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We have studied lethal mutations in the single calmodulin gene (Cam) of Drosophila to gain insight into the in vivo functions of this important calcium sensor. As a result of maternal calmodulin (CaM) in the mature egg, lethality is delayed until the postembryonic stages. Prior to death in the first larval instar, Cam nulls show a striking behavioral abnormality (spontaneous backward movement) whereas a mutation, Cam7, that results in a single amino acid change (V91G) produces a very different phenotype: short indented pupal cases and pupal death with head eversion defects. We show here that the null behavioral phenotype originates in the nervous system and involves a CaM function that requires calcium binding to all four sites of the protein. Further, backward movement can be induced in hypomorphic mutants by exposure to high light levels. In contrast, the V91G mutation specifically affects the musculature and causes abnormal calcium release in response to depolarization of the muscles. Genetic interaction studies suggest that failed regulation of the muscle calcium release channel, the ryanodine receptor, is the major defect underlying the Cam7 phenotype.

Calcineurin is a Ca2+-calmodulin-activated, Ser-Thr protein phosphatase that is essential for the translation of Ca2+ signals into changes in cell function and development. We carried out a dominant modifier screen in the Drosophila eye using an activated form of the catalytic subunit to identify new targets, regulators, and functions of calcineurin. An examination of 70,000 mutagenized flies yielded nine specific complementation groups, four that enhanced and five that suppressed the activated calcineurin phenotype. The gene canB2, which encodes the essential regulatory subunit of calcineurin, was identified as a suppressor group, demonstrating that the screen was capable of identifying genes relevant to calcineurin function. We demonstrated that a second suppressor group was sprouty, a negative regulator of receptor tyrosine kinase signaling. Wing and eye phenotypes of ectopic activated calcineurin and genetic interactions with components of signaling pathways suggested a role for calcineurin in repressing Egf receptor/Ras signal transduction. On the basis of our results, we propose that calcineurin, upon activation by Ca2+-calmodulin, cooperates with other factors to negatively regulate Egf receptor signaling at the level of sprouty and the GTPase-activating protein Gap1.

Inositol 1,4,5-trisphosphate (IP3) receptors are ligand-gated channels that release intracellular Ca2+ stores in response to the second messenger, IP3. We investigated the potential role of IP3 receptors during nuclear envelope assembly in vitro, using Xenopus egg extracts. Previous work suggested that Ca2+ mobilization is required for nuclear vesicle fusion and implicated IP3 receptor activity. To test the involvement of IP3 receptors using selective reagents, we obtained three distinct polyclonal antibodies to the type 1 IP3 receptor. Pretreatment of membranes with two of the antibodies inhibited IP3-stimulated CA2+ release in vitro and also inhibited nuclear vesicle fusion. One inhibitory serum was directed against 420 residues within the "coupling" domain, which includes several potential regulatory sites. The other inhibitory serum was directed against 95 residues near the C terminus and identifies an inhibitory epitope(s) in this region. The antibodies had no effect on receptor affinity for IP3. Because nuclear vesicle fusion was inhibited by antibodies that block Ca2+ flux, but not by control and preimmune antibodies, we concluded that the activation of IP3 receptors is required for fusion. The signal that activates the channel during fusion is unknown.

We have investigated the role of the ryanodine receptor in Drosophila development by using pharmacological and genetic approaches. We identified a P element insertion in the Drosophila ryanodine receptor gene, Ryanodine receptor 44F ( Ryr ), and used it to generate the hypomorphic allele Ryr 16 . An examination of hypodermal, visceral, and circulatory muscle showed that, in each case, muscle contraction was impaired in Ryr 16 larvae. Treatment with the drug ryanodine, a highly specific modulator of ryanodine receptor channel activity, also inhibited muscle function, and, at high levels, completely blocked hypodermal muscle contraction. These results suggest that the ryanodine receptor is required for proper muscle function and may be essential for excitation-contraction coupling in larval body wall muscles. Nonmuscle roles of Ryr were also investigated. Ryanodine-sensitive Ca 2+ stores had previously been implicated in phototransduction; to address this, we generated Ryr 16 mutant clones in the adult eye and performed whole-cell, patch–clamp recordings on dissociated ommatidia. Our results do not support a role for Ryr in normal light responses.

At the onset of mitosis in higher eukaryotes the nuclear envelope, ER, and Golgi complex are partially or completely converted to small vesicles. After mitosis, these biochemically distinct vesicles must fuse selectively to reassemble each organelle. The potential similarities between this process and the process of vesicular transport through the secretory pathway were pointed out by Warren (1985). Secretion involves the regulated formation of coated vesicles at one compartment and their fusion with a specific target membrane (Rothman and Orci, 1992). Secretion ceases during mitosis (Hesketh et al., 1984; Colman et al., 1985), indicating that vesicle formation and/or vesicle fusion are temporarily inhibited. Warren suggested that a mitotic block to vesicle fusion would be sufficient to cause organelles to vesiculate. Newport and Spann (1987) showed that mitotic disassembly of nuclear membranes required an unknown stoichiometric factor (s), perhaps coat proteins. However, direct evidence for the mechanism of mitotic disassembly of the nuclear envelope, or its relationship to membrane traffic, was lacking.

We studied the fusion of nuclear vesicles bound to chromatin in Xenopus egg extracts. Fusion was inhibited by 5 mM BAPTA, a Ca2+ buffer that suppresses cytosolic [Ca2+] gradients. The BAPTA-inhibited step in fusion was biochemically distinct from, and occurred later than, the GTP gamma S-sensitive step mediated by the monomeric GTPase, ADP-ribosylation factor. Exogenous inositol 1,4,5-trisphosphate (IP3), which triggers Ca2+ release from lumenal stores via IP3 receptors, stimulated fusion in the presence of BAPTA. This rescue was specific, because inositol 1,3,4-trisphosphate had no effect. Heparin, a potent antagonist of IP3 receptors, independently blocked fusion in an IP3-reversible manner. We suggest that phosphoinositide signaling may regulate nuclear vesicle fusion.