Unique biochemical and behavioral alterations in Drosophila shibire(ts1) mutants imply a conformational state affecting dynamin subcellular distribution and synaptic vesicle cycling

Biological Sciences, Western Illinois University, MQB, Illinois, United States
Journal of Neurobiology (Impact Factor: 3.84). 11/2002; 53(3):319-29. DOI: 10.1002/neu.10101
Source: PubMed


Dynamin is a GTPase protein that is essential for clathrin-mediated endocytosis of synaptic vesicle membranes. The Drosophila dynamin mutation shi(ts1) changes a single residue (G273D) at the boundary of the GTPase domain. In cell fractionation of homogenized fly heads without monovalent cations, all dynamin was in pellet fractions and was minimally susceptible to Triton-X extraction. Addition of Na(+) or K(+) can extract dynamin to the cytosolic (supernatant) fraction. The shi(ts1) mutation reduced the sensitivity of dynamin to salt extraction compared with other temperature-sensitive alleles or wild type. Sensitivity to salt extraction in shi(ts1) was enhanced by GTP and nonhydrolyzable GTP-gammaS. The shi(ts1) mutation may therefore induce a conformational change, involving the GTP binding site, that affects dynamin aggregation. Temperature-sensitive shibire mutations are known to arrest endocytosis at restrictive temperatures, with concomitant accumulation of presynaptic collared pits. Consistent with an effect upon dynamin aggregation, intact shi(ts1) flies recovered much more slowly from heat-induced paralysis than did other temperature-sensitive shibire mutants. Moreover, a genetic mutation that lowers GTP abundance (awd(msf15)), which reduces the paralytic temperature threshold of other temperature-sensitive shibire mutations that lie closer to consensus GTPase motifs, did not reduce the paralytic threshold of shi(ts1). Taken together, the results may link the GTPase domain to conformational shifts that influence aggregation in vitro and endocytosis in vivo, and provide an unexpected point of entry to link the biophysical properties of dynamin to physiological processes at synapses.

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    • "In contrast, during more intense (10 Hz) stimulation, Dynamin photoinactivation (2 min of 508-nm light) results in a fast drop of the EJP amplitude and most recordings reach 0 in <5 min after photoinactivation (Fig. 1 H), very similar to recordings made from (shi ts1 ) that were stimulated at restrictive temperature show a depletion of synaptic vesicles and numerous deeply invaginated pits that are stuck at the plasma membrane (Koenig and Ikeda, 1989; Chen et al., 1991, 2002; van der Bliek and Meyerowitz, 1991). Likewise, in mammalian neurons in which either one or two of the three Dynamin isoforms were deleted, a massive accumulation of invaginated pits is observed (Ferguson et al., 2007; Hayashi et al., 2008; Raimondi et al., 2011; Park et al., 2013). "
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    ABSTRACT: Dynamin is a well-known regulator of synaptic endocytosis. Temperature-sensitive dynamin (shi(ts1)) mutations in Drosophila melanogaster or deletion of some of the mammalian Dynamins causes the accumulation of invaginated endocytic pits at synapses, sometimes also on bulk endosomes, indicating impaired membrane scission. However, complete loss of dynamin function has not been studied in neurons in vivo, and whether Dynamin acts in different aspects of synaptic vesicle formation remains enigmatic. We used acute photoinactivation and found that loss of Dynamin function blocked membrane recycling and caused the buildup of huge membrane-connected cisternae, in contrast to the invaginated pits that accumulate in shi(ts1) mutants. Moreover, photoinactivation of Dynamin in shi(ts1) animals converted these pits into bulk cisternae. Bulk membrane retrieval has also been seen upon Clathrin photoinactivation, and superresolution imaging indicated that acute Dynamin photoinactivation blocked Clathrin and α-adaptin relocalization to synaptic membranes upon nerve stimulation. Hence, our data indicate that Dynamin is critically involved in the stabilization of Clathrin- and AP2-dependent endocytic pits.
    Full-text · Article · Mar 2014 · The Journal of Cell Biology
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    • "The shi ts1 allele is known to be phenotypically stronger than the shi ts2 allele. Animals carrying the shi ts1 allele show abnormal distribution and stability of Dynamin complexes even at the permissive temperature whereas the shi ts2 mutation does not have an obvious biochemical phenotype at the permissive temperature (Chen et al., 2002). Earlier studies have shown that even at the larval stage the different potencies of these alleles are apparent. "
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    ABSTRACT: A prevailing hypothesis is that the set of genes that underlie the endophenotypes of alcoholism overlap with those responsible for the addicted state. Functional ethanol tolerance, an endophenotype of alcoholism, is defined as a reduced response to ethanol caused by prior ethanol exposure. The neuronal origins of functional rapid tolerance are thought to be a homeostatic response of the nervous system that counters the effects of the drug. Synaptic proteins that regulate neuronal activity are an important evolutionarily conserved target of ethanol. We used mutant analysis in Drosophila to identify synaptic proteins that are important for the acquisition of rapid tolerance to sedation with ethanol. Tolerance was assayed by sedating flies with ethanol vapor and comparing the recovery time of flies after their first sedation and their second sedation. Temperature-sensitive paralytic mutants that alter key facets of synaptic neurotransmission, such as the propagation of action potentials, synaptic vesicle fusion, exocytosis, and endocytosis, were tested for the ability to acquire functional tolerance at both the permissive and restrictive temperatures. The shibire gene encodes Drosophila Dynamin. We tested 2 temperature-sensitive alleles of the gene. The shi(ts1) allele blocked tolerance at both the permissive and restrictive temperatures, while shi(ts2) blocked only at the restrictive temperature. Using the temperature-sensitive property of shi(ts2) , we showed that Dynamin function is required concomitant with exposure to ethanol. A temperature-sensitive allele of the Syntaxin 1A gene, Syx1A(3-69), also blocked the acquisition of ethanol tolerance. We have shown that shibire and Syntaxin 1A are required for the acquisition of rapid functional tolerance to ethanol. Furthermore, the shibire gene product, Dynamin, appears to be required for an immediate early response to ethanol that triggers a cellular response leading to rapid functional tolerance.
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    • "However, this does not mean that these alleles are temperature sensitive for all phenotypes. At the permissive temperature, the shi ts1 allele has been biochemically shown to cause the production of dynamin complexes that are substantially more stable than the wild-type complex (Chen et al. 2002). Furthermore, at the permissive temperature, the shi ts1 mutation alters the latency of the giant fiber escape pathway (Hummon and Costello 1989). "
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    ABSTRACT: The hypnotic effects of anesthetics are caused by their interactions with neuronal components vital for proper signaling. An understanding of the adaptive mechanisms that lead to the development of anesthetic tolerance can offer insight into the regulation of neuroexcitability and plasticity that alter behavioral output. Here we use genetic and pharmacological manipulation of Drosophila to investigate the mechanisms of tolerance to benzyl alcohol. The mutants tested were temperature-sensitive paralytics that interfere with neuronal signaling: two mutations in dynamin that affect vesicle recycling, shi (ts1) and shi (ts2), and one that affects the voltage-activated Na(+) channel, para (ts1). We also used N-ethylmaleimide (NEM) to pharmacologically interfere with synaptic function. We found that blocking the generation of action potentials using a temperature-sensitive paralytic mutation does not induce nor prevent the development of functional tolerance to benzyl alcohol, but that disruption of synaptic signaling using mutations in the dynamin gene or by NEM treatment inhibits the induction of tolerance.
    Full-text · Article · Feb 2011 · Behavior Genetics
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