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BackgroundAbelson tyrosine kinase (Abl) plays a key role in axon guidance in linking guidance receptors to actin dynamics. The long C-terminal domain (CTD) of Drosophila Abl is important for this role, and previous work identified the ‘first quarter’ (1Q) of the CTD as essential. Here, we link the physical interactions of 1Q binding partners to Abl’s function in axon guidance.MethodsProtein binding partners of 1Q were identified by GST pulldown and mass spectrometry, and validated using axon guidance assays in the embryonic nerve cord and motoneurons. The role of 1Q was assessed genetically, utilizing a battery ofAbltransgenes in combination with mutation or overexpression of the genes of pulled down proteins, and their partners in actin dynamics. The set ofAbltransgenes had the following regions deleted: all of 1Q, each half of 1Q (‘eighths’, 1E and 2E) or a PxxP motif in 2E, which may bind SH3 domains.ResultsGST pulldown identified Hem and Sra-1 as binding partners of 1Q, and our genetic analyses show that both proteins function with Abl in axon guidance, with Sra-1 likely interacting with 1Q. As Hem and Sra-1 are part of the actin-polymerizing WAVE regulatory complex (WRC), we extended our analyses to Abi and Trio, which interact with Abl and WRC members. Overall, the 1Q region (and especially 2E and its PxxP motif) are important for Abl’s ability to work with WRC in axon guidance. These areas are also important for Abl’s ability to function with the actin regulator Enabled. In comparison, 1E contributes to Abl function with the WRC at the midline, but less so with Enabled.ConclusionsThe 1Q region, and especially the 2E region with its PxxP motif, links Abl with the WRC, its regulators Trio and Abi, and the actin regulator Ena. Removing 1E has specific effects suggesting it may help modulate Abl’s interaction with the WRC or Ena. Thus, the 1Q region of Abl plays a key role in regulating actin dynamics during axon guidance.

Functional studies of the methuselah/methuselah-like (mth/mthl) gene family have focused on the founding member mth, but little is known regarding the developmental functions of this receptor or any of its paralogs. We undertook a comprehensive analysis of developmental expression and sequence divergence in the mth/mthl gene family. Using in situ hybridization techniques, we detect expression of six genes (mthl1, 5, 9, 11, 13, and 14) in the embryo during gastrulation and development of the gut, heart, and lymph glands. Four receptors (mthl3, 4, 6, and 8) are expressed in the larval central nervous system, imaginal discs, or both, and two receptors (mthl10 and mth) are expressed in both embryos and larvae. Phylogenetic analysis of all mth/mthl genes in five Drosophila species, mosquito and flour beetle structured the mth/mthl family into several subclades. mthl1, 5, and 14 are present in most species, each forming a separate clade. A newly identified Drosophila mthl gene (CG31720; herein mthl15) formed another ancient clade. The remaining Drosophila receptors, including mth, are members of a large “superclade” that diversified relatively recently during dipteran evolution, in many cases within the melanogaster subgroup. Comparing the expression patterns of the mth/mthl “superclade” paralogs to the embryonic expression of the singleton ortholog in Tribolium suggests both subfunctionalization and acquisition of novel functionalities. Taken together, our findings shed novel light on mth as a young member of an adaptively evolving developmental gene family. J. Exp. Zool. (Mol. Dev. Evol.) 318B:368-387, 2012. © 2012 Wiley Periodicals, Inc.

The life span of model organisms can be modulated by environmental conditions that influence cellular metabolism, oxidation, or DNA integrity. The yeast nicotinamidase gene pnc1 was identified as a key transcriptional target and mediator of calorie restriction and stress-induced life span extension. PNC1 is thought to exert its effect on yeast life span by modulating cellular nicotinamide and NAD levels, resulting in increased activity of Sir2 family class III histone deacetylases. In Caenorhabditis elegans, knockdown of a pnc1 homolog was shown recently to shorten the worm life span, whereas its overexpression increased survival under conditions of oxidative stress. The function and regulation of nicotinamidases in higher organisms has not been determined. Here, we report the identification and biochemical characterization of the Drosophila nicotinamidase, D-NAAM, and demonstrate that its overexpression significantly increases median and maximal fly life span. The life span extension was reversed in Sir2 mutant flies, suggesting Sir2 dependence. Testing for physiological effectors of D-NAAM in Drosophila S2 cells, we identified oxidative stress as a primary regulator, both at the transcription level and protein activity. In contrast to the yeast model, stress factors such as high osmolarity and heat shock, calorie restriction, or inhibitors of TOR and phosphatidylinositol 3-kinase pathways do not appear to regulate D-NAAM in S2 cells. Interestingly, the expression of D-NAAM in human neuronal cells conferred protection from oxidative stress-induced cell death in a sirtuin-dependent manner. Together, our findings establish a life span extending the ability of nicotinamidase in flies and offer a role for nicotinamide-modulating genes in oxidative stress regulated pathways influencing longevity and neuronal cell survival.

Frazzled is a Netrin-dependent chemoattractive receptor required for axon pathway formation in the developing Drosophila embryonic CNS. The cytoplasmic domain is important and contains three conserved P-motifs (P1, P2, and P3) thought to initiate intracellular signaling cascades and to crosstalk with other receptors during axon pathway formation. Here, we rescue homozygous frazzled embryos by pan-neurally expressing a series of mutants lacking either the cytoplasmic domain or one of the conserved P-motifs and assess the ability of these mutants to rescue frazzled defects in commissural, longitudinal and motor axon pathways. Surprisingly, while the cytoplasmic domain is required, removal of an individual P-motif does not prevent gross formation of commissures. However, removal of P3 from Fra does prevent eagle-expressing commissural axons from crossing the midline in the posterior commissure suggesting that some neurons have a stronger requirement for P3-dependent signaling. Indeed, axons within the longitudinal connective as well as a small subset of motor neurons within the ISNb pathway also specifically require P3 to project to their targets correctly. In these latter axon projections, deleting the P1-motif appears to de-regulate the receptor's activity, actually increasing the frequency of motor neuron projection errors and inducing ectopic midline crossing errors. Collectively, these data demonstrate the critical nature of both the P1 and the P3-motifs to Frazzled function in vivo during axon pathway formation.

Frazzled (Fra) is a chemoattractive guidance receptor regulating the cytoskeletal dynamics underlying growth cone steering at the Drosophila embryonic midline. Here, by genetically evaluating the role of Rho GTPases in Fra signaling in vivo, we uncover a Rho-dependent pathway apparently regulating conventional myosin II activity. Midline crossing errors induced by expressing activated Cdc42(v12) or Rac(v12) are suppressed by a heterozygous loss of fra(4) signaling but, in a Fra(wt) gain-of-function condition, no interaction is detected. In contrast, the frequency of crossovers is enhanced approximately 5-fold when Fra(wt) is co-expressed with activated Rho(v14) and this interaction specifically requires the cytoplasmic P3 motif of Fra. Expression of Rho(v14) and activated MLCK (ctMLCK) synergistically increase ectopic crossovers and both require phosphorylation of the regulatory light chain (Sqh) of myosin II. Abelson tyrosine kinase may also help regulate myosin II activity. Heterozygous abl(4) abolishes the midline crossing errors induced by ctMLCK alone or in combination with Fra(wt); suppression of Rho(v14) crossovers is not observed. Interestingly, an interaction between Fra and an activated Abl (Bcr-Abl) also specifically requires the P3 motif. Therefore, the P3 motif of Frazzled appears to initiate Rho and Abl dependent signals to directly or indirectly regulate myosin II activity in growth cones.

G-protein signaling is known to be required for cell-cell contacts during the development of the Drosophila dorsal vessel. However, the identity of the G protein-coupled receptor (GPCR) that regulates this signaling pathway activity is unknown. Here we describe the identification of a novel cardiac specific GPCR, called Gia, for "GPCR in aorta". Gia is the only heart-specific GPCR identified in Drosophila to date and it is specifically expressed in cardioblasts that fuse at the dorsal midline to become the aorta. Gia is the only Drosophila gene so far identified for which expression is entirely restricted to cells of the aorta. Deletion of Gia led to a broken-hearted phenotype, characterized by pericardial cells dissociated from cardioblasts and abnormal distribution of cell junction proteins. Both phenotypes were similar to those observed in mutants of the heterotrimeric cardiac G proteins. Lack of Gia also led to defects in the alignment and fusion of cardioblasts in the aorta. Gia forms a protein complex with G-αo47A, the alpha subunit of the heterotrimeric cardiac G proteins and interacts genetically with G-αo47A during cardiac morphogenesis. Our study identified Gia as an essential aorta-specific GPCR that functions upstream of cardiac heterotrimeric G proteins and is required for morphological integrity of the aorta during heart tube formation. These studies lead to a redefinition of the bro phenotype, to encompass morphological integrity of the heart tube as well as cardioblast-pericardial cell spatial interactions.

Rho family GTPases are ideal candidates to regulate aspects of cytoskeletal dynamics downstream of axon guidance receptors. To examine the in vivo role of Rho GTPases in midline guidance, dominant negative (dn) and constitutively active (ct) forms of Rho, Drac1, and Dcdc42 are expressed in the Drosophila CNS. When expressed alone, only ctDrac and ctDcdc42 cause axons in the pCC/MP2 pathway to cross the midline inappropriately. Heterozygous loss of Roundabout enhances the ctDrac phenotype and causes errors in embryos expressing dnRho or ctRho. Homozygous loss of Son-of-Sevenless (Sos) also enhances the ctDrac phenotype and causes errors in embryos expressing either dnRho or dnDrac. CtRho suppresses the midline crossing errors caused by loss of Sos. CtDrac and ctDcdc42 phenotypes are suppressed by heterozygous loss of Profilin, but strongly enhanced by coexpression of constitutively active myosin light chain kinase (ctMLCK), which increases myosin II activity. Expression of ctMLCK also causes errors in embryos expressing either dnRho or ctRho. Our data confirm that Rho family GTPases are required for regulation of actin polymerization and/or myosin activity and that this is critical for the response of growth cones to midline repulsive signals. Midline repulsion appears to require down-regulation of Drac1 and Dcdc42 and activation of Rho.

The formation of axon trajectories requires integration of local adhesive interactions with directional information from attractive and repulsive cues. Here, we show that these two types of information are functionally integrated; activation of the transmembrane receptor Roundabout (Robo) by its ligand, the secreted repulsive guidance cue Slit, inactivates N-cadherin-mediated adhesion. Loss of N-cadherin-mediated adhesion is accompanied by tyrosine phosphorylation of beta-catenin and its loss from the N-cadherin complex, concomitant with the formation of a supramolecular complex containing Robo, Abelson (Abl) kinase and N-cadherin. Local formation of such a receptor complex is an ideal mechanism to steer the growth cone while still allowing adhesion and growth in other directions.

Coordinated regulation of actin cytoskeletal dynamics is critical to growth cone movement. The intracellular molecules calmodulin and profilin actively regulate actin-based motility and participate in the signaling pathways used to steer growth cones. Here we show that in the developing Drosophila embryo, calmodulin and profilin convey complimentary information that is necessary for appropriate growth cone advance. Reducing calmodulin activity by expression of a dominant inhibitor (KA) stalls axon extension of pioneer neurons within the CNS, while a partial loss of profilin function decreases extension of motor axons in the periphery. Yet, surprisingly, when calmodulin and profilin are simultaneously reduced, the ability of both CNS pioneer axons and motor axons to extend beyond the choice points is restored. In the CNS, at the time when growth cones must decide whether to cross or not to cross the midline, a reduction in calmodulin and/or roundabout signaling causes axons to cross the midline inappropriately. These inappropriate crossings are suppressed when profilin activity is simultaneously reduced. Interestingly, the mutual suppression of calmodulin and profilin activity requires a minimal level of profilin. In KA combinations with profilin null alleles, defects in axon extension and midline guidance are synergistically enhanced rather than suppressed. Together, our data indicate that the growth cone must coordinate the activity of both calmodulin and profilin in order to advance past selected choice points, including those dictating midline crossovers. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 26–38, 2001

Background: Calmodulin (CaM) is the major calcium-dependent regulator of a large variety of important intracellular processes in eukaryotes. The structure of CaM consists of two globular calcium-binding domains joined by a central 28-residue α helix. This linker helix has been hypothesized to act as a flexible tether and is crucial for the binding and activation of numerous target proteins. Although the way in which alterations of the central helix modulate the molecular recognition mechanism is not known exactly, the relative orientation of the globular domains seems to be of great importance. The structural analysis of central helix mutants may contribute to a better understanding of how changes in the conformation of CaM effect its function. Results: We have determined the crystal structure of a calcium-saturated mutant of chicken CaM (mut-2) that lacks two residues in the central helix, Thr79 and Asp80, at 1.8 A resolution. The mutated shorter central helix is straight, relative to that of the wild-type structure. The loss of a partial turn of the central α helix causes the C-terminal domain to rotate 220° around the helix axis, with respect to the N-terminal domain. This rotation places the two domains on the same side of the central helix, in a cis orientation, rather than in the trans orientation found in wild-type structures. Conclusions: The deletion of two residues in the central helix of CaM does not distort or cause a bending of the linker α helix. The main consequence of the mutation is a change in the relative orientation of the two globular calcium-binding domains, causing the hydrophobic patches in these domains to be closer and much less accessible to interact with the target enzymes. This may explain why this mutant of CaM shows a marked decrease in its ability to activate some enzymes while the mutation has little or no effect on its ability to activate others.

Fasciclin II (Fas II), an NCAM-like cell adhesion molecule in Drosophila, is expressed on a subset of embryonic axons and controls selective axon fasiculation. Fas II is also expressed in imaginal discs. Here we use genetic analysis to show that Fas II is required for the control of proneural gene expression. Clusters of cells in the eye-antennal imaginal disc express the achaete proneural gene and give rise to mechanosensory neurons; other clusters of cells express the atonal gene and give rise to ocellar photoreceptor neurons. In fasII loss-of-function mutants, the expression of both proneural genes is absent in certain locations, and, as a result, the corresponding sensory precursors fail to develop. In fasII gain-of-function conditions, extra sensory structures arise from this same region of the imaginal disc. Mutations in the Abelson tyrosine kinase gene show dominant interactions with fasII mutations, suggesting that Abl and Fas II function in a signaling pathway that controls proneural gene expression.

Ca 2+ -calmodulin (CaM) function was selectively disrupted in a specific subset of growth cones in transgenic Drosophila embryos in which a specific enhancer element drives the expression of the kinesin motor domain fused to a CaM antagonist peptide (kinesin-antagonist or KA, which blocks CaM binding to target proteins) or CaM itself (kinesin-CaM or KC, which acts as a Ca 2+ -binding protein). In both KA and KC mutant embryos, specific growth cones exhibit dosage-dependent stalls in axon extension and errors in axon guidance, including both defects in fasciculation and abnormal crossings of the midline. These results demonstrate an in vivo function for Ca 2+ -CaM signaling in growth cone extension and guidance and suggest that Ca 2+ -CaM may in part regulate specific growth cone decisions, including when to defasciculate and whether or not to cross the midline.

Functional studies of the methuselah/methuselah-like (mth/mthl) gene family have focused on the founding member mth, but little is known regarding the developmental functions of this receptor or any of its paralogs. We undertook a comprehensive analysis of developmental expression and sequence divergence in the mth/mthl gene family. Using in situ hybridization techniques, we detect expression of six genes (mthl1, 5, 9, 11, 13, and 14) in the embryo during gastrulation and development of the gut, heart, and lymph glands. Four receptors (mthl3, 4, 6, and 8) are expressed in the larval central nervous system, imaginal discs, or both, and two receptors (mthl10 and mth) are expressed in both embryos and larvae. Phylogenetic analysis of all mth/mthl genes in five Drosophila species, mosquito and flour beetle structured the mth/mthl family into several subclades. mthl1, 5, and 14 are present in most species, each forming a separate clade. A newly identified Drosophila mthl gene (CG31720; herein mthl15) formed another ancient clade. The remaining Drosophila receptors, including mth, are members of a large "superclade" that diversified relatively recently during dipteran evolution, in many cases within the melanogaster subgroup. Comparing the expression patterns of the mth/mthl "superclade" paralogs to the embryonic expression of the singleton ortholog in Tribolium suggests both subfunctionalization and acquisition of novel functionalities. Taken together, our findings shed novel light on mth as a young member of an adaptively evolving developmental gene family.

Activation of four target enzymes by two series of calmodulin Ca2+ binding site mutants has been examined. In each mutant, the conserved bidentate glutamate of one of the Ca2+ binding sites is mutated to glutamine or lysine. The enzymes studied were smooth and skeletal muscle myosin light chain kinases, adenylylcyclase, and plasma membrane Ca(2+)-ATPase. For the first three enzymes, the activation patterns with the two mutant series were very similar: mutation of site 4 was most deleterious, then site 2, site 3, and site 1. This ranking was observed previously in Ca2+ binding and Ca(2+)-induced conformational studies of these mutants. Thus the response of these enzymes is probably determined by the extent to which each mutant's competence to interact with target binding regions has been compromised. In contrast, for Ca(2+)-ATPase, mutants of sites 3 and 4 were much poorer activators than those of sites 1 and 2. Events beyond calmodulin binding and related to enzyme activation probably dictate this unusual activation pattern and also the anomalously poor activation of skeletal muscle myosin light chain kinase by site 1 mutant B1Q. Site 1 mutant B1K showed wild type activation of all four enzymes suggesting that in site 1, the lysine substitution can evoke the conformational changes associated with Ca2+ binding.

TaM-BMI is a genetically engineered chimeric protein consisting of the first 55 amino acids of cardiac troponin C (but with the normally inactive first Ca2+ binding domain reactivated by site- directed mutagenesis) ligated to the last three domains of chicken calmodulin (George, S.E., VanBerkum, M.F., Ono, T., Cook, R., Hanley, R.M., Putkey, J.A., and Means, A. R. (1990) J. Biol. Chem. 265, 9228-9235). This protein binds chicken smooth muscle myosin light chain kinase (smMLCK) but fails to activate the enzyme, thus functioning as a potent competitive inhibitor (Ki = 66 nM). We have created 29 mutants of calmodulin designed to identify the minimal number of alterations which must be introduced in the first domain to convert the protein to a competitive inhibitor of smMLCK. Alterations of three amino acids predicted to lie on the external surface of calmodulin (E14A, T34K, S38M) recapitulated the phenotype of TaM-BMI and exhibited a Ki of 38 nM. Both the triple mutant and TaM-BMI activated phosphodiesterase and bound a synthetic peptide analog of the calmodulin binding region of smMLCK with an affinity similar to that of native calmodulin (Kact and Kd values of approximately 2 and 3 nM respectively). When a synthetic peptide analog of the myosin light chain phosphorylation site was used as substrate rather than the 20-kDa light chains, TaM-BMI and the triple mutant were partial agonists: the Km for peptide substrate was increased 100- and 60-fold, and catalytic activity was 45 and 60%, respectively, relative to calmodulin. These data suggest TaM-BMI and E14A/T34K/S38M may interact with the calmodulin binding domain of smMLCK in a manner similar to calmodulin. However, alterations in electrostatic and hydrophobic interactions created by the three amino acid substitutions prevent the conformational change in the enzyme usually produced by calmodulin binding. Lack of such changes results in loss of catalytic activity and light chain binding. Additionally, our results show that altering only 3 amino acids residues converts calmodulin to an enzyme-selective antagonist, thus demonstrating the ability to separate calmodulin binding to smMLCK from calmodulin-induced activation of the enzyme.

To evaluate the role of domain I of calmodulin (CaM) in the activation of target enzymes, a series of CaM mutants was constructed in which domain I (49 amino acids) was substantially deleted, or was exchanged with the homologous region (58 amino acids) of cardiac troponin C (cTnC). The proteins are 1) aM, a mutant CaM in which domain I has been deleted; 2) TaM, first domain of cTnC, last three domains of CaM; 3) TaM-BMI, same as TaM, except the nonfunctional first Ca2(+)-binding domain has been restored by mutagenesis; 4) CaT, first domain of CaM, last three domains of cTnC. These proteins were evaluated for Ca2+ binding properties and as activators of three CaM target enzymes, CaM-dependent phosphodiesterase (PDE), smooth muscle myosin light chain kinase (MLCK), and CaM-dependent multifunctional protein kinase (CaM kinase II). The chimeric proteins containing four domains bound Ca2+ in the manner expected from the number and nature of EF hands. In contrast, aM bound only two Ca2+, suggesting that deletion of domain I may have disrupted binding in one of the remaining three domains, and did not activate the three enzymes. The kinetics of activation of PDE by CaM, TaM, and TaM-BMI were identical. Although cTnC and CaT could maximally activate PDE, the Kact for these mutants were greater than 2000 times than for CaM. All mutated proteins except CaT were poor activators of CaM kinase II and this protein activated the kinase to 65% that of CaM, with a nearly identical Kact. CaT and TaM, were poor agonists of MLCK. Activation of Ca2(+)-binding site I in TaM (TaM-BMI), completely prevented activation of MLCK. In addition, TaM-BMI was a potent competitive inhibitor of MLCK activation by CaM (Ki = 66 nM). We conclude 1) a domain I is necessary to activate these target enzymes, and the substitution of the corresponding region of cTnC into CaM leads to differential effects; 2) an active first Ca2(+)-binding site is not essential for activation of PDE and the primary sequence of the first domain of CaM need not be highly conserved; 3) for CaM kinase II, determinants in the first domain are critical whereas more flexibility exists for the remaining three domains; 4) since TaM-BMI acts as a potent competitive inhibitor of MLCK binding of CaM to a target enzyme and activation can be dissociable events.

The central helical region of calmodulin (CaM) includes amino acids 65-92 and serves to separate the two pairs of Ca2(+)-binding sites. This region may impart conformational flexibility and also interact with target proteins. The functional effects of deleting two, three, five, or eight amino acids from the central helix were monitored by examining the activation of phosphodiesterase, smooth muscle myosin light chain (MLC) kinase, and Ca2+/CaM-dependent protein kinase II (CaM kinase II). CaMDM(-8), a calmodulin-deletion mutant with 8 amino acids deleted from the middle of the central helix, failed to activate MLC kinase, phosphodiesterase, or CaM kinase II at physiologically significant concentrations of activator but also had altered electrophoretic mobility and tyrosine fluorescence properties suggesting major changes in the structure of this mutant. Deletion of five amino acids (77-81) resulted in an increase in apparent Kact for phosphodiesterase (150-fold), CaM kinase II (25-fold), and MLC kinase (5-fold) relative to CaM. The maximal autophosphorylation activity of CaM kinase II was also diminished 70% with CaMDM(-5). For phosphodiesterase activation, CaMDM(-2) has a 15-fold increase in apparent Kact while CaMDM(-3) had an apparent Kact value only 3-fold higher than native CaM. In contrast, the activation of MLC kinase by the two (79-80)- and three (79-81)-amino acid deletion mutants were indistinguishable from each other or native CaM. CaMDM(-2) and CaMDM(-3) stimulated CaM kinase II autophosphorylation to 85 and 70%, respectively, of native CaM with less than a 2-fold increase in Kact. Therefore, all deletions in the central helix of CaM reduce the efficiency of phosphodiesterase activation as reflected by substantial alterations in Kact. MLC kinase activation, however, is relatively insensitive to small two or three amino acid deletions. CaM kinase II interacts with the central helix deletion mutants in a complex manner with alterations in both the Kact and the maximum activity. The data suggest the central helix of CaM may serve as a flexible tether for MLC kinase (and to a lesser extent CaM kinase II) but that an extended conformation of CaM, as predicted from the crystal structure, may be required for phosphodiesterase activation.

Calmodulin and Abelson tyrosine kinase are key signaling molecules transducing guidance cues at the Drosophila embryonic midline. A reduction in the signaling strength of either pathway alone induces ectopic midline crossing errors in a few segments. When Calmodulin and Abelson signaling levels are simultaneously reduced, the frequency of ectopic crossovers is synergistically enhanced as all segments exhibit crossing errors. But as the level of signaling is further reduced, commissures begin to fuse and large gaps form in the longitudinal connectives. Quantitative analysis suggests that the level of Abelson activity is particularly important. Like Calmodulin, Abelson interacts with son-of-sevenless to increase ectopic crossovers suggesting all three contribute to midline repulsive signaling. Axons cross the midline in almost every segment if Frazzled is co-overexpressed with the Calmodulin inhibitor, but the crossovers induced by the Calmodulin inhibitor itself do not require endogenous Frazzled. Thus, Calmodulin and Abelson tyrosine kinase are key signaling molecules working synergistically to transduce both midline attractive and repulsive cues. While they may function downstream of specific receptors, the emergence of commissural and longitudinal connective defects point to a novel convergence of Calmodulin and Abelson signaling during the regulation of actin and myosin dynamics underlying a guidance decision.

Tyrosine phosphorylation-dependent signaling cascades play key roles in determining the formation of an axon pathway. The cytoplasmic Abelson tyrosine kinase participate in several signaling pathways that orchestrate both growth cone advance and steering in response to guidance cues. Here, a genetic approach is used to evaluate the role for Abelson in growth cones during a decision to cross or not to cross the Drosophila embryonic midline. Our data indicate that both loss- and gain-of-function conditions for Abl cause neurons within the pCC/MP2 pathway to project across the midline incorrectly. The frequency of abnormal crossovers is enhanced by mutations in the genes encoding the midline repellent, Slit, or its receptor, Roundabout. In comm mutants, where repulsive signals remain elevated, increasing or decreasing Abl activity partially rescues commissure formation. Thus, both too much and too little Abl activity causes axons to cross the midline inappropriately, indicating that Abl plays a critical role in transducing midline repulsive cues. How Abl functions in this role is not yet clear, but we suggest that Abl may help regulate cytoskeletal dynamics underlying a growth cone's response to midline cues. © 2003 Wiley Periodicals, Inc. J Neurobiol 57: 15–30, 2003

Conventional myosin II activity provides the motile force for axon outgrowth, but to achieve directional movement during axon pathway formation, myosin activity should be regulated by the attractive and repulsive guidance cues that guide an axon to its target. Here, evidence for this regulation is obtained by using a constitutively active Myosin Light Chain Kinase (ctMLCK) to selectively elevate myosin II activity in Drosophila CNS neurons. Expression of ctMLCK pan-neurally or in primarily pCC/MP2 neurons causes these axons to cross the midline incorrectly. This occurs without altering cell fates and is sensitive to mutations in the regulatory light chains. These results confirm the importance of regulating myosin II activity during axon pathway formation. Mutations in the midline repulsive ligand Slit, or its receptor Roundabout, enhance the number of ctMLCK-induced crossovers, but ctMLCK expression also partially rescues commissure formation in commissureless mutants, where repulsive signals remain high. Overexpression of Frazzled, the receptor for midline attractive Netrins, enhances ctMLCK-dependent crossovers, but crossovers are suppressed when Frazzled activity is reduced by using loss-of-function mutations. These results confirm that proper pathway formation requires careful regulation of MLCK and/or myosin II activity and suggest that regulation occurs in direct response to attractive and repulsive cues.

The establishment of axon trajectories is ultimately determined by the integration of intracellular signaling pathways. Here, a genetic approach in Drosophila has demonstrated that both Calmodulin and Son of sevenless signaling pathways are used to regulate which axons cross the midline. A loss in either signaling pathway leads to abnormal projection of axons across the midline and these increase with roundabout or slit mutations. When both Calmodulin and Son of sevenless are disrupted, the midline crossing of axons mimics that seen in roundabout mutants, although Roundabout remains expressed on crossing axons. Calmodulin and Son of sevenless also regulate axon crossing in a commissureless mutant. These data suggest that Calmodulin and Son of sevenless signaling pathways function to interpret midline repulsive cues which prevent axons crossing the midline.

Background In the Drosophila embryonic nerve cord, the formation of commissures require both the chemoattractive Netrin receptor Frazzled (Fra) and the Abelson (Abl) cytoplasmic tyrosine kinase. Abl binds to the cytoplasmic domain of Fra and loss-of-function mutations in abl enhance fra-dependent commissural defects. To further test Abl's role in attractive signaling, we over-expressed Abl in Fra mutants anticipating rescue of commissures. Methodology/Principal Findings The Gal4-UAS system was used to pan-neurally over-express Abl in homozygous fra embryos. Surprisingly, this led to a significant decrease in both posterior and anterior commissure formation and induced some commissural and longitudinal axons to project beyond the CNS/PNS border. Re-expressing wild-type Fra, or Fra mutants with a P-motif deleted, revert both commissural and exiting phenotypes, indicating that Fra is required but not a specific P-motif. This is supported by S2 cell experiments demonstrating that Abl binds to Fra independent of any specific P-motif and that Fra continues to be phosphorylated when individual P-motifs are removed. Decreasing midline repulsion by reducing Robo signaling had no effect on the Abl phenotype and the phenotypes still occur in a Netrin mutant. Pan-neural over-expression of activated Rac or Cdc42 in a fra mutant also induced a significant loss in commissures, but axons did not exit the CNS. Conclusion/Significance Taken together, these data suggest that Fra activity is required to correctly regulate Abl-dependent cytoskeletal dynamics underlying commissure formation. In the absence of Fra, increased Abl activity appears to be incorrectly utilized downstream of other guidance receptors resulting in a loss of commissures and the abnormal projections of some axons beyond the CNS/PNS border.

The interaction between the peptide corresponding to the calmodulin-binding domain of the smooth muscle myosin light-chain kinase and (Ca2+)4-calmodulin has been studied by multinuclear and multidimensional nuclear magnetic resonance methods. The study was facilitated by the use of 15N-labeled peptide in conjunction with 15N-edited and 15N-correlated 1H spectroscopy. The peptide forms a 1:1 complex with calcium-saturated calmodulin which is in slow exchange with free peptide. The 1H and 15N resonances of the bound have been assigned. An extensive set of structural constraints for the bound peptide has been assembled from the analysis of nuclear Overhauser effects and three-bond coupling constants. The backbone conformation of the bound peptide has been determined using these constraints by use of distance geometry and related computational methods. The backbone conformation of the peptide has been determined to high precision and is generally indicative of helical secondary structure. Nonhelical backbone conformations are seen in the middle and at the C-terminal end of the bound peptide. These studies provide the first direct confirmation of the amphiphilic helix model for the structure of peptides bound to calcium-saturated calmodulin.

Calmodulin is a Ca2+ binding protein present in all eukaryotic cells that serves as the primary intracellular receptor for Ca2+. This 148 amino acid protein is involved in activation of more than 20 enzymes which mediate a wide variety of physiological processes. Many of these enzymes are inhibited in an intramolecular manner and the Ca(2+)-calmodulin complex relieves this inhibition. Calmodulin is essential for life as disruption of the gene in genetically tractable organisms is lethal. This protein plays important regulatory roles in cell proliferation and is required at multiple points in the cell cycle. The mechanism of enzyme activation by calmodulin and its importance in cell growth regulation are reviewed.

Whereas Ca2+ serves as the primary intracellular messenger for regulation of contraction in all types of muscle, the underlying molecular mechanisms are different. Striated muscles utilize troponin C as the Ca2+ receptor and contain an array of cell specific proteins involved in maintenance of Ca2+ homeostasis. Phosphorylation reactions are important in regulation of contraction but phosphorylation of myosin light chains does not play a major role in force generation (Sweeney and Stull, 1990). Smooth muscles utilize calmodulin as the Ca2+ receptor and phosphorylation of myosin light chains is the rate-limiting reaction in contractility (Kamm and Stull, 1985). Since myosin light chain kinase is the primary enzyme that catalyzes light chain phosphorylation, analysis of this complex activation process is crucial to the understanding of how smooth muscles move.

The 3-A crystal structure of calmodulin indicates that it has a polarized tertiary arrangement in which calcium binding domains I and II are separated from domains III and IV by a long central helix consisting of residues 65-92. To investigate the functional significance of the central helix, mutated calmodulins were engineered with alterations in this region. Using oligonucleotide-primed site-directed mutagenesis, Thr-79 was converted to Pro-79 to generate CaMPM. CaMPM was further mutated by insertion of Pro-Ser-Thr-Asp between Asp-78 and Pro-79 to yield CaMIM. Calmodulin, CaMPM, and CaMIM were indistinguishable in their ability to activate calcineurin and Ca2+-ATPase. All mutated calmodulins would also maximally activate cGMP-phosphodiesterase and myosin light chain kinase, however, the concentrations of CaMPM and CaMIM necessary for half-maximal activation (Kact) were 2- and 9-fold greater, respectively, than CaM23. Conversion of the 2 Pro residues in CaMIM to amino acids that predict retention of helical secondary structure did not restore normal calmodulin activity. To investigate the nature of the interaction between mutated calmodulins and target enzymes, synthetic peptides modeled after the calmodulin binding region of smooth and skeletal muscle myosin light chain kinase were prepared and used as inhibitors of calmodulin-dependent cGMP-phosphodiesterase. The data suggest that the different kinetics of activation of myosin light chain kinase by CaM23 and CaMIM are not due to differences in the ability of the activators to bind to the calmodulin binding site of this enzyme. These observations are consistent with a model in which the length but not composition of the central helix is more important for the activation of certain enzymes. The data also support the hypothesis that calmodulin contains multiple sites for protein-protein interaction that are differentially recognized by its multiple target proteins.

Of all the known members of the superfamily of proteins that utilize the EF-hand helix-loop-helix configuration to bind Ca++, calmodulin is unique. This intracellular receptor is ubiquitous in eukaryotes and is highly conserved at the primary amino acid sequence level. In vertebrates only a single conservative amino acid substitution exists between fish and humans 1. Even between primitive eukaryotes such as yeasts and higher vertebrate species, the proteins show at least 80% amino acid identity. In addition calmodulin serves as the obligatory Ca++-dependent activator of a variety of enzymes, exists in enzyme and organelle complexes in the Ca free state and is associated with several intracellular structural proteins. These characteristics are in sharp contrast to other members of this superfamily such as troponin C, calbindins, S-100, calretinin, calcineurin B and myosin light chains. The function of these proteins, when known, tends to be highly specific. Distribution is largely restricted to vertebrates and even within members of this phylum, is found only in selected cell types. The one exception to this generalization is calcineurin B which enjoys a much broader species distribution. However this breadth may also be related to calmodulin since calcineurin is the only known Ca /calmodulin-dependent protein phosphatase. Indeed some calmodulin-dependent enzymes are more widely distributed among phyla and between cell types of a given organism than are the other members of the calmodulin superfamily 2.