discs large in the Drosophila testis: An old player on a new task

Gamete development requires a coordinated soma-germ line interaction that ensures renewal and differentiation of germline and somatic stem cells. The physical contact between the germline and somatic cell populations is crucial because it allows the exchange of diffusible signals among them. The tumor suppressor gene discs large (dlg) encodes a septate junction protein with functions in epithelial cell polarity, asymmetric neuroblast division and formation of neuromuscular junctions. Our recent work reveals a new role of dlg in the Drosophila testis, as mutations in dlg lead to testis defects and cell death. Dlg is required throughout spermatogenesis in the somatic lineage and its localization changes from a uniform distribution along the plasma membrane of somatic cells in the testis apex, to a restricted localization on the distally located somatic cell in growing cysts. The extensive defects in dlg testis underline the importance of the somatic cells in the establishment and maintenance of the male stem cell niche and somatic cell differentiation. Here, we discuss our latest findings on the role of dlg in the Drosophila testis, supporting the view that junction proteins are dynamic structures, which can provide guiding cues to recruit scaffold proteins or other signaling molecules.Key words: dlg, Drosophila melanogaster, germ cell differentiation, septate junctions, somatic stem cells, somatic cyst cellsThe discovery of mutations causing neoplasia during Drosophila development¹ followed by the molecular characterization of these genes has shown that cell polarity is critically affected in the tumor cells. Three of these genes, lethal (2) giant larvae (lgl), discs large-1 (dlg) and scribble (scrib), encode scaffolding proteins, associated with either the cytoskeleton matrix or septate junctions.^2–9 Analysis over the last decades revealed that these proteins act as more than just static barriers limiting the diffusion of other components along the cortical cell domains. In particular, they function as dynamic organizing centers, targeting site-specific proteins in discrete domains and provide guiding cues for signaling molecules and insertion of membrane components.¹⁰ Nowadays, several studies place Dlg as a key player in numerous tissues at different time points throughout development, contributing to epithelial polarity establishment, polarized membrane insertion, asymmetric neuroblast division, formation of neuromuscular junctions (NMJ) and planar cell polarity in vertebrates.^{4,7,9,11–15} Interestingly, the four mammalian homologs of the Drosophila dlg are also involved in cell polarity and become downregulated in a series of human cancers. Moreover, a mammalian dlg-1 transgene can substitute a defective dlg gene in Drosophila and rescue the development of dlg mutant animals.^8,9,16 Therefore, it is perfectly plausible to envisage that Drosophila functions uncovered in other tissues may be similarly conserved in other species.Similar to vertebrates, the Drosophila testis consists of germ cells and somatic cells. The somatic cells of the hub form the organizing center at the apex of the testis and recruit germline stem cells (GSCs) (Fig. 1A), giving rise to the male stem cell niche. Each GSC attached to the hub is surrounded by two somatic stem cells (SSCs). Upon asymmetric stem cell division, each GSC produces a new GSC attached to the hub and a distally located gonialblast, whereas each SSC pair divides to generate two SSCs and two somatic cyst cells (SCCs).^17–19 The gonialblast divides mitotically four more times to give rise to 16 interconnected spermatogonial cells, forming a cyst surrounded by the two SCCs.²¹ Then, the spermatogonial cyst grows markedly in size and differentiates to primary spermatocytes that enter the pre-meiotic phase (Fig. 1A, B and D–F).¹⁹ We have recently investigated a new role of dlg in the Drosophila testis.²⁰ In contrast to the overgrowth phenotypes observed in imaginal discs and brain hemispheres,^4,6,21 dlg inactivation leads to testis degeneration during early larval development. The dlg testes are extremely small and contain a reduced number of GSCs loosely attached to the hub (Fig. 1C).²⁰ In addition, the few spermatogonial cysts, which become formed, fully degenerate during the second and third larval instars.Open in a separate windowFigure 1(A) Diagram depicting early spermatogenesis in Drosophila. The red line indicates the Dlg distribution in the hub, SSCs, early and late SCCs. GSCs, germline stem cells; SCCs, somatic cyst cells; SSCs, somatic stem cells. (B) Apex of wild-type 3^rd instar larval testis and (C) dlg^m52 3^rd instar larval testis displaying a reduced number of GSCs, spermatogonial and spermatocyte cysts. In mutant dlg^m52 testes, SSCs and early SCCS positively stained for Traffic-jam are still present. However, late SCCs identified by staining for Eye absent remain undetectable.²⁰ Vasa (red), Traffic-jam (green) and Arm + α-Spectrin (blue). (D–F) Pattern of Dlg distribution in 3^rd instar larval testis. (E and F) are enlargements at different optical sections of the testis shown in (D), displaying Dlg staining in the hub region and growing spermatocyte cysts, respectively. Testis hub is oriented towards the left.Recent advances in Drosophila spermatogenesis and the male stem cel niche have clearly shown that the intrinsic signals of the germ cells are important but not sufficient to support stem cell homeostasis. Signals emanating from SSCs and SCCs are also required for testis development. Physical contacts among the cell populations in the Drosophila testis allow the exchange of signals, which promote tissue survival and set the balance between stem cell identity and differentiation.¹⁸ Interestingly, the Dlg protein is present in all somatic cells including the hub, SSCs and SCCs (Fig. 1D–F) and a specific requirement of dlg in these cells is further supported by the finding that the mutant phenotype could be reverted by expressing dlg in somatic cells but not in germ cells.²⁰ Further analysis points out that the mutant GSCs are significantly larger than in wild-type, lower in number and loosely attached to the hub.²⁰ Preliminary results indicate a defective orientation of the daughter centrosome and absence of mitotic spindle in dividing GSCs, which together with the increased GSC size, allows us to speculate that GSCs may grow but fail to undergo mitosis. Similar phenotypes are observed in mutations affecting the insulin pathway,²² further stressing the importance of cell communication between germ cells and somatic cells. However, a functional connection between dlg and the insulin pathway remains yet to be experimentally determined. The defects detected in the dlg mutant testis place dlg as a key regulator in the early development of spermatogonial cysts.During testis differentiation, the Dlg protein displays a dynamic change in its intracellular localization. First, Dlg is uniformly associated with the plasma membrane on all somatic cells in the male stem cell niche and early spermatogonial cysts, and then becomes restricted to the most proximal SCC in late spermatogonial cysts and growing spermatocyte cysts (Fig. 1D–F). The transition from a uniform to a restricted distribution is achieved between the 8- to 16-cell cyst stages, when one of the two SCCs caps the distal side of the growing cyst. Interestingly, the capping corresponds to the axis of cyst growth and points out the direction of cyst expansion. A restoration of nearly normal testis morphology can be obtained by expressing a dlg transgene in SSCs and early SCCs. In contrast, expression of a dlg transgene in later SCCs can still restore the development of already formed cysts, some of which may reach an advanced post-meiotic stage, but the testis is generally depleted in early cysts.²⁰ These data indicate that dlg is required for the differentiation of the somatic cell lineage and, therefore, the early differentiation of the germline into spermatogonial cells. Results of RNAi experiments provide also evidence that dlg silencing in late SSCs results in a fragmentation of the cysts in advanced stages.²⁰ The specific recruitment of Dlg on the membrane of distal SCCs remains an open question, although it is possible to envisage that phosphorylation of Dlg by the PAR-1 kinase may play a role, as it has been shown in the case of postsynaptic targeting of Dlg in NMJs.²³Therefore, Dlg may exert different functions in the somatic cells that are required for (1) GSC attachment to the hub and proper asymmetric GSC division, (2) the architecture and early differentiation of the spermatogonial cysts and (3) the expansion and growth of the spermatocyte cysts. Presumably, dlg is required for establishing and maintaining a tight connection between GSCs and SSCs around the hub. The connection between gonialblast and SCC is also maintained during the mitotic divisions. In SSCs and early SCCs, dlg acts critically to establish a normal cyst structure, whereas in further spermatogonial and spermatocyte stages dlg is critical for the survival, growth and expansion of the cyst. Our rescue experiments further suggest that if proper cyst architecture is not established when the two stem cell populations move away from the hub, it cannot be re-established at later stages. Moreover, the restricted Dlg localization in the distal SCC suggests that dlg may be necessary for the polarized growth of spermatocyte cysts and thus act as a critical factor for planar cell polarity. In the second phase, dlg is involved in spermatogonial and spermatocyte cyst growth, viability and differentiation. Further RNA silencing experiments using GAL4-drivers that target dlg in SCCs during late spermatocyte growth, meiosis and post-meiotic stages may further provide insights into dlg requirement during the whole spermatogenesis. Preliminary results indicate that Dlg is similarly produced and localized on the distal SCC in spermatocyte and spermatid cysts of adult testes, suggesting that dlg may be required from the early stages, from the establishment of male stem cell niche and SCC survival, up to the later stages of sperm formation.An unexpected finding of our analysis deals with the formation of wavy and ruffled plasma membrane in dlg overexpressing cells capping the spermatocyte cysts. One way to interpret this result would be to consider that Dlg regulates the intensity of germ cell encapsulation through the Egfr pathway, which is the major signaling pathway active at the microenvironment of the spermatogonial cysts.^24,25 Membrane ruffling, detected in somatic cells upon dlg overexpression, is highly reminiscent of the formation of lammellipodia-like structures formed upon upregulation of Rac1 in SCCs.²⁶ Rac1 is a downstream component of the Egfr pathway and acts antagonistically to Rho to regulate germ cell encapsulation. As the Dlg protein plays a central role in the organization of epithelial junctions and in signal transduction at sites of cell-cell contact, it is possible to envisage that the C-terminal tail of Egfr interacts with one of the PDZ domains of Dlg.⁹ In this way, dlg inactivation would result in a disruption of the Egfr protein complexes, block the Egfr pathway and impair Rac1 function. Based on these data, we hypothesize that Dlg may act on the cytoskeleton of the somatic cells to mediate cell-shape changes leading to either cellular extensions over the spermatogonial and spermatocyte cysts or reinforcing cell-to-cell contact with the growing germ cells.A second possibility would imply a general role for Dlg in membrane proliferation and expansion of the SCCs. It has already been shown that Dlg regulates membrane proliferation in a subset of NMJ in a dose-dependent fashion.²⁷ Recent focus on membrane growth during cellularization indicates again that Dlg is an important player in the process of polarized membrane insertion.^11,28–30 Up to now, there is no mechanism describing how SCCs in Drosophila testis expand, elongate and envelop germ cell cysts, and how the SCCs direct sperm differentiation and individualization. Membrane proliferation during tissue spreading and cell surface extensions is frequently associated with the formation of membrane ruffles.^31,32 The finding that dlg overexpression in the distal SCC leads to membrane ruffling indicates that Dlg may mediate membrane growth and membrane extension over the cysts but not necessarily at the expense of the proximal SCC devoid of Dlg. Therefore, there should be a physical limitation in the expansion of the dlg-expressing cell, independent of the amount of synthesized Dlg. Further analyses of components at the junctions between the distal and proximal SCCs or components exhibiting a complementary distribution to Dlg may provide ways to identify further regulators of testis morphogenesis.If Dlg defines sites of membrane addition it may provide a link between membrane trafficking and insertion of polarized membrane components. In NMJs, the postsynaptic distribution of the t-SNARE protein Gtaxin depends on its direct interaction to the Dlg GUK domain,¹² whereas in early embryogenesis Dlg genetically interacts with Exo84.³³ Moreover, the Dlg-Strabismus complex recruits membrane associated proteins and lipids from internal membranes to sites of new plasma membrane formation.¹¹ The occurrence of similar proteins in testis was reported in humans where the SNARE-associated component Snapin binds Pumilio2 and Nanos1 proteins in the male germ cells.³⁴ It would be interesting to know whether Dlg plays a similar role in Drosophila testis, in guiding t-SNARE proteins and components of the exocyst complex into intracellular membranes, either directly or indirectly by regulating the distribution of their direct binding partners. Although Dlg may bind to different proteins in epithelial cells, neuroblasts and NMJ according to the protein availability in these tissues, the function of the Dlg protein may be still conserved in a broader sense. Through its PDZ domains Dlg may bind to numerous transmembrane proteins and receptors, and may link them to the cytoskeleton or signaling pathways. The knowledge gained on the role of Dlg in these systems will allow us to study how Dlg mediates membrane proliferation in the early germ cells in male gonads.Recent work has showed that Zero population growth (Zpg), the Drosophila gap junction Innexin 4, is localized to the spermatogonia surface, primarily on the sides adjacent to SCCs³⁵ and is required for the survival and differentiation of early germ cells in both sexes.^35–37 In zpg testes, the spermatogonia are unable to differentiate and are progressively lost, leading to the formation of tiny testes containing a small number of GSCs and germline clusters devoid of branching fusome,³⁵ resembling the dlg phenotype. In contrast, the SCCs that die through apoptosis in dlg testes are present in zpg, indicating that Dlg acts primarily on SCCs and Zpg on the germ cells.^20,35 Moreover, zpg testes display often a considerably enlarged hub. However, a direct comparison of the effect of the two proteins on the hub cannot be made because the null dlg^m52 allele produces a truncated non-functional Dlg protein that could still be detected in the hub.²⁰ Apparently this protein, which contains the PDZ1 and PDZ2 domains, could be recognized by a monoclonal antibody against the PDZ2 domain (data not shown).²⁰ This observation raises the possibility that the truncated Dlg protein may maintain some of its binding properties, which prevents the hub structure from falling apart. Further studies will be performed to determine the requirement of dlg in hub formation and structure.Our results, complementary to current researches conducted in this field, point out the importance of the somatic cell contribution in the organization of the Drosophila testis and the differentiation of the male germline. In mammals, spermatogenesis depends also on interactions between somatic Sertoli cells and germ cells. Sertoli cells act as supportive somatic cells and contain junction proteins with a high degree of similarity to Dlg. These proteins play a critical role in mammalian spermatogenesis.^38,39 Furthermore, the identification of mammalian genes with known function in Drosophila spermatogenesis and the evolutionary conservation among the Dlg proteins suggests that the pathways regulating the balance between stem cell renewal and differentiation might be similarly conserved. Interestingly, recent observations in mammals indicate that Dlg homologs play a role in the formation of mouse gonads and interact with gap junction proteins.^13,40 In addition, Dlg is required for smooth muscle orientation in the mouse ureter¹³ and interacts with the gap protein Connexin 32,⁴¹ whereas ZO-1, a MAGUK protein bearing similarity to Dlg and associated with tight junctions in mammalian Sertoli cells,³⁹ binds also to gap junction proteins, among them connexin 43, which is the predominant gap junction protein in the testis.^38,39,42 All these observations point out to functional similarities between Drosophila and vertebrate Dlg and provide strong indications that our findings in Drosophila may be extended to higher organisms.     

Binding of the mammalian homolog of the Drosophila discs large tumor suppressor protein to the ribosome receptor

DLG, the mammalian homolog of the Drosophila Discs Large suppressor protein, functions as a scaffolding protein that facilitates the transmission of diverse downstream signals. In the present study, we attempted to identify partner proteins for DLG, and found that DLG interacts through its PDZ domains with the ribosome receptor. The ribosome receptor is an integral endoplasmic reticulum protein that has been suggested to be involved in secretion. Our finding raises the possibility that DLG plays a role in the regulation of secretion by interacting with the ribosome receptor.     

Zonula adherens formation in Caenorhabditis elegans requires dlg-1, the homologue of the Drosophila gene discs large

The correct assembly of junction components, such as E-cadherin and beta-catenin, into the zonula adherens is fundamental for the function of epithelia, both in flies and in vertebrates. In C. elegans, however, the cadherin-catenin system is not essential for general adhesion, raising the question as to the genetic basis controlling junction morphogenesis in nematodes. Here we show that dlg-1, the C. elegans homologue of the Drosophila tumour-suppressor gene discs-large, plays a crucial role in epithelial development. DLG-1 is restricted to adherens junctions of all embryonic epithelia, which contrasts with the localisation of the Drosophila and vertebrate homologues in septate and tight junctions, respectively. Proper localisation of DLG-1 requires the basolateral LET-413 protein, but is independent of the cadherin-catenin system. Embryos in which dlg-1 activity was eliminated by RNA-mediated interference fail to form a continuous belt of junction-associated antigens and arrest development. Loss of dlg-1 activity differentially affects localisation of proteins normally enriched apically to the zonula adherens. While the distribution of an atypical protein kinase C (PKC-3) and other cytoplasmic proteins (PAR-3, PAR-6) is not affected in dlg-1 (RNAi) embryos, the transmembrane protein encoded by crb-1, the C. elegans homologue of Drosophila crumbs, is no longer concentrated in this domain. In contrast to Drosophila, however, crb-1 and a second crb-like gene are not essential for epithelial development in C. elegans. Together the data indicate that several aspects of the spatial organisation of epithelial cells and its genetic control differ between flies, worms, and vertebrates, while others are conserved. The molecular nature of DLG-1 makes it a likely candidate to participate in the organisation of a protein scaffold that controls the assembly of junction components into the zonula adherens.     

Bazooka is a permissive factor for the invasive behavior of discs large tumor cells in Drosophila ovarian follicular epithelia

Drosophila Bazooka and atypical protein kinase C are essential for epithelial polarity and adhesion. We show here that wild-type bazooka function is required during cell invasion of epithelial follicle cells mutant for the tumor suppressor discs large. Clonal studies indicate that follicle cell Bazooka acts as a permissive factor during cell invasion, possibly by stabilizing adhesion between the invading somatic cells and their substratum, the germline cells. Genetic epistasis experiments demonstrate that bazooka acts downstream of discs large in tumor cell invasion. In contrast, during the migration of border cells, Bazooka function is dispensable for cell invasion and motility, but rather is required cell-autonomously in mediating cell adhesion within the migrating border cell cluster. Taken together, these studies reveal Bazooka functions distinctly in different types of invasive behaviors of epithelial follicle cells, potentially by regulating adhesion between follicle cells or between follicle cells and their germline substratum.     

The embryonic origin of imaginal discs in Drosophila

The thoracic imaginal discs of Drosophila melanogaster can be observed during embryogenesis as clusters of cells with particular shapes, sizes and behaviours. These structures can be detected soon after germ band shortening and their development appears to be tightly linked to that of the larval epidermis.     

Lumenal transmission of decapentaplegic in Drosophila imaginal discs

Drosophila imaginal discs are sac-like appendage primordia comprising apposed peripodial and columnar cell layers. Cell survival in disc columnar epithelia requires the secreted signal Decapentaplegic (DPP), which also acts as a gradient morphogen during pattern formation. The distribution mechanism by which secreted DPP mediates global cell survival and graded patterning is poorly understood. Here we report detection of DPP in the lumenal cavity between apposed peripodial and columnar cell layers of both wing and eye discs. We show that peripodial cell survival hinges upon DPP signal reception and implicate DPP-dependent viability of the peripodial epithelium in growth of the entire disc. These results are consistent with lumenal transmission of the DPP survival signal during imaginal disc development.     

Imaginal discs regulate developmental timing in Drosophila melanogaster

The regulation of body size in animals involves mechanisms that terminate growth. In holometabolous insects growth ends at the onset of metamorphosis and is contingent on their reaching a critical size in the final larval instar. Despite the importance of critical size in regulating final body size, the developmental mechanisms regulating critical size are poorly understood. Here we demonstrate that the developing adult organs, called imaginal discs, are a regulator of critical size in larval Drosophila. We show that damage to, or slow growth of, the imaginal discs is sufficient to retard metamorphosis both by increasing critical size and extending the period between attainment of critical size and metamorphosis. Nevertheless, larvae with damaged and slow growing discs metamorphose at the same size as wild-type larvae. In contrast, complete removal of all imaginal tissue has no effect on critical size. These data indicate that both attainment of critical size and the timely onset of metamorphosis are regulated by the imaginal discs in Drosophila, and suggest that the termination of growth is coordinated among growing tissues to ensure that all organs attain a characteristic final size.     

Segmental differences in the protein content of Drosophila imaginal discs

The protein content of various Drosophila imaginal discs was analysed by two-dimensional electrophoresis followed by silver-staining. Three proteins, identified as tropomyosins α and β and actin I, are more abundant in the metathoracic discs (haltere and third leg) than in the mesothoracic discs (wing and second leg). In the case of the wing disc, these proteins are probably contributed by the adepithelial (muscle precursor) cells, as indicated by their non-uniform localisation within the disc. Mutations in the bithorax complex have no effect on the difference between second and third leg discs. We conclude that there is a segmental difference in the protein content of homologous discs, that this difference is probably localized in the adepithelial cells, and that it is not under the direct control of known alleles of the bithorax complex.     

Metamorphosis of imaginal discs of Drosophila melanogaster

Summary Imaginal discs ofDrosophila melanogaster larvae, 24–53 hrs after oviposition, were transplanted into mature immobile larval hosts. The transplants did not respond to the hormonal stimuli of metamorphosis, but instead completed their larval development. When reinjected into mature larval hosts, they now differentiated the full set of their presumptive imaginal structures. The process of acquiring competence for metamorphosis appears to be independent of the hormonal conditions.Supported by a credit of the Swiss National Foundation granted to Prof. Dr. E. Hadorn. I thank Dr. R. Nöthiger for his valuable criticism during this investigation.     

Extracellular protease production by Drosophila imaginal discs

We are investigating the role of extracellular proteases in imaginal disc eversion to understand the mechanism that controls cell rearrangements within epithelia. We have identified three cation-dependent neutral proteases released by Drosophila leg discs everting in culture. Serine protease inhibitors block disc eversion and inhibit activity of disc proteases. The pattern of extracellular proteases changes when eversion is blocked with added protease inhibitors. Changes in protease activity occur when released disc proteases are treated with trypsin. Trypsin treatment of intact imaginal discs releases protease and inhibitor activities to the medium, indicating their presence on the cell surface before release. Our results suggest that extracellular proteases are required for imaginal disc morphogenesis and are regulated by more than one mechanism.     

Human homologue of the Drosophila discs large tumor suppressor protein forms an oligomer in solution. Identification of the self-association site

The human homologue of the Drosophila discs large tumor suppressor protein (hDlg), a member of the membrane-associated guanylate kinase (MAGUK) superfamily, interacts with K(+) channels, N-methyl-d-aspartate receptors, calcium ATPase, adenomatous polyposis coli, and PTEN tumor suppressor proteins, and several viral oncoproteins through its PDZ domains. MAGUKs play pivotal roles in the clustering and aggregation of receptors, ion channels, and cell adhesion molecules at the synapses. To investigate the physiological basis of hDlg interactions, we examined the self-association state of full-length hDlg as well as defined segments of hDlg expressed as recombinant proteins in bacteria and insect Sf9 cells. Gel permeation chromatography of full-length hDlg revealed that the purified protein migrates as a large particle of size >440 kDa. Similar measurements of defined domains of hDlg indicated that the anomalous mobility of hDlg originated from its amino-terminal domain. Ultrastructural analysis of hDlg by low angle rotary shadow electron microscopy revealed that the full-length hDlg protein as well as its amino-terminal domain exhibits a highly flexible irregular shape. Further evaluation of the self-association state of hDlg using sedimentation equilibrium centrifugation, matrix-assisted laser desorption/ionization mass spectrometry, and chemical cross-linking techniques confirmed that the oligomerization site of hDlg is contained within its amino-terminal domain. This unique amino-terminal domain mediates multimerization of hDlg into dimeric and tetrameric species in solution. Sedimentation velocity experiments demonstrated that the oligomerization domain exists as an elongated tetramer in solution. In vitro mutagenesis was used to demonstrate that a single cysteine residue present in the oligomerization domain of hDlg is not required for its self-association. Understanding the oligomerization status of hDlg may help to explicate the mechanism of hDlg association with multimeric K(+) channels and dimeric adenomatous polyposis coli tumor suppressor protein. Our findings, therefore, begin to rationalize the role of hDlg in the clustering of membrane channels and formation of multiprotein complexes necessary for signaling and cell proliferation pathways.     

GAKIN, a novel kinesin-like protein associates with the human homologue of the Drosophila discs large tumor suppressor in T lymphocytes

Reorganization of the cortical cytoskeleton is a hallmark of T lymphocyte activation. Upon binding to antigen presenting cells, the T cells rapidly undergo cytoskeletal re-organization thus forming a cap at the cell-cell contact site leading to receptor clustering, protein segregation, and cellular polarization. Previously, we reported cloning of the human lymphocyte homologue of the Drosophila Discs Large tumor suppressor protein (hDlg). Here we show that a novel protein termed GAKIN binds to the guanylate kinase-like domain of hDlg. Affinity protein purification, peptide sequencing, and cloning of GAKIN cDNA from Jurkat J77 lymphocytes identified GAKIN as a novel member of the kinesin superfamily of motor proteins. GAKIN mRNA is ubiquitously expressed, and the predicted amino acid sequence shares significant sequence similarity with the Drosophila kinesin-73 motor protein. GAKIN sequence contains a motor domain at the NH(2) terminus, a central stalk domain, and a putative microtubule-interacting sequence called the CAP-Gly domain at the COOH terminus. Among the MAGUK superfamily of proteins examined, GAKIN binds to the guanylate kinase-like domain of PSD-95 but not of p55. The hDlg and GAKIN are localized mainly in the cytoplasm of resting T lymphocytes, however, upon CD2 receptor cross-linking the hDlg can translocate to the lymphocyte cap. We propose that the GAKIN-hDlg interaction lays the foundation for a general paradigm of coupling MAGUKs to the microtubule-based cytoskeleton, and that this interaction may be functionally important for the intracellular trafficking of MAGUKs and associated protein complexes in vivo.     

Binding of the human homolog of the Drosophila discs large tumor suppressor protein to the mitochondrial ribosomal protein MRP-S34

The human homolog of the Drosophila discs large tumor suppressor protein (hDLG) functions as a scaffolding protein that facilitates the transmission of diverse downstream signals. Here we show that hDLG interacts through its PDZ domains with the carboxy-terminal S/TXV motif of the mitochondrial ribosomal protein S-34 (MRP-S34). Our results suggest that hDLG interacts with MRP-S34 prior to entry of MRP-S34 into the mitochondria and may be involved in the trafficking of MRP-S34.     

Localization of the Antennapedia protein in Drosophila embryos and imaginal discs

Antibodies have been raised against a fusion protein containing the 3' region of the coding sequence of the Antennapedia (Antp) gene fused to β-galactosidase. The distribution of the protein on whole mount embryos and imaginal discs of third instar larvae was examined by immunofluorescence. In young embryos, expression of the Antp protein was limited to the thoracic segments in the epidermis, whereas it was found in all neuromeres of head, thorax and abdomen. At the end of embryogenesis, the Antp protein mainly accumulated in the ventral nervous system in certain parts of the thoracic neuromeres, from posterior T1 to anterior T3, with a gap in posterior T2. Comparison of Antp protein distribution in nervous systems from wild-type and Df P9 embryos, lacking the genes of the Bithorax-complex (BX-C), revealed a pattern of expression which indicated that the BX-C represses Antp in the posterior segments with the exception of the last abdominal neuromeres (A8-9) which are regulated independently. The protein pattern in nervous systems from Sex combs reduced(Scr^xF9) mutant embryos was indistinguishable from that found in wild-type embryos; thus, neurogenic expression of Antp in T1 and the more anterior segments does not appear to be under the control of Scr⁺. All imaginal discs derived from the three thoracic segments express Antp protein. The distribution was distinct in each disc; strongest expression was observed in the proximal parts of the discs. In the leg discs the protein distribution seemed to be compartmentally restricted, whereas in the wing disc this was not the case. Antp protein was not detected in the eye-antennal disc. In embryos, as well as in imaginal discs, the protein is localized in the nucleus.     

Pyridine nucleotide metabolism in imaginal discs of Drosophila melanogaster

The pyridine nucleotide metabolism of imaginal discs of Drosophila melanogaster has been studied in vitro by incubating discs with labeled nicotinic acid in the presence and absence of ecdysterone. The major labeled compounds found within the discs are NAD, NADP, and nicotinic acid. There is preferential uptake of nicotinamide over nicotinic acid, although the Priess-Handler pathway is used exclusively. The presence of ecdysterone produces a small increase in the NADP/NAD ratio, and an increase in NAD synthesis, probably to compensate for increased NAD turnover.Supported by Grant GB 43569 from the National Science Foundation.