Complex Proteolytic Processing Acts on Delta, a Transmembrane Ligand for Notch, during Drosophila Development

Delta functions as a cell nonautonomous membrane-bound ligand that binds to Notch, a cell-autonomous receptor, during cell fate specification. Interaction between Delta and Notch leads to signal transduction and elicitation of cellular responses. During our investigations to further understand the biochemical mechanism by which Delta signaling is regulated, we have identified four Delta isoforms in Drosophila embryonic and larval extracts. We have demonstrated that at least one of the smaller isoforms, Delta S, results from proteolysis. Using antibodies to the Delta extracellular and intracellular domains in colocalization experiments, we have found that at least three Delta isoforms exist in vivo, providing the first evidence that multiple forms of Delta exist during development. Finally, we demonstrate that Delta is a transmembrane ligand that can be taken up by Notch-expressing Drosophila cultured cells. Cell culture experiments imply that full-length Delta is taken up by Notch-expressing cells. We present evidence that suggests this uptake occurs by a nonphagocytic mechanism.

Tissue-specific Expression of Dominant Negative Mutant Drosophila HSC70 Causes Developmental Defects and Lethality

The Drosophila melanogaster HSC3 and HSC4 genes encode Hsc70 proteins homologous to the mammalian endoplasmic reticulum (ER) protein BiP and the cytoplasmic clathrin uncoating ATPase, respectively. These proteins possess ATP binding/hydrolysis activities that mediate their ability to aid in protein folding by coordinating the sequential binding and release of misfolded proteins. To investigate the roles of HSC3 (Hsc3p) and HSC4 (Hsc4p) proteins during development, GAL4-targeted gene expression was used to analyze the effects of producing dominant negatively acting Hsc3p (D231S, K97S) and Hsc4p (D206S, K71S) proteins, containing single amino acid substitutions in their ATP-binding domains, in specific tissues of Drosophila throughout development. We show that the production of each mutant protein results in lethality over a range of developmental stages, depending on the levels of protein produced and which tissues are targeted. We demonstrate that the functions of both Hsc3p and Hsc4p are required for proper tissue establishment and maintenance. Production of mutant Hsc4p, but not Hsc3p, results in induction of the stress-inducible Hsp70 at normal temperatures. Evidence is presented that lethality is caused by tissue-specific defects that result from a global accumulation of misfolded protein caused by lack of functional Hsc70. We show that both mutant Hsc3ps are defective in ATP-induced substrate release, although Hsc3p(D231S) does undergo an ATP-induced conformational change. We believe that the amino acid substitutions in Hsc3p interfere with the structural coupling of ATP binding to substrate release, and this defect is the basis for the mutant proteins’ dominant negative effects in vivo.

Muscle LIM Proteins Are Associated with Muscle Sarcomeres and Require dMEF2 for Their Expression during Drosophila Myogenesis

A genetic hierarchy of interactions, involving myogenic regulatory factors of the MyoD and myocyte enhancer-binding 2 (MEF2) families, serves to elaborate and maintain the differentiated muscle phenotype through transcriptional regulation of muscle-specific target genes. Much work suggests that members of the cysteine-rich protein (CRP) family of LIM domain proteins also play a role in muscle differentiation; however, the specific functions of CRPs in this process remain undefined. Previously, we characterized two members of the Drosophila CRP family, the muscle LIM proteins Mlp60A and Mlp84B, which show restricted expression in differentiating muscle lineages. To extend our analysis of Drosophila Mlps, we characterized the expression of Mlps in mutant backgrounds that disrupt specific aspects of muscle development. We show a genetic requirement for the transcription factor dMEF2 in regulating Mlp expression and an ability of dMEF2 to bind, in vitro, to consensus MEF2 sites derived from those present in Mlp genomic sequences. These data suggest that the Mlp genes may be direct targets of dMEF2 within the genetic hierarchy controlling muscle differentiation. Mutations that disrupt myoblast fusion fail to affect Mlp expression. In later stages of myogenic differentiation, which are dedicated primarily to assembly of the contractile apparatus, we analyzed the subcellular distribution of Mlp84B in detail. Immunofluorescent studies revealed the localization of Mlp84B to muscle attachment sites and the periphery of Z-bands of striated muscle. Analysis of mutations that affect expression of integrins and α-actinin, key components of these structures, also failed to perturb Mlp84B distribution. In conclusion, we have used molecular epistasis analysis to position Mlp function downstream of events involving mesoderm specification and patterning and concomitant with terminal muscle differentiation. Furthermore, our results are consistent with a structural role for Mlps as components of muscle cytoarchitecture.

Mutations in Drosophila Enabled and Rescue by Human Vasodilator-stimulated Phosphoprotein (VASP) Indicate Important Functional Roles for Ena/VASP Homology Domain 1 (EVH1) and EVH2 Domains

Drosophila Enabled (Ena) was initially identified as a dominant genetic suppressor of mutations in the Abelson tyrosine kinase and, more recently, as a member of the Ena/human vasodilator-stimulated phosphoprotein (VASP) family of proteins. We have used genetic, biochemical, and cell biological approaches to demonstrate the functional relationship between Ena and human VASP. In addition, we have defined the roles of Ena domains identified as essential for its activity in vivo. We have demonstrated that VASP rescues the embryonic lethality associated with loss of Ena function in Drosophila and have shown that Ena, like VASP, is associated with actin filaments and focal adhesions when expressed in cultured cells. To define sequences that are central to Ena function, we have characterized the molecular lesions present in two lethal ena mutant alleles that affected the Ena/VASP homology domain 1 (EVH1) and EVH2. A missense mutation that resulted in an amino acid substitution in the EVH1 domain eliminated in vitro binding of Ena to the cytoskeletal protein zyxin, a previously reported binding partner of VASP. A nonsense mutation that resulted in a C-terminally truncated Ena protein lacking the EVH2 domain failed to form multimeric complexes and exhibited reduced binding to zyxin and the Abelson Src homology 3 domain. Our analysis demonstrates that Ena and VASP are functionally homologous and defines the conserved EVH1 and EVH2 domains as central to the physiological activity of Ena.

Drosophila coracle, a Member of the Protein 4.1 Superfamily, Has Essential Structural Functions in the Septate Junctions and Developmental Functions in Embryonic and Adult Epithelial Cells

Although extensively studied biochemically, members of the Protein 4.1 superfamily have not been as well characterized genetically. Studies of coracle, a Drosophila Protein 4.1 homologue, provide an opportunity to examine the genetic functions of this gene family. coracle was originally identified as a dominant suppressor of EgfrElp, a hypermorphic form of the Drosophila Epidermal growth factor receptor gene. In this article, we present a phenotypic analysis of coracle, one of the first for a member of the Protein 4.1 superfamily. Screens for new coracle alleles confirm the null coracle phenotype of embryonic lethality and failure in dorsal closure, and they identify additional defects in the embryonic epidermis and salivary glands. Hypomorphic coracle alleles reveal functions in many imaginal tissues. Analysis of coracle mutant cells indicates that Coracle is a necessary structural component of the septate junction required for the maintenance of the transepithelial barrier but is not necessary for apical–basal polarity, epithelial integrity, or cytoskeletal integrity. In addition, coracle phenotypes suggest a specific role in cell signaling events. Finally, complementation analysis provides information regarding the functional organization of Coracle and possibly other Protein 4.1 superfamily members. These studies provide insights into a range of in vivo functions for coracle in developing embryos and adults.

Identification of TER94, an AAA ATPase Protein, as a Bam-dependent Component of the Drosophila Fusome

The Drosophila fusome is a germ cell-specific organelle assembled from membrane skeletal proteins and membranous vesicles. Mutational studies that have examined inactivating alleles of fusome proteins indicate that the organelle plays central roles in germ cell differentiation. Although mutations in genes encoding skeletal fusome components prevent proper cyst formation, mutations in the bag-of-marbles gene disrupt the assembly of membranous cisternae within the fusome and block cystoblast differentiation altogether. To understand the relationship between fusome cisternae and cystoblast differentiation, we have begun to identify other proteins in this network of fusome tubules. In this article we present evidence that the fly homologue of the transitional endoplasmic reticulum ATPase (TER94) is one such protein. The presence of TER94 suggests that the fusome cisternae grow by vesicle fusion and are a germ cell modification of endoplasmic reticulum. We also show that fusome association of TER94 is Bam-dependent, suggesting that cystoblast differentiation may be linked to fusome reticulum biogenesis.

Class VI Unconventional Myosin is Required for Spermatogenesis in Drosophila

We have identified partial loss of function mutations in class VI unconventional myosin, 95F myosin, which results in male sterility. During spermatogenesis the germ line precursor cells undergo mitosis and meiosis to form a bundle of 64 spermatids. The spermatids remain interconnected by cytoplasmic bridges until individualization. The process of individualization involves the formation of a complex of cytoskeletal proteins and membrane, the individualization complex (IC), around the spermatid nuclei. This complex traverses the length of each spermatid resolving the shared membrane into a single membrane enclosing each spermatid. We have determined that 95F myosin is a component of the IC whose function is essential for individualization. In wild-type testes, 95F myosin localizes to the leading edge of the IC. Two independent mutations in 95F myosin reduce the amount of 95F myosin in only a subset of tissues, including the testes. This reduction of 95F myosin causes male sterility as a result of defects in spermatid individualization. Germ line transformation with the 95F myosin heavy chain cDNA rescues the male sterility phenotype. IC movement is aberrant in these 95F myosin mutants, indicating a critical role for 95F myosin in IC movement. This report is the first identification of a component of the IC other than actin. We propose that 95F myosin is a motor that participates in membrane reorganization during individualization.

The fourth chromosome of Drosophila melanogaster: Interspersed euchromatic and heterochromatic domains

The small fourth chromosome of Drosophila melanogaster (3.5% of the genome) presents a puzzle. Cytological analysis suggests that the bulk of the fourth, including the portion that appears banded in the polytene chromosomes, is heterochromatic; the banded region includes blocks of middle repetitious DNA associated with heterochromatin protein 1 (HP1). However, genetic screens indicate 50–75 genes in this region, a density similar to that in other euchromatic portions of the genome. Using a P element containing an hsp70-white gene and a copy of hsp26 (marked with a fragment of plant DNA designated pt), we have identified domains that allow for full expression of the white marker (R domains), and others that induce a variegating phenotype (V domains). In the former case, the hsp26-pt gene shows an accessibility and heat-shock-inducible activity similar to that seen in euchromatin, whereas in the latter case, accessibility and inducible expression are reduced to levels typical of heterochromatin. Mapping by in situ hybridization and by hybridization of flanking DNA sequences to a collection of cosmid and bacterial artificial chromosome clones shows that the R domains (euchromatin-like) and V domains (heterochromatin-like) are interspersed. Examination of the effect of genetic modifiers on the variegating transgenes shows some differences among these domains. The results suggest that heterochromatic and euchromatic domains are interspersed and closely associated within this 1.2-megabase region of the genome.

LexA chimeras reveal the function of Drosophila Fos as a context-dependent transcriptional activator

The transcriptional activation potential of proteins can be assayed in chimeras containing a heterologous DNA-binding domain that mediates their recruitment to reporter genes. This approach has been widely used in yeast and in transient mammalian cell assays. Here, we applied it to assay the transactivation potential of proteins in transgenic Drosophila embryos. We found that a chimera between the DNA-binding bacterial LexA protein and the transactivation domain from yeast GAL4 behaved as a potent synthetic activator in all embryonic tissues. In contrast, a LexA chimera containing Drosophila Fos (Dfos) required an unexpected degree of context to function as a transcriptional activator. We provide evidence to suggest that this context is provided by Djun and Mad (a Drosophila Smad), and that these partner factors need to be activated by signaling from Jun N-terminal kinase and decapentaplegic, respectively. Because Dfos behaves as an autonomous transcriptional activator in more artificial assays systems, our data suggest that context-dependence of transcription factors may be more prevalent than previously thought.

Activation of protein kinase A-independent pathways by Gsα in Drosophila

One of the best-described transmembrane signal transduction mechanisms is based on receptor activation of the α subunit of the heterotrimeric G protein Gs, leading to stimulation of adenylyl cyclase and the production of cAMP. Intracellular cAMP is then thought to mediate its effects largely, if not entirely, by activation of protein kinase A and the subsequent phosphorylation of substrates which in turn control diverse cellular phenomena. In this report we demonstrate, by two different methods, that reduction or elimination of protein kinase A activity had no effect on phenotypes generated by activation of Gsα pathways in Drosophila wing epithelial cells. These genetic studies show that the Gsα pathway mediates its primary effects by a novel pathway in differentiating wing epithelial cells. This novel pathway may in part be responsible for some of the complex, cell-specific responses observed following activation of this pathway in different cell types.

Transfected Drosophila cells as a probe for defining the minimal requirements for stimulating unprimed CD8+ T cells

Stimulation of naive T cells by antigen-presenting cells (APC) is thought to involve two qualitatively different signals: signal one results from T-cell receptor (TCR) recognition of antigenic peptides bound to major histocompatibility complex (MHC) molecules, whereas signal two reflects contact with one or more costimulatory molecules. The requirements for stimulating naive T cells were studied with MHC class I-restricted CD8+ T cells from a T-cell receptor transgenic line, with defined peptides as antigen and transfected Drosophila cells as APC. Three main findings are reported. First, stimulation of naive T cells via signal one alone (MHC plus peptide) was essentially nonimmunogenic; thus T cells cultured with peptides presented by MHC class I-transfected Drosophila APC lacking costimulatory molecules showed little or no change in their surface phenotype. Second, cotransfection of two costimulatory molecules, B7-1 and intercellular adhesion molecule 1 (ICAM-1), converted class I+ Drosophila cells to potent APC capable of inducing strong T-proliferative responses and cytokine (interleukin 2) production. Third, B7-1 and ICAM-1 acted synergistically, indicating that signal two is complex; synergy between B7-1 and ICAM-1 varied from moderate to extreme and was influenced by both the dose and affinity of the peptide used and the parameter of T-cell activation studied. Transfected Drosophila cells are thus a useful tool for examining the minimal APC requirements for naive T cells.

The gcm-motif: A novel DNA-binding motif conserved in Drosophila and mammals

In the Drosophila nervous system, the glial cells missing gene (gcm) is transiently expressed in glial precursors to switch their fate from the neuronal default to glia. It encodes a novel 504-amino acid protein with a nuclear localization signal. We report here that the GCM protein is a novel DNA-binding protein and that its DNA-binding activity is localized in the N-terminal 181 amino acids. It binds with high specificity to the nucleotide sequence, (A/G)CCCGCAT, which is a novel sequence among known targets of DNA-binding proteins. Eleven such GCM-binding sequences are found in the 5′ upstream region of the repo gene, whose expression in early glial cells is dependent on gcm. This suggests that the GCM protein is a transcriptional regulator directly controlling repo. We have also identified homologous genes from human and mouse whose products share a highly conserved N-terminal region with Drosophila GCM. At least one of these was shown to have DNA-binding activity similar to that of GCM. By comparing the deduced amino acid sequences of these gene products, we were able to define the “gcm motif,” an evolutionarily conserved motif with DNA-binding activity. By PCR amplification, we obtained evidence for the existence of additional gcm-motif genes in mouse as well as in Drosophila. The gcm-motif, therefore, forms a family of novel DNA-binding proteins, and may function in various aspects of cell fate determination.

Similar DNA recognition properties of alternatively spliced Drosophila POU factors

The POU-IV or “Brn-3” class of POU-domain transcription factors is represented in Drosophila by I-POU and twin-of-I-POU, alternative splice products of the I-POU gene. I-POU has been previously reported to inhibit DNA binding by the POU-III class factor drifter/Cf1a via the formation of heterodimeric complexes. Here we report that expression of the I-POU/tI-POU message is maximal late in the embryonic phase of Drosophila development, and I-POU is the preferred splice variant. Although I-POU lacks two basic amino acid residues in the POU-homeodomain found in tI-POU and Brn-3.0, these three POU-IV class proteins exhibit very similar DNA-binding specificity. In contrast to previously published reports, the results presented here show no effect of I-POU on DNA binding by drifter, and no evidence for I-POU/drifter dimerization. These results suggest that the I-POU/tI-POU gene products function by transcriptional mechanisms similar to those of the homologous POU-IV class factors expressed in other species, not by a unique inhibitory mechanism.

Circadian modulation of dopamine receptor responsiveness in Drosophila melanogaster

We investigated the circadian function of Drosophila dopamine receptors by using a behaviorally active decapitated preparation that allows for direct application of drugs to the nerve cord. Quinpirole, a D2-like dopamine receptor agonist, induces reflexive locomotion in decapitated flies. We show that the amount of locomotion induced changes as a function of the time of day, with the highest responsiveness to quinpirole during the subjective night. Furthermore, dopamine receptor responsiveness is under circadian control and depends on the normal function of the period gene. The head pacemaker is at least partly dispensable for the circadian modulation of quinpirole-induced locomotion, because changes in agonist responsiveness persist in decapitated flies that are aged for 12 h. This finding suggests a role for the period-dependent molecular oscillators in the body in the modulation of amine receptor responsiveness.

Prolactin is not a juvenile hormone in Xenopus laevis metamorphosis

Prolactin (PRL) is widely considered to be the juvenile hormone of anuran tadpoles and to counteract the effects of thyroid hormone (TH), the hormone that controls amphibian metamorphosis. This putative function was concluded mainly from experiments in which mammalian PRL was injected into tadpoles or added to cultured tadpole tissues. In this study, we show that overexpression of ovine or Xenopus laevis PRL in transgenic X. laevis does not prolong tadpole life, establishing that PRL does not play a role in the life cycle of amphibians that is equivalent to that of juvenile hormone in insect metamorphosis. However, overexpression of PRL produces tailed frogs by reversing specifically some but not all of the programs of tail resorption and stimulating growth of fibroblasts in the tail. Whereas TH induces muscle resorption in tails of these transgenics, the tail fibroblasts continue to proliferate resulting in a fibrotic tail that is resistant to TH.