Miyanishi, Yosuke et al. (2011) International Worm Meeting "Functional imaging of neuronal activity for 2-nonanone stimulation."

Nakai, Junichi, Kimura, Kotaro, Miyanishi, Yosuke

[International Worm Meeting, 2011]

To better understand the neural basis that regulates a worm's sensory behavior and its modulation by learning, we are studying avoidance behavioral responses to 2-nonanone. We previously reported that the avoidance behavior to 2-nonanone is enhanced, rather than reduced, after preexposure to the odor, and this enhancement is acquired as a non-associative dopamine-dependent learning (Kimura et al., J. Neurosci., 2010; Fujita and Kimura, this abstract). In addition, we observed that worms respond to a spatial gradient of 2-nonanone (Yamazoe and Kimura, CE Neuro, 2010), which cannot be simply explained by the pirouette or weathervane strategies. 2-nonanone is mainly sensed by the AWB neurons, which have been shown to exhibit odor-OFF response in aqueous step stimulation with 2-nonanone (Troemel et al., Cell 1997; Ha et al., Neuron 2010). To understand how the neuronal circuits of worms regulate the characteristic 2-nonanone behavioral response, we are monitoring calcium changes in the AWB and downstream neurons using G-CaMP 4 (Shindo et al., PLoS ONE, 2010). We thank Drs. S. Oda, K. Yoshida, and Y. Iino (U. Tokyo) for suggestions on microfluidics; M. Hendricks and Y. Zhang (Harvard) for aqueous 2-nonanone stimulation; and E. Busch and M. de Bono (MRC) for gaseous microfluidic stimulation.

Kimura, Akatsuki (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Quantiative measurement and modeling of organelle positioning in the one-cell stage C. elegans embryo"

Kimura, Akatsuki

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The intracellular positioning of organelles and other components is important for cells to function. The one-cell stage embryo of C. elegans is a good and well-studied model to address the mechanics underlying organelle positioning. We have been focusing on the positioning of the centrosomes. By calculating the expected forces exerted by microtubules and comparing the forces with the position and velocity of the movements of centrosomes in vivo, we were able to propose feasible models to explain the positioning of the centrosomes during pronuclear centration [1], asymmetric spindle displacement [2], and spindle elongation [3]. For better understanding of the organelle positioning inside the cell, the physical properties of the cytoplasm is critical. We are starting to construct a physical model to describe dynamic behaviors of the cytoplasm. [References] 1. A. Kimura & S. Onami (2005) Computer simulations and image processing reveal length-dependent pulling force as the primary mechanism for C. elegans male pronuclear migration. Dev. Cell., 8: 765-775. 2. A. Kimura & S. Onami (2007) Local cortical pulling-force repression switches centrosomal centration and posterior displacement in C. elegans. J. Cell Biol., 179: 1347-1354. 3. Y. Hara & A. Kimura (2009) Cell-size-dependent spindle elongation in the Caenorhabditis elegans early embryo. Curr. Biol., 19: 1549-1554.

Tomoko Aso et al. (2004) East Asia Worm Meeting "The effect of oxidized fatty acid on C.elegans antioxidant status measured by electron spin resonance (ESR)."

Shinya Matsumoto, Akiko Souma, Tomoko Aso, Yasuki Matsumura, Rie Kimura, Tomohiko Mori

[East Asia Worm Meeting, 2004]

Unsaturated fatty acids are easily oxidized because of the oxygen susceptibility of unsaturated bonds in the molecules. Many studies have shown that oxidized fatty acids have variety of adverse effects on organisms as oxidative stress, e.g., cell membrane oxidation and DNA damage. It is known that oxidative stress induces aging and shortens lifespan, but the mechanisms are remained largely unanswered. We asked whether the fed oxidized fatty acid have changed redox status in C. elegans by instrumental analysis.We used electron spin resonance (ESR), which is a useful analysis for free radical studies. ESR, which is a popular and traditional instrument in the antioxidation analysis, can detect free radical and gives specific spectrum of the radical. When nitroxide radicals such as Tempol are administered to an animal, they are reduced by hosts' redox systems and lose ESR signals. So far, this measurement has not been applied on C.elegans . We examined changes in the ESR signal of Tempol administered to C.elegans and measured of the reduced rate of Tempol as intensity of the reduction activity of worms. We fed wild type worms with hydroperoxides and the oxidized fatty acids. Then, we examined the effects of the oxidants on redox status of C.elegans . In this analysis, we observed reduction of Tempol in wild type. In C.elegans which was fed bacteria together with oxidized fatty acids and hydroperoxides, enhanced reduction activity had been observed in comparison with wild type. These results showed that it is possible to measure the reduction activity of C.elegans with ESR and suggested that the antioxidant activity of C.elegans fed with the oxidants was enhanced because of the oxidative stress

Shinya Matsumoto et al. (2005) International Worm Meeting "Effect of oxidized fatty acids on C. elegans redox status."

Tomoko Aso, Rie Kimura, Shinya Matsumoto, Tomohiko Mori, Akiko Soma, Yasuki Matsumura

[International Worm Meeting, 2005]

Oxidized fatty acids, when orally administered, cause toxic effect to organism. Although the adverse effect of oxidative stress upon cell and cellular components such as plasma membrane, protein and DNA, has been intensively studied, the exact molecular mechanism concerning toxic effect of oral administration of oxidized fatty acids is not fully understood. To evaluate the effect of oxidized fatty acids from the stand point of nutrition and bio-safety, we fed C.elegans with oxidized fatty acids and observed the effect of the oxidized fatty acids upon fertility, life span and the expression of genes which are implicated on antioxidative stress. Oxidized oleic (18:1), linoleic (18:2), arachidonic (20:4), icosapentaenoic (20:5) and docosahexaenoic acid (22:6) were incorporated into debris of OP50 membrane and seeded with live OP50 on NGM plate. N2 eggs were laid on the plate and the hatched worms were exposed to the oxidized fatty acids throughout their development. The number of progeny derived from the oxidized fatty acids-fed worms showed little change compared with that of normal OP50-fed worms, indicating oxidized fatty acids had no effect of the fertility of worms. Oxidized fatty acids induced increased death around 7-10 days after hatch, which was not observed in OP50-fed worms. The average life span (50% death length) and the maximum life span were less influenced by the oxidized fatty acids. This indicates that oxidized fatty acid susceptibility increased temporarily or stage-specifically during worm life time. The expression of SOD and catalase have shown to be enhanced during this stage. We then asked whether the fed oxidized fatty acid have changed redox status in C. elegans by electron spin resonance (ESR). ESR detect free radical compound based on the existence of unpaired electron in the molecule and gives specific spectrum of the radical. When stable nitroxide radicals such as Tempol are administered to an animal, they are reduced either by hosts' redox systems or endogenous free radicals and consequently the Tempol signal intensity is weakened. Therefore, by comparing the decrease rate of Tempol signal intensity, the endogenous redox status of the animal can be evaluated. We fed N2 worms with hydroperoxides and the oxidized fatty acids. Tempol was added to the worms and the effects of the oxidants on redox status of the worms was examined by ESR. In worms which were fed bacteria together with oxidized fatty acids and hydroperoxides, enhanced reduction activity of Tempol had been observed in comparison with worms fed without oxidants. These results may indicate the oxidized fatty acids may induce oxidative stress on worm by increasing the endogenous free radicals.

Shinya Matsumoto et al. (2004) East Asia Worm Meeting "Effect of oxidized fatty acids on viability of C.elegans."

Akiko Souma, Rie Kimura, Yasuki Matsumura, Tomohiko Mori, Tomoko Aso, Shinya Matsumoto

[East Asia Worm Meeting, 2004]

Oxidized fatty acids, when orally administered, cause toxic effect to organism, in the worst case, to the death. Although the adverse effect of oxidative stress upon cell or cellular components, such as plasma membrane, protein and DNA, has been intensively studied, the exact molecular mechanism concerning toxic effect of oral administration of oxidized fatty acids is not fully understood. To evaluate the effect of oxidized fatty acids from the stand point of nutrition and bio-safety, we fed C.elegans with oxidized fatty acids and observed the effect of the oxidized fatty acids upon fertility, life span and the expression of genes which are implicated on antioxidative stress.Oxidized oleic (18:1), linoleic (18:2), arachidonic (20:4), icosapentaenoic (20:5) and docosahexaenoic acid (22:6) were incorporated into debris of OP50 membrane and seeded with live OP50 on NGM plate. N2 eggs were laid on the plate and the hatched worms were exposed to the oxidized fatty acids throughout their life-time. The number of progeny derived from the oxidized fatty acids-fed worms showed little change compared to those of normal OP50-fed worms, indicating oxidized fatty acids had no effect of the fertility of worms. Oxidized fatty acids induced increased death around 7-10 days after hatch, which was not observed in OP50-fed worms. The average life span (50% death length) and the maximum life span were less influenced by the oxidized fatty acids. This indicates that oxidized fatty acid susceptibility increased temporarily or stage-specifically during worm life time. The expression of SOD and catalase have shown to be enhanced during this stage.

Akatsuki Kimura et al. (2006) East Asia C. elegans Meeting "A micro-movement analysis on dynamic positioning of centrosomes in one-cell embryo"

Shuichi Onami, Akatsuki Kimura

[East Asia C. elegans Meeting, 2006]

Positioning of the centrosomes in a cell is critical for the intracellular organization of organelles and cell division plane. We are studying the mechanisms of centrosome positioning in C. elegans one-cell embryo. Following fertilization, the sperm-supplied centrosomes migrate centripetally from a periphery to the center of the embryo. Later, centrosomes migrate centrifugally from the center towards a posterior position, which results in asymmetric first cell division. For the centripetal migration, we provided evidence that the length-dependent pulling force is the primary force [1]. For the centrifugal migration, the cortical pulling force is considered critical [2]. However, little is known about how these forces are controlled spatially and temporary to enable successive migrations of the centrosomes towards different directions. To clarify the spatial distribution of forces acting on the centrosomes, we have devised an image-processing-based approach. The movements of the centrosomes within a few seconds were quantified using image processing. We call these tiny movements that are difficult to evaluate by human eye, ""micro-movements"". The measurement of the micro-movements in wild-type and several gene-knockdown embryos indicated that the micro-movements reflect the spatial distribution of forces. Time-lapse analyses of the micro-movements succeeded to clarify a change in spatial distribution of the forces during transition from the centripetal to centrifugal movement. Based on the results, we propose a model of force distribution during the first cell cycle that explains successive migrations of the centrosomes towards centripetal and centrifugal directions. Computer simulation studies confirmed that the model is consistent with the dynamic positioning of the centrosomes in wild-type, goa-1;gpa-16(RNAi) and let-99(RNAi) embryos. [1] Kimura A, Onami S. Dev Cell 8, 765-775 (2005) [2] Grill et al. Nature 409, 630-633 (2001) *Present institution of A. Kimura: National Institute of Genetics, Japan

Brousseau D et al. (1998) East Coast Worm Meeting "A Remarkably Large and Diverse Group of Insulin Superfamily Genes in C. elegans"

Devadhar S, Homburger S, Heller J, Buchman A, Platt D, Costa M, Quinn S, Reddy B, Ferguson KC, Doberstein S, Brousseau D

[East Coast Worm Meeting, 1998]

We are using combined genetic and genomic analyses to identify new C. elegans genes involved in insulin receptor signaling. As a starting point, we have characterized a group of insulin-related genes that is considerably larger and more diverse than that recognized in other organisms. The insulin superfamily genes analyzed to date are expressed primarily in subsets of neurons, where they might function as neuromodulators or hormones. Kimura et al., 1997 (Science 277: 942) have shown that the daf-2 gene, which is required to prevent dauer formation, encodes an insulin receptor homolog. We are in the process of generating loss- and gain-of-function phenotypes for the insulin homologs, and determining whether any of these genes may encode ligands for the DAF-2 receptor. In addition, we have used RNA-mediated interference to identify new genes acting downstream of the DAF-2 receptor in the dauer formation pathway. Vertebrate orthologs of these genes might represent novel drug targets for the treatment of type II diabetes.

Hidehito KUROYANAGI et al. (2005) International Worm Meeting "CaM-K cascade and CREB regulate C. elegans dauer formation."

Masatoshi Hagiwara, Yoshishige Kimura, Sachiko Yoshimura, Hidehito KUROYANAGI

[International Worm Meeting, 2005]

CREB is involved in multiple biological phenomena such as long-term memory. However, signaling pathways leading to CREB activation in the whole animal context remain to be elucidated. CaM-K cascade in C. elegans, CaM kinase kinase (CKK-1) and CaM kinase I/IV (CMK-1), activates CRE-mediated gene expression via CREB (CRH-1) [KIMURA et al. 2002]. Disruption mutants of ckk-1, cmk-1 and crh-1 showed dauer-like larva formation constitutive phenotype. Epistatic analyses revealed that ckk-1 and crh-1 function upstream of daf-12 encoding a nuclear hormone receptor. Cell-type specific expression of CRH-1 in XXX cells, which is considered to prevent dauer formation by releasing steroid hormone(s), rescued the crh-1 mutant phenotype. These results suggests that CaM-K-CREB pathway forms a novel branch of dauer regulatory pathways, and function in XXX cells to negatively regulate dauer formation via endocrine signaling.

Brousseau D et al. (1998) Midwest Worm Meeting "A Remarkably Large and Diverse Group of Insulin Superfamily Genes in C. elegans"

Heller J, Homburger S, Quinn S, Brousseau D, Platt D, Costa M, Ferguson KC, Devadhar S, Buchman A, Reddy B, Doberstein S

[Midwest Worm Meeting, 1998]

We are using combined genetic and genomic analyses to identify new C. elegans genes involved in insulin receptor signaling. As a starting point, we have characterized a group of insulin-related genes that is considerably larger and more diverse than that recognized in other organisms. The insulin superfamily genes analyzed to date are expressed primarily in subsets of neurons, where they might function as neuromodulators or hormones. Kimura et al., 1997 (Science 277: 942) have shown that the daf-2 gene, which is required to prevent dauer formation, encodes an insulin receptor homolog. We are in the process of generating loss- and gain-of-function phenotypes for the insulin homologs, and determining whether any of these genes may encode ligands for the DAF-2 receptor. In addition, we have used RNA-mediated interference to identify new genes acting downstream of the DAF-2 receptor in the dauer formation pathway. Vertebrate orthologs of these genes might represent novel drug targets for the treatment of type II diabetes.

Kenyon CJ et al. (2000) West Coast Worm Meeting "Expression and regulation of daf-16::gfp constructs"

Kenyon CJ, Lin K-C

[West Coast Worm Meeting, 2000]

Wild-type C.elegans ages rapidly, undergoing development, senescence, and death in less than 3 weeks. In contrast, mutants with reduced activity of daf-2, a homolog of the insulin and insulin-like growth factor (IGF-1) receptors (Kimura et al., 1997), age more slowly and live more than twice as long (Kenyon et al., 1993). Wild-type DAF-2 activity also promotes growth to adulthood and prevents dauer formation, since severe loss of daf-2 function causes the animals to become dauers in the presence of food. Both the lifespan extension and dauer-constitutive phenotypes caused by daf-2 mutations are dependent on the activity of daf-16, which encodes an HNF-3/forkhead family member (Ogg et al., 1997; Lin et al., 1997). We have created GFP-tagged daf-16 constructs and used them to study the expression and regulation of daf-16. We will report our progress on this study.