Essential function of Drosophila Sec6 in apical exocytosis of epithelial photoreceptor cells

Polarized exocytosis plays a major role in development and cell differentiation but the mechanisms that target exocytosis to specific membrane domains in animal cells are still poorly understood. We characterized Drosophila Sec6, a component of the exocyst complex that is believed to tether secretory vesicles to specific plasma membrane sites. sec6 mutations cause cell lethality and disrupt plasma membrane growth. In developing photoreceptor cells (PRCs), Sec6 but not Sec5 or Sec8 shows accumulation at adherens junctions. In late PRCs, Sec6, Sec5, and Sec8 colocalize at the rhabdomere, the light sensing subdomain of the apical membrane. PRCs with reduced Sec6 function accumulate secretory vesicles and fail to transport proteins to the rhabdomere, but show normal localization of proteins to the apical stalk membrane and the basolateral membrane. Furthermore, we show that Rab11 forms a complex with Sec5 and that Sec5 interacts with Sec6 suggesting that the exocyst is a Rab11 effector that facilitates protein transport to the apical rhabdomere in Drosophila PRCs.     

Direct allosteric regulation between the GAF domain and catalytic domain of photoreceptor phosphodiesterase PDE6

Photoreceptor cGMP phosphodiesterase (PDE6) is the central enzyme in the visual transduction cascade. The PDE6 catalytic subunit contains a catalytic domain and regulatory GAF domains. Unlike most GAF domain-containing cyclic nucleotide phosphodiesterases, little is known about direct allosteric communication of PDE6. In this study, we demonstrate for the first time direct, inter-domain allosteric communication between the GAF and catalytic domains in PDE6. The binding affinity of PDE6 for pharmacological inhibitors or for the C-terminal region of the inhibitory gamma subunit (Pgamma), known to directly inhibit PDE6 catalysis, was increased approximately 2-fold by ligands binding to the GAF domain. Binding of the N-terminal half of Pgamma to the GAF domains suffices to induce this allosteric effect. Allosteric communication between GAF and catalytic domains is reciprocal, in that drug binding to the catalytic domain slowed cGMP dissociation from the GAF domain. Although cGMP hydrolysis was not affected by binding of Pgamma1-60, Pgamma lacking its last seven amino acids decreased the Michaelis constant of PDE6 by 2.5-fold. Pgamma1-60 binding to the GAF domain increased vardenafil but not cGMP affinity, indicating that substrate- and inhibitor-binding sites do not totally overlap. In addition, prolonged incubation of PDE6 with vardenafil or sildenafil (but not 3-isobutyl-1-methylxanthine and zaprinast) induced a distinct conformational change in the catalytic domain without affecting the binding properties of the GAF domains. We conclude that although Pgamma-mediated regulation plays the dominant role in visual excitation, the direct, inter-domain allosteric regulation described in this study may play a feedback role in light adaptational processes during phototransduction.     

Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells

A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. Here, we show that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of gamma-tubulin and other centrosomal components to the centrosome. The NH2-terminal half of CNN interacts with gamma-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring gamma-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.     

Maintaining the proper connection between the centrioles and the pericentriolar matrix requires Drosophila centrosomin

Centrosomes consist of two centrioles surrounded by an amorphous pericentriolar matrix (PCM), but it is unknown how centrioles and PCM are connected. We show that the centrioles in Drosophila embryos that lack the centrosomal protein Centrosomin (Cnn) can recruit PCM components but cannot maintain a proper attachment to the PCM. As a result, the centrioles "rocket" around in the embryo and often lose their connection to the nucleus in interphase and to the spindle poles in mitosis. This leads to severe mitotic defects in embryos and to errors in centriole segregation in somatic cells. The Cnn-related protein CDK5RAP2 is linked to microcephaly in humans, but cnn mutant brains are of normal size, and we observe only subtle defects in the asymmetric divisions of mutant neuroblasts. We conclude that Cnn maintains the proper connection between the centrioles and the PCM; this connection is required for accurate centriole segregation in somatic cells but is not essential for the asymmetric division of neuroblasts.     

Mutations in centrosomin reveal requirements for centrosomal function during early Drosophila embryogenesis.

BACKGROUND: Although centrosomes serve as the primary organizing centers for the microtubule-based cytoskeleton in animal cells, various studies question the requirements for these organelles during the formation of microtubule arrays and execution of microtubule-dependent processes. Using a genetic approach to interfere with centrosomal function, we present an assessment of this issue, in the context of early embryogenesis of the fruit fly Drosophila melanogaster. RESULTS: We identified mutant alleles of the centrosomin (cnn) locus, which encodes a core component of centrosomes in Drosophila. The cnn mutant flies were viable but sterile. The normal course of early embryonic development was arrested in all progeny of cnn mutant females. Our analysis identified a failure to form functional centrosomes and spindle poles as the primary mutant phenotype of cnn embryos. Various aspects of early development that are dependent on cytoskeletal control were disrupted in cnn mutant embryos. In particular, structural rearrangements of cortical microfilaments were strongly dependent on proper centrosomal function. CONCLUSIONS: Centrosomin is an essential core component of early embryonic centrosomes in Drosophila. Microtubule-dependent events of early embryogenesis display differential requirements for centrosomal function.     

Hormonal regulation of mummy is needed for apical extracellular matrix formation and epithelial morphogenesis in Drosophila

Many epithelia produce apical extracellular matrices (aECM) that are crucial for organ morphogenesis or physiology. Apical ECM formation relies on coordinated synthesis and modification of constituting components, to enable their subcellular targeting and extracellular assembly into functional matrices. The exoskeleton of Drosophila, the cuticle, is a stratified aECM containing ordered chitin polysaccharide lamellae and proteinaceous layers, and is suited for studies of molecular functions needed for aECM assembly. Here, we show that Drosophila mummy (mmy) mutants display defects in epithelial organisation in conjunction with aberrant deposition of the cuticle and an apical matrix needed for tracheal tubulogenesis. We find that mmy encodes the UDP-N-acetylglucosamine pyrophosphorylase, which catalyses the production of UDP-N-acetylglucosamine, an obligate substrate for chitin synthases as well as for protein glycosylation and GPI-anchor formation. Consequently, in mmy mutants GlcNAc-groups including chitin are severely reduced and modification and subcellular localisation of proteins designated for extracellular space is defective. Moreover, mmy expression is selectively upregulated in epithelia at the time they actively deposit aECM, and is altered by the moulting hormone 20-Hydroxyecdysone, suggesting that mmy is part of a developmental genetic programme to promote aECM formation.     

Role of spectraplakin in Drosophila photoreceptor morphogenesis

Background

Crumbs (Crb), a cell polarity gene, has been shown to provide a positional cue for the apical membrane domain and adherens junction during Drosophila photoreceptor morphogenesis. It has recently been found that stable microtubules in developing Drosophila photoreceptors were linked to Crb localization. Coordinated interactions between microtubule and actin cytoskeletons are involved in many polarized cellular processes. Since Spectraplakin is able to bind both microtubule and actin cytoskeletons, the role of Spectraplakin was analyzed in the regulations of apical Crb domain in developing Drosophila photoreceptors.

Methodology/Principal Findings

The localization pattern of Spectraplakin in developing pupal photoreceptors showed a unique intracellular distribution. Spectraplakin localized at rhabdomere terminal web which is at the basal side of the apical Crb or rhabdomere, and in between the adherens junctions. The spectraplakin mutant photoreceptors showed dramatic mislocalizations of Crb, adherens junctions, and the stable microtubules. This role of Spectraplakin in Crb and adherens junction regulation was further supported by spectraplakin''s gain-of-function phenotype. Spectraplakin overexpression in photoreceptors caused a cell polarity defect including dramatic mislocalization of Crb, adherens junctions and the stable microtubules in the developing photoreceptors. Furthermore, a strong genetic interaction between spectraplakin and crb was found using a genetic modifier test.

Conclusions/Significance

In summary, we found a unique localization of Spectraplakin in photoreceptors, and identified the role of spectraplakin in the regulation of the apical Crb domain and adherens junctions through genetic mutational analysis. Our data suggest that Spectraplakin, an actin-microtubule cross-linker, is essential in the apical and adherens junction controls during the photoreceptors morphogenesis.     

The centrosomin protein is required for centrosome assembly and function during cleavage in Drosophila.

Centrosomin is a 150 kDa centrosomal protein of Drosophila melanogaster. To study the function of Centrosomin in the centrosome, we have recovered mutations that are viable but male and female sterile (cnnmfs). We have shown that these alleles (1, 2, 3, 7, 8 and hk21) induce a maternal effect on early embryogenesis and result in the accumulation of low or undetectable levels of Centrosomin in the centrosomes of cleavage stage embryos. Hemizygous cnn females produce embryos that show dramatic defects in chromosome segregation and spindle organization during the syncytial cleavage divisions. In these embryos the syncytial divisions proceed as far as the twelfth cycle, and embryos fail to cellularize. Aberrant divisions and nuclear fusions occur in the early cycles of the nuclear divisions, and become more prominent at later stages. Giant nuclei are seen in late stage embryos. The spindles that form in mutant embryos exhibit multiple anomalies. There is a high occurrence of apparently linked spindles that share poles, indicating that Centrosomin is required for the proper spacing and separation of mitotic spindles within the syncytium. Spindle poles in the mutants contain little or no detectable amounts of the centrosomal proteins CP60, CP190 and (gamma)-tubulin and late stage embryos often do not have astral microtubules at their spindle poles. Spindle morphology and centrosomal composition suggest that the primary cause of these division defects in mutant embryos is centrosomal malfunction. These results suggest that Centrosomin is required for the assembly and function of centrosomes during the syncytial cleavage divisions.     

Genetic requirements of vestigial in the regulation of Drosophila wing development

The gene vestigial has been proposed to act as a master gene because of its supposed capacity to initiate and drive wing development. We show that the ectopic expression of vestigial only induces ectopic outgrowths with wing cuticular differentiation and wing blade gene expression patterns in specific developmental and genetic contexts. In the process of transformation, wingless seems to be an essential but insufficient co-factor of vestigial. vestigial ectopic expression alone or vestigial plus wingless co-expression in clones differentiate 'mixed' cuticular patterns (they contain wing blade trichomes and chaetae characteristic of the endogenous surrounding tissue) and express wing blade genes only in patches of cells within the clones. In addition, we have found that these clones, in the wing imaginal disc, may cause autonomous as well as non-autonomous cuticular transformations and wing blade gene expression patterns. These non-autonomous effects in surrounding cells result from recruitment or 'inductive assimilation' of vestigial or wingless-vestigial overexpressing cells.     

Genetic regulation of glucose 6-phosphate dehydrogenase activity in Drosophila melanogaster

Studies on two variants of X-linked enzyme, G6PD, in several inbred and outbred strains of Drosophila melanogaster suggest that (1) there is dosage compensation at this locus; (2) males have 20–33% more activity than females, due to enzyme-deficient eggs in the latter; (3) outcrossing Drosophila strains results in a significant rise in G6PD specific activity in such a way as to suggest the presence of two or more nonlinked loci specific in their effect on G6PD activity (the effect is twice as great in males as it is in females); (4) there is less A enzyme than B enzyme activity/mg protein in males, but they are equal in females; (5) the presence or absence of X-linked regulators for G6PD could not be ascertained.Aided by National Institutes of Health grants HD 00004, HD00486, and GM 14155.     

Task-specific association of photoreceptor systems and steering parameters in Drosophila

Visual motion processing enables moving fruit flies to stabilize their course and altitude and to approach selected objects. Earlier attempts to identify task-specific pathways between two photoreceptor systems (peripheral retinula cells 1-6, and central retinula cells 7 + 8) and three steering parameters (wingstroke asymmetry, abdomen deflection, hindleg deflection) attributed course control and object fixation to peripheral retinula cells 1-6-mediated simultaneous reactions of these parameters. The present investigation includes first results from fixed flying or freely walking ninaE17 mutants which cannot synthesize the peripheral retinula cells 1-6 photoreceptor-specific opsin. Retention of about 12% of the normal course control and about 58% of the object fixation in these flies suggests partial input sharing for both responses and, possibly, a specialization for large-field (peripheral retinula cells 1-6) and small-field (central retinula cells 7 + 8) motion. Such signals must be combined to perceive relative motion between an object and its background. The combining links found in larger species might explain a previously neglected interdependence of course control and object fixation in Drosophila. -Output decomposition revealed an unexpected orchestration of steering. Wingstroke asymmetry and abdomen deflection do not contribute in fixed proportions to the yaw torque of the flight system. Different steering modes seem to be selected according to their actual efficiency under closed-loop conditions and to the degree of intended turning. An easy experimental access to abdominal steering is introduced.     

Cdc42 and Par proteins stabilize dynamic adherens junctions in the Drosophila neuroectoderm through regulation of apical endocytosis

Cell rearrangements require dynamic changes in cell–cell contacts to maintain tissue integrity. We investigated the function of Cdc42 in maintaining adherens junctions (AJs) and apical polarity in the Drosophila melanogaster neuroectodermal epithelium. About one third of cells exit the epithelium through ingression and become neuroblasts. Cdc42-compromised embryos lost AJs in the neuroectoderm during neuroblast ingression. In contrast, when neuroblast formation was suppressed, AJs were maintained despite the loss of Cdc42 function. Loss of Cdc42 function caused an increase in the endocytotic uptake of apical proteins, including apical polarity factors such as Crumbs, which are required for AJ stability. In addition, Cdc42 has a second function in regulating endocytotic trafficking, as it is required for the progression of apical cargo from the early to the late endosome. The Par complex acts as an effector for Cdc42 in controlling the endocytosis of apical proteins. This study reveals functional interactions between apical polarity proteins and endocytosis that are critical for stabilizing dynamic basolateral AJs.     

Light-induced voltage noise in the photoreceptor of Drosophila melanogaster

The Drosophila photoreceptor potential is thought to be composed of discrete unit potentials called bumps. The steady-state receptor potential and the accompanying voltage fluctuations were recorded intracellularly under steady illumination. The occurrence rate, effective amplitude, and duration of the bumps were deduced by assuming a shot noise model. Over a wide range of light intensity, the duration of bumps remained essentially constant (25-30 ms). Below the saturation intensity for the receptor potential, the bump rate was roughly proportional to the intensity, and the adjustment of bumps to smaller size at higher intensity was mainly responsible for the nonlinear behavior of the receptor potential. The reduction in size of bumps at increasing light intensity was found to be due mainly to the diminishing magnitude of the bump current, and not to some other secondary effects. The bump rate saturated at about 3 x 105-106 events/s.     

Two-color in vivo imaging of photoreceptor apoptosis and development in Drosophila

We report a new two-color fluorescent imaging system to visualize the mosaic adult photoreceptor neurons (PRs) in real-time. Using this method, we examined a collection of 434 mutants and identified genes required for PR survival, planar cell polarity (PCP), patterning and differentiation. We could track the progression of PR degeneration in living flies. By introducing the expression of p35, a caspase inhibitor, we found mutations that specifically activate caspase-dependent death. Moreover, we showed that grh is required in R3 for correct PCP establishment. The “Tomato/GFP-FLP/FRT” method allows high-throughput, rapid and precise identification of survival and developmental pathways in living adult PRs at single-cell resolution.     

The organization of INAD-signaling complexes by a multivalent PDZ domain protein in Drosophila photoreceptor cells ensures sensitivity and speed of signaling

Phototransduction in Drosophila has emerged as an attractive model system for studying the organization of signaling cascades in vivo. In photoreceptor neurons, the multivalent PDZ protein INAD serves as a scaffold to assemble different components of the phototransduction pathway, including the effector PLC, the light-activated ion channel TRP, and a protein kinase C involved in deactivation of the light response. INAD is required for organizing and maintaining signaling complexes in the rhabdomeres of photoreceptors. This macromolecular organization endows photoreceptors with many of their signaling properties, including high sensitivity, fast activation and deactivation kinetics, and exquisite feedback regulation by small localized changes in [Ca2+]i. Assembly of transduction components into signaling complexes is also an important cellular strategy for ensuring specificity of signaling while minimizing unwanted cross-talk. In this report, we review INAD's role as a signal transduction scaffold and its role in the assembly and localization of photoreceptor complexes.     

Analysis of Drosophila photoreceptor axon guidance in eye-specific mosaics

During development of the adult Drosophila visual system, axons of the eight photoreceptors in each ommatidium fasciculate together and project as a single bundle towards the optic lobes of the brain. Within the brain, individual photoreceptor axons from each bundle then seek specific targets in distinct layers of the optic lobes. The axons of photoreceptors R1-R6 terminate in the lamina, while R7 and R8 axons pass through the lamina to terminate in separate layers of the medulla. To identify genes required for photoreceptor axon guidance, including those with essential functions during early development, we have devised a strategy for the simple and efficient generation of genetic mosaics in which mutant photoreceptor axons innervate a predominantly wild-type brain. In a large-scale saturation mutagenesis performed using this system, we recovered new alleles of the gene encoding the receptor tyrosine phosphatase PTP69D. PTP69D has previously been shown to function in the correct targeting of motor axons in the embryo and R1-R6 axons in the visual system. Here, we show that PTP69D is also required for correct targeting of R7 axons. Whereas mutant R1-R6 axons occasionally extend beyond their normal targets in the lamina, mutant R7 axons often fail to reach their targets in the medulla, stopping instead at the same level as the R8 axon. These targeting errors are difficult to reconcile with models in which PTP69D plays an instructive role in photoreceptor axon targeting, as previously proposed. Rather, we suggest that PTP69D plays a permissive role, perhaps reducing the adhesion of R1-R6 and R7 growth cones to the pioneer R8 axon so that they can respond independently to their specific targeting cues.     

Genetic regulation of the replication pattern of polytene chromosomes in interspecific hybrids of Drosophila

The sixth chromosome (microchromosome) of D. littoralis changed its replication pattern in nuclei of the salivary gland cells in reciprocal F₁ hybrids between D. virilis and D. littoralis. — To locate the factor (or factors) in the D. virilis chromosomes, which may influence the replication pattern of the sixth chromsome of D. littoralis, we produced hybrid stocks with synthetic karyotypes characterized by different combinations of D. littoralis homologous chromosomes and hybrid chromosomes. Based on autoradiographical studies of DNA synthesis in synthetic karyotypes, it may be concluded that the dominant factor (or factors), which influences the replication of the sixth chromosome of D. littoralis, is located on the homeologous microchromosomes of D. virilis. The possible interrelation between the changed replication pattern of D. littoralis sixth chromosome and its atypical behaviour at early embryogenesis in (D. virilis x D. littoralis) F₁ hybrids is discussed.