The Research Of The Molecular Mechanism Of Sensory Transduction In C.elegans,&Identification And Screening The Genes Required For Synaptic Vesicle Cycle

Animals have many modes of sensation such as hearing,touch and proprioception medicated by the mechanosensitive channels.Neurons transduce mechanical stimuli into electrical impulses through mechanosensitive channels within the molecular conformation of their plasma membranes,and then the pass the signal of the sensory system to the downstream.In metazoan,there are few mechnosensitive channel confirmed,so finding the new mechnosensitive channel is an important question in the future.In this study,using the patch clamp technique in C.elegans,we found that the male-specific neuron CEM can response to the mechanical force for the first time,and the mechanosensitive channel in the CEM neuron is a chloride selective channel.Furthermore,we found that the islolated single CEM neuron also could produce the mechano-gated current,and the properties of the current were same to the CEM neurons in vivo.And the mechanical force can produce a robust calcium transient in the CEM neurons,so we speculate that in the physiological status,the concentration of the chloride intracellular is higher than the extracellular.And the hypothesis is confirmed under expressing the histamine gated chloride channel in the CEM neurons.At the same time,the pharmacology experiments show that the current evoked by the mechanical force in the CEM neurons can be inhibited by the NFA and the DCPIB.After a series of the selection and identification,we got some candidates,but we need the further research to find the mechano-gated channel in the CEM neurons.We identify a chloride selective channel medicated the mechanical force in metazoan for the first time.Our work lay a foundation on the identification of the new chloride selective mechanosensitive channels.Animals utilize specialized sensory neurons to detect various painful stimuli,including toxic chemicals and harsh touch.However,how these neurons responding to and discriminating different kinds of stimuli is poorly understood.By combining in vivo calcium imaging and molecular genetic manipulation,here we investigated the responsiveness of the C.elegans phasmid neurons PHA/PHB to a variety of sensory stimuli and its underlying molecular mechanisms.Our data shown that PHA/PHB neurons are polymodal sensory neurons responsible to harmful chemicals,including 1-octanol,SDS,glycerol and high concentration of IAA,and painful harsh touch.We found that both a TRPV channel subunit OSM-9 and a CNG channel subunit TAX-4 were essential for chemo-sensation in PHA/PHB neurons.Furthermore,PHA/PHB were inhibited indirectly by copper,a heavy ion repellent,with neuropeptide released from other neurons.We further shown that OSM-9,but not known mechano-gated channels in C.elegans,was involved in mechano-sensation in PHA/PHB neurons.Over all,our work demonstrate that how the polymodal neurons PHA/PHB are activated and modulated by multiple environmental stimuli,and lay a foundation of further understanding of the mechanisms of polymodal signaling such as nociception in more complex organisms.Synaptic vesicles(synaptic vesicles,SV)storage neurotransmitters,when the neuron is stimulated,the synaptic vesicles will be fusion to the presynaptic membrane,and then the neurotransmitter is released,and bind to the postsynaptic receptor,causing the action potential of the postsynaptic cells.Then the SV can be recycle by the endocytosis,and begin to the next recycle.Although the research of the SV is too much,but the mechanisms of the exocytosis and endocytosis are not known clearly.In this paper,using the C.elegans as a model,we want to find more new genes which regulating the SV recycle by the whole genome RNAi screening.We use the RNAi sensitive strain as a probe,which express SNB-1::pHluorin(SpH)fused protein in the nervous system,and perform the high throughput fluorescence screening by the COPAS system.After many times screening,we got 177 up-regulated genes and 97 down-regulated genes,and 76%genes is conserved.We chose a unknown functions gene B0035.1/ZNF207 to investigate the function in the SV recycle.b0035.1 is a conserved gene which has a C2H2 zinc finger domain,and according to the express pattern of the B0035.1,we find that b0035.1 is expressed in the nucleus of the nervous system and part of the muscle cells.B0035.1 mutant worms have a lot of deficient phenotypes,such as the abnormal locomotion and the lower locomotion speed,and they have a significant Aldicarb resistant and a much stronger fluorescence of the SpH,otherwise,the endogenous EPSC and the evoked EPSC of the mutant worms is decreased compared to the wt.according to the CHIP-seq and RNA-seq,we perform the qPCR test,and we find the RNA expression level of the rab-11.1 in the b0035.1 mutant worms is decreased,and the fluorescence intensity of the rab-11.1 RNAi strains is similar to the b0035.1 RNAi strains.When we perform the two genes together,we don't find the overlay effect.So rab-11.1 and b0035.1 play a role in the endocytosis in the same pathway.All these data indicate that the B0035.1 may function in the endocytosis by regulating the function of rab-11.1.