Calmodulin UAS-Constructs and the In Vivo Roles ofCalmodulin: Analysis of a Muscle-Specific PhenotypeBo Wang, Clare Bolduc, and Kathy Beckingham*Department of Biochemistry and Cell Biology, Rice University, Houston, Texas

Received 5 June 2002; Accepted 10 July 2002

The vast range of intracellular processes that are regu-lated by Ca2� fluxes has only recently begun to beappreciated. The Ca2�-binding protein calmodulin(CaM) is recognized as a central player in Ca2� signaling,although its precise roles, particularly in developmentalprocesses, are largely undefined. CaM is a dumbbell-shaped molecule, with terminal globular domains eachcontaining a pair of “EF-hand” type Ca2� binding sites(Nelson and Chazin, 1998). The conformational changesassociated with Ca2� binding allow CaM to interact withdifferent targets in its Ca2�-free (apo-) and Ca2�-bound(holo-) forms and thus to execute its role as a Ca2�

sensor (Zhang et al., 1995).Although the N- and C-terminal domains of CaM are

very similar, the C-terminal binding sites (3 and 4) havea higher Ca2� affinity than N-terminal sites 1 and 2.Moreover, there is now evidence that the two domainshave different regulatory roles. Most strikingly, in Para-mecium, mutations in the N-terminal lobe of CaM affectCa2�-activated Na� channel currents, resulting in slug-gish, unresponsive behavior, whereas C-terminal lobemutations alter Ca2�-activated K� currents (Kink et al.,1990), producing hyperexcitability. Thus, the structuralbipartition of CaM may permit functional bipartition interms of regulating Ca2�-sensitive targets (Ling et al.,1994).

In connection with our studies of the roles of CaM inDrosophila, we have generated a series of UAS-CaMconstructs. These constructs express 1) Drosophilawild-type CaM, or mutated CaMs with either 2) the twoN-terminal sites, 3) the two C-terminal sites, or 4) all fourCa2� binding sites inactivated. The mutations used toinactivate the sites all involve a glutamate-to-glutaminemutation at a conserved residue critical to Ca2� binding(Maune et al., 1992). Studies with the recombinant mu-tated proteins, termed B12Q, B34Q, and B1234Q, re-spectively, have established that they fold correctly andthat Ca2� binding at the mutated sites is essentiallyundetectable (Maune et al., 1992; Mukherjea et al.,1996). This mutant CaM series thus allows unique in vivostudies of the ability of CaM, with or without boundCa2�, to rescue or influence any phenotype of interestand to determine the individual roles of the two Ca2�-binding domains in any effects thus identified.

We demonstrate the usefulness of these constructsthrough in vivo analysis of a particular Drosophila Cam

mutation. This mutation, Cam7, produces a single aminoacid change (V91G) in the C-terminal domain of CaMthat generates a unique phenotype, distinct from thatproduced by Cam nulls or other point mutations to theCam gene (Nelson et al., 1997). The mutant larvae aresluggish and form short “Michelin Man” pupal cases withdeep indentations at the larval segment boundaries, re-sulting from excessive shortening during pupariation.Although the mutants develop adult body forms, noneever eclose and defects in head eversion, sometimesproducing “inside-out” heads, are common.

In other analyses (Wang et al., in preparation), wehave established that 1) the Cam7 pupal defects alloriginate in the larval musculature, and 2) defectiveregulation of the major Ca2� channel of the muscleintracellular stores, the ryanodine receptor (RyR) isprobably a major source of these defects. CaM regulationof RyR is biphasic: at very low [Ca2�], apo-CaM binds toRyR, sensitizing the channel to opening by small in-creases in [Ca2�]; at high [Ca2�], holo-CaM inhibits RyRopening (Rodney et al., 2001). In in vitro experimentswe have established that V91G CaM binds the CaMbinding region of Drosophila RyR (Takeshima et al.,1994) with altered properties predicted to lead to i)enhanced channel opening at low [Ca2�], and ii) failureof channel closure at high [Ca2�]. These changes wouldlead to increased Ca2� flux into the muscles and thus toexcessive contraction, as seen in Cam7 mutant animals.We have also shown that removing half the functionalRyR molecules in Cam7 animals (by making them het-erozygous for the strong, hypomorphic Ryr mutation,Ryr16 (Sullivan et al., 2000), dramatically rescues theCam7 phenotype.

Contract grant sponsors: NASA Specialized Center of Research and Train-ing (NSCORT) in Gravitational Biology at Rice University and the Robert A.Welch Foundation of Texas.

Present address for Clare Bolduc: Dept. of Biochemistry and MolecularBiology, MD Anderson Cancer Center, 1515 Holcombe, Box 117, Houston,TX 77030.

* Correspondence to: Kathy Beckingham, Department of Biochemistryand Cell Biology, Rice University, 6100 Main Street, Houston, TX 77251.

E-mail: kate@rice.eduPublished online 00 Month 2002 inWiley InterScience (www.interscience.wiley.com)DOI: 10.1002/gene.10141

© 2002 Wiley-Liss, Inc. genesis 34:86–90 (2002)

For mammalian skeletal RyR, the biphasic regulationof RyR activity by CaM reflects functional partitioningwithin CaM (Rodney et al., 2001). The inhibitory effect

of CaM at high [Ca2�] resides in the C-terminal lobe andrequires Ca2� binding to this lobe. Thus, the mutant CaMwith both C-terminal binding sites inactivated (B34Q

FIG. 1. Effects on the Cam7 mutant phenotype of expressing various mutant CaMs in the larval musculature A: Pupal phenotypes. TheCam7 mutation produces pupal cases that are shorter than wild-type (compare a and b), with deeply indented rings at the larval segmentboundaries, The following cross was used to express exogenous CaMs in the muscles of hemizygous Cam7 larvae. y w; Cam7/CyO(y�);24B-Gal4 � y w; Camn339/CyO(y�); UAS-Cam. Camn339 is a Cam RNA null allele (Heiman et al., 1996). The required progeny were identifiedby their y- mouth hooks, Wild-type CaM rescues the Cam7 phenotype to a wild-type structure (c). Expression of B34Q CaM causes 100%lethality in the larval stage (see B, below), Expression of B12Q CaM results in the formation of long, thin pupal cases with incompleteretraction of the anterior segments. B1234Q CaM has little effect on the pupal case phenotype. Genotypes: (a) Control: y w; �/CyO(y�);24B-Gal4/�; (b) V91G: y w; Cam7/Camn339; 24B-Gal4/�; (c–e) V91G with exogenous CaMs: y w; Cam7/Camn339; 24B-Gal4/UAS-Cam. B:B34Q Larval phenotype. Cam7 larvae (that is, Cam7 hemizygotes) are sluggish but morphologically indistinguishable from wild-type larvae orCam7 heterozygotes (a). Expression of UAS-B34Q CaM in Cam7 larval muscles produces a constant state of contraction, generating indentationsat the segment boundaries and severe folding of the two longitudinal tracheal tracts (b). These larvae are highly compromised in locomotionand die before pupariation, Genotypes: (a) Control: y w; Cam7/�; 24B-Gal4/�; (b) B34Q CaM: y w; Cam7/Camn339; 24B-Gal4/UAS-B34Q.

87CALMODULIN UAS-CONSTRUCTS

CaM) loses this inhibition but not the enhanced sensiti-zation at low [Ca2�]. In contrast, mutation of the N-terminal Ca2� binding sites produces no effects on eithersensitization or inactivation of RyR.

To test further our hypothesis that the muscle pheno-type of Cam7 reflects defective interaction with RyR, we

compared the rescue effects of the mutant CaMs de-scribed above to those of the wild-type protein whenexpressed in larval muscles. We predicted that the B12Qmutant would behave like wild-type CaM and rescue theCam7 phenotype, whereas both B34Q CaM and B1234QCaM would fail to rescue, and perhaps exacerbate, thephenotype.

The GAL4 driver 24B was used for muscle-specificexpression of UAS-CaM constructs (Brand and Perrimon,1993). Expression of wild-type Drosophila CaM undercontrol of this driver rescued both the developmentaldefects (pupal case wrinkles, head eversion defects) andthe lethality of the Cam7 mutation (see Fig. 1A, Table 1).Expression of B34Q CaM not only failed to rescue theCam7 phenotype, but produced more extreme muscleproblems. As larvae, the animals were severely con-tracted (Fig. 1B) and had difficulty producing cyclicwaves of contraction/expansion for locomotion. All an-imals died in the larval stages. This phenotype is consis-tent with the previous findings that Ca2� binding at theC-terminal sites is critical for limiting release of Ca2� viathe RyR channels (Rodney et al., 2001).

Surprisingly, the effects of B12Q CaM on the Cam7

muscle phenotype were not identical to those of wild-type CaM. Although B12Q CaM, like the wild-type pro-tein, rescued the overcontraction of the larval bodyduring pupariation and produced smooth pupal cases,these pupal cases were longer and narrower than thoseproduced by wild-type CaM (Fig. 1A, Table 1). Further,the anterior larval segments did not fully retract duringpupariation in these animals. B12Q CaM thus producesan abnormal degree of relaxation of the body wall mus-cles during pupariation, indicating a role for Ca2� bind-

FIG. 2. Length/width ratios for pupae of Cam7 mutants expressingvarious mutant CaMs in their musculature. For genotypes, seeFigure 1. **Values that differ from the Cam7 (V91G) control with P �0.0001. *Value with P � 0.0012. Statistical analyses were performedwith Statview software (v. 4.51, Abacus Concepts, Berkeley, CA).

Table 1Rescue Effect of CaM Mutants on the Cam7 Phenotype

EndogenousCaM

ExogenousmuscleCaM # Pupae % W% M% H% N% Eclosed %

V91G CaM — 90 78 41 13 6.7 16 0V91G CaM WT CaM 88 94 3.3 0 0 0 91V91G CaM B12Q CaM 117 86 11 0 0 65 9.4V91G CaM B34Q CaM 90 0 0 0 0 0 0V91G CaM B1234Q

CaM87 31 18 1.1 0 9.2 2.3

V91G CaM B12Q CaMand B34Q

CaM

60 0 0 0 0 0 0

V91G CaM V91G CaM 80 72.5 25 8.75 2.5 36.5 0

#: number of first instar larvae collected.Pupae %: percentage of larvae that pupariated.W%: percentage of animals that had well-formed pupal heads but failed to eclose.M%: percentage of animals that had malformed pupal head structures.H%: percentage of animals that had no head eversion.N%: percentage of animals that had no recognizable pharate adult body structure.Eclosed%: percentage of animals that eclose.For definitions of B12Q CaM, B34Q CaM, and B1234Q CaM, see text. cDNAs for wild-type and Ca2� binding mutant CaMs (Mukherjea

et al., 1996) were transferred as Bam HI–Sal I fragments into the Bgl II–Xho I sites of vector pUAST (Brand and Perrimon, 1993). The V91GCaM cDNA (Nelson et al., 1997) was transferred as a Xho I–Xba fragment into the same sites of pUAST. At least two transformant lines onthe second and two on the third (or fourth) chromosome are available for each construct. Where tested, different insertions gave essentiallyidentical results. The 24B-Gal4 line was used to drive expression in the muscles (Brand and Perrimon, 1993).

88 WANG ET AL.

ing to the N-terminal sites in the muscle relaxation/contraction cycle. Roles for these N-terminal bindingsites in subsequent pupal muscle development are alsoindicated from consideration of the later fate of Cam7

pupae with exogenous muscle B12Q CaM. Althoughexpression of wild-type CaM and B12Q CaM result in asimilar number of animals surviving to become pupae,most of these pupae produce viable adults with wild-type CaM, whereas only a small fraction eclose as adultswhen B12Q CaM is expressed (Table 1). Further, most ofthe B12Q CaM pupae fail to develop recognizable adultstructures (Table 1).

Additional evidence for a function for the N-terminalsites in muscle relaxation comes from expression ofB1234Q CaM in the Cam7 larval muscles. The effects aredistinctly different from those produced by B34Q CaM:the extreme larval muscle contraction is not producedand although a smaller percentage of larvae survive tothe pupal stage, those that do form pupal cases similar tothose produced by Cam7 alone. Thus, a sparing of theeffects of B34Q CaM is seen when the N-terminal lobecannot bind Ca2�. Like B12Q CaM, B1234Q CaM rescuesthe Cam7 phenotype sufficiently to allow some animalsto survive to adulthood, although this is a smaller per-centage than seen with the B12Q CaM alone (Table 1).

As a control, we also generated a UAS construct ex-pressing Cam7 mutant CaM (V91G CaM) and used it toexpress additional V91G CaM in Cam7 mutant muscles.This additional V91G CaM expression produced a some-what greater contraction of the pupal cases that provedstatistically significant (Fig. 2).

The studies described so far address the ability of themodified CaMs to affect the phenotype of the Cam7

mutation in the hemizygous condition. To determine ifany of the mutant CaMs had dominant effects, we ex-pressed them in the muscles of animals heterozygous forCam7 (that is, with one copy of Cam�) and also in Camwild-type (Cam�/Cam�) animals. These animals weregenerated as siblings from the same cross (see Legend,Fig. 3). In the phenotypically wild-type Cam7/Cam�

heterozygous animals, mutant B12Q CaM produced noeffects on pupal case structure or survival (Fig. 3A).Some minor wrinkling of the pupal cases was seen withB1234Q CaM, but overall survival to adulthood was un-affected. In contrast, B34Q CaM severely affected devel-opment. Less than half of the animals pupariated (Fig.3A) and some pupae had body wrinkles comparable tothe Cam7 hemizygotes. Dominant effects of the B34QCaM were even seen in the Cam�/Cam� animals (Fig.3B): there was significant death before pupariation, withformation of abnormal pupal cases in some survivors.Overall, �75% of the animals died before adulthood.None of the other CaM constructs produced effects inCam�/Cam� animals.

We investigated one final aspect of CaM function invivo with these constructs. Given the evidence pre-sented here, and elsewhere, for distinct functions of theN- and C-terminal domains, we addressed the question ofhow much the effects reported here for a single mutated

FIG. 3. Assays for dominant effects of UAS-CaM constructs. A,B:B34Q CaM has dominant effects. The mutated CaMs were ex-pressed in the musculature of Cam7/Cam� heterozygotes (A) andCam�/Cam� animals (B). For each construct, the following crosswas performed, y w; Cam7/CyO(y�); 24B-Gal4 � y w; Cam�/Cam�;UAS-Cam. For (A), 30 y� first instar larvae (genotype y w; Cam7/Cam�; 24B-Gal4/UAS-Cam) were collected, transferred to a singlefood vial, and observed. For (B), 30 y� sibling first instar larvae(genotype y w; Cam�/CyO(y�); 24B-Gal4/UAS-Cam) were similarlytreated. Control larvae with no expression of exogenous CaM for (A)were y w; Cam7/Cam�; 24B-Gal4/�; and for (B) were y w; Cam�/CyO(y�); 24B-Gal4/�. Pupae: percentage of larvae that pupariate.Eclosed: percentage of adult survivors. Only the B34Q CaM con-struct produces effects in the presence of wild-type CaM. C: Failureof mutant B12Q CaM to rescue the dominant effects of B34Q CaM.A version of the cross detailed for Figure 1 giving expression ofB34Q CaM in the muscles was performed in parallel with the fol-lowing cross, which gave expression of both B12Q CaM and B34QCaM in the muscles. y w; Cam7/CyO(y�); 24B-Gal4 � y w; Camn339

UAS-B12Q/CyO(y�); UAS-B34Q. Comparison of the y- larvae(Cam7/Camn339) from these two crosses addressed whether B12QCaM could rescue the effects of B34Q CaM in Cam7 hemizygotes.The results are shown in Table 1. Comparison of the y� siblings fromthe two crosses (Cam�/(Cam7 or Camn339) genotypes) addressedwhether B12Q CaM could reverse the dominant effects of B34QCaM. For each comparison, 30 first instar larvae were studied. Asshown here, in the presence of wild-type Cam coexpression ofB12Q CaM with B34Q CaM was much more detrimental than ex-pression of B34Q CaM alone.

89CALMODULIN UAS-CONSTRUCTS

domain depend on the presence, within the same pro-tein molecule, of a normally functioning partner domain.If the two domains truly act independently, then simul-taneous expression of B12Q CaM (which has a func-tional C-terminal domain) and B34Q CaM (which has afunctional N-terminal domain) should be equivalent toexpressing wild-type CaM. That is, Cam7 rescue abilitycomparable to wild-type protein should be detected.When tested, however, coexpression of the two mutantproteins in Cam7 hemizygotes caused 100% lethality aslarvae, as for B34Q alone (Table 1). It seems clear, atleast for the C-terminal domain, that correct functioningcan only occur in the context of the intact protein. Infact, analysis of the effects of coexpression of B12Q CaMand B34Q CaM in animals with one copy of Cam� (seeLegend, Fig. 3C) suggests that not only does B12Q CaMfail to rescue the B34Q CaM effects, it produces a moresevere phenotype (Fig. 3C).

The most surprising finding from these studies is theopposing effects on muscle contraction produced byloss of Ca2� binding in the individual terminal domainsof CaM. This suggests discrete and opposing roles for thetwo domains in the generation of proper muscle re-sponses, and is strikingly similar to the opposing effectsof N- and C-terminal CaM mutations on behavioral re-sponses in Paramecium (Kink et al., 1990).

As discussed above, the effects of the C-terminal mu-tant B34Q CaM probably reflect a failure to inhibit Ca2�

flux from RyR channels. Defective regulation of the volt-age-sensitive L-type channels, which initiate Ca2� fluxinto the muscles, may also be involved, since the C-terminal domain of CaM also mediates closure of thesechannels at high [Ca2�] (Peterson et al., 1999). As men-tioned previously, CaM sensitizes RyR channels to open-ing at low [Ca2�], an effect that is presumably requiredfor generation of full contractile force. It is worth con-sidering, therefore, whether the opposing effect ofB12Q CaM reflects a loss of this regulation. As discussed,in vitro studies with mammalian skeletal RyR detectedno loss of this sensitization with either B12Q or B34QCaMs, but established that B12Q CaM binds more weaklyto RyR than wild-type CaM (Rodney et al., 2001). If theN-terminal domain were involved in sensitization of RyR,this weaker binding of the B12Q mutant would producethe change in behavior of the RyR channels required forthe in vivo response we detect. Thus, relevant issues are:1) Have our in vivo studies exposed a regulatory inter-action with the RyR that is lost in vitro?, and 2) Does therole of CaM in RyR sensitization in Drosophila involve afunction of the N-terminal domain not seen for mamma-lian skeletal muscle RyR? Of course, the alternative ex-planation, that the effects of B12Q CaM involve interac-tion(s) with other target(s) essential for musclecontraction, is clearly a possibility.

The dominant-negative effect of B34Q CaM was unex-pected, but in vivo studies of CaM regulation of L-typechannels (Peterson et al., 1999) provide insight into thisfinding. Apo-CaM is constitutively tethered to L-typechannels via its C-terminal domain, and in wild-typecells, mutants comparable to B34Q CaM can competewith wild-type CaM for binding to the channels anddisrupt function. It seems possible that a comparableinteraction of B34Q with RyR could underlie the domi-nant effects we have detected.

ACKNOWLEDGMENT

We thank Drs. Susan Hamilton and George Rodney forhelpful discussions.

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