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K S Krishnan

Publications and source records attributed to K S Krishnan.

At least 37 records · Page 2Linked to original sources
PubMed1999

Phenotypic interaction between temperature-sensitive paralytic mutants comatose and paralytic suggests a role for N-ethylmaleimide-sensitive fusion factor in synaptic vesicle cycling in Drosophila.

The temperature-induced paralysis of comatose (comt) mutants of Drosophila is suggestive of a function for N-ethylmaleimide-sensitive fusion factor (NSF) in the CNS. Mutations in the para gene encoding the subunit of the voltage-gated sodium channel also result in a similar phenotype. We show that paralysis in comt flies is activity-dependent, and in the doubly mutant comt para flies comt-like paralysis does not set in until the effects of para are reversed by shifting to permissive temperatures. During recording from the thoracic flight muscles, we observed that comt flies showed a burst of spontaneous activity at restrictive temperature. This has been reported earlier as a unique characteristic of comt paralysis. The comt para double mutant showed this burst of activity not at restrictive but only on shifting back to permissive temperature. The unusual behavior and electrophysiology of the doubly mutant flies reported here indicates a role for NSF in synaptic vesicle cycling.

Animals↗
PubMed1998

Probable mechanisms underlying interallelic complementation and temperature-sensitivity of mutations at the shibire locus of Drosophila melanogaster.

The shibire locus of Drosophila melanogaster encodes dynamin, a GTPase required for the fission of endocytic vesicles from plasma membrane. Biochemical studies indicate that mammalian dynamin is part of a complex containing multiple dynamin subunits and other polypeptides. To gain insight into sequences of dynamin critical for its function, we have characterized in detail a collection of conditional and lethal shi alleles. We describe a probable null allele of shi and show that its properties are distinct from those of two classes of lethal alleles (termed I and II) that show intergroup, interallelic complementation. Sequenced class I alleles, which display dominant properties, carry missense mutations in conserved residues in the GTPase domain of dynamin. In contrast, the sequenced class II alleles, which appear completely recessive, carry missense mutations in conserved residues of a previously uncharacterized "middle domain" that lies adjacent to the GTPase region. These data suggest that critical interactions mediated by this middle domain are severely affected by the class II lethal mutations; thus, the mutant sequences should be very useful for confirming the in vivo relevance of interactions observed in vitro. Viable heteroallelic combinations of shi lethals show rapid and reversible temperature-sensitive paralytic phenotypes hitherto only described for the ts alleles of shi. When taken together with the molecular analysis of shi mutations, these observations suggest that the GTPase domain of dynamin carries an intrinsically temperature-sensitive activity: hypomorphic mutations that reduce this activity at low temperatures result in conditional temperature-sensitive phenotype. These observations explain why screens for conditional paralytic mutants in Drosophila inevitably recover ts alleles of shi at high frequencies.

Alleles↗
PubMed1997

A genetic and mosaic analysis of a locus involved in the anesthesia response of Drosophila melanogaster.

We describe a genetic and behavioral analysis of several alleles of har38, a mutant with altered sensitivity to the general anesthetic halothane. We obtained a P-element-induced allele of har38 and generated several excision alleles by remobilizing the P element. The mutants narrow abdomen (na) and har85 are confirmed to be allelic to har 38. Besides a decreased sensitivity to halothane, all mutant alleles of this locus cause a characteristic walking behavior in the absence of anesthetics. We have quantified this behavior using a geotaxis apparatus. Responses of the mutant alleles to different inhalational anesthetics were tested. The results strongly favor a multipathway model for the onset of anesthesia. Mosaic flies were tested for their response to halothane and checked for their abnormal walking behavior. The analysis suggests that both the behaviors are exhibited only by such mosaics as have the entire head of mutant origin. It is likely that this focus represents an element of a common pathway in the anesthetic response to several inhalational anesthetics but not all. This result is the first demonstration of regional specificity in the CNS of any animal for general anesthetic action.

Alleles↗
PubMed1996

Traffic of dynamin within individual Drosophila synaptic boutons relative to compartment-specific markers.

Presynaptic terminals contain several specialized compartments, which have been described by electron microscopy. We show in an identified Drosophila neuromuscular synapse that several of these compartments-synaptic vesicle clusters, presynaptic plasma membrane, presynaptic cytosol, and axonal cytoskeleton-labeled by specific reagents may be resolved from one another by laser scanning confocal microscopy. Using a panel of compartment-specific markers and Drosophila shibire(ts1) mutants to trap an intermediate stage in synaptic vesicle recycling, we have examined the localization and redistribution of dynamin within single synaptic varicosities at the larval neuromuscular junction. Our results suggest that dynamin is not a freely diffusible molecule in resting nerve terminals; rather, it appears localized to synaptic sites by association with yet uncharacterized presynaptic components. In shi(ts1) nerve terminals depleted of synaptic vesicles, dynamin is quantitatively redistributed to the plasma membrane. It is not, however, distributed uniformly over presynaptic plasmalemma; instead, fluorescence images show "hot spots" of dynamin on the plasma membrane of vesicle-depleted nerve terminals. We suggest that these dynamin-rich domains may mark the active zones for synaptic vesicle endocytosis first described at the frog neuromuscular junction.

Animals↗
PubMed1996

Alleviation of the temperature-sensitive paralytic phenotype of shibire(ts) mutants in Drosophila by sub-anesthetic concentrations of carbon dioxide.

Cellular mechanisms involved in general anesthesia are unknown. We report here that sub-anesthetic concentrations of carbon dioxide specifically suppress the temperature-sensitive paralytic phenotype of Drosophila shibire(ts) mutants that have a conditional block in synaptic vesicle recycling. Carbon dioxide not only suppresses the onset of temperature-sensitive paralysis, but also rapidly reverses paralysis induced at the restrictive temperature. This effect of CO2 is most pronounced at about 35% in air, and depends on the absolute concentration of available carbon dioxide rather than on the ratio of oxygen to CO2. Other general anesthetics, halothane, N2 or argon do not suppress the paralytic phenotype of shibire significantly at concentrations we tested. Paralysis of the other temperature sensitive paralytic mutants in our collection is not suppressed by carbon dioxide. These behavioral observations are discussed in the light of possible mechanisms underlying paralysis of shi(ts) flies. We suggest that spontaneous seizures induced in shi(ts) flies held at their restrictive temperatures cause vesicle depletion at critical synapses and consequent behavioral paralysis. The effect of subanesthetic concentrations of CO2 may be to depress spontaneous CNS activity, thus raising the threshold temperature at which synaptic vesicle depletion occurs. In support of this model, we show that the threshold temperature for paralysis is reduced in shi(ts) flies when CNS activity is increased by pharmacological or genetic manipulations, and that subanesthetic concentrations of CO2 aggravate, rather than alleviate, the t.s. paralytic phenotype of hypoactive parats flies defective in axonal voltage-gated sodium channels.

Alleles↗
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

Mutations that affect ion channels change the sensitivity of Drosophila melanogaster to volatile anesthetics.

We have quantitated the response of D. melanogaster to general anesthetics with a device, the inebriometer, that assays the fly's geotactic and postural behavior. When alleles of several loci that encode or regulate subunits of ion channels were compared with control stocks, several ion channel mutants clearly increased the anesthetic sensitivity of Drosophila. The effects were specific in that: a) for several alleles, genetic tests indicated that the anesthesia phenotype was due to the ion channel mutation and not to extraneous genetic differences between the stocks; b) a given ion channel mutation often affected the response to one anesthetic but not another; and c) the behavior of decapitated flies in the inebriometer indicated that the anesthetic phenotype of several mutants did not merely reflect a global change in the fly's physiology. These results provide support for the idea that ion channels are on the pathway(s) influenced by anesthetics and that different anesthetics use different pathways. They also provide perspective on the behavior of previously isolated mutations (har) that decrease the sensitivity of Drosophila to anesthetics in the inebriometer.

Anesthetics, Inhalation↗
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↗
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↗
PubMed1990

A genetic study of the anesthetic response: mutants of Drosophila melanogaster altered in sensitivity to halothane.

In an attempt to identify genes that control or encode the targets of general anesthetics, we have chemically mutagenized fruit flies and selected four lines that show an abnormal response to the volatile anesthetic halothane. Specifically, about 2-fold higher concentrations of halothane are required to induce the loss of motor control in the mutant flies. Fine mapping of two isolates indicates that they alter a previously uncharacterized gene of Drosophila. In the absence of anesthetics, these mutants display alterations of behavior that imply changes in the adult and the larval neuromuscular system.

Animals↗
PubMed1987

Thermal gels: a procedure for determination of heat-inactivation temperature of enzymes.

A facile procedure for the study of inactivation temperatures of enzymes that could be stained for activity after electrophoresis is described. This is particularly suitable for enzymes that have multiple electrophoretic forms. The results of studies on Drosophila dehydrogenases and acetylcholinesterase are presented. The procedure may be useful in the study of genetic variants.

Acetylcholinesterase↗
PubMed1987

Monoclonal antibodies to synaptic macromolecules of Drosophila melanogaster.

We have obtained monoclonal antibodies to Drosophila acetylcholinesterase, glutamate dehydrogenase as well as other unknown macromolecules which may have some relevance in synaptic function. The majority of antibodies against acetylcholinesterase recognised common epitopes on all four subunits--but one (MA2) was specific to a 110 kDa dimer. Antibodies to unknown synaptic macromolecules were identified by their selective staining in immunofluorescence studies. F2A3 stains sensory neurons and their synapses in the visual and olfactory systems.

Acetylcholinesterase↗
PubMed1985

Analysis of the effect of car size on accident injury probability using automobile insurance data.

The Highway Loss Data Institute (HLDI) compiles injury and collision claim information from major insurance companies. These data have indicated that a disproportionately large number of injury claims are made for small cars. As a result, it might be concluded that small cars increase injury risk to their occupants. Recent advertisements by a major automobile manufacturer suggest a similar conclusion should be drawn from these data. In this paper it is shown that the apparent higher injury risk attributed to small cars can reflect the behavior of persons driving small cars. The number of injury claim accidents per collision claim accident is examined as an alternative measure of injury risk. Possible problems in interpretation are discussed.

Accidents, Traffic↗
PubMed1983

Molecular properties of Drosophila acetylcholinesterase.

Two distinct classes of acetylcholinesterase (AChE) from the fruit fly Drosophila melanogaster are reported: a soluble species that shows heterogeneity of forms and a particulate species. The subunit composition of the particulate enzyme was studied using the active site label [3H]diisopropylfluorophosphate. Comparison of the electrophoretic patterns on nondenaturing gels using the activity stain and the active site label shows that the label is specific to AChE. The smallest active site-containing subunit of the enzyme is a monomer of approximately 60,000 daltons MW. Two such units are linked by disulphide bonds to produce a dimer of about 110,000 daltons. Another monomeric form of MW approximately 64,000 daltons, although present, does not participate in the dimerisation. The particulate enzyme when solubilised exists as a 9-10S species as determined by sucrose gradient centrifugation. This species has a MW greater than 200,000, as shown by its behaviour on a coarse-bead Sephadex-G200 column. Electrophoretic analysis suggests a MW of nearly 250,000 daltons for this form. Thus, this species is likely to be a tetramer. One possibility is that this tetramer is made up of two units of 64,000 daltons each and a dimer of 110,000 daltons. Preliminary data on mutant enzymes that support such a possibility are also presented.

Acetylcholinesterase↗
PubMed1980

Isolation and characterization of membranes from Drosophila melanogaster.

As a prerequisite for examining the membranes of neurological mutants, we have undertaken to fractionate and characterize membranes derived from heads of adult and whole larvae of wild type Canton-S (C-S) Drosophila. Of particular interest to us are membrane fractions rich in putative brain membrane marker enzyme acetylcholinesterase.

Animals↗