Kimura KD et al. (1996) East Coast Worm Meeting "CLONING the daf-2 GENE"

Ruvkun GB, Tissenbaum HA, Kimura KD

[East Coast Worm Meeting, 1996]

A number of dauer-constitutive (Daf-c) and dauer-defective (Daf-d) mutants have been isolated, and most of them are ordered into neuronal signal transduction pathways. daf-2 and daf-23 are the only two daf-c genes which are positioned either in a separate branch of the pathway or downstream of daf-12. In addition, weak daf-2 and daf-23 mutants cause prolonged life span of the adult worms, which is not caused by any other daf-c mutants (ref. 1-3). These data suggest that daf-2 and daf-23 may play different role(s) from the other daf genes. Recently, daf-23 has been cloned in our lab (see abstract of J. Morris et al., this meeting) and found to be a homologue of the catalytic subunit of mammalian PI 3-kinase. Because daf-2 is positioned at the same place of the genetic epistatic pathway as daf-23, it could be part of the PI 3-kinase signaling cascade. Cloning of daf-2 could help to elucidate the function of PI 3-kinase signaling in dauer formation and aging. We have mapped daf-2 gene by RFLP analysis to a region between mgP34 and mgP35 on the left arm of chromosome III. We have detected a daf-2 allele-specific polymorphism associated with a uv-induced allele using YACs as a probe. We are currently cloning the polymorphic genomic fragment and screening cDNA library using the YAC probes. We are also doing microinjection of the YACs to rescue the daf-2 phenotype. References: (1) Kenyon et al. (1993) Nature 366, 461-464, (2) Larsen et al. (1995) Genetics 139, 1567-1583, (3) Dorman et al. (1995) Genetics 141, 1399-1406

Kotaro Kimura et al. (2005) International Worm Meeting "Different types of plasticity of avoidance behaviors."

Isao Katsura, Kotaro Kimura

[International Worm Meeting, 2005]

Although C. elegans exhibits several types of behavioral plasticity (ex. Rankin, 2004 Curr. Biol., 14, R617), we know little about plasticity of its avoidance behaviors. Because avoidance cues are toxic and/or hazardous signals, we speculated that the animals may exhibit plasticity other than adaptation in avoidance behaviors. We studied changes in avoidance behaviors of N2 animals after persistent stimulation using higher osmolarity, 1-octanol or 2-nonanone, which are sensed mainly by different sensory neuron classes respectively. For osmolarity assay, we used quadrant plates to obtain temporally more stable stimulus than the traditional "ring" assay; For repellent odor assay, we used 9 cm round plates and scored the average relative distance of the animals from the odor source. Osmolarity: Avoidance behavior to higher osmolarity was suppressed after starvation as well as after pre-exposure to the same stimulus for 1 hr. This suppression after pre-exposure is likely due to the adaptation of ASH neuron (Hilliard et al., 2005 EMBO J., 24, 63). 1-octanol: After starvation, avoidance behavior to 1-octaol (6 - 600 nL) was also suppressed, as similarly reported by Chao et al. (2004 PNAS, 101, 15512). However, after animals had been pre-exposed to 90 nL of the same odor during starvation, the avoidance behavior was not suppressed, and its magnitude was similar to that of naive animals. This modulation of avoidance behavior by pre-exposure could be explained either by suppression of the starvation-induced suppression of avoidance behavior, or by enhancement of the suppressed avoidance behavior. 2-nonanone: In contrast to 1-octanol, avoidance behavior to 2-nonanone was not suppressed by starvation. Still, pre-exposure to 30 nL of 2-nonanone during starvation significantly enhanced the avoidance behavior: Magnitude of the avoidance behavior to 200 nL of 2-nonanone of the pre-exposed N2 animals was enhanced (p < 0.001) and comparable to that of the behavior to 600 nL of 2-nonanone of unexposed animals. This result suggests that the pre-exposure may enhance the sensitivity of the sensory neuron to 2-nonanone by about 3-fold. Alternatively, the enhancement could be due to modulation of the locomotion. The pre-exposure in the presence of food also enhanced the avoidance behavior although it was not statistically significant. We are interested in these pre-exposure-induced modulations of avoidance behaviors because the plasticity of avoidance behaviors may be much more complicated than we expected. We are currently studying the plasticity in the following aspects: (1) its specificity and persistence, (2) responsible sensory neurons, and (3) genes required for the phenomenon.

Kimura K et al. (2011) Bioarchitecture "A novel mechanism of microtubule length-dependent force to pull centrosomes toward the cell center."

Kimura K, Kimura A

[Bioarchitecture, 2011]

The centrosome is a major microtubule-organizing center in animal cells, and its intracellular positioning is critical for defining intracellular architecture. The centrosome positions itself at the cell center. Centrosome centration depends on the microtubule cytoskeleton. To accomplish robust centration regardless of the cell size or cell shape, it has been assumed that the force mediated by the microtubules depends on microtubule length. However, a concrete mechanism to generate forces to pull the centrosome in a microtubule length-dependent manner has been elusive. Recently, we successfully demonstrated that centrosome-directed movement of intracellular organelles along microtubules drives centrosome centration in the Caenorhabditis elegans early embryo. Based on this observation, we proposed the centrosome-organelle mutual pulling model in which the reaction forces of organelle transport generated along microtubules act as a driving force that pulls the centrosomes toward the cell center. This is the first experiment-based model that accounts for the microtubule length-dependent pulling force generated in the cytoplasm contributing to centrosome centration. Intriguingly, this model is consistent with a recent estimation that the pulling force is proportional to the cubic length of microtubules.

Kimura, K. et al. (2009) International Worm Meeting "A tug-of-war model for the centrosome centering in Caenorhabditis elegans early embryo."

Kimura, A., Kimura, K.

[International Worm Meeting, 2009]

The centrosome is a major microtubule organizing center in animal cells. The centrosome is generally positioned in the cell center by driving forces through the microtubules. Such positioning is critical for proper microtubule-dependent cellular activities such as cell division and organelle distribution. It has been widely assumed that the centering is powered by the microtubule pulling force, which depends on the minus-end directed motor protein, cytoplasmic dynein. To pull the centrosome via microtubules toward the center, DHC (dynein heavy chain), the subunit responsible for the motor activity, should be anchored to some intracellular structures. However, such structures remain unknown. Here, we propose a tug-of-war model that intracellular organelles associated with DHC have anchoring function to pull microtubules. Three lines of examinations support our model. We used C. elegans embryo at a pronuclear migration stage, a typical example of centrosome centering. First, we performed screening for genes involved in the anchoring function of the structures and identified dyrb-1 encoding a light chain subunit of dynein. Live cell imaging of GFP fused DYRB-1 during the centering suggested that substantial fraction of DYRB-1 with the structure move along astral microtubules. Second, we analyzed organelle transport along astral microtubules to verify the model, and found that intracellular organelles such as early endosomes move along astral microtubules in a DHC and DYRB-1 dependent manner. Moreover, we found a strong correlation between organelle movement along astral microtubules and centrosome centering. Third, to test whether the organelle movements are required for centrosome centering, we attempted to decrease the organelle movements by knockdown of membrane traffic regulators and observed whether the centering is impaired. We found that the centering was delayed in embryos with reduced organelle movement. We confirmed that other events such as spindle rocking movement that are dependent on microtubules and DHC function are not impaired in these embryos. These results suggest that centrosome-directed movements of intracellular organelles produce a counteracting force against the movement that pulls the centrosome toward each moving organelles.

Kimura, Yoshishige et al. (2017) International Worm Meeting "Imaging mass spectrometry for C. elegans."

Kaneko, Tomomi, Kimura, Yoshishige, Aoyagi, Satoka

[International Worm Meeting, 2017]

Imaging mass spectrometry (IMS) is a two-dimensional mass spectrometry to visualize the spatial distribution of biomolecules, which does not need either separation or purification of target molecules. The free-living soil nematode C. elegans is a common model organism, extensively used in life science research. Though, various investigations have been performed for metabolomic profiling of worms, the information of a single worm has been lost by? the conventional mass spectrometry (MS) techniques. Thus, the development of a label-free, non-targeted MS technique for molecular mapping in C. elegans has been required. We have previously performed MALDI imaging of C. elegans. However, the resolution was not enough to analyze cellular or subcellular level of biomoleculer distribution. Thus, we next tried the application of TOF-SIMS (Time-of-Flight Secondary Mass Spectrometry) system for C. elegans, which enables us to obtain subcellular distribution of metabolites. By comparison of several sample preparation methods, the frozen sections of C. elegans fixed by paraformaldehyde (PFA) were suitable for TOF-SIMS analysis. By sputtering of Ar gas cluster ion beam (Ar-GCIB), the sensitivity to fatty acids (e.g. stearic acid (SA), oleic acid (OA), and eicosapentaenoic acid (EPA)) was significantly enhanced, and high-resolution images of biomolecules were acquired. Further modification to prepare C. elegans samples for TOF-SIMS imaging is in progress. This is promising to obtain the cellular and subcellular distributions of the various biomolecules easily and efficiently.

Kimura A et al. (2010) Methods Cell Biol "Modeling microtubule-mediated forces and centrosome positioning in Caenorhabditis elegans embryos."

Kimura A, Onami S

[Methods Cell Biol, 2010]

Microtubules and associated motor proteins are the major generators and mediators of the forces that organize the functional positioning of intracellular structures. The positioning of the centrosomes is a primary target for microtubule-mediated organization. The positioning of the centrosomes further defines the positionings of nucleus, mitotic spindles, and other organelles. Numerical modeling is an effective means by which we can further understand the physical mechanisms underlying microtubule-mediated centrosome positioning. Here, we summarize how we formulated the biophysical properties of microtubules in order to construct a numerical model of centrosome positioning in Caenorhabditis elegans embryos. Microtubules elongate and shrink in a stochastic manner, in a process known as "dynamic instability." Upon association with the cell cortex or motor proteins, microtubules mediate pushing and pulling forces. These forces move the centrosome, which is located at the minus-end of the microtubules, to the right place with the right timing. We discuss how the modeling efforts complement experimental knowledge and allow us to evaluate the sufficiency of various candidate hypotheses.

Kimura A et al. (2007) J Cell Biol "Local cortical pulling-force repression switches centrosomal centration and posterior displacement in C. elegans."

Onami S, Kimura A

[J Cell Biol, 2007]

Centrosome positioning is actively regulated by forces acting on microtubules radiating from the centrosomes. Two mechanisms, center-directed and polarized cortical pulling, are major contributors to the successive centering and posteriorly displacing migrations of the centrosomes in single-cell-stage Caenorhabditis elegans. In this study, we analyze the spatial distribution of the forces acting on the centrosomes to examine the mechanism that switches centrosomal migration from centering to displacing. We clarify the spatial distribution of the forces using image processing to measure the micrometer-scale movements of the centrosomes. The changes in distribution show that polarized cortical pulling functions during centering migration. The polarized cortical pulling force directed posteriorly is repressed predominantly in the lateral regions during centering migration and is derepressed during posteriorly displacing migration. Computer simulations show that this local repression of cortical pulling force is sufficient for switching between centering and displacing migration. Local regulation of cortical pulling might be a mechanism conserved for the precise temporal regulation of centrosomal dynamic positioning.

Kimura A et al. (2000) J Biol Chem "Both PCE-1/RX and OTX/CRX interactions are necessary for photoreceptor-specific gene expression."

Wawrousek EF, Kimura A, Nakamura M, Singh D, Shinohara T, Kikuchi M

[J Biol Chem, 2000]

RX, a homeodomain-containing protein essential for proper eye development (Mathers, P. H. Grinberg, A., Mahon, K. A., and Jamrich, M. (1997) Nature 387, 603-607), binds to the photoreceptor conserved element-1 (PCE-1/Ret 1) in the photoreceptor cell-specific arrestin promoter and stimulates gene expression. RX is found in many retinal cell types including photoreceptor cells. Another homeodomain-containing protein, CRX, which binds to the OTX element to stimulate promoter activity, is found exclusively in photoreceptor cells (Chen, S., Wang, Q. L., Nie, Z., Sun, H., Lennon, G., Copeland, N. G., Gillbert, D. J. Jenkins, N. A., and Zack, D. J. (1997) Neuron 19, 1017-1030; Furukawa, T., Morrow, E. M., and Cepko, C. L. (1997) Cell 91, 531-541). Binding assay and cell culture studies indicate that both PCE-1 and OTX elements and at least two different regulatory factors RX and CRX are necessary for high level, photoreceptor cell-restricted gene expression. Thus, photoreceptor specificity can be achieved by multiple promoter elements interacting with a combination of both photoreceptor-specific regulatory factors and factors present in closely related cell lineages.

Kenji Kimura et al. (2008) "A tug-of-war model for the centrosomal centration in C. elegans early embryo."

Kenji Kimura, Akatsuki Kimura

[2008]

"The centrosome is the microtubule (MT) organizing center and critical for MT dependent cellular functions in animal cells. The position of the centrosome is actively maintained at the cell center. Male pronuclear migration (PNM), the migration of male pronucleus-centrosome complex from periphery to center of the cell after fertilization, is a typical example of the centrosomal centration. As a primary centrosomal centration mechanism, it has reported that the centration requires the MT pulling forces by the minus-end directed motor protein, cytoplasmic dynein. However, it remains unclear where the MT is pulled and how the pulling force is generated by the dynein during PNM. Here, we propose a ""tug-of-war model"" that the dynein dependent minus-end directed organelle movement along the MT generates the MT pulling force for the centration. Three lines of observations during PNM in C. elegans support this model. First, we observed the subcellular localization of the dynein using GFP-fused dynein subunits to examine where the dynein functions during PNM. These reporter proteins localized to cytoplasm and the MT elongated from the centrosome during PNM. We found that the localization on the MT is dependent on dhc-1 which encodes dynein heavy chain subunit and is essential for motor activity. The result suggests that the dynein on the MT are generating the pulling force during PNM. Second, we quantified transport of some organelles along the MT during PNM to analyze the correlation between the dynein dependent organelle movement and PNM. We found that early endosome (EE) moved straightly toward the centrosome in a dhc-1 dependent manner. In addition, the direction of total vector of EE movements is opposite to the direction of PNM. The result suggests that an inverse correlation exists between EE movement and PNM. Third, we addressed whether PNM is inhibited by reduction of EE movement to verify our model. We found that the rate of PNM was delayed by knockdown of some transport regulators. These results suggest that dynein dependent movements of organelles toward the centrosome generate a counteracting force as the MT pulling force, and contribute to the centrosomal centration during PNM in C. elegans."

Kotaro Kimura et al. (2008) C.elegans Neuronal Development Meeting "Tracking Analysis of the Enhancement of 2-Nonanone Avoidance"

Kotaro Kimura, Isao Katsura

[C.elegans Neuronal Development Meeting, 2008]

C. elegans exhibits multiple types of behavioral plasticity. The plasticity of its avoidance behaviors, however, has not been studied enough. We have reported that preexposure to the repulsive odors of 2-nonanone or 1-octanol caused enhancement, rather than adaptation, of avoidance behavior of N2 animals to these odors (''05 IWM #487C, ''06 CeNeuro #54, ''07 IWM #252). A 1-h preexposure to 90 nL of 2-nonanone significantly enhanced the average migration distance of the worms from the odor source, and the memory of preexposure was maintained for at least an additional 1 h after odor removal. Further, we found that dopamine signaling regulated the enhancement of 2-nonanone avoidance. To our knowledge, this is the first example in worms of feeding status-independent regulation of behavioral plasticity by dopamine. We are currently working to identify the dopamine receptor involved in the enhancement. To understand which aspect of the avoidance behavior is modulated in the enhancement of 2-nonanone avoidance, we analyzed the animal''s locomotion by using a tracking system with a fixed high-resolution CCD camera at 0.5 Hz. Tracks of the animals during the 2-nonanone avoidance appeared to be composed of pirouettes and runs, as in salt chemotaxis (Pierce-Shimomura et al., JNS 1999). Based on the shapes of the tracks, we defined the periods in a worm''s track of 2-nonanone avoidance as runs if the following 2 conditions continued for 12 s or longer: (1) dTheta for 4 s (2 frames) was 100deg and (2) the average velocity during 4 s was 0.05 mm/s. The other periods were pirouettes. We then analyzed the effect of preexposure on the pirouettes and runs and found that the preexposed animals exhibited significant increases in run duration as compared to naive and mock-treated animals. Interestingly, the preexposure significantly increased the frequency of the longer runs (duration, =90 s ) and decreased that of the shorter runs (duration, 18-22 s), while the frequency of runs lasting for less than 18 s was unchanged. The results suggest that the enhancement of 2-nonanone avoidance is caused by increases in run duration and that stable and plastic run modes exsist in 2-nonanone avoidance.