Lu-Lu Shen

The phenotypic and behavioral defects can be transferred from zinc-exposed nematodes to their progeny

In despite of the essential functions of zinc as trace element for humans, toxic effects will be pronounced while organisms are exposed to high levels of zinc. However, whether the multiple biological toxicities caused by zinc exposure could be transferred to progeny is still largely unknown. In the present study, we explored the model organism, Caenorhabditis elegans, to analyze the multiple toxicities from zinc exposure and their possibly transferable properties. Our results indicate that the zinc exposure could result in multiple biological defects by affecting the endpoints of life span, development, reproduction, locomotion behavior and chemotaxis plasticity. Zinc exposure could specially cause the appearance of uncoordinated (UNC) animals. In addition, Zn exposure could result in the stress responses in most of the tissues in exposed nematodes. Moreover, these phenotypic and behavioral toxicities could be transferred from zinc-exposed nematodes to their progeny. The fold changes of hsp16-2-gfp expression in embryos of progeny animals suggest that the zinc toxicity might be deposited in eggs of nematodes. We summarized these defects into two groups according to their transferable properties. Therefore, our data suggest that zinc exposure can result in multiple toxicities and these multiple biological defects can be largely transferred to progeny in C. elegans.

Toxicity evaluation in nematode Caenorhabditis elegans after chronic metal exposure

In this study, specific developmental stage for adults from day 1 to day 10 was selected to evaluate the chronic metal toxicity, because the population of dead nematodes and the accumulation of intestinal autofluorescence increased sharply after day 10. Chronic exposure to Cr, Pb, Cu, and Hg caused a significant elevation in fractions of dead animals after day 4, and resulted in a significant induction of hsp-16.2::gfp expression at all assayed metal concentrations. Moreover, chronic exposure to Ag, Cr, Pb, Cu, Hg, and Cd would induce a more severe stress response than exposure to Zn and Mn in intestine, and chronic exposure to Pb, Hg, Cr, Zn, and Mn would induce a more severe stress response than exposure to Ag, Cu and Cd in head neurons. Therefore, in determining the usefulness of animals in metal toxicity assessment, this study established a method using nematodes in testing the chronic metal toxicity.

Regulation of longevity by genes required for the functions of AIY interneuron in nematode Caenorhabditis elegans.

In Caenorhabditis elegans, functional ttx-3, sra-11, ceh-10, and ceh-23 genes are required for the functions of AIY interneuron. Compared to wild-type N2, mutations in ttx-3 and ceh-10 significantly decreased lifespan, whereas mutations in sra-11 and ceh-23 did not obviously influence nematode lifespan. Mutations in ttx-3 and ceh-10 were associated closely with lower pumping rates at adult day 8 and caused a more rapid accumulated intestinal autofluorescence than wild-type N2. Mutations in ceh-10 remarkably affected fertility and egg number in the uterus. The regulation of ttx-3 and ceh-10 on longevity was not temperature-dependent, and ttx-3, and ceh-10 mutants all formed very few dauers at 27°C. The shortened lifespan of the ttx-3 or ceh-10 mutants was completely or largely rescued by expression of TTX-3 or CEH-10 in AIY interneurons. Moreover, the long-lived phenotype of the daf-2 mutant could be suppressed by both the ttx-3 and the ceh-10 mutations. Furthermore, ablation of AIY interneurons shortened the longevity of wild-type and the daf-2 mutant. Therefore, ttx-3 and ceh-10 regulate the longevity through influencing the insulin/IGF signaling pathway in C. elegans.

Involvement of genes required for synaptic function in aging control in C. elegans

ObjectiveTo identify new genes required for neurosecretory control of aging in C. elegans.MethodsIn view of the importance of nervous system in aging regulation, we performed the screen for genes involved in the aging regulation from genetic loci encoding synaptic proteins by lifespan assay and accumulation of lipofuscin autofluorescence. We further investigated the dauer formation phenotypes of their corresponding mutants and whether they were possibly up-regulated by the insulin-like signaling pathway.ResultsThe genetic loci of unc-10, syd-2, hlb-1, dlk-1, mkk-4, scd-2, snb-1, ric-4, nrx-1, unc-13, sbt-1 and unc-64 might be involved in the aging control. In addition, functions of unc-10, syd-2, hlb-1, dlk-1, mkk-4, scd-2, snb-1, ric-4 and nrx-1 in regulating aging may be opposite to those of unc-13, sbt-1 and unc-64. The intestinal autofluorescence assay further indicated that the identified long-lived and short-lived mutants were actually due to the suppressed or accelerated aging. Among the identified genes, syd-2, hlb-1, mkk-4, scd-2, snb-1, ric-4 and unc-64 were also involved in the control of dauer formation. Moreover, daf-2 mutation positively regulated the expression of syd-2 and hlb-1, and negatively regulated the expression of mkk-4, nrx-1, ric-4, sbt-1, rpm-1, unc-10, dlk-1 and unc-13. The daf-16 mutation positively regulated the expression of syd-2 and hlb-1, and negatively regulated the expression of mkk-4, nrx-1, sbt-1, rpm-1, unc-10, dlk-1 and unc-13.ConclusionThese data suggest the possibly important status of the synaptic transmission to the animal’s life-span control machinery, as well as the dauer formation control.摘要目的鉴定参与线虫衰老的神经内分泌调控的新基因。方法鉴于神经系统在衰老调控中的重要作用, 通过寿命分析和脂褐质自发荧 光的检测, 从编码突触蛋白的遗传位点中选参与衰老调控的基因。 我们还进一步检查了这些遗传位点相应的突变体的永久性幼虫形成情况, 探讨它们是否可能受胰岛素样信号通路的调控。结果遗传位点 unc-10, syd-2, hlb-1, dlk-1, mkk-4, scd-2, snb-1, ric-4, nrx-1, unc-13, sbt-1, unc-64 可能参与线虫衰老的调控。 而且在衰老的调控中, unc-10, syd-2, hlb-1, dlk-1, mkk-4, scd-2, snb-1, ric-4, nrx-1 的功能可能与 unc-13, sbt-1, unc-64 相反。 肠道脂褐质自发荧光的检测进一步证明了选出的各基因对应突变体的长寿或短寿表型, 是由减慢或缩短的组织衰老所致。 在选出的基因中, syd-2, hlb-1, mkk-4, scd-2, snb-1, ric-4, unc-64 也参与了永久性幼虫形成的调控。 另外, daf-2 突变增强了 syd-2 和 hlb-1 的表达, 降低了 mkk-4, nrx-1, ric-4, sbt-1, rpm-1, unc-10, dlk-1, unc-13 的表达。 daf-16 突变提高了 syd-2 和 hlb-1 的表达, 降低了 mkk-4, nrx-1, sbt-1, rpm-1, unc-10, dlk-1, unc-13 的表达。结论突触功能可能在个体寿命和永久性幼虫形成的调控机制中具有重要的作用。

Confirmation of combinational effects of calcium with other metals in a paper recycling mill effluent on nematode lifespan with toxicity identification evaluation method

We used toxicity identification evaluation (TIE) method to confirm the combinational effects of identified toxic metals in a paper recycling mill effluent in inducing the decreased lifespan in nematode Caenorhabditis elegans. Exposure to Ca + Al caused more severely decreased lifespan than that exposed to Ca, or Al; and exposure to Ca + Fe induced more severely decreased lifespan than that exposed to Ca, or Fe. Exposure to Ca+Al+Fe caused more severely decreased lifespan than that exposed to Ca, or Ca+Fe. Moreover, the baseline toxicity on lifespan was doubled by doubling the concentration of combined metals (Ca+Al+Fe) in spiking test in original effluent (oe), and lifespan defects in oe+Ca+Al+Fe exposed nematodes were more severe than that in Ca+Al+Fe exposed nematode. Therefore, Ca+Al+Fe exposure may largely explain the formation of decreased lifespan induced by the examined industrial effluent. Furthermore, the observed reduction of lifespan induced by the combination of high level of Ca with other metals may be at least partially independent of the insulin-like pathway.

Genes required for the functions of olfactory AWA neuron regulate the longevity of Caenorhabditis elegans in an insulin/IGF signaling-dependent fashion

ObjectiveTo investigate the interaction between the genes required for the functions of AWA olfactory neuron and insulin/IGF signaling in regulating the longevity of nematode Caenorhabditis elegans (C. elegans).MethodsThe mutants that had loss-of-function mutation of the genes required for AWA, AWC, ASE, and AFD sensory neurons were employed. Lifespan, the speed of pharynx pumping, the intestinal autofluorescence, the dauer formation, and the brood size were examined. Rescue experiments were performed to confirm the role of the genes required for the functions of AWA neuron in regulating lifespan. Moreover, genetic interactions between genes required for the functions of AWA neuron and insulin/IGF signaling were investigated.ResultsMutations of odr-7, odr-2, and odr-3 genes required for the functions of AWA neuron significantly increased the mean lifespan of nematodes and slowed the accumulation of intestinal autofluorescence. Besides, these mutations were closely associated with higher pumping rates during aging. However, mutation of odr-7, odr-2, or odr-3 did not obviously affect the brood size or the dauer formation, and the regulation of longevity by odr-7, odr-2, and odr-3 was temperature-independent. In contrast, mutations of genes required for the functions of ASE, AWC, and AFD sensory neurons did not influence the nematode lifespan. Moreover, expression of odr-7, odr-2 and odr-3 in AWA neuron could completely or largely restore the altered lifespan in odr-7, odr-2 and odr-3 mutants. Furthermore, genetic interaction assay demonstrated that the extended lifespan in odr-7 mutant could be suppressed by daf-16 mutation and enhanced by daf-2 or age-1 mutation, whereas mev-1 and pha-4 were not required for the long lifespan of odr-7 mutant.ConclusionThe genes required for the function of AWA sensory neuron could regulate the nematode longevity in an insulin/IGF signaling-dependent fashion in C. elegans.摘要目的研究嗅觉神经元AWA 功能必需基因与胰岛素信号之间在调控秀丽线虫衰老上的关系。方法测定AWA、 AWC、 ASE 与AFD 感觉神经元的功能必需基因突变后线虫的寿命、 咽泵运动速率、 肠道荧光、 永久性幼虫形成与后代数目的变化。 此外, 进行基因功能的恢复实验以确认AWA功能必需基因在调控衰老中的作用。 最后, 对AWA功能必需基因与胰岛素信号间在调控衰老上的遗传关系进行了分析。结果AWA功能必需基因odr-7 、 odr-2与odr-3突变能显著延长动物寿命, 并在衰老过程中诱导产生相对于野生型更高的咽泵运动速率和更少的肠道脂褐质积累。 然而, odr-7、 odr-2与odr-3的基因突变并不影响线虫的后代数目与永久性幼虫的形成, 且odr-7、 odr-2与odr-3基因对于寿命的调控并未呈现出明显的温度依赖性。 比较而言, 感觉神经元ASE、 AWC与AFD的功能必需基因的突变并未显著影响动物寿命。 而且, 在嗅觉神经元AWA 表达odr-7、 odr-2 与odr-3 基因可以完全或很大程度上恢复对应突变体的长寿表现型。 进一步遗传分析表明, odr-7突变体的长寿表现型可被daf-16基因突变所抑制, 被daf-2或age-1基因突变所增强, 而mev-1与pha-4基因突变对此并不产生影响。结论嗅觉神经元AWA功能必需基因以胰岛素信号依赖的方式调控秀丽线虫的衰老。To investigate the interaction between the genes required for the functions of AWA olfactory neuron and insulin/IGF signaling in regulating the longevity of nematode Caenorhabditis elegans (C. elegans). The mutants that had loss-of-function mutation of the genes required for AWA, AWC, ASE, and AFD sensory neurons were employed. Lifespan, the speed of pharynx pumping, the intestinal autofluorescence, the dauer formation, and the brood size were examined. Rescue experiments were performed to confirm the role of the genes required for the functions of AWA neuron in regulating lifespan. Moreover, genetic interactions between genes required for the functions of AWA neuron and insulin/IGF signaling were investigated. Mutations of odr-7, odr-2, and odr-3 genes required for the functions of AWA neuron significantly increased the mean lifespan of nematodes and slowed the accumulation of intestinal autofluorescence. Besides, these mutations were closely associated with higher pumping rates during aging. However, mutation of odr-7, odr-2, or odr-3 did not obviously affect the brood size or the dauer formation, and the regulation of longevity by odr-7, odr-2, and odr-3 was temperature-independent. In contrast, mutations of genes required for the functions of ASE, AWC, and AFD sensory neurons did not influence the nematode lifespan. Moreover, expression of odr-7, odr-2 and odr-3 in AWA neuron could completely or largely restore the altered lifespan in odr-7, odr-2 and odr-3 mutants. Furthermore, genetic interaction assay demonstrated that the extended lifespan in odr-7 mutant could be suppressed by daf-16 mutation and enhanced by daf-2 or age-1 mutation, whereas mev-1 and pha-4 were not required for the long lifespan of odr-7 mutant. The genes required for the function of AWA sensory neuron could regulate the nematode longevity in an insulin/IGF signaling-dependent fashion in C. elegans. 研究嗅觉神经元AWA 功能必需基因与胰岛素信号之间在调控秀丽线虫衰老上的关系。 测定AWA、 AWC、 ASE 与AFD 感觉神经元的功能必需基因突变后线虫的寿命、 咽泵运动速率、 肠道荧光、 永久性幼虫形成与后代数目的变化。 此外, 进行基因功能的恢复实验以确认AWA功能必需基因在调控衰老中的作用。 最后, 对AWA功能必需基因与胰岛素信号间在调控衰老上的遗传关系进行了分析。 AWA功能必需基因odr-7 、 odr-2与odr-3突变能显著延长动物寿命, 并在衰老过程中诱导产生相对于野生型更高的咽泵运动速率和更少的肠道脂褐质积累。 然而, odr-7、 odr-2与odr-3的基因突变并不影响线虫的后代数目与永久性幼虫的形成, 且odr-7、 odr-2与odr-3基因对于寿命的调控并未呈现出明显的温度依赖性。 比较而言, 感觉神经元ASE、 AWC与AFD的功能必需基因的突变并未显著影响动物寿命。 而且, 在嗅觉神经元AWA 表达odr-7、 odr-2 与odr-3 基因可以完全或很大程度上恢复对应突变体的长寿表现型。 进一步遗传分析表明, odr-7突变体的长寿表现型可被daf-16基因突变所抑制, 被daf-2或age-1基因突变所增强, 而mev-1与pha-4基因突变对此并不产生影响。 嗅觉神经元AWA功能必需基因以胰岛素信号依赖的方式调控秀丽线虫的衰老。

UNC-64 and RIC-4, the plasma membrane-associated SNAREs syntaxin and SNAP-25, regulate fat storage in nematode Caenorhabditis elegans

ObjectiveTo investigate whether genes required for synaptogenesis and synaptic function are also involved in fat storage control in Caenorhabditis elegans.MethodsFat storage was examined in mutants of genes affecting the synaptogenesis and synaptic function. In addition, the genetic interactions of SNAREs syntaxin/unc-64 and SNAP-25/ric-4 with daf-2, daf-7, nhr-49, sbp-1 and mdt-15 in regulating fat storage were further investigated. The tissue-specific activities of unc-64 and ric-4 were investigated to study the roles of unc-64 and ric-4 in regulating fat storage in the nervous system and/or the intestine.ResultsMutations of genes required for the formation of presynaptic neurotransmission site did not obviously influence fat storage. However, among the genes required for synaptic function, the plasma membrane-associated SNAREs syntaxin/unc-64 and SNAP-25/ric-4 genes were involved in the fat storage control. Fat storage in the intestinal cells was dramatically increased in unc-64 and ric-4 mutants as revealed by Sudan Black and Nile Red strainings, although the fat droplet size was not significantly changed. Moreover, in both the nervous system and the intestine, expression of unc-64 significantly inhibited the increase in fat storage observed in unc-64 mutant. And expression of ric-4 in the nervous system completely restored fat storage in ric-4 mutant. Genetic interaction assay further indicated that both unc-64 and ric-4 regulated fat storage independently of daf-2 [encoding an insulin-like growth factor-I (IGF-I) receptor], daf-7 [encoding a transforming growth factor-β (TGF-β) ligand], and nhr-49 (encoding a nuclear hormone receptor). Besides, mutation of daf-16 did not obviously affect the phenotype of increased fat storage in unc-64 or ric-4 mutant. Furthermore, unc-64 and ric-4 regulated fat storage probably through the ARC105/mdt-15- and SREBP/sbp-1-mediated signaling pathways. In addition, fat storage in unc-64; ric-4 was higher than that in either unc-64 or ric-4 single mutant nematodes, suggesting that unc-64 functions in parallel with ric-4 in regulating fat storage.ConclusionThe plasma membrane-associated SNAREs syntaxin/unc-64 and SNAP-25/ric-4 function in parallel in regulating fat storage in C. elegans, probably through the ARC105/mdt-15- and SREBP/sbp-1-mediated signaling pathways.摘要目的研究神经突触组装与功能调控相关基因是否参与秀丽线虫的脂肪积累调节。方法分析神经突触组装与功能调控相关基因突变体的脂肪积累变化, 进而观察SNAREs syntaxin/unc-64和SNAP-25/ric-4基因与daf-2、 daf-7、 nhr-49、 sbp-1 及mdt-15所介导的信号通路在调控脂肪积累上的遗传关系。 对unc-64 与ric-4基因进行组织特异性活性分析, 以确定它们在神经系统与肠道内对脂肪积累的影响。结果突触前为神经突触组装所必需的基因的突变并未明显影响脂肪积累。 对调控神经突触功能的基因进行分析的结果显示, SNAREs syntaxin/unc-64与SNAP-25/ric-4基因均参与了脂肪积累的调节。 利用苏丹黑染色与尼罗红标记法观察到unc-64与ric-4突变体肠道中脂肪积累显著增加, 而unc-64与ric-4突变体中积累的脂肪颗粒并未出现尺寸的显著变化。 在神经系统与肠道中, unc-64基因的表达均能显著降低unc-64突变体动物的脂肪积累, 而基因ric-4在神经系统的表达则可以完全恢复ric-4突变体动物的脂肪积累。 遗传分析表明, unc-64 与ric-4对脂肪积累的调控独立于daf-2 (IGF-I受体基因)、 daf-7 (TGF-β配体基因)和nhr-49(核激素受体基因), 且不受daf-16基因突变的影响。 进一步的研究结果显示, unc-64 与ric-4可能经由ARC105/mdt-15与SREBP/sbp-1介导的信号通路来调节动物的脂肪积累进程。 此外, unc-64; ric-4双突变体的脂肪积累水平要显著高于unc-64或ric-4单突变体的脂肪积累水平, 提示这两个基因通过平行的遗传通路调控脂肪积累。结论质膜相关的SNAREs syntaxin/unc-64和SNAP-25/ric-4基因通过平行的遗传通路并经由ARC105/mdt-15与SREBP/sbp-1介导的信号通路来调控秀丽线虫的脂肪积累。To investigate whether genes required for synaptogenesis and synaptic function are also involved in fat storage control in Caenorhabditis elegans. Fat storage was examined in mutants of genes affecting the synaptogenesis and synaptic function. In addition, the genetic interactions of SNAREs syntaxin/unc-64 and SNAP-25/ric-4 with daf-2, daf-7, nhr-49, sbp-1 and mdt-15 in regulating fat storage were further investigated. The tissue-specific activities of unc-64 and ric-4 were investigated to study the roles of unc-64 and ric-4 in regulating fat storage in the nervous system and/or the intestine. Mutations of genes required for the formation of presynaptic neurotransmission site did not obviously influence fat storage. However, among the genes required for synaptic function, the plasma membrane-associated SNAREs syntaxin/unc-64 and SNAP-25/ric-4 genes were involved in the fat storage control. Fat storage in the intestinal cells was dramatically increased in unc-64 and ric-4 mutants as revealed by Sudan Black and Nile Red strainings, although the fat droplet size was not significantly changed. Moreover, in both the nervous system and the intestine, expression of unc-64 significantly inhibited the increase in fat storage observed in unc-64 mutant. And expression of ric-4 in the nervous system completely restored fat storage in ric-4 mutant. Genetic interaction assay further indicated that both unc-64 and ric-4 regulated fat storage independently of daf-2 [encoding an insulin-like growth factor-I (IGF-I) receptor], daf-7 [encoding a transforming growth factor-β (TGF-β) ligand], and nhr-49 (encoding a nuclear hormone receptor). Besides, mutation of daf-16 did not obviously affect the phenotype of increased fat storage in unc-64 or ric-4 mutant. Furthermore, unc-64 and ric-4 regulated fat storage probably through the ARC105/mdt-15- and SREBP/sbp-1-mediated signaling pathways. In addition, fat storage in unc-64; ric-4 was higher than that in either unc-64 or ric-4 single mutant nematodes, suggesting that unc-64 functions in parallel with ric-4 in regulating fat storage. The plasma membrane-associated SNAREs syntaxin/unc-64 and SNAP-25/ric-4 function in parallel in regulating fat storage in C. elegans, probably through the ARC105/mdt-15- and SREBP/sbp-1-mediated signaling pathways. 研究神经突触组装与功能调控相关基因是否参与秀丽线虫的脂肪积累调节。 分析神经突触组装与功能调控相关基因突变体的脂肪积累变化, 进而观察SNAREs syntaxin/unc-64和SNAP-25/ric-4基因与daf-2、 daf-7、 nhr-49、 sbp-1 及mdt-15所介导的信号通路在调控脂肪积累上的遗传关系。 对unc-64 与ric-4基因进行组织特异性活性分析, 以确定它们在神经系统与肠道内对脂肪积累的影响。 突触前为神经突触组装所必需的基因的突变并未明显影响脂肪积累。 对调控神经突触功能的基因进行分析的结果显示, SNAREs syntaxin/unc-64与SNAP-25/ric-4基因均参与了脂肪积累的调节。 利用苏丹黑染色与尼罗红标记法观察到unc-64与ric-4突变体肠道中脂肪积累显著增加, 而unc-64与ric-4突变体中积累的脂肪颗粒并未出现尺寸的显著变化。 在神经系统与肠道中, unc-64基因的表达均能显著降低unc-64突变体动物的脂肪积累, 而基因ric-4在神经系统的表达则可以完全恢复ric-4突变体动物的脂肪积累。 遗传分析表明, unc-64 与ric-4对脂肪积累的调控独立于daf-2 (IGF-I受体基因)、 daf-7 (TGF-β配体基因)和nhr-49(核激素受体基因), 且不受daf-16基因突变的影响。 进一步的研究结果显示, unc-64 与ric-4可能经由ARC105/mdt-15与SREBP/sbp-1介导的信号通路来调节动物的脂肪积累进程。 此外, unc-64; ric-4双突变体的脂肪积累水平要显著高于unc-64或ric-4单突变体的脂肪积累水平, 提示这两个基因通过平行的遗传通路调控脂肪积累。 质膜相关的SNAREs syntaxin/unc-64和SNAP-25/ric-4基因通过平行的遗传通路并经由ARC105/mdt-15与SREBP/sbp-1介导的信号通路来调控秀丽线虫的脂肪积累。

Regulation of aging by unc-13 and sbt-1 in Caenorhabditis elegans is temperature-dependent

To investigate the role of environmental factor—temperature in the regulation of aging process by unc-13 and sbt-1 in Caenorhabditis elegans. The lifespan, the speed of pharynx pumping, and the intestinal autofluorescence of unc-13 and sbt-1 mutants were examined at different temperature conditions. In addition, to exclude the possible influences from other factors in unc-13 and sbt-1 mutants, the dauer formation, the thermotaxis, the brood size and the population percentage of the mutants expressing hsp16.2-gfp were further investigated. Mutations of unc-13 and sbt-1 significantly increased the mean and the maximum lifespans of nematodes cultured at 20 °C and 25 °C, while no noticeable increase was found at 15 °C in either the mean or the maximum lifespan. Investigations on the speed of pharynx pumping and the intestinal autofluorescence suggested that at 20 °C and 25 °C, mutations of unc-13 and sbt-1 could slow the aging process and delay the accumulation of aging-related cellular damage. Meanwhile, mutations of unc-13 or sbt-1 did not affect the dauer formation or the thermotaxis to different temperatures in nematodes. In contrast, at 20 °C and 25 °C conditions, mutations of unc-13 and sbt-1 significantly decreased the brood size and the percentage of nematodes expressing hsp16.2-gfp, while no such differences were detected at 15 °C. Moreover, the thermotolerance of unc-13 and sbt-1 mutants could be greatly strengthened after the 16-h heat shock at 35 °C. The regulation of aging by unc-13 and sbt-1 is temperaturedependent. And the alterations in reproduction capability and stress response may be associated with the formation of this temperature-dependent property. 研究环境因素—温度对秀丽线虫 unc-13 和 sbt-1 基因调控衰老作用的影响。 在不同培养温度下,测定 unc-13 和 sbt-1 突变线虫的寿命、 咽泵运动速率和肠道荧光。 此外, 为排除其他可能的影响因素, 对 unc-13 和 sbt-1 突变体的永久性幼虫形成率、 对培养温度的趋向性、 子代数目以及表达 hsp16.2-gfp 线虫在群体中的比例进行了测定。 在20 °C 和25 °C 培养条件下, unc-13 和 sbt-1 突变能显著提高线虫的平均寿命和最高寿命; 然而在15 °C 条件下, unc-13 和 sbt-1 突变体的平均寿命和最高寿命与野生型相似。 咽泵运动速率和肠道脂褐质自发荧光分析进一步验证了在20 °C 和25 °C 培养条件下, unc-13 和 sbt-1 突变可以减慢线虫衰老或延缓衰老相关的细胞损伤累积。 同时, unc-13 和 sbt-1 突变不影响永久性幼虫发生率以及动物对不同温度的趋向性。 相反, 在20 °C和25 °C 培养条件下, unc-13 和 sbt-1 突变能显著降低秀丽线虫的子代数目和群体中表达 hsp16.2-gfp 的线虫比率, 而在15 °C 条件下未出现此种差异。 此外, unc-13 和 sbt-1 突变体经35 °C 热激16 h 后, 其耐热性得到增强。 unc-13 和 sbt-1 对衰老的调节具有温度依赖性。 unc-13 和 sbt-1 突变体的生殖功能和应激反应的改变可能与此温度依赖性的形成有关。ObjectiveTo investigate the role of environmental factor—temperature in the regulation of aging process by unc-13 and sbt-1 in Caenorhabditis elegans.MethodsThe lifespan, the speed of pharynx pumping, and the intestinal autofluorescence of unc-13 and sbt-1 mutants were examined at different temperature conditions. In addition, to exclude the possible influences from other factors in unc-13 and sbt-1 mutants, the dauer formation, the thermotaxis, the brood size and the population percentage of the mutants expressing hsp16.2-gfp were further investigated.ResultsMutations of unc-13 and sbt-1 significantly increased the mean and the maximum lifespans of nematodes cultured at 20 °C and 25 °C, while no noticeable increase was found at 15 °C in either the mean or the maximum lifespan. Investigations on the speed of pharynx pumping and the intestinal autofluorescence suggested that at 20 °C and 25 °C, mutations of unc-13 and sbt-1 could slow the aging process and delay the accumulation of aging-related cellular damage. Meanwhile, mutations of unc-13 or sbt-1 did not affect the dauer formation or the thermotaxis to different temperatures in nematodes. In contrast, at 20 °C and 25 °C conditions, mutations of unc-13 and sbt-1 significantly decreased the brood size and the percentage of nematodes expressing hsp16.2-gfp, while no such differences were detected at 15 °C. Moreover, the thermotolerance of unc-13 and sbt-1 mutants could be greatly strengthened after the 16-h heat shock at 35 °C.ConclusionThe regulation of aging by unc-13 and sbt-1 is temperaturedependent. And the alterations in reproduction capability and stress response may be associated with the formation of this temperature-dependent property.摘要目的研究环境因素—温度对秀丽线虫 unc-13 和 sbt-1 基因调控衰老作用的影响。方法在不同培养温度下,测定 unc-13 和 sbt-1 突变线虫的寿命、 咽泵运动速率和肠道荧光。 此外, 为排除其他可能的影响因素, 对 unc-13 和 sbt-1 突变体的永久性幼虫形成率、 对培养温度的趋向性、 子代数目以及表达 hsp16.2-gfp 线虫在群体中的比例进行了测定。结果在20 °C 和25 °C 培养条件下, unc-13 和 sbt-1 突变能显著提高线虫的平均寿命和最高寿命; 然而在15 °C 条件下, unc-13 和 sbt-1 突变体的平均寿命和最高寿命与野生型相似。 咽泵运动速率和肠道脂褐质自发荧光分析进一步验证了在20 °C 和25 °C 培养条件下, unc-13 和 sbt-1 突变可以减慢线虫衰老或延缓衰老相关的细胞损伤累积。 同时, unc-13 和 sbt-1 突变不影响永久性幼虫发生率以及动物对不同温度的趋向性。 相反, 在20 °C和25 °C 培养条件下, unc-13 和 sbt-1 突变能显著降低秀丽线虫的子代数目和群体中表达 hsp16.2-gfp 的线虫比率, 而在15 °C 条件下未出现此种差异。 此外, unc-13 和 sbt-1 突变体经35 °C 热激16 h 后, 其耐热性得到增强。结论unc-13 和 sbt-1 对衰老的调节具有温度依赖性。 unc-13 和 sbt-1 突变体的生殖功能和应激反应的改变可能与此温度依赖性的形成有关。

UNC-64 and RIC-4, the plasma membrane-associated SNAREs syntaxin and SNAP-25, regulate fat storage in nematode Caenorhabditis elegans@@@质膜相关的 SNAREs syntaxin/UNC-64 与 SNAP-25/RIC-4蛋白调控秀丽线虫的脂肪积累

ObjectiveTo investigate whether genes required for synaptogenesis and synaptic function are also involved in fat storage control in Caenorhabditis elegans.MethodsFat storage was examined in mutants of genes affecting the synaptogenesis and synaptic function. In addition, the genetic interactions of SNAREs syntaxin/unc-64 and SNAP-25/ric-4 with daf-2, daf-7, nhr-49, sbp-1 and mdt-15 in regulating fat storage were further investigated. The tissue-specific activities of unc-64 and ric-4 were investigated to study the roles of unc-64 and ric-4 in regulating fat storage in the nervous system and/or the intestine.ResultsMutations of genes required for the formation of presynaptic neurotransmission site did not obviously influence fat storage. However, among the genes required for synaptic function, the plasma membrane-associated SNAREs syntaxin/unc-64 and SNAP-25/ric-4 genes were involved in the fat storage control. Fat storage in the intestinal cells was dramatically increased in unc-64 and ric-4 mutants as revealed by Sudan Black and Nile Red strainings, although the fat droplet size was not significantly changed. Moreover, in both the nervous system and the intestine, expression of unc-64 significantly inhibited the increase in fat storage observed in unc-64 mutant. And expression of ric-4 in the nervous system completely restored fat storage in ric-4 mutant. Genetic interaction assay further indicated that both unc-64 and ric-4 regulated fat storage independently of daf-2 [encoding an insulin-like growth factor-I (IGF-I) receptor], daf-7 [encoding a transforming growth factor-β (TGF-β) ligand], and nhr-49 (encoding a nuclear hormone receptor). Besides, mutation of daf-16 did not obviously affect the phenotype of increased fat storage in unc-64 or ric-4 mutant. Furthermore, unc-64 and ric-4 regulated fat storage probably through the ARC105/mdt-15- and SREBP/sbp-1-mediated signaling pathways. In addition, fat storage in unc-64; ric-4 was higher than that in either unc-64 or ric-4 single mutant nematodes, suggesting that unc-64 functions in parallel with ric-4 in regulating fat storage.ConclusionThe plasma membrane-associated SNAREs syntaxin/unc-64 and SNAP-25/ric-4 function in parallel in regulating fat storage in C. elegans, probably through the ARC105/mdt-15- and SREBP/sbp-1-mediated signaling pathways.摘要目的研究神经突触组装与功能调控相关基因是否参与秀丽线虫的脂肪积累调节。方法分析神经突触组装与功能调控相关基因突变体的脂肪积累变化, 进而观察SNAREs syntaxin/unc-64和SNAP-25/ric-4基因与daf-2、 daf-7、 nhr-49、 sbp-1 及mdt-15所介导的信号通路在调控脂肪积累上的遗传关系。 对unc-64 与ric-4基因进行组织特异性活性分析, 以确定它们在神经系统与肠道内对脂肪积累的影响。结果突触前为神经突触组装所必需的基因的突变并未明显影响脂肪积累。 对调控神经突触功能的基因进行分析的结果显示, SNAREs syntaxin/unc-64与SNAP-25/ric-4基因均参与了脂肪积累的调节。 利用苏丹黑染色与尼罗红标记法观察到unc-64与ric-4突变体肠道中脂肪积累显著增加, 而unc-64与ric-4突变体中积累的脂肪颗粒并未出现尺寸的显著变化。 在神经系统与肠道中, unc-64基因的表达均能显著降低unc-64突变体动物的脂肪积累, 而基因ric-4在神经系统的表达则可以完全恢复ric-4突变体动物的脂肪积累。 遗传分析表明, unc-64 与ric-4对脂肪积累的调控独立于daf-2 (IGF-I受体基因)、 daf-7 (TGF-β配体基因)和nhr-49(核激素受体基因), 且不受daf-16基因突变的影响。 进一步的研究结果显示, unc-64 与ric-4可能经由ARC105/mdt-15与SREBP/sbp-1介导的信号通路来调节动物的脂肪积累进程。 此外, unc-64; ric-4双突变体的脂肪积累水平要显著高于unc-64或ric-4单突变体的脂肪积累水平, 提示这两个基因通过平行的遗传通路调控脂肪积累。结论质膜相关的SNAREs syntaxin/unc-64和SNAP-25/ric-4基因通过平行的遗传通路并经由ARC105/mdt-15与SREBP/sbp-1介导的信号通路来调控秀丽线虫的脂肪积累。To investigate whether genes required for synaptogenesis and synaptic function are also involved in fat storage control in Caenorhabditis elegans. Fat storage was examined in mutants of genes affecting the synaptogenesis and synaptic function. In addition, the genetic interactions of SNAREs syntaxin/unc-64 and SNAP-25/ric-4 with daf-2, daf-7, nhr-49, sbp-1 and mdt-15 in regulating fat storage were further investigated. The tissue-specific activities of unc-64 and ric-4 were investigated to study the roles of unc-64 and ric-4 in regulating fat storage in the nervous system and/or the intestine. Mutations of genes required for the formation of presynaptic neurotransmission site did not obviously influence fat storage. However, among the genes required for synaptic function, the plasma membrane-associated SNAREs syntaxin/unc-64 and SNAP-25/ric-4 genes were involved in the fat storage control. Fat storage in the intestinal cells was dramatically increased in unc-64 and ric-4 mutants as revealed by Sudan Black and Nile Red strainings, although the fat droplet size was not significantly changed. Moreover, in both the nervous system and the intestine, expression of unc-64 significantly inhibited the increase in fat storage observed in unc-64 mutant. And expression of ric-4 in the nervous system completely restored fat storage in ric-4 mutant. Genetic interaction assay further indicated that both unc-64 and ric-4 regulated fat storage independently of daf-2 [encoding an insulin-like growth factor-I (IGF-I) receptor], daf-7 [encoding a transforming growth factor-β (TGF-β) ligand], and nhr-49 (encoding a nuclear hormone receptor). Besides, mutation of daf-16 did not obviously affect the phenotype of increased fat storage in unc-64 or ric-4 mutant. Furthermore, unc-64 and ric-4 regulated fat storage probably through the ARC105/mdt-15- and SREBP/sbp-1-mediated signaling pathways. In addition, fat storage in unc-64; ric-4 was higher than that in either unc-64 or ric-4 single mutant nematodes, suggesting that unc-64 functions in parallel with ric-4 in regulating fat storage. The plasma membrane-associated SNAREs syntaxin/unc-64 and SNAP-25/ric-4 function in parallel in regulating fat storage in C. elegans, probably through the ARC105/mdt-15- and SREBP/sbp-1-mediated signaling pathways. 研究神经突触组装与功能调控相关基因是否参与秀丽线虫的脂肪积累调节。 分析神经突触组装与功能调控相关基因突变体的脂肪积累变化, 进而观察SNAREs syntaxin/unc-64和SNAP-25/ric-4基因与daf-2、 daf-7、 nhr-49、 sbp-1 及mdt-15所介导的信号通路在调控脂肪积累上的遗传关系。 对unc-64 与ric-4基因进行组织特异性活性分析, 以确定它们在神经系统与肠道内对脂肪积累的影响。 突触前为神经突触组装所必需的基因的突变并未明显影响脂肪积累。 对调控神经突触功能的基因进行分析的结果显示, SNAREs syntaxin/unc-64与SNAP-25/ric-4基因均参与了脂肪积累的调节。 利用苏丹黑染色与尼罗红标记法观察到unc-64与ric-4突变体肠道中脂肪积累显著增加, 而unc-64与ric-4突变体中积累的脂肪颗粒并未出现尺寸的显著变化。 在神经系统与肠道中, unc-64基因的表达均能显著降低unc-64突变体动物的脂肪积累, 而基因ric-4在神经系统的表达则可以完全恢复ric-4突变体动物的脂肪积累。 遗传分析表明, unc-64 与ric-4对脂肪积累的调控独立于daf-2 (IGF-I受体基因)、 daf-7 (TGF-β配体基因)和nhr-49(核激素受体基因), 且不受daf-16基因突变的影响。 进一步的研究结果显示, unc-64 与ric-4可能经由ARC105/mdt-15与SREBP/sbp-1介导的信号通路来调节动物的脂肪积累进程。 此外, unc-64; ric-4双突变体的脂肪积累水平要显著高于unc-64或ric-4单突变体的脂肪积累水平, 提示这两个基因通过平行的遗传通路调控脂肪积累。 质膜相关的SNAREs syntaxin/unc-64和SNAP-25/ric-4基因通过平行的遗传通路并经由ARC105/mdt-15与SREBP/sbp-1介导的信号通路来调控秀丽线虫的脂肪积累。