embryos, longitudinal cortical flow contributes to contractile ring formation, while rotational cortical flow is additionally induced in uniaxially loaded embryos. Rotational flow depends on astral microtubule signals and is required for the redistribution of the actomyosin cortex in loaded embryos. Rupture of longitudinally aligned cortical fibers during cortex rotation releases tension, initiates orthogonal longitudinal flow and thereby contributes to furrowing in loaded embryos. Moreover, actomyosin regulators involved in RhoA regulation, cortical polarity and chirality are all required for rotational flow and become essential for cytokinesis under mechanical stress. In sum, our findings extend the current framework of mechanical stress response during cell division and show scaling of orthogonal cortical flows to the amount of mechanical stress.

Singh D et al. (2022) J Photochem Photobiol B "Imaging of lipid droplets using coumarin fluorophores in live cells and C. elegans."

Soppina P, Kanvah S, Singh D, Soppina V, Regar R

[J Photochem Photobiol B, 2022]

Fluorescent probes offer incredibly effective tools for visualizing the dynamic morphology of lipid droplets (LDs) and investigating their physiological interactions. In this work, we have utilized solvatochromic coumarin probes bearing nitrile and ester substituents for live-cell imaging. The fluorescence probes are characterized by a donor (diethylamino) and acceptor (nitrile and/or ester) substituents and a rotatable double bond. The designed architecture allows investigation of environmental sensitivity apart from providing excellent ability to target sub-cellular organelles. The synthesized fluorophores showed low cytotoxicity and excellent localization within the lipid droplets. Further, the fluorophores were also utilized to study viscosity changes within the LDs induced by Nystatin. More importantly, we also demonstrate imaging of LDs in multi-cellular animal models such as C. elegans.

Singh D et al. (2014) Dev Cell "Coupling of rotational cortical flow, asymmetric midbody positioning, and spindle rotation mediates dorsoventral axis formation in C. elegans."

Singh D, Pohl C

[Dev Cell, 2014]

Cortical flows mediate anteroposterior polarization in Caenorhabditis elegans by generating two mutually exclusive membrane domains. However, factors downstream of anteroposterior polarity that establish the dorsoventral axis remain elusive. Here, we show that rotational cortical flow orthogonal to the anteroposterior axis during the division of the AB blastomere in the two-cell embryo positions the cytokinetic midbody remnant of the previous division asymmetrically at the future ventral side of the embryo. In the neighboring P1 blastomere, astral microtubules contact a transient PAR-2-dependent actin coat that forms asymmetrically onto the midbody remnant-P1 interface. Ablation of the midbody remnant or perturbation of rotational cortical flow reveals that microtubule-midbody remnant contacts are crucial for P1 spindle rotation and dorsoventral axis formation. Thus, our findings suggest a mechanism for dorsoventral patterning that relies on coupling of anteroposterior polarity, rotational cortical flow, midbody remnant positioning, and spindle orientation.

Sakoguchi H et al. (2016) Biosci Biotechnol Biochem "Screening of biologically active monosaccharides: growth inhibitory effects of d-allose, d-talose, and l-idose against the nematode Caenorhabditis elegans."

Izumori K, Yoshihara A, Sakoguchi H, Sato M

[Biosci Biotechnol Biochem, 2016]

We compared the growth inhibitory effects of all aldohexose stereoisomers against the model animal Caenorhabditis elegans. Among the tested compounds, the rare sugars d-allose (d-All), d-talose (d-Tal), and l-idose (l-Ido) showed considerable growth inhibition under both monoxenic and axenic culture conditions. 6-Deoxy-d-All had no effect on growth, which suggests that C6-phosphorylation by hexokinase is essential for inhibition by d-All.

Sakoguchi H et al. (2016) Bioorg Med Chem Lett "Growth inhibitory effect of d-arabinose against the nematode Caenorhabditis elegans: Discovery of a novel bioactive monosaccharide."

Shintani T, Izumori K, Sato M, Sakoguchi H, Okuma K, Yoshihara A

[Bioorg Med Chem Lett, 2016]

Biological activities of unusual monosaccharides (rare sugars) have largely remained unstudied until recently. We compared the growth inhibitory effects of aldohexose stereoisomers against the animal model Caenorhabditis elegans cultured in monoxenic conditions with Escherichia coli as food. Among these stereoisomers, the rare sugar d-arabinose (d-Ara) showed particularly strong growth inhibition. The IC50 value for d-Ara was estimated to be 7.5mM, which surpassed that of the potent glycolytic inhibitor 2-deoxy-d-glucose (19.5mM) used as a positive control. The inhibitory effect of d-Ara was also observed in animals cultured in axenic conditions using a chemically defined medium; this excluded the possible influence of E. coli. To our knowledge, this is the first report of biological activity of d-Ara. The d-Ara-induced inhibition was recovered by adding either d-ribose or d-fructose, but not d-glucose. These findings suggest that the inhibition could be induced by multiple mechanisms, for example, disturbance of d-ribose and d-fructose metabolism.

Yoshihara A et al. (2019) Bioorg Med Chem Lett "Growth inhibition by 1-deoxy-d-allulose, a novel bioactive deoxy sugar, screened using Caenorhabditis elegans assay."

Sakoguchi H, Fleet GWJ, Izumori K, Shintani T, Sato M, Yoshihara A

[Bioorg Med Chem Lett, 2019]

The biological activities of deoxy sugars (deoxy monosaccharides) have remained largely unstudied until recently. We compared the growth inhibition by all 1-deoxyketohexoses using the animal model Caenorhabditis elegans. Among the eight stereoisomers, 1-deoxy-d-allulose (1d-d-Alu) showed particularly strong growth inhibition. The 50% inhibition of growth (GI₅₀) concentration by 1d-d-Alu was estimated to be 5.4mM, which is approximately 10 times lower than that of d-allulose (52.7mM), and even lower than that of the potent glycolytic inhibitor, 2-deoxy-d-glucose (19.5mM), implying that 1d-d-Alu has a strong growth inhibition. In contrast, 5-deoxy- and 6-deoxy-d-allulose showed no growth inhibition of C. elegans. The inhibition by 1d-d-Alu was alleviated by the addition of d-ribose or d-fructose. Our findings suggest that 1d-d-Alu-mediated growth inhibition could be induced by the imbalance in d-ribose metabolism. To our knowledge, this is the first report of biological activity of 1d-d-Alu which may be considered as an antimetabolite drug candidate.

Katane M et al. (2020) Biochim Biophys Acta Proteins Proteom "Biochemical characterization of d-aspartate oxidase from Caenorhabditis elegans: Its potential use in the determination of free D-glutamate in biological samples."

Katane M, Sekine M, Nakayama K, Homma H, Kuwabara H, Saitoh Y, Miyamoto T

[Biochim Biophys Acta Proteins Proteom, 2020]

d-Aspartate oxidase (DDO) is a flavin adenine dinucleotide (FAD)-containing flavoprotein that stereospecifically acts on acidic D-amino acids (i.e., free d-aspartate and D-glutamate). Mammalian DDO, which exhibits higher activity toward d-aspartate than D-glutamate, is presumed to regulate levels of d-aspartate in the body and is not thought to degrade D-glutamate in vivo. By contrast, three DDO isoforms are present in the nematode Caenorhabditis elegans, DDO-1, DDO-2, and DDO-3, all of which exhibit substantial activity toward D-glutamate as well as d-aspartate. In this study, we optimized the Escherichia coli culture conditions for production of recombinant C. elegans DDO-1, purified the protein, and showed that it is a flavoprotein with a noncovalently but tightly attached FAD. Furthermore, C. elegans DDO-1, but not mammalian (rat) DDO, efficiently and selectively degraded D-glutamate in addition to d-aspartate, even in the presence of various other amino acids. Thus, C. elegans DDO-1 might be a useful tool for determining these acidic D-amino acids in biological samples.

Shintani T et al. (2019) J Appl Glycosci (1999) "D-Allose, a Stereoisomer of D-Glucose, Extends the Lifespan of Caenorhabditis elegans via Sirtuin and Insulin Signaling."

Izumori K, Sakoguchi H, Yoshihara A, Shintani T, Sato M

[J Appl Glycosci (1999), 2019]

D-Allose (D-All), C-3 epimer of D-glucose, is a rare sugar known to suppress reactive oxygen species generation and prevent hypertension. We previously reported that D-allulose, a structural isomer of D-All, prolongs the lifespan of the nematode Caenorhabditis elegans. Thus, D-All was predicted to affect longevity. In this study, we provide the first empirical evidence that D-All extends the lifespan of C. elegans. Lifespan assays revealed that a lifespan extension was induced by 28 mM D-All. In particular, a lifespan extension of 23.8 % was achieved (p< 0.0001). We further revealed that the effects of D-All on lifespan were dependent on the insulin gene daf-16 and the longevity gene sir-2.1, indicating a distinct mechanism from those of other hexoses, such as D-allulose, with previously reported antiaging effects.