RESUMEN
UNC-31 or its mammalian homologue, Ca(2+)-dependent activator protein for secretion (CAPS), is indispensable for exocytosis of dense core vesicle (DCV) and synaptic vesicle (SV). From N- to the C-terminus, UNC-31 contains putative functional domains, including dynactin 1 binding domain (DBD), C2, PH, (M)UNC-13 homology domain (MHD) and DCV binding domain (DCVBD), the last four we examined in this study. We employed UNC-31 null mutant C. elegans worms to examine whether UNC-31 functions could be rescued by ectopic expression of full length UNC-31 vs each of these four domain-deleted mutants. Full length UNC-31 cDNA rescued the phenotypes of C. elegans null mutants in response to Ca(2+)-elevation in ALA neurons. Surprisingly, MHD deletion also rescued UNC-31 exocytotic function in part because the relatively high Ca(2+) level (pre-flash Ca(2+) was 450 nM) used in the capacitance study could bypass the MHD defect. Nonetheless, the three other domain-truncation cDNAs had almost no rescue on Ca(2+) evoked secretion. Importantly, this genetic null mutant rescue strategy enabled physiological studies at levels of whole organism to single cells, such as locomotion assay, pharmacological study of neurotransmission at neuromuscular junction, in vivo neuropeptide release measurement and analysis of vesicular docking. Our results suggest that each of these UNC-31 domains support distinct sequential molecular actions of UNC-31 in vesicular exocytosis, including steps in vesicle tethering and docking that bridge vesicle with plasma membrane, and subsequently priming vesicle by initiating the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core complex.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Unión al Calcio/metabolismo , Neuronas/metabolismo , Vesículas Secretoras/metabolismo , Transmisión Sináptica , Animales , Proteínas de Caenorhabditis elegans/genética , Proteínas de Unión al Calcio/genética , Exocitosis , Estructura Terciaria de Proteína/genética , Eliminación de SecuenciaRESUMEN
Animals integrate various environmental stimuli within the nervous system to generate proper behavioral responses. However, the underlying neural circuits and molecular mechanisms are largely unknown. The insulin-like signaling pathway is known to regulate dauer formation, fat metabolism, and longevity in Caenorhabditis elegans (C. Elegans). Here, we show that this highly conserved signaling pathway also functions in the integrative response to an olfactory diacetyl and a gustatory Cu(2+) stimuli. Worms of wild-type N2 Bristol displayed a strong avoidance to the Cu(2+) barrier in the migration pathway to the attractive diacetyl. Mutants of daf-2 (insulin receptor), daf-18 (PTEN lipid phosphatase), pdk-1 (phosphoinositide-dependent kinase), akt-1/-2 (Akt/PKB kinase) and sgk-1 (serum- and glucocorticoid-inducible kinase) show severe defects in the elusion from the Cu(2+). Mutations in DAF-16, a forkhead-type transcriptional factor, suppress the integrative defects of daf-2 and akt-1/-2 mutants. We further report that neither cGMP nor TGFß pathways, two other dauer formation regulators, likely plays a role in the integrative learning. These results suggest that the insulin-like signaling pathway constitutes an essential component for sensory integration and decision-making behavior plasticity.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/fisiología , GMP Cíclico/fisiología , Insulina/metabolismo , Olfato/fisiología , Gusto/fisiología , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Quimiotaxis/genética , Quimiotaxis/fisiología , Cobre/fisiología , GMP Cíclico/genética , Diacetil/metabolismo , Longevidad , Transducción de Señal , Olfato/genética , Gusto/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Modulation of the Ca2+ sensitivity and cooperativity of secretion is an important means of regulating neurotransmission and hormone secretion. Employing high-time resolution measurement of membrane capacitance (Cm) stimulated by step-like or ramp [Ca2+]i elevation, we have identified the co-existence of both a high and low Ca2+-sensitive exocytosis in an immortal pituitary gonadotrope cell line, LbetaT2. Ramp [Ca2+]i generated by slow uncaging elicited a biphasic C(m) response. The first phase of response, which represents a highly Ca2+-sensitive pool (HCSP) of vesicles, began to secrete at low [Ca2+]i concentration (<1 microM) with low Ca2+ cooperativity. In contrast, the second phase, which represents a lowly Ca2+-sensitive pool (LCSP) of vesicles, only exocytozed at higher [Ca2+]i (>5 microM) and displayed a steep Ca2+ cooperativity. The co-existence of vesicle populations with different Ca2+ sensitivities was further confirmed by flash photolysis stimuli. The size of the HCSP was approximately 30 fF under resting conditions, but was dramatically increased (approximately threefold) by application of phorbol-12-myristate-13-acetate (PMA, an activator of protein kinase C). Forskolin (an activator of protein kinase A), however, exerted no significant effect on the size of both HCSP and LCSP. GnRH (gonadotropin releasing hormone) augmented the size of both pools to a larger extent (5- and 1.7-fold increase for HCSP and LCSP, respectively). The heterogeneity of Ca2+ sensitivity from different pools of vesicles and its differential modulation by intracellular signals suggests that LbetaT2 cells are an ideal model to further unravel the mechanism underlying the modulation of Ca2+-sensing machineries for exocytosis.