Biomedical subjects

M Ramaswami

Publications and source records attributed to M Ramaswami.

27 records · Page 2Linked to original sources

PubMed1995

Distinct roles for N-ethylmaleimide-sensitive fusion protein (NSF) suggested by the identification of a second Drosophila NSF homolog.

The N-ethylmaleimide-sensitive fusion protein (NSF) is a cytoplasmic protein implicated in the fusion of intracellular transport vesicles with their target membranes. NSF is thought to function in the fusion of essentially all types of vesicles, including endoplasmic reticulum, Golgi, and endocytic vesicles, as well as secretory vesicles undergoing regulated fusion (for review see Rothman, J.E. (1994) Nature 372, 55-63). However, little experimental evidence exists to address the possibility that organisms might have multiple NSF proteins serving distinct functions in the same or different cells. We previously cloned a neurally expressed Drosophila homolog, dNSF-1 (Ordway, R.W., Pallanck, L., and Ganetzky, B. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 5715-5719), and have subsequently identified mutations in this gene that confer an apparent failure of synaptic transmission at elevated temperature (Pallanck, L., Ordway, R.W., and Ganetzky, B. (1995) Nature, 376, 25; Siddiqi, O., and Benzer, S. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3253-3257). Here we report that 1) Drosophila contains a second NSF homolog, termed dNSF-2, that exhibits 84% amino acid identity to dNSF-1, 2) dNSF-1 and dNSF-2 display overlapping but different temporal expression, and 3) multiple transcripts are derived from the dNSF-2 gene. These findings raise the possibility that different NSF gene products serve distinct or overlapping functions with the organism.

Amino Acid Sequence↗

PubMed1995

Redistribution of synaptic vesicles and their proteins in temperature-sensitive shibire(ts1) mutant Drosophila.

From an extract of Drosophila melanogaster head homogenates, a membrane fraction can be isolated that has the same sedimentation properties as vertebrate synaptic vesicles and contains Drosophila synaptotagmin. The fraction disappears from homogenates of temperature-sensitive (ts) mutant shibire(ts1) (shi(ts1)) flies paralyzed by exposure to non-permissive temperatures, and reappears on return to permissive temperatures. Since reversible, temperature-dependent depletion of synaptic vesicles is known to occur in shibire(ts1) flies, we conclude that the fraction we have identified contains synaptic vesicles. We have examined the fate of synaptic vesicle membrane proteins in shibire flies at nonpermissive temperatures and found that all of these vesicle antigens are transferred to rapidly sedimenting membranes and codistribute with a plasma membrane marker by both glycerol velocity and metrizamide density sedimentation and by confocal microscopy. Three criteria were used to establish that other neuron-specific antigens--neuronal synaptobrevin and cysteine-string proteins--are legitimate components of synaptic vesicles: cosedimentation with Drosophila synaptotagmin, immunoadsorption, and disappearance of these antigens from the vesicle fractions in paralyzed shibire flies.

Amino Acid Sequence↗

PubMed1994

The Drosophila easily shocked gene: a mutation in a phospholipid synthetic pathway causes seizure, neuronal failure, and paralysis.

We have characterized easily shocked (eas), a Drosophila "band-sensitive" paralytic mutant. Electrophysiological recordings from flight muscles in the giant fiber pathway of adult eas flies reveal that induction of paralysis with electrical stimulation results in a brief seizure, followed by a failure of the muscles to respond to giant fiber stimulation. Molecular cloning, germline transformation, and biochemical experiments show that eas mutants are defective in the gene for ethanolamine kinase, which is required for a pathway of phosphatidylethanolamine synthesis. Assays of phospholipid composition reveal that total phosphatidylethanolamine is decreased in eas mutants. The data suggest that eas bang sensitivity is due to an excitability defect caused by altered membrane phospholipid composition.

Amino Acid Sequence↗

PubMed1994

Intermediates in synaptic vesicle recycling revealed by optical imaging of Drosophila neuromuscular junctions.

We show that uptake and release of the styryl dye FM1-43 may be used to monitor synaptic vesicle exocytosis and recycling at Drosophila larval neuromuscular junctions. At Drosophila nerve terminals, FM1-43 specifically labels subsynaptic domains enriched in synaptotagmin, in a manner that requires Ca2+, membrane depolarization, and shibire (shi) function. Endocytosis rates, very low in unstimulated synapses, are induced severalfold by the exocytosis of synaptic vesicles. Using shi(ts)1 mutant synapses to separate synaptic vesicle fusion and recycling temporally, we show that recycling events subsequent to the shi block do not require extracellular Ca2+. We suggest that two distinct intermediate stages in vesicle recycling may be trapped and analyzed at Drosophila neuromuscular junctions.

Animals↗

PubMed1994

Facile formation of heteromultimeric potassium channels by expression of cloned human cDNAs.

Complementary DNAs representing three voltage-gated potassium channels of human origin have previously been expressed in Xenopus laevis oocytes by injecting RNA transcribed in vitro [Ramaswami, M., Gautam, M., Kamb, A., Rudy, B., Tanouye, M. A. & Mathew, M. K. (1990) Mol. Cell. Nueorsci 1, 214-223]. We have coinjected RNAs for pairs of K(+)-channel genes into Xenopus oocytes. Analysis of the kinetics of the evoked currents, their voltage dependence and pharmacological sensitivities demonstrate that channels formed on coinjection of RNA pairs have properties distinct from those evoked by either channel type alone. We conclude that these currents arise from heteromultimeric aggregates of the subunits encoded by the individual RNAs. Quantitative analysis of the currents indicate that at least 60% of the current seen can be ascribed to heteromultimeric channels demonstrating their facile formation. Given that there are a large number of primary transcripts present in the nervous system, the demonstration of pharmacologically distinct heteromultimers may complicate the extension of studies on single, cloned K(+)-channels in heterologous systems to neuronal cells.

Cloning, Molecular↗

PubMed1993

Genetic studies on dynamin function in Drosophila.

The shibire(ts2) mutation of Drosophila melanogaster causes a temperature sensitive inhibition of endocytosis; this in turn leads to synaptic-vesicle depletion and consequent paralysis. Heat-pulses delivered during development of shibire(ts2) individuals affect the morphology of a number of adult structures. A simple screening protocol has been used to isolate several mutations that partially suppress the temperature-sensitive paralytic phenotype of shibire(ts2) mutant animals. All of these mutations very tightly linked to shibire and are likely to be second site intragenic mutations that restore partial activity to the shibire(ts2) product. The mutations suppress both behavioral, and easily-scored developmental phenotypes of shibire(ts2) characterized in this paper. Our results suggest that defects in endocytosis, and not in microtubule interactions, are responsible for all of the phenotypes of shibire(ts2) mutant Drosophila examined in this study.

Animals↗

PubMed1991

A role for hydrophobic residues in the voltage-dependent gating of Shaker K+ channels.

A leucine heptad repeat is well conserved in voltage-dependent ion channels. Herein we examine the role of the repeat region in Shaker K+ channels through substitution of the leucines in the repeat and through coexpression of normal and truncated products. In contrast to leucine-zipper DNA-binding proteins, we find that the subunit assembly of Shaker does not depend on the leucine heptad repeat. Instead, we report that substitutions of the leucines in the repeat produce large effects on the observed voltage dependence of conductance voltage and prepulse inactivation curves. Our results suggest that the leucines mediate interactions that play an important role in the transduction of charge movement into channel opening and closing.

Amino Acid Sequence↗

PubMed1989

Two sodium-channel genes in Drosophila: implications for channel diversity.

We describe two Drosophila melanogaster transcription units that are highly homologous to a rat Na+-channel cDNA. They appear to encode the major subunits of two distinct Na+-channel proteins. One of these maps to the second chromosome and is identical to a Na+-channel gene whose partial sequence has been previously reported [Salkoff, L., Butler, A., Wei, A., Scavarda, N., Giffen, K., Ifune, K., Goodman, R. & Mandel, G. (1987) Science 237, 744-749]. The other transcription unit maps to position 14C/D, on the X chromosome, close to the paralyzed (para) gene. Mutations in para affect membrane excitability in Drosophila neurons [Ganetzky, B. & Wu, C.F. (1986) Annu. Rev. Genet. 20, 13-44]. Sequence comparisons suggest that two Na+-channel genes arose early in evolution, before the divergence of vertebrate and invertebrate lines.

Amino Acid Sequence↗

PubMedDate not supplied

The organization of extrinsic neurons and their implications in the functional roles of the mushroom bodies in Drosophila melanogaster Meigen.

Although the importance of the Drosophila mushroom body in olfactory learning and memory has been stressed, virtually nothing is known about the brain regions to which it is connected. Using Golgi and GAL4-UAS techniques, we performed the first systematic attempt to reveal the anatomy of its extrinsic neurons. A novel presynaptic reporter construct, UAS-neuronal synaptobrevin-green fluorescent protein (n-syb-GFP), was used to reveal the direction of information in the GAL4-labeled neurons. Our results showed that the main target of the output neurons from the mushroom body lobes is the anterior part of the inferior medial, superior medial, and superior lateral protocerebrum. The lobes also receive afferents from these neuropils. The lack of major output projections directly to the deutocerebrum's premotor pathways discourages the view that the role of the mushroom body may be that of an immediate modifier of behavior. Our data, as well as a critical evaluation of the literature, suggest that the mushroom body may not by itself be a "center" for learning and memory, but that it can equally be considered as a preprocessor of olfactory signals en route to "higher" protocerebral regions.

Animals↗