Src is a 60 kDa tyrosine kinase that is expressed in most of animal tissues. Src has three splice variants, C-src, which is ubiquitously expressed, and N1- and N2-src, which are neuronal specific splice variants. The srcs are differentially spliced at their SH3 domains, therefore the hypothesis is that this splicing allows them to have different binding partners and perform different roles in neurons. The aim of this project is to identify new interactions for the three src splice variants in neurons and their possible functional roles. The SH3 domains, kinase active truncated proteins ( 80) and kinase dead mutant full length versions of the three splice variants of src were cloned from a rat brain cDNA library into bacterial expression vectors. GST-pull downs from nerve terminal lysates showed that different src splice variants had different binding partners. These partners were identified by mass spectrometry and confirmed by western blotting. C-src binding partners included dynamin, synapsin, and synaptojanin, while N2-src binding partners included synaptophysin, Munc18-1, and NSF. The interaction between N2-src and Munc 18-1 was characterized further; however a number of in vitro interaction assays and kinase assays showed that Munc 18-1 may not be a direct binding partner for N2-src or substrate. N1-src displayed a stimulation-dependent interaction with dynamin I. This was shown to be phosphorylation-dependent in contrast to C-src binding. The major phosphorylation sites on dynamin I, S774 and S778, were not involved in the regulation of N1-src binding. The binding site for N1-src on dynamin I was different to C-src, with extensive mutagenesis studies suggesting that the interaction site is at the tail of the dynamin I xa splice variant, which has an additional two phosphorylation sites.

The effect of 1,2,3,4-tetrahydro-9-aminoacridine (THA) on quantal transmitter release was examined at the frog neuromuscular junction. THA (3 μM) caused an increase in m (no. of quanta released) as measured by K+-evoked miniature endplate potential (MEPP) frequency. This was due to an increase in p (probability of release), as n (no. of functional release sites) was unchanged. The increase in p was dose-dependent over a range of 0.3-10 μM. By contrast, physostigmine (3 μM) caused a decrease in p, and neostigmine, which does not cross the nerve membrane, had no consistent effect on p. At the postsynaptic site, neostigmine produced the largest increase in MEPP size (79.2%), and THA produced the smallest (17.5%). The divergent effects of THA and physostigmine on p indicate a fundamental difference in their actions at the nerve terminal.

The mechanism by which ionic lanthanum (La3+) increases and subsequently decreases spontaneous transmitter release was investigated by recording miniature endplate potentials (MEPPs) at frog neuromuscular junctions. Addition of tetrodotoxin and Co2+ delayed the onset of MEPP frequency increase but did not otherwise prevent the response. Dinitrophenol substantially reduced but did not eliminate the increase, whereas 3,4,5-trimethoxybenzoic acid8-(diethylamino) octyl ester (TMB-8) completely abolished it. Thus, La3+ does not act by depolarizing the terminal or by substituting for Ca2+ at transmitter release sites. Instead, it appears to enter the terminal through Na+ channels and promote Ca2+ release from intracellular organelles. The profound depletion of transmitter with time may be due to the high turnover of transmitter coupled with the inhibition of metabolic processes by La3+.

Orientador : Maria Julia Marques / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-03T23:03:55Z (GMT). No. of bitstreams: 1 Mendes_ZarifTorresRehder_M.pdf: 784934 bytes, checksum: dac9ec18f5f84c47d0194a64020fd1d8 (MD5) Previous issue date: 2004 / Resumo: No presente trabalho estudamos o padrão de distribuição dos receptores de acetilcolina (AChRs) e terminais nervosos em animais C57BL/10, após longo período de regeneração muscular. A degeneração-regeneração muscular foi induzida pelo anestésico local cloridrato de lidocaína no músculo esternomastóideo. Após 90 e 150 dias, os músculos foram retirados e os AChRs e terminais nervosos marcados com rodamina-a-bungarotoxina e anti-neurofilamento, respectivamente e observados através de microscopia de fluorescência confocal. Em todas as junções superficiais da fibra regenerada observada (n=416 junções) os receptores estavam distribuídos em ilhas e a fibra muscular apresentava núcleo central, caracterizando sua regeneração. Os terminais possuíam finas arborizações com dilatações em suas extremidades, situadas no centro das ilhas dos receptores. O padrão de distribuição da acetilcolinesterase foi semelhante ao observado para os AChRs. Nas fibras musculares regeneradas e desnervadas (n=900 junções) os AChRs não estavam distribuídos em ilhas. Esses resultados mostram que os receptores colinérgicos, após longo tempo de regeneração muscular, mantêm o padrão de distribuição em ilhas e o terminal nervoso parece determinar este padrão, provavelmente em consequência de brotamentos intraterminais que ocorrem em presença de regeneração muscular / Abstract: Mdx mice have deficiency of dystrophin and cycles of muscle fiber regeneration. Changes in the distribution of acetylcholine receptors have been reported at the neuromuscular junction of mdx and may be a consequence of muscle fiber regeneration. At the present, we verified whether the distribution of receptors is still altered in long-term regenerated muscle fibers from C57Bl/10 mice. The left sternomastoid muscle of adult mice was injected with 60 µl of lidocaine hydrochloride to induce degeneration-regeneration. Some animals had the STN denervated at the time of lidocaine injection. After 90 and 150 days, receptors were labeled with rhodamine-a-bungarotoxin for confocal observation. For both periods studied, receptors were distributed into clusters. In denervated-regenerated fibers, receptors were distributed as branches, similar to normal denervated muscles. This suggests that nerve-dependent mechanisms are involved in the changes in receptor distribution seen in regenerated muscle fibers, after lidocaine treatment and a similar phenomenon might explain the changes in receptor distribution seen in dystrophic muscle fibers / Mestrado / Anatomia / Mestre em Biologia Celular e Estrutural

The effects of Hg2+, methyl mercury, and flufenamic acid, all of which inhibit mitochondria, were examined at frog motor nerve terminals. Unbiased estimates of m (no. of transmitter quanta released), n (no. of functional release sites), p (probability of release), and vars p (spatial variance in p) were obtained using K+-induced asynchronous neurosecretion (m, n and p not having the same definitions as with nerve-evoked release). Transient but significant increases in m, n, p and vars p were found with all three agents. These findings indicate that mitochondrial inhibition and release of sequestered Ca2+ can be detected as a real-time increase in vars p. The results also suggest that changes in vars p might be used to differentiate between cellular (membrane) and subcellular (organellar) actions of drugs at the nerve terminal.