Biophysical And Molecular Principles Underlying Odor Detection And Discrimination In C.Elesans,&Inhibition Of Glioma Cell Lysosome Exocytosis Inhibits Glioma Invasion

Abstract:The nematode Caenorhabditis elegans (C. elegans) are widely used in the study of sensory function due to their short life cycle, convenient genetic manipulation, the already known synaptic connection and various sensory abilities. The olfactory sensation is one of the most representative sensory abilities in C. elegans which is closely related to feeding and mating behaviors. Using EMS forward genetic screening, laser ablation and chemotaxis assays, previous studies have identified five pairs olfactory neurons, including attractive neurons AWA and AWC and repulsive neurons AWB, ASH and ADL, and a bunch of olfactory-related genes in C. elegans. However, direct evidence about how these genes participate in the sensation of odorants and how the odorant signals are transmitted in each of these olfactory neurons are still lacking. Here, using in vivo electrophysiology combined with molecular genetics and pharmacological analysis, we found AWC neurons can be hyperpolarized by either low concentration10-4) or high concentration(10-2) of isoamyl alcohol(IAA) and depolarized by removal of1AA. Further studies demonstrated that hyperpolarization of AWC was due to closure of a CNG channel TAX-2/TAX-4. Inhibition of transmembrane GC ODR-1/DAF-11mediated by Ga ODR-3and GPA-3decreases of intracellular cGMP, thereby closing TAX-2/TAX-4. AWB neurons respond differentially to10-2and10-4IAA.10-4IAA elicited inward currents from AWB.10-2IAA induced similar responses from AWB with those from AWC. Different Ga and their targets mediated the different olfactory responses.10-4IAA response was due to increased cGMP level through inhibition of PDE-1,2,3,5by GPA-3, while10-2IAA response was due to decreased cGMP level through inhibition of GC by ODR-3. AWA neurons have similar inward response to10-2and10-4IAA, both of which are mediated by TRP channel OSM-9. The polymodel nonciceptor ASH only responds to10-2IAA with inward currents carried by OSM-9. ADL neurons have no response to either10-2or10-4IAA. We postulate that different IAA receptors are expressed in these olfactory neurons and determined the different olfactory responses. AWB neurons may express both high IAA receptor and low IAA receptor, while ASH neurons only have high IAA receptor. ADL neuron do not express IAA receptor. Thus, high IAA was sensed by AWA, AWB, AWC and ASH neurons, and low IAA was sensed by AWA, AWB and AWC neurons. Taken together, our studies provided the direct evidence for the signal transduction process of odorant sensation in each olfactory neurons of C. elegans, and at the same time elucidated how multiple signaling cascades are elaborately employed by a combination of olfactory neurons and intracellular signaling pathways to detect and process different olfactory cues and initiate complex behaviors. Abstract:Secretory lysosome can secrete their content to perform specific functions in physiological or pathological conditions in addition to their conventional functions of degrading proteins. In cancer cells, lysosome often transfer from perinuclear to peripheral regions. Numerous proteases are sequestered in lysosome and increasing studies demonstrated that many of these proteases play important roles in the progress of cancer partly due to their degradation of extracelluar matrix. Thus, whether proteases secreted from lysosome contribute to the metastasis of cancer need to be further determined. We found the migration and invasion of C6glioma cell were inhibited after lysosome was disrupted by either GPN or vacuolin-1in both transwell and scratch experiments. We specifically knocked-down the expression of Rab27a which is a Rab GTPase required for lysosome exocytosis by RNAi also reduced the migration of glioma cell. These experiments indicate that lysosome exocytosis may be involved in the glioma cell migration. Further studies revealed that cathepsin D, a lysosomal enzyme, participate in migration. Inhibiting cathepsin D with pepstatin A inhibited glioma cell migration. Surface staining and FACS assay of lysosome marker LAMP1revealed that the surface level of LAMP1in glioma cell was much higher than that in astrocyte, suggesting the lysosome of glioma cell is much more prone to be exocytosed and facilitate the invasion of glioma. Our study revealed that cathepsin D released by Rab27a mediated lysosome exocytosis contribute to the migration of glioma cell and also suggest that inhibition of lysosome exocytosis may be used as a cancer therapy.