See the embryonic expression pattern of cac at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site
There are three peaks of cac expression during development. The first peak begins to rise in mid-to-late embryo stages and
reaches a peak during the first larval instar. Expression then declines over the remaining larval instars but begins to rise again after
pupariation. There is a second peak in midpupal stages and a final peak in late pupae just before adult eclosion. cac RNA is
expressed widely in the embryonic nervous system. Intense, dark staining is seen in the dorsal cerebral hemispheres as well as throughout the ventral
nerve cord. In addition, lightly stained nerves can be seen extending anteriorly from the CNS toward the region of the antennomaxillary complex at the extreme anterior end of the animal (Smith, 1996).
Presynaptic calcium channels play a central role in chemical synaptic
transmission by providing the calcium trigger for evoked neurotransmitter release. These voltage-gated calcium channels are composed of a primary structural subunit,
alpha1, as well as auxiliary ß and alpha2delta
subunits. The cacophony (cac) gene encodes a primary presynaptic calcium channel alpha1 subunit in
Drosophila. Transgenic
expression of a cac-encoded alpha1 subunit fused with enhanced
green fluorescent protein efficiently rescues cac
lethal mutations and allows in vivo analysis of calcium channel localization at active zones. The results reported in this study further characterize the primary role of cac-encoded calcium channels in neurotransmitter
release. In addition, these studies provide a unique
genetic tool for live imaging of functional presynaptic
calcium channels in vivo and define a molecular marker for immunolocalization of other presynaptic proteins relative to active zones. These findings are
expected to facilitate additional analysis of synaptic
development and function in this important model system (Kawasaki, 2004).
To characterize the presynaptic localization of cac-encoded calcium channels, confocal immunofluorescence imaging was used at larval neuromuscular synapses to examine the distribution of EGFP-tagged 1 subunits. Double labeling using a monoclonal anti-GFP antibody and a neuronal plasma membrane-specific antibody, anti-HRP, was performed in a cac lethal mutant rescued by neural expression of CAC1-EGFP. These experiments confirm the presence of calcium channel puncta within presynaptic boutons of rescued larvae but not wild-type controls lacking the transgene. The likely possibility that these CAC1-EGFP puncta correspond to active zones was examined by double labeling with an antibody against DPAK, a well characterized marker for the postsynaptic densities closely apposed to presynaptic active zones. These experiments revealed extensive colocalization of CAC1-EGFP and DPAK, confirming active zone localization of cac-encoded calcium channel 1 subunits. Consistent with previous studies defining the three-dimensional ultrastructure of larval neuromuscular boutons and the surrounding postsynaptic membrane, active zones exhibited an approximately even distribution over the entire surface of a bouton. This was revealed in a series of optical sections through the z-axis. This study therefore confirms that transgenic expression of a EGFP-tagged calcium channel 1 subunit is targeted to active zones and also retains its function in neurotransmitter release (Kawasaki, 2004).
The question of the roles of the two main parts of the insect brain, the mushroom bodies and the central complex, in controlling motor coordination and triggering a variety of behavioral programs, including sound production, remains controversial. With the aim of improving understanding of this question, the parameters of songs used by five-day-old males during courtship for fertilized wild-type females (Canton-S, C-S) were studied over 5-min periods at 25°C; males were of two wild-type Drosophila melanogaster lines (Berlin and C-S). Berlin males lacking mushroom bodies because of treatment with hydroxyurea during development (chemical removal of the mushroom bodies) were used, along with two mutants with defects in the mushroom bodies (mbm1 and mud1), two mutants with defects in the central complex (ccbKS127 and cexKS181), and mutant cxbN71 with defects in both the mushroom bodies and the central complex. The experiments reported here show that courtship songs in males lacking mushroom bodies were virtually identical to those of wild-type males. The main parameters of pulsatile song in mutants mbm1 and mud1 (interpulse interval and train duration) were insignificantly different from those of the songs of wild-type flies, though the stability of the pulse oscillator was the same. Flies of these lines were no different from wild-type flies in terms of courtship success (percentage of copulating pairs in 10-min tests). Conversely, the songs of mutants with defects in the central complex differed from those of wild-type males: (1) there was degradation of the stability of the pulse oscillator and interpulse intervals were very variable; (2) pulses were often significantly longer and appeared multicyclic, as in the well-known cacophony mutant, while the mean train duration was significantly shorter. Males of the line cexKS181 usually courted very intensely, though abnormal sounds were generally emitted. Mutants cexKS181 and ccbKS127 were significantly less successful in courtship than wild-type flies. These data show that the central complex appears to play a very important role in controlling song, while the mushroom bodies are not related to this function (Popov, 2003).
Phenogenetic analysis reveals that cac specifies an essential gene product that is involved in the operation of the visual system and thoracic neuromuscular systems as well as being required for specific behavioral and physiological functions. The cac transcription unit maps to l(1)L13-associated chromosomal lesions, and the cacS and cacH18 mutants have sequence polymorphisms that cause significant changes in the predicted Cac protein (Smith, 1998).
It is still conceivable that this nbA (H18) mutation and the cac one (S) define two different functions (or even genes); but the fact that both are mutated in the same ORF, which encodes a protein that can be considered highly relevant to both song-related and visual-system functions, increases the weight of evidence in favor of these two different kinds of mutants having identified the same molecular-genetic entity. Furthermore, the phenotypic interrelatedness of cac and nbA-defined functions has been boosted by elements of the current results. Thus, several genetic combinations involving cacP73 (originally isolated on the basis of visual defects) give reduced courtship-song pulse amplitude, and several genotypes involving the cacS mutation (identified initially with respect to an anomalous courtship song) have subtle ERG defects. The coupling of courtship-song and visual phenotypes, previously thought to be strictly separated between mutually exclusive classes of these interacting mutants, further suggests that all these mutations are allelic. It is concluded that the cac gene encodes the Dmca1A calcium channel a1 subunit protein, that this protein is an important factor mediating behaviorally-related functions of excitable cells, and that mutations in this gene are responsible for the various phenotypes of cac mutants (Smith, 1998)
The cacS mutation does not lead to pathological abnormalities in the courtship song, but causes quantitative changes in elements of the song, leaving these acoustical signals nicely patterned. The cacS mutation causes analogous (meaning nonpathological) changes in visual system physiology: it reduces the amplitude of the ERG transients (but does not eliminate them) and causes a novel but low amplitude aberration in the ERG. Also, the cacS/cacL-6 heteroallelic combination has defective Y-tube phototaxis. The cacS mutation is in Dmca1A exon 19; this exon seems not to be subject to alternative splicing, so it would be expected to be included in all products expressed from the cac locus. These results imply that the cacS mutation damages Dmca1A channel functions common to physiological processes underlying the generation of courtship song and of a normal ERG, but not sufficiently to disrupt most visually-mediated behaviors (Smith, 1998).
Mutations in ion-channel genes have often been associated with temperature-sensitive phenotypes such as paralysis. In this context, an additional cacophony phenotype, temperature-sensitive convulsions, was recently discovered to be a feature of the 'song allele' cacS but not the 'vision alleles' cacH18 and cacEE171 (Peixoto, 1998). This raised the question of whether other temperature-sensitive ion-channel mutants would sing abnormally. Indeed, mutations at the slowpoke (slo) locus in D. melanogaster, which encodes a calcium-activated potassium channel cause severe song defects. Calcium and potassium currents are involved in the function of pacemaker cells. A simple, speculative scheme is presented for how the products of cac and slo could work together to form a pacemaker that would underlie the tone-pulse component of Drosophila's courtship song (Smith, 1998).
The lovesongs of these flies are thought to be involved in species recognition as well as stimulation of females to copulate and hence are hypothesized to be a component of prezygotic isolation during speciation. It is intriguing that changes in courtship song caused by the cacS mutation -- specifically affecting the number of cycles per pulse, the interpulse interval, and the pulse amplitude -- are similar to the differences in song between several closely related species. The quasi-separability of cac phenotypes implies that it might be possible to 'tune' the courtship song with relatively small evolutionary changes in this one gene. It will be interesting to examine the homologous channel protein from species known to differ in their song components for differences that might contribute to evolutionary divergences of these species' signatures. A gene such as slo may harbor song-related interspecific variations as well (Smith, 1998).
A phenylalanine (analogous to the cacS-mutated residue) in the transmembrane domain of the minK subunit of the IsK potassium channel has been subjected to in vitro mutagenesis (Wilson, 1994). This protein change (F to C at residue 57) led to an IsK potassium current that was normal in terms of half-maximal activation voltage and effect on membrane potential; that is, indistinguishable in these parameters from IsK currents stemming from the expression of molecularly unaltered transcripts. However, the evolutionary and structural divergence of the minK protein makes it difficult to extrapolate these results to the Dmca1A calcium channel. These results draw attention to the role of this transmembrane region (IIIS6) (and in particular to the cac-defined phenylalanine within it, which is now accessible for electrophysiological bioassay via analysis of calcium currents in cacS mutant flies) in the function of calcium channels in general (Smith, 1998).
The cacH18 mutant has defects in most visual phenotypes assayed but no defects in courtship song, and it carries a mutation that creates a stop codon within the alternative exon I/IIa, indicating that Dmca1A isoforms containing at least this variant motif are necessary for and specific to normal visual function. In this context 'variant' means this exon encodes a stretch of amino acids that is hypothesized not to be able to interact with the typical ß subunit (Smith, 1998).
What might be the specific etiology of the vision defects in cac mutants? The mutations could cause developmental or degenerative defects in the optic ganglia or in retinula cells; indeed, degeneration has been reported for many mutations that affect visual transduction. The presence was verified of a deep pseudopupil, an indicator of intact eye structure, during preparation for ERG recording in all genotypes tested. However, a subtle morphological or degenerative defect in the genotypes examined in this report cannot be ruled out (Smith, 1998).
The absence or reduced amplitude of ERG transients in cac mutants, even in genotypes with robust (if aberrant) light coincident receptor potentials, indicates a probable defect in transmission from the retinula cells to postsynaptic cells in the lamina. Synaptic neurotransmitter secretion is known to be dependent on calcium influx mediated by voltage-dependent calcium channels. While the defect in transmission could be pre- or postsynaptic, an attractive hypothesis is that a class of synapse-specific Dmca1A channel isoforms is affected by these mutations (Smith, 1998).
That the light coincident receptor potential in the most severely affected cac mutants is almost completely eliminated implies that such a genotype (visual mutation heterozygous with a lethal) causes an almost total failure of photoreceptor excitation -- with the proviso that an increased stimulus intensity might have coaxed a small degree of depolarization from these mutant types. In Drosophila and other invertebrates, photoexcitation of rhodopsin molecules leads to G-protein-mediated activation of phospholipase-C and generation of inositol phosphates, followed by a light-activated inward current carried predominantly by Ca2+ and thought to be mediated by cation channels formed by the transient receptor potential (TRP) and TRP-like (TRPL) proteins. Ca2+-CaM-regulated Ca2+ release from ryanodine-sensitive stores is believed to be involved in generation of the light-activated current. Inactivation of phototransduction appears to require an influx of extracellular Ca2+, hypothetically involving calcium-regulated phosphorylation mechanisms. Adaptation, or variation of the gain of phototransduction in varying light levels, is controlled by light-dependent changes in intracellular calcium levels, likely mediated by an eye-specific protein kinase C encoded by the inaC gene. Given the regulatory role of calcium in phototransduction, it seems that aberrant calcium regulation due to defective Dmca1A calcium channel function could disrupt phototransduction. Indeed, the ERG of trp mutants exhibit a transient near-normal light coincident receptor potential followed by a rapid decay, and intense light stimulation has been shown to completely but reversibly inactivate trp-mutant photoreceptors; these phenotypes have been suggested to be due to exhaustion of intracellular Ca2+ stores secondary to the defect in TRP-mediated calcium influx. Regardless of etiology, it is clear that defects in proteins involved in calcium influx can have profound effects on phototransduction (Smith, 1998).
Voltage-activated Dmca1A-encoded calcium currents should now be considered as contributing under physiological conditions to the predominantly calcium-mediated light-activated current, subsequent to the light-dependent initiation of retinula depolarization, which is mediated by TRP and TRPL currents. The current phenogenetic and molecular results suggest that further experiments -- in particular, the analysis of Dmca1A and other ion currents in photoreceptors of cac-mutant flies -- could decipher the contributions of Dmca1A calcium currents to membrane excitability or calcium regulation of phototransduction (Smith, 1998).
The multiple phenotypes, complicated genetic interactions, and extensive intragenic complementation imply that cac mutations affect Dmca1A channel functions that are at least partially separable. Consideration of complementation patterns between viable and lethal cac alleles supports this idea. A deletion that removes the cac locus fails to complement all viable alleles for all phenotypes assayed. The cacL-6 allele has allele-specific effects on light coincident receptor potential amplitude, in that it dramatically worsens the amplitude defect of cacH18 and cacEE171 but complements the amplitude defect of cacP73. The cacL-6 allele also partially complements the Y-tube phototaxis defect of cacP73, and its heteroallelic combination with cacEE171 caused blindness in the Y-tube phototaxis assay rather than negative phototaxis. The cacL-10 allele complements cacH18 for phototaxis only. The effects of the cacL-13 allele are identical to those of the deletion, indicating that cacL-13 is null for all functions assayed. The cacL-20 allele partially complements cacP73 for optomotor behavior and fully complements cacP73 in both phototaxis assays. The cacL-24 allele complements every visual phenotype, but not courtship song (Smith, 1998).
The ensemble of these phenogenetic analyses indicates that (1) cacH18 and cacP73 are hypomorphic for phototaxis, in that homozygotes with two copies of the mutant gene give normal behavior, but one copy of either (when heteroallelic with the deletion) reveals a mutant phenotype; (2) only one of these lethal cac alleles (cacL-13) is null, in that the others each complement cac phenotypes that the deletion does not; (3) the lethal cac alleles (except cacL-13) must each have different, putatively separable undamaged functions, in that they each are able to complement (and therefore retain functions required for) different subsets of phenotypes and of the viable cac alleles; and, as a corollary, (4) the viable cac alleles must have different separable damaged functions, in that they each exhibit a different pattern of complementation by the several lethal alleles (Smith, 1998).
Other results also reveal the separability of these phenotypes. The cacH18 mutation leaves courtship song and light coincident receptor potential kinetics intact. There are heteroallelic genotypes that cause aberrant courtship song and ERG transients but normal LCRP amplitude and kinetics (cacS, when heterozygous with any of several cac lethal alleles) or aberrant kinetics but normal LCRP amplitude and normal courtship song (cacP73/cacL-6). Similar examples exist for most of the assayed phenotypes. While the complexity of the interactions precludes a simple definition of functional classes, it is clear that the etiology of the various phenotypes must involve multiple Dmca1A functions that are at least partially separable (Smith, 1998).
Previous analyses of Dmca1A transcripts identified pairs of mutually exclusive alternative exons at two different sites and a third site that generates four transcript variants by differential inclusion of three- and six-bp exons; additional transcript complexity is thought to be generated by RNA editing at 11 identified nucleotides in the transcript. Functional complexity could be mediated by several mechanisms, including temporal, tissue-specific, or subcellular spatial regulation of Dmca1A expression; one imagines that at least some distinct cac-mediated functions might correspond directly to distinct Dmca1A isoforms. Indeed, the cacH18 mutation creates a stop codon within the intriguingly variant alternative exon I/IIa, indicating that Dmca1A isoforms containing this variant motif are required for normal visual function, but not for viability or normal courtship song. Additional molecular and phenogenetic analyses will continue to unravel the links between the molecular complexity and the varied and functionally separable biological functions of these Dmca1A calcium channels (Smith, 1998).
When exposed to high temperatures (37 degrees), cac flies show frequent convulsions and pronounced locomotor defects. This TS phenotype seems consistent with the idea that cac is a mutation in a calcium-channel gene; it maps to the same X-chromosomal
locus that encodes the polypeptide comprising the alpha-1 subunit of this membrane protein: Dmca1A. Previously, only one other voltage-sensitive calcium channel (Dmca1D) was
known in Drosophila, but no behavioral defects have as yet been associated with variations at the autosomal locus encoding Dmca1D. Analysis of the courtship song of some other TS
physiological mutants that are independent of cac shows that slowpoke mutations, which affect a calcium-activated potassium channel, cause severe song
abnormalities. Certain additional TS mutants, in particular paralytic (parats1) and no-action-potential (napts1), exhibit subtler song defects. The results therefore
suggest that genes involved in ion-channel function are a potential source of intraspecific genetic variation for song parameters, such as
the number of cycles present in 'pulses' of tone or the rate at which pulses are produced by the male's wing vibrations during courtship. The
implications of these findings from the perspective of interspecific lovesong variations in Drosophila are discussed. cacophony is one of the most interesting song mutations from an evolutionary point of view, at least in part because its abnormal pulses are
nicely patterned, as in the case of wild-type males from various Drosophila species, and do not appear to be pathologically defective. A similar statement is possible
about the songs of slowpoke males, although perhaps some of these mutant song bouts are more properly categorized as erratic and messy. Nevertheless, it is hard to believe
that the song produced by double mutants cac;slo1/slo1 comes from D. melanogaster males, so striking are the differences from the wild-type patterns (Peixoto, 1998).
cacophony is a temperature-sensitive mutant:
When exposed to high temperatures (~37ƒ) cac flies show frequent convulsions and pronounced locomotor defects. This convulsion phenotype is characterized by
flies turning upside-down or on their sides, shaking their legs for a few seconds, and then turning right-side up. The flies also curl their abdomen severely, either when
on their backs or when walking, and twist their bodies at the same time. In addition, occasionally the cac adults will walk sideways, spin around on the same spot for
a couple of seconds (apparently completely disoriented), leap across the chamber, or jump and tumble up and down out of control. There was no obvious sequence
in the occurrence of these phenotypes. After long exposures at 37ƒ, cac flies spend more and more time on their backs, shaking their legs until they seem to collapse.
This typically requires more than 1 hr of heating for 1-day-old flies, but much less for older ones. As long as leg movement is still occurring, the
mutant individuals usually recover in a few minutes after transfer to room temperature (Peixoto, 1998).
Only pulse song was examined in this report (courtship hums, or sine-song, being another type of song). Usually, all the pulses of the song of a given fly are logged, that is, marked for storage in the relevant file using the computer as an
event-recorder, while scanning the visual record of the song along with the video image of the flies' behavior. Logging of some songs extended for only 2 min, and
more than 500 pulses were typically logged. Songs with less than 40 pulses were not included in the analysis. Four parameters of the flies' pulse song were measured: interpulse interval (IPI), Cycles-per-Pulse (CPP), amplitude, and intrapulse frequency (IPF). CPP
and IPF values can vary together among Drosophila types, but there is no way to predict one value from knowledge of the
other; thus, these were treated as separate song parameters. The pulse amplitude measurements were attempts to quantify a song's loudness. This is difficult to
measure reliably, and the units specified are arbitrary (Peixoto, 1998).
To examine the effects that temperature variation might have on the pulse song produced by cac, a song analysis of cac and wild-type flies was carried out at
temperatures ranging from 15 to 30 degrees in steps of 2.5 degrees. Also included in this analysis was the mutant parats1, because a preliminary analysis had found it to have an effect on song at 25 degrees. Four pulse-song parameters were examined: amplitude of sound, IPI, CPP, and IPF. Temperature has a major effect on amplitude and IPI of all three genotypes, although it is far less clear in the case of CPP and IPF, even though the temperature effect is
significant for the latter. Significant genotype differences were observed for amplitude, IPI, and CPP but not for IPF. The results also show
the basic differences between cac mutation songs and wild-type (normal) songs, that is, higher amplitude and CPP, as well as longer IPIs in the former
compared to the latter. IPFs are similar between these two genotypes.
Although the overall trend observed for amplitude and IPI is similar for wild-type, cac, and para (as the temperature rises, there is an increase in the former and a
decrease in the latter), differences were revealed in the way the various types of males react to temperature. These differences are responsible for the significant
genotype x temperature interactions observed. The difference in IPI between cac and wild type shows a significant negative correlation with temperature. The difference is actually larger at
lower temperatures, a result that is somewhat counterintuitive if one considers that the convulsion phenotype of this mutant occurs at elevated temperatures. It is
possible that this reflects in part the nonlinear nature of the IPI change with temperature. No significant correlation with temperature was observed for the amplitude
differences. The difference in IPI between parats1 and wild type shows the opposite trend observed for cac mutants. There is a significant positive correlation of temperature with the larger IPI difference at 30ƒ. In the case of amplitude, however, the differences between parats1 and wild type show a significant negative correlation (Peixoto, 1998).
Genetic analysis of synaptic mechanisms in Drosophila has identified a temperature-sensitive paralytic mutant of the voltage-gated calcium channel alpha1 subunit gene, cacophony (cac). Electrophysiological studies in this mutant, designated cacTS2, indicate cac encodes a primary calcium channel alpha1 subunit functioning in neurotransmitter release. To further examine the functions and interactions of cac-encoded calcium channels, a genetic screen was performed to isolate new mutations that modify the cacTS2 paralytic phenotype. The screen recovered 10 mutations that enhance or suppress cacTS2, including second-site mutations in cac (intragenic modifiers) as well as mutations mapping to other genes (extragenic modifiers). Molecular characterization of three intragenic modifiers is reported and the consequences of these mutations for temperature-sensitive behavior, synaptic function, and processing of cac pre-mRNAs, is reported. These mutations may further define the structural basis of calcium channel alpha1 subunit function in neurotransmitter release (Brooks, 2003).
The mutant alleles slo1 and slo2
define slowpoke as a new courtship-song gene. The pulse songs produced by these two mutants are clearly aberrant and they are in fact
often difficult to log due to the low-amplitude or polycyclic nature of pulses (at a given moment of singing). Using the same criteria and IPI cutoffs used
with the other mutants, all four song parameters examined are affected by these two slo alleles, which cause somewhat distinct song abnormalities. Males
homozygous for the slo1 mutation produce very low-amplitude songs with long IPIs, and low CPP and IPF values. Isolated putative pulses, usually monocyclic
signals, often occur in slo1 song records; however, they were not logged because they did not occur in pulse trains. In the case
of the slo2 allele, the IPIs of homozygous mutant males are not as long, and the sound amplitude not as low, as in the case of slo1. A train of pulses in the song
produced by flies homozygous for slo2 often ends with a highly polycyclic pulse. In fact, the mean number of cycles per pulse of slo2 flies is higher than the
wild-type control. Isolated pulses were also often observed, but in this case (cf. slo1) they are usually highly polycyclic. Heterozygous flies slo1/slo2 show effects intermediate between the two homozygotes. The
differences in the phenotypes between the two mutants obviously suggest differences in the molecular nature of the lesions that are unknown. slo1 is a chemically
induced mutation, while slo2 was generated using gamma rays (Peixoto, 1998). Neither shows any gross chromosomal rearrangements (N. S.
Atkinson, personal communication to Peixoto, 1998).
A fair fraction of the song mutants resulting from changes in genes that have been characterized at the molecular level involve membrane excitability. Not surprisingly,
these basic functions, when mutated, lead to grossly appreciable defects in behavior. Only some of these mutants are song-defective as well. cacophony now finds itself in this category, that is, the courtship variant mutant that started out as a song
mutant but is now known to have other phenotypic defects, such as heat-induced convulsions. This kind of general impairment could be at least as detrimental to
fitness as the song abnormalities produced by cac mutants. Other pleiotropic song mutants with molecular correlates involve the regulation of gene expression (considered in general terms: transcription or RNA processing). In
addition to the period and dissonance mutants in this category, consider the fruitless gene and its
mutants. These courtship mutations defined a locus encoding a transcription factor. fru mutations affect courtship
song, as well as other aspects of the fly's reproductive behavior, including fertility. Pleiotropies of these sorts place important
constraints on the evolution of these behavioral genes (Peixoto, 1998 and references).
Genetic variation for features of the Drosophila courtship song have been reported from natural populations.
It is possible that the level of genetic variability
observed is influenced not only by sexual selection acting on the song parameters themselves, but also by selection on the pleiotropic effects of these putative song
genes. These pleiotropic effects could even include other aspects of the mate recognition system. For example, there are smellblind mutations at the para locus that affect the response of males to female
pheromones. It is also conceivable that directional selection acting on some of these pleiotropic effects,
for example, selection for temperature tolerance and ion-channel genes, could drive changes in the song repertoire that could eventually lead to reproductive isolation
between different populations (Peixoto, 1998 and references).
While the constraints associated with pleiotropy certainly do not prevent the rapid evolution of Drosophila courtship songs, it might explain why there is little evidence for genes with major effects on song found in crosses between closely related species. It is likely that the lovesong differences between most such species are based on the cumulative effect of very mild and subtle
changes in several genes, at least a handful of them involving, for example, interspecific variations at the cac, slo, and mle (nap) loci. The major innovations in song production in the genus Drosophila seem to have occurred among Hawaiian flies for which founder-effect models of speciation have been proposed. These include, for
example, the idea of fixation of a mutation in a major locus, via genetic drift, followed by selection for modifiers on its deleterious effects. Pleiotropy and epistasis
have major roles in these models. Epistasis between conspecific genes is a key component of this sexually related phenotype. Epistatic interactions among song genes, such as the one
found between cac and napts1 within D. melanogaster, could also have important implications for sexual selection on the phenotypes they control and on their
potential role in speciation. Because of the role acoustic signals (such as the Drosophila's lovesong) play in female receptivity, mating preferences, and sexual isolation between species, song factors are among the best candidates for the so-called 'speciation genes'.
The behavioral analysis presented here reveals that mutations in loci affecting ion-channel function might be a source of genetic variation in the fly's lovesong.
Because of their enormous diversity, channel genes might turn out to be among the most common classes of song genes (Peixoto, 1998 and references).
The N-ethylmaleimide-sensitive fusion protein (NSF) has been implicated in vesicle trafficking in perhaps all
eukaryotic cells. The Drosophila comatose (comt) gene encodes an NSF homolog, dNSF1. Work
with temperature-sensitive (TS) paralytic alleles of comt has revealed a function for dNSF1 at synapses, where it
appears to prime synaptic vesicles for neurotransmitter release. To further examine the molecular basis of dNSF1
function and to broaden the analysis of synaptic transmission to other gene products, a genetic
screen was performed for mutations that interact with comt. Four mutations that modify TS paralysis in comt are described, including two
intragenic modifiers (one enhancer and one suppressor) and two extragenic modifiers (both enhancers). The intragenic mutations will contribute to
structure-function analysis of dNSF1 and the extragenic mutations identify gene products with related functions in synaptic transmission. Both
extragenic enhancers result in TS behavioral phenotypes when separated from comt, and both map to loci not previously identified in screens for TS
mutants. One of these mutations is a TS paralytic allele of the calcium channel 1-subunit gene, cacophony (Dellinger, 2000).
Identification of cacTS2 as a modifier of comt raises a number of interesting issues. The original cacophony mutant (now known as cacS) was named on the basis of an aberrant male courtship song. The courtship song is produced by a patterned beating of the wings, and this pattern as well as the wing beat amplitude are altered in cacS mutants. The finding that cac-encoded calcium channels function in neurotransmitter release suggests that impairment of central synapses may contribute to altered song patterning in cacS. Given that cac-encoded alpha1-subunits function at flight muscle neuromuscular synapses, peripheral synaptic defects may contribute to the song phenotype as well. A second issue is whether the genetic interaction of cacTS2 and comt reflects direct or indirect interactions of the encoded gene products. Electrophysiological analysis indicates that the cac-encoded alpha1-subunit mediates fast neurotransmitter release and that dNSF1 functions in maintaining the readily releasable pool of synaptic vesicles. Thus the observed genetic interaction may reflect simply that both the comt and cac gene products function in neurotransmitter release. Alternatively, the genetic interaction may result from the well-characterized biochemical interactions of SNAREs with both NSF and calcium channels. While this issue remains unresolved, two observations favor the former possibility. (1) No sequence homology has been detected between the cac-encoded alpha1-subunit and SYNPRINT sequences thought to mediate direct interactions with other synaptic proteins. (2) Preliminary synaptic electrophysiology in cacTS2 comtST17 double mutants is consistent with independent actions of comt and cac mutations in neurotransmitter release (Dellinger, 2000).
The cacophony locus of Drosophila, which encodes a calcium-channel subunit, has been mutated to cause courtship-song defects or abnormal responses to visual stimuli. However, the most recently isolated cac mutant was identified as an enhancer of a comatose mutation's effects on general locomotion. The cacTS2 mutation was analyzed in terms of its intragenic molecular change and its effects on behaviors more complex than the fly's elementary ability to move. The molecular etiology of this mutation is a nucleotide substitution that causes a proline-to-serine change in a region of the polypeptide near its EF hand. Given that this motif is involved in channel inactivation, it was intriguing that cacTS2 males generate song pulses containing larger-than-normal numbers of cycles -- provided that such males are exposed to an elevated temperature. Similar treatments caused only mild visual-response abnormalities and generic locomotor sluggishness. These results are discussed in the context of calcium-channel functions that subserve certain behaviors and of defects exhibited by the original cacophony mutant. Despite its different kind of amino-acid substitution, compared with that of cacTS2, cacS males sing abnormally in a manner that mimics the new mutant's heat-sensitive song anomaly (Chan, 2002).
The newest cacophony mutant is a courtship variant, as is the original cac mutant. Thus, cacTS2 males are somewhat impaired in their overall courtship performance, including mating ability. However, cacTS2 males courted more vigorously and effectively than one might expect from monitoring their generic locomotor activity. One component of the courtship performance of cacTS2 males implies a behavioral problem that goes beyond the nature of the sounds they communicate to females. They performed worse than wingless wild-type males did, which indicates that this mutant is more pleiotropically defective than a 'song only' variant (Chan, 2002).
Nevertheless, the most sharply defined courtship defect exhibited by a cacTS2 male is its heat-sensitive anomalies of tone pulses that emanate from the wing vibrations it directs at a female. These abnormalities of cycles per pulse and pulse amplitude were found to be similar to the nonconditional courtship-song peculiarities exhibited by the original cacS mutant. That the respective mutant phenotypes are alike is important, because cacTS2 males did not have to exhibit any kind of singing eccentricity: inasmuch as the isolation of this mutant involved behavioral criteria that had nothing to do with courtship, the outcome of song-testing cacTS2 could have left cacS as the only singing variant associated with this gene. But both the original and the newest cacophony mutations cause courtship-song peculiarities, and it is interesting that the anomalously loud and polycyclic pulses produced by both cacS and cacTS2 males do not involve an appreciable derangement of such sounds: each mutant type remains nicely patterned with respect to the qualities of individual 'clicks' and their rate of production. Once again, if cacTS2 turned out to be song defective it was not a foregone conclusion that such males would produce these sounds in a manner more salutary than that of other singing variants, such as those expressing slowpoke (slo) mutations. In this regard, slo potassium-channel mutants were identified using generic behavioral criteria (as was cacTS2) and were found later to sing aberrantly and to exhibit erratic patterns of anomalous tone pulses (Chan, 2002).
This brings us to the question of why it might be that the songs of cacS and of cacTS2 (at 30° C) males are not only song defective, but also similarly so in their tone-pulse qualities. As was introduced in conjunction with documenting cacTS2's intragenic site change, this amino-acid substitution is very near the EF hand within Dmca1A, directly C-terminal to the IVS6 transmembrane domain. The highly conserved EF hand and adjacent residues among calcium-channel alpha1 subunits of various species are involved in channel inactivation mediated by Ca2+ binding. Thus, this form of inactivation involves a calcium-influenced conformational change that occurs via cation binding within the EF hand's helix-loop-helix. Given the P-to-S substitution in cacTS2 immediately C-terminal to the EF hand (where this evolutionarily conserved proline is changed to a polar serine that has more conformational freedom) one imagines that the local three-dimensional structure in which the EF hand finds itself is altered in the mutant. The function of this domain would be altered accordingly but not ruined at permissive temperatures. Thus, the amino-acid substitution in cacTS2 near the EF hand suggests that this protein change could cause the Dmca1A calcium channel to exhibit altered inactivation kinetics. Whereas inactivation features of the alpha1 subunit encoded by cac are unknown, it is reasonable to speculate that that process becomes less robust than normal in the cacTS2 mutant as the flies are heated from 20° to 30°. Why the dynamics of inactivation may be subtly heat sensitive over the temperature range just stated is difficult to surmise, although perhaps it is the case that this process can barely occur at all at 37°, accounting for the grossly subnormal synaptic neurotransmission that occurs at that extreme temperature (Chan, 2002).
This hypothesis, as it relates to cacTS2's behavioral phenotype within a 'physiological' range of temperatures, goes on to suggest that anomalously polycyclic pulses in the songs of males expressing this mutation smack of a channel-inactivation change that would alter the contribution of calcium currents to the overall behavioral process in question. Thus, the repetitive-pattern phenotype, which is a reasonable descriptor for trains of Drosophila song pulses, would not have the intrapulse cycles inactivated as 'tightly' as in wild type (Chan, 2002).
What about the songs of cacS males, whose pulses are similarly polycyclic (albeit without the temperature sensitivity that accompanies the cacTS2 phenotype)? The cacS mutant is accounted for by an amino-acid substitution within the sixth membrane-embedded region of the penultimate intra-Dmca1A repeat, a.k.a. IIIS6. Certain types of calcium channels prevent excessive influx of calcium when the channel opens by voltage-mediated inactivation. Pore-forming S6 transmembrane domains play a role in modulating voltage-dependent calcium-channel inactivation. This has been revealed (1) by creating chimeric alpha1-subunit polypeptides in which portions of IIIS6 from fast-inactivating channels replaced those of a slow-inactivating one, leading to inactivation kinetics characteristic of the donor calcium-channel type and (2) by physiological disruptions of channel functions that are pointed to by the etiology of certain patho-physiological mutants in humans; certain such S6 mutations slow and others accelerate the development of inactivation. Therefore, a mnemonic device for apprehending the song abnormality exhibited by cacS mutant males is, again, subnormal inactivation of intratone-pulse sounds, owing to their inappropriate polycyclicity. However, in this case the putative inactivation defect would have a different mechanistic etiology compared with that hypothesized for the cacTS2-mutated polypeptide (Chan, 2002).
Rieckhof, G. E., Yoshihara, M., Guan. Z. and Littleton, J. T. (2003). Presynaptic N-type calcium channels regulate synaptic growth. J. Biol. Chem. 278: 41099-41108. 12896973
Voltage-gated calcium channels couple changes in membrane potential to neuronal functions regulated by calcium, including neurotransmitter release. Presynaptic N-type calcium channels not only control neurotransmitter release but also regulate synaptic growth at Drosophila neuromuscular junctions. In a screen for behavioral mutants that disrupt synaptic transmission, an allele of the N-type calcium channel locus (Dmca1A) was identified that caused synaptic undergrowth. The underlying molecular defect was identified as a neutralization of a charged residue in the third S4 voltage sensor. RNA interference reduction of N-type calcium channel expression also reduced synaptic growth. Hypomorphic mutations in syntaxin-1A or n-synaptobrevin, which also disrupt neurotransmitter release, did not affect synapse proliferation at the neuromuscular junction, suggesting calcium entry through presynaptic N-type calcium channels, not neurotransmitter release per se, is important for synaptic growth. The reduced synapse proliferation in Dmca1A mutants is not due to increased synapse retraction but instead reflects a role for calcium influx in synaptic growth mechanisms. These results suggest N-type channels participate in synaptic growth through signaling pathways that are distinct from those that mediate neurotransmitter release. Linking presynaptic voltage-gated calcium entry to downstream calcium-sensitive synaptic growth regulators provides an efficient activity-dependent mechanism for modifying synaptic strength (Rieckhof, 2003).
Dmca1A is abundantly expressed in the Drosophila nervous system and encodes the presynaptic N-type calcium channel responsible for calcium influx that triggers synaptic vesicle fusion. Null mutations in Dmca1A are embryonic lethal [lethal(1)L13], whereas partial loss-of-function mutations disrupt synaptic transmission, leading to defects in various behaviors, including courtship (cacaphony) and phototaxis (nightblind-A). In addition to defects in neurotransmitter release, Dmca1A hypomorphic mutants show altered morphology at the mature larval NMJ. There is a decrease in both terminal branching and varicosity number compared with wild-type controls and hypomorphic alleles of syntaxin and n-synaptobrevin. The reduced synaptic proliferation is not secondary to defective synaptic transmission because syntaxin and synaptobrevin show more profound defects in transmitter release but have normal synaptic proliferation. Similar results have also been reported in synaptotagmin mutants as well as SNARE mutants. Thus, mutations in Dmca1A affect calcium-regulated synaptic pathways separate from those that regulate transmitter release (Rieckhof, 2003).
No evidence was found for a role of presynaptic calcium entry through either N- or L-type calcium channels in the early stages of synapse formation during late embryogenesis. It was also determined that the morphological defects in Dmca1A mutants are not due to an increase in terminal retraction, suggesting active growth rather than synapse stability is defective. Therefore, the structural defects observed in Dmca1A mutants occur between the establishment of the initial synaptic field and its final larval maturation. Previous work has demonstrated that the overall shape and branching pattern at the Drosophila NMJ is established early in development. Subsequent growth largely requires the addition of new varicosities to previously formed terminal branches. It is within this second activity-dependent growth phase that is thought to be presynaptic that calcium entry is required to promote synaptic maturation. The 35% reduction observed in varicosity number in Dmca1ANT27 mutants at the end of larval development is likely an underestimate of the actual contribution of presynaptic calcium entry to synaptic growth regulation. (1) The initial activity-independent elaboration of synapses during late embryogenesis does not require calcium channel function, allowing the establishment of the initial synaptic field. (2) The Dmca1ANT27 mutant is a hypomorphic allele, reducing calcium channel function but not eliminating it. Previously isolated alleles of the Dmca1A locus that are more severe than Dmca1ANT27 are embryonic lethal, preventing an analysis of the activity-dependent phase of synaptic growth in more severe alleles. Further studies with mosaic animals will be required to fully characterize the persistence of synaptic growth mechanisms in the complete absence of presynaptic calcium influx (Rieckhof, 2003).
The opening of presynaptic N-type channels during robust synaptic activity may allow calcium to influence varicosity sprouting mechanisms to locally control synaptic remodeling. Changes in intracellular calcium have been shown to affect growth cone motility and neurite outgrowth. Indeed, filopodial protrusions from neuronal growth cones are triggered by altered calcium concentrations. Synaptic activity results in calcium-dependent CaMKII activation via binding of calcium/calmodulin and subsequent auto-phosphorylation. Activated CaMKII phosphorylates the synaptic MAGUK protein DLG, causing release of FAS2 from its synaptic scaffold and subsequent modulation of synaptic growth in Drosophila. In addition, CaMKII activation also regulates the activity of the ether-a-go-go (eag) family of potassium channels in Drosophila, altering aspects of nerve excitability that could contribute to synaptic growth. Intracellular calcium levels directly regulate cAMP signaling through the activation of adenylate cyclase by calmodulin, enhancing cAMP-dependent pathways implicated in synaptic growth. It is likely that disruptions in presynaptic calcium entry in Dmca1ANT27 mutants leads to alterations in several presynaptic signaling cascades that modulate growth. Further genetic analysis should begin to elucidate how the regulation of calcium entry modulates these activity-dependent synaptic growth pathways (Rieckhof, 2003).
Voltage-gated calcium channels consist of four repeated units (I-IV) containing six alpha-helical transmembrane segments (S-S6). The fourth transmembrane segment (S4) of voltage-gated ion channels has been shown to function as a voltage sensor. It is thought that the S4 sensor, a transmembrane alpha-helix in which every third or fourth residue is basic and carries a positive charge, undergoes conformational changes during depolarization that result in channel opening. Sequence analysis of Dmca1ANT27 identified a charge-neutralizing mutation in a highly conserved arginine residue in the S4 voltage sensor, supporting an essential role for S4 helix movement during channel gating and subsequent calcium influx. The altered S4 charged amino acid likely explains the TS phenotype of the mutant; channel gating requires conformational changes in the S4 helix that are temperature-dependent. Mutations in mammalian Dmca1A homologs have been linked to a variety of neuronal disorders, including episodic and spinocerebellar ataxias, hemiplegic migraine, blindness, hypokalemic periodic paralysis, and epilepsy. Although it seems paradoxical that a hyperexcitability phenotype such as seizures could arise from a reduction in calcium channel function, the Drosophila giant fiber pathway is extremely sensitive to changes in both inhibition and excitation. The decrease in calcium channel function in the inhibitory pathways may bear more weight in the overall output of the circuit, even though the excitatory outputs have reduced release as well. Similar seizure defects are seen in mammalian N-type calcium channel mutants (tottering, lethargic, and rocker) where overall calcium channel function is also reduced. Similar to what is observed in Drosophila N-type mutants, mutations in the mouse alpha1a calcium channel locus (rocker) that disrupt the pore region of the channel and reduce calcium influx cause a profound reduction in Purkinje cell dendritic arborization. In addition, pharmacological disruption of calcium channel function in salamander rod photoreceptors has been shown to inhibit varicosity formation. The reduced varicosity number in calcium channel mutants suggests an important role for normal levels of calcium entry during synaptic activity for the proper modulation of synaptic growth, in addition to its well established role in neurotransmitter release. Modulation of N-type calcium channel function via alterations in the rates of presynaptic action potential firing could provide an efficient mechanism for the regulation of activity-dependent synaptic growth (Rieckhof, 2003).
Endocytosis of synaptic vesicles follows exocytosis, and both processes require external Ca2+. However, it is not known whether Ca2+ influx through one route initiates both processes. At larval Drosophila neuromuscular junctions, exocytosis and endocytosis were separately measured using the fluorescent dye FM1-43. In a temperature-sensitive Ca2+ channel mutant, cacophonyTS2, exocytosis induced by high K+ decreases at nonpermissive temperatures, while endocytosis remains unchanged. In wild-type larvae, a spider toxin Ca2+ blocker, PLTXII, preferentially inhibits exocytosis, whereas the T-type Ca2+ channel blocker flunarizine and the blocker La3+ selectively depresses endocytosis. None of these blockers affect exocytosis or endocytosis induced by a Ca2+ ionophore. Evoked synaptic potentials are depressed regardless of stimulus frequency in cacophonyTS2 at nonpermissive temperatures and in wild-type by PLTXII, whereas flunarizine or La3+ gradually depressed synaptic potentials only during high-frequency stimulation, suggesting depletion of synaptic vesicles due to blockade of endocytosis. In shibirets1, a dynamin mutant, flunarizine or La3+ inhibit assembly of clathrin at the plasma membrane during stimulation without affecting dynamin function (Kuromi, 2004).
To maintain synaptic transmission during intense neuronal activities, synaptic vesicles (SVs) are effectively recycled by endocytosis. Ca2+ influx through voltage-gated Ca2+ channels plays a crucial role in exocytosis. It has also been found, in early studies at frog neuromuscular junctions, that external Ca2+ is essential for SV recycling. Subsequent studies have confirmed that Ca2+ influx is also required for endocytosis in various types of central synapses and secretory cells. In contrast, endocytosis has been shown to occur even in the absence of external Ca2+ after cessation of stimulation in rat hippocampal neurons and in presynaptic boutons of a Drosophila temperature-sensitive mutant, shibirets1 (shits1), at room temperature after depletion of SVs at nonpermissive temperatures. These studies suggest that endocytosis can be triggered independently of the Ca2+ influx that initiates exocytosis. To reconcile these seemingly contradictory findings, it has been postulated that Ca2+ influx during stimulation that causes exocytosis also triggers the formation of intermediates for SV recycling, and once they are formed, the following steps of endocytosis proceed without Ca2+. However, because of difficulties in separating Ca2+ influx routes for exocytosis and endocytosis during stimulation, this hypothesis remains unproven (Kuromi, 2004 and references therein).
Immunostaining studies suggest that sites for endocytosis are distinct from those for exocytosis at nerve terminals. It is then possible that Ca2+ influx routes for these two processes are separate. Along the line of this idea, multiple subtypes of Ca2+ channels are demonstrated in nerve terminals. Those subtypes of Ca2+ channels are spatially segregated in presynaptic terminals, and their roles in transmitter release have been subject to speculation. Specific roles of these Ca2+ channel subtypes in exocytosis and endocytosis, however, have not been identified (Kuromi, 2004).
Does Ca2+ influx through one route trigger both exocytosis and endocytosis? To address this question, a temperature-sensitive Ca2+ channel mutant, cacophonyTS2 (cacTS2), and various Ca2+ channel blockers have been used at larval Drosophila neuromuscular junctions. A fluorescent dye, FM1-43, is incorporated into SVs in nerve terminals by endocytosis, and FM1-43 loaded in SVs is released by exocytosis. By measuring the amount of FM1-43 released from or taken up into nerve terminals, exocytosis and endocytosis were separately determined. Thus, it has been revealed that distinct Ca2+ influx routes separately regulate exocytosis and endocytosis. Taking advantage of drugs that selectively block endocytosis, it has been further shown in shits1 that selective blockade of the Ca2+ influx route linked to endocytosis inhibits clathrin assembly on the plasma membrane of nerve terminals. It is suggested that Ca2+ influx during stimulation through this route forms an intermediate complex, which leads to endocytosis (Kuromi, 2004).
A widely accepted model of endocytosis is that the clathrin coat assembles first on the presynaptic membrane, forming a shallow coated pit, which then invaginates to generate a bud with a constricted neck and eventually a free clathrin-coated vesicle by fission of the neck. A model has been proposed with two steps in SV recycling in which a Ca2+-dependent step (step I), which occurs during stimulation, is followed by a Ca2+-independent, shibire-dependent step (step II). In shits1 it has been shown that when FNZ or La3+ is added after high K+ stimulation, endocytosis at permissive temperatures occurs normally, indicating that FNZ or La3+ have no effect on step II. In contrast, when FNZ or La3+ is present during high K+ stimulation, endocytosis is not observed although exocytosis is unaffected. These observations strongly support the hypothesis that FNZ or La3+ selectively block Ca2+ influx through the route designated for endocytosis (step I) (Kuromi, 2004).
Immunostaining experiments with shits1 at nonpermissive temperatures reveal that synaptotagmin I is transferred to the plasma membrane during high K+ stimulation regardless of the presence of La3+, confirming that La3+ has no effect on exocytosis. However, it was noted that in the absence of La3+, clusters of clathrin immmunoreactivity are detected at the periphery of boutons after high K+ stimulation, while in the presence of La3+, clathrin remains in the cytosol of boutons after high K+ stimulation. These observations suggest that La3+ inhibits clathrin assembly at the plasma membrane. Dynamin plays an essential role in the fission of a clathrin-coated bud, and this process occurs in the absence of external Ca2+. It is suggested that the part of Ca2+ influx sensitive to FNZ or La3+ during stimulation (step I), plays a crucial role in clathrin assembly at the plasma membrane (Kuromi, 2004).
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date revised: 15 March 2008
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