Maintenance of Rhodopsin levels in Drosophila photoreceptor and phototransduction requires Protein Kinase D

ABSTRACT

During Drosophila phototransduction, the G protein coupled receptor (GPCR) Rhodopsin (Rh1) transduces photon absorption into electrical signal via G-protein coupled activation of phospholipase C (PLC). Rh1 levels in the plasma membrane are critical for normal sensitivity to light. In this study, we report that Protein Kinase D (dPKD) regulates Rh1 homeostasis in adult photoreceptors. Although eye development and retinal structure are unaffected in the dPKD hypomorph (dPKD^H), it exhibited elevated levels of Rh1. Surprisingly, despite having elevated levels of Rh1, no defect was observed in the electrical response to light in these flies. By contrast the levels of another transmembrane protein of the photoreceptor plasma membrane, Transient receptor potential (TRP) was not altered in dPKD^H. Our results indicate that dPKD is dispensable for eye development but is required for maintaining Rh1 levels in adult photoreceptors.     

藻胆体核亚基ApcD定点突变及体内重组功能研究

在对Anabaena sp.PCC7120藻胆体核亚基ApcD结合色素PCB的体内重组中,发现色素蛋白在提纯前后最大吸收峰和荧光峰发生了红移,从提纯前的605nm及633nm变为提纯后的650nm及665nm.为了研究该现象的原因,构建了ApcD的8个突变体,重组结果显示:突变体ApcD(Y88I)色素蛋白在提纯后的吸收光谱和荧光光谱较提纯前均多出一个峰,分别为668nm,690nm;ApcD(W59Q)、ApcD(Y73A)、ApcD(W87E)色素蛋白在提纯前后的吸收光谱和荧光光谱一致;ApcD(M126S)、ApcD(Y116S)、ApcD(M160T)色素蛋白在提纯前后的吸收光谱一致,而提纯后的荧光峰位置较提纯前分别红移了5nm、7nm和10nm;ApcD(M115I)色素蛋白在提纯前后的吸收光谱和荧光光谱均发生了红移,从提纯前的605nm和633nm变为提纯后的638nm和655nm.这些色素蛋白在酸性尿素溶液变性条件下的最大吸收峰始终在662nm,表明辅基色素仍然是藻蓝胆素;在对PCB-ApcD、PCB-ApcD(Y116S)及PCB-ApcD(M160T)的圆二色谱分析发现,该两个氨基酸的突变均...     

Multiple Mechanisms of Photoreceptor Spectral Tuning in Heliconius Butterflies

The evolution of color vision is often studied through the lens of receptor gain relative to an ancestor with fewer spectral classes of photoreceptor. For instance, in Heliconius butterflies, a genus-specific UVRh opsin duplication led to the evolution of UV color discrimination in Heliconius erato females, a rare trait among butterflies. However, color vision evolution is not well understood in the context of loss. In Heliconius melpomene and Heliconius ismenius lineages, the UV2 receptor subtype has been lost, which limits female color vision in shorter wavelengths. Here, we compare the visual systems of butterflies that have either retained or lost the UV2 photoreceptor using intracellular recordings, ATAC-seq, and antibody staining. We identify several ways these butterflies modulate their color vision. In H. melpomene, chromatin reorganization has downregulated an otherwise intact UVRh2 gene, whereas in H. ismenius, pseudogenization has led to the truncation of UVRh2. In species that lack the UV2 receptor, the peak sensitivity of the remaining UV1 photoreceptor cell is shifted to longer wavelengths. Across Heliconius, we identify the widespread use of filtering pigments and co-expression of two opsins in the same photoreceptor cells. Multiple mechanisms of spectral tuning, including the molecular evolution of blue opsins, have led to the divergence of receptor sensitivities between species. The diversity of photoreceptor and ommatidial subtypes between species suggests that Heliconius visual systems are under varying selection pressures for color discrimination. Modulating the wavelengths of peak sensitivities of both the blue- and remaining UV-sensitive photoreceptor cells suggests that Heliconius species may have compensated for UV receptor loss.     

视紫红质及RPE65蛋白在光致视网膜变性疾病中的作用机制

视紫红质是感光细胞中的一种视色素,在光线的接收和视觉电位的产生方面具有重要的生理作用,由视紫红质介导的过度光信号传导是光性视网膜变性的主要原因。近年的研究表明,视网膜色素上皮细胞中的RPE65蛋白作为影响视紫红质再生的关键因素,与视网膜光损伤的易感性密切相关。就视紫红质和RPE65蛋白在光致视网膜变性中的作用机理作一探讨。     

Kinesin II is required for cell survival and adherens junction positioning in Drosophila photoreceptors

Photoreceptor morphogenesis requires specific and coordinated localization of junctional markers at different stages of development. Here, we provide evidence that Drosophila Klp64D, a homolog of Kif3A motor subunit of the heterotrimeric Kinesin II complex, is essential for viability of developing photoreceptors and localization of junctional proteins. Genetic analysis of mutant clones shows that absence of Klp64D protein in early larval eye disc does not affect initial differentiation, but results in abnormal nuclear position in differentiating photoreceptors. These cells eventually die in the pupal stage, indicating klp64D's role in cell viability. The function of Klp64D protein is cell type specific because the p35 cell death inhibitor can rescue cell death in cone cells but not photoreceptors. In contrast to early induction of mutant clones, late induction during third instar larval stage just prior to pupation allows survival of single‐ or few‐celled clones of klp64D mutant cells. Analysis of these lately induced clones shows that Klp64D function is essential for Bazooka (Par‐3 homolog) and Armadillo localization to the adherens junction (AJ) in pupal photoreceptors. These findings suggest that Kinesin II complex plays a cell type‐specific function in the localization of AJ and cell polarity proteins in the developing retina, thereby contributing to photoreceptor morphogenesis. genesis 48:522–530, 2010. © 2010 Wiley‐Liss, Inc.     

Early duplication and functional diversification of the opsin gene family in insects

Recent analysis of the complete mosquito Anopheles gambiae genome has revealed a far higher number of opsin genes than for either the Drosophila melanogaster genome or any other known insect. In particular, the analysis revealed an extraordinary opsin gene content expansion, whereby half are long wavelength-sensitive (LW) opsin gene duplicates. We analyzed this genomic data in relationship to other known insect opsins to estimate the relative timing of the LW opsin gene duplications and to identify "missing" paralogs in extant species. The inferred branching patterns of the LW opsin gene family phylogeny indicate at least one early gene duplication within insects before the emergence of the orders Orthoptera, Mantodea, Hymenoptera, Lepidoptera, and Diptera. These data predict the existence of one more LW opsin gene than is currently known from most insects. We tested this prediction by using a degenerate PCR strategy to screen the hymenopteran genome for novel LW opsin genes. We isolated two LW opsin gene sequences from each of five bee species, Bombus impatiens, B. terrestris, Diadasia afflicta, D. rinconis, and Osmia rufa, including 1.1 to 1.2 kb from a known (LW Rh1) and 1 kb from a new opsin gene (LW Rh2). Phylogenetic analysis suggests that the novel hymenopteran gene is orthologous to A. gambiae GPRop7, a gene that is apparently missing from D. melanogaster. Relative rate tests show that LW Rh2 is evolving at a slower rate than LW Rh1 and, therefore, may be a useful marker for higher-level hymenopteran systematics. Site-specific rate tests indicate the presence of several amino acid sites between LW Rh1 and LW Rh2 that have undergone shifts in selective constraints after duplication. These sites and others are discussed in relationship to putative structural and functional differences between the two genes.     

抗体重链可变区框架Ⅰ区对抗体分泌的影响

抗体重链可变区框架Ⅰ区(FR-Ⅰ)对抗体在原核细胞中的分泌表达具有显著的影响,单个氨基酸的改变即可导致抗体分子分泌能力的丧失.为了探索抗体在哺乳动物细胞中的高效表达,我们对一株不能有效分泌的人源抗狂犬病病毒抗体pCMV-RV/VH的FR-Ⅰ区氨基酸编码基因进行定点突变研究.实验显示,抗体重链可变区FR-Ⅰ区H6位的氨基酸由Glu突变为Gin之后,抗体的分泌表达水平得到了显著的提高,并且与抗原特异性结合的能力也明显增强.通过免疫荧光法对抗体在细胞内的转运过程进行了初步的探讨,发现能够有效分泌的抗体与无分泌表达的抗体都能够在细胞内进行正常的转录和翻译,进入内质网并转运至高尔基体,而且胞内表达水平基本一致.我们认为分泌能力的不同可能是FR-Ⅰ区影响抗体分子的折叠与装配所致,其中该区H6位氨基酸的性质能够显著影响抗体在哺乳动物细胞中的分泌.本研究以抗体重链可变区FR-Ⅰ区氨基酸为焦点,对基因工程抗体在真核细胞中高效表达的影响因素进行了探索,为改进抗体规模化生产提供了依据.     

Functional Analysis of Conserved Histidines in Choline Acetyltransferase by Site-Directed Mutagenesis

Abstract: The choline acetyltransferase (ChAT) reaction involves the transfer of the acetyl group of acetyl-CoA to choline, in which an active site histidine is believed to act as a general acid/base catalyst. A comparison of the deduced amino acid sequences of the enzyme from Drosophila , pig, rat, and Caernohabditis elegans revealed three conserved histidines: Drosophila His²⁶⁸, His³⁹³, and His⁴²⁶. Each of these histidines was replaced by a leucine and a glutamine, and the kinetic properties of each of the recombinant mutant enzymes were determined. The mutations yielded active His²⁶⁸Leu-ChAT, His^Z68Gln-ChAT, and His³⁹³Gln-ChAT and inactive His³⁹³Leu-ChAT, His⁴²⁶Leu- ChAT, and His⁴²⁶Gln-ChAT. The kinetic constants K_m(CoA), K_{m(acetyloholine)}. and V_max were essentially the same for all of the active mutants. When the integrity of the CoASAc binding site was investigated in the inactive mutants, the data suggested that the binding site in His³⁹³Leu-ChAT is disrupted but conserved in His⁴²⁶Leu-ChAT and His⁴²⁶Gln- ChAT. These results suggest that His⁴²⁶ is an essential catalytic residue and could serve as an acid/base catalyst.     

L78部分氨基酸残基对SERCA1a钙转运功能的影响

跨肌浆网膜的钙离子ATP酶SERCA1a可在水解ATP的同时逆浓度梯度从胞浆转运钙离子入肌浆网,引发收缩的骨骼肌细胞舒张.目前对SERCA1a结构与功能的研究,要领先于对其他P型离子转运ATP酶的研究.为了解SERCA1a跨膜螺旋M7与M8膜内侧连接部Linker78(L78)的功能,我们评估了L78部分氨基酸残基突变对SERCA1a反应循环的影响,发现:除G864A之外的所有突变体均可不同程度地降低钙离子转运速率;G862或P863氨基酸残基的突变导致SERCA1a的ATP酶活性显著降低或丧失,并引起由ATP或无机磷酸Pi生成的磷酸化酶中间体EP量显著减少;A893被P取代对SERCA1a的影响与G862、P863突变相似;与野生型SERCA1a相比,G864A与FMQ873-875单突变体ATP酶活性及EP生成量无明显差别.上述结果表明,M7、M8膜内侧连接部L78不仅与跨膜区钙离子转运过程密切相关,而且L78在GPG862-864处及A893附近的正确转折对胞浆结构域P的磷酸化也具有关键的远距离调控作用,提示L78的结构及柔度对SERCA1a构象正常的周期性变化至关重要.     

Color Processing in the Early Visual System of Drosophila

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Identification of Catalytic and Substrate-binding Site Residues in Bacillus cereus ATCC7064 Oligo-1,6-glucosidase

Three active site residues (Asp199, Glu255, Asp329) and two substrate-binding site residues (His103, His328) of oligo-1,6-glucosidase (EC 3.2.1.10) from Bacillus cereus ATCC7064 were identified by site-directed mutagenesis. These residues were deduced from the X-ray crystallographic analysis and the comparison of the primary structure of the oligo-1,6-glucosidase with those of Saccharomyces carlsbergensis α-glucosidase, Aspergillus oryzae α-amylase and pig pancreatic α-amylase which act on α-1,4-glucosidic linkages. The distances between these putative residues of B. cereus oligo-1,6-glucosidase calculated from the X-ray analysis data closely resemble those of A. oryzae α-amylase and pig pancreatic α-amylase. A single mutation of Asp199→Asn, Glu255→Gln, or Asp329→Asn resulted in drastic reduction in activity, confirming that three residues are crucial for the reaction process of α-1,6-glucosidic bond cleavage. Thus, it is identified that the basic mechanism of oligo-1,6-glucosidase for the hydrolysis of α-1,6-glucosidic linkage is essentially the same as those of other amylolytic enzymes belonging to Family 13 (α-amylase family). On the other hand, mutations of histidine residues His103 and His328 resulted in pronounced dissimilarity in catalytic function. The mutation His328→Asn caused the essential loss in activity, while the mutation His103→Asn yielded a mutant enzyme that retained 59% of the κ₀/K_m of that for the wild-type enzyme. Since mutants of other α-amylases acting on α-1,4-glucosidic bond linkage lost most of their activity by the site-directed mutagenesis at their equivalent residues to His103 and His328, the retaining of activity by Hisl03→Asn mutation in B. cereus oligo-1,6-glucosidase revealed the distinguished role of His103 for the hydrolysis of α-1,6-glucosidic bond linkage.     

Role of Positively Charged Residues Lys267, Lys270, and Arg411 of Cytochrome P450scc (Cyp11A1) in Interaction with Adrenodoxin

Cytochrome P450scc and adrenodoxin are redox proteins of the electron transfer chain of the inner mitochondrial membrane steroid hydroxylases. In the present work site-directed mutagenesis of the charged residues of cytochrome P450scc and adrenodoxin, which might be involved in interaction, was used to study the nature of electrostatic contacts between the hemeprotein and the ferredoxin. The target residues for mutagenesis were selected based on the theoretical model of cytochrome P450scc-adrenodoxin complex and previously reported chemical modification studies of cytochrome P450scc. In the present work, to clarify the molecular mechanism of hemeprotein interaction with ferredoxin, we constructed cytochrome P450scc Lys267, Lys270, and Arg411 mutants and Glu47 mutant of adrenodoxin and analyzed their possible role in electrostatic interaction and the role of these residues in the functional activity of the proteins. Charge neutralization at positions Lys267 or Lys270 of cytochrome P450scc causes no significant effect on the physicochemical and functional properties of cytochrome P450scc. However, cytochrome P450scc mutant Arg411Gln was found to exhibit decreased binding affinity to adrenodoxin and lower activity in the cholesterol side chain cleavage reaction. Studies of the functional properties of Glu47Gln and Glu47Arg adrenodoxin mutants indicate that a negatively charged residue in the loop covering the Fe2S2 cluster, being important for maintenance of the correct architecture of these structural elements of ferredoxin, is not directly involved in electrostatic interaction with cytochrome P450scc. Moreover, our results indicate the presence of at least two different binding (contact) sites on the proximal surface of cytochrome P450scc with different electrostatic input to interaction with adrenodoxin. In the binary complex, the positively charged sites of the proximal surface of cytochrome P450scc well correspond to the two negatively charged sites of adrenodoxin: the "interaction" domain site and the "core" domain site.     

Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings

Intracellular recording is a powerful technique used to determine how a single cell may respond to a given stimulus. In vision research, intracellular recording has historically been a common technique used to study sensitivities of individual photoreceptor cells to different light stimuli that is still being used today. However, there remains a dearth of detailed methodology in the literature for researchers wishing to replicate intracellular recording experiments in the eye. Here we present the insect as a model for examining eye physiology more generally. Insect photoreceptor cells are located near the surface of the eye and are therefore easy to reach, and many of the mechanisms involved in vision are conserved across animal phyla. We describe the basic procedure for in vivo intracellular recording of photoreceptor cells in the eye of a butterfly, with the goal of making this technique more accessible to researchers with little prior experience in electrophysiology. We introduce the basic equipment needed, how to prepare a live butterfly for recording, how to insert a glass microelectrode into a single cell, and finally the recording procedure itself. We also explain the basic analysis of raw response data for determining spectral sensitivity of individual cell types. Although our protocol focuses on determining spectral sensitivity, other stimuli (e.g., polarized light) and variations of the method are applicable to this setup.     

定点突变技术研究苏云金杆菌Cry1类晶体蛋白结构与功能的进展

近年来利用定点突变技术研究苏云金杆菌（Bacillus thuringiesis,Bt）杀虫晶体蛋白（Insecticidal crystal proteins,ICP）作用机制已取得良好进展.杀虫晶体蛋白不同结构域上氨基酸残基的突变将影响其稳定性,与受体的结合,不可逆的昆虫中肠膜插入及离子通道活性的强弱等.突变研究表明,结构域Ⅰ参与不可逆结合及插入昆虫中肠膜过程;结构域Ⅱ参与受体结合,包括初始结合与不可逆结合;结构域Ⅲ在杀虫特异性和维持三维结构的稳定性方面起重要作用,同时,可能参与离子通道的形成,受体结合和插入昆虫中肠膜过程.利用各种定点突变技术对各位点进行突变可以研究单一位点的功能,到目前为止,已有很多关于这方面的研究,并且筛选到了毒力提高的工程菌株.     

Dissection and Immunohistochemistry of Larval,Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 ². The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 ^{3 4}. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes⁵: In the ''p'' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the ''y'' subtype, Rh4 in yR7s is associated with Rh6 in yR8s ^{6 7 8}.Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc⁹ and initiates the assembly of undifferentiated cells into ommatidia^10-11. The ''founder cell'' R8 is specified first and recruits R1-6 and then R7 ^12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes ¹⁵, including rhabdomere formation, synaptogenesis and eventually rh expression.In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).     

Evolution of wing pigmentation in Drosophila: Diversity,physiological regulation,and cis-regulatory evolution

Fruit flies (Drosophila and its close relatives, or “drosophilids”) are a group that includes an important model organism, Drosophila melanogaster, and also very diverse species distributed worldwide. Many of these species have black or brown pigmentation patterns on their wings, and have been used as material for evo-devo research. Pigmentation patterns are thought to have evolved rapidly compared with body plans or body shapes; hence they are advantageous model systems for studying evolutionary gains of traits and parallel evolution. Various groups of drosophilids, including genus Idiomyia (Hawaiian Drosophila), have a variety of pigmentations, ranging from simple black pigmentations around crossveins to a single antero-distal spot and a more complex mottled pattern. Pigmentation patterns are sometimes obviously used for sexual displays; however, in some cases they may have other functions. The process of wing formation in Drosophila, the general mechanism of pigmentation formation, and the transport of substances necessary for pigmentation, including melanin precursors, through wing veins are summarized here. Lastly, the evolution of the expression of genes regulating pigmentation patterns, the role of cis-regulatory regions, and the conditions required for the evolutionary emergence of pigmentation patterns are discussed. Future prospects for research on the evolution of wing pigmentation pattern formation in drosophilids are presented, particularly from the point of view of how they compare with other studies of the evolution of new traits.     

Genetical analysis of visual system disorganizer (vid), a new gene involved in normal development of eye and optic lobe of the brain in Drosophila melanogaster

A neuroanatomical screening of a collection of P-element mutagenized flies has been carried out with the aim of finding new mutants affecting the optic lobe of the adult brain in Drosophila melanogaster. We have identified a new gene that is involved in the development of the adult axon array in the optic ganglia and in the ommatidia assembly. We have named this locus visual system disorganizer (vid). Reversional mutagenesis demonstrated that the vid mutant was the result of a P-element insertion in the Drosophila genome and allowed us to generate independent alleles, some of which resulted in semilethality, like the vid original mutant, while the others were completely lethal. A genetic somatic mosaic analysis indicated that the vid gene is required in the eye for its normal development by inductive effects. This analysis also suggests an inductive effect of the vid gene on the distal portion of the optic lobe, particularly the lamina and the first optic chiasma. Moreover, the absence of mutant phenotype in the proximal region of the optic ganglia, including the medulla, the second optic chiasma, and the lobula complex underlying mosaic eyes, is suggestive of an autonomously acting mechanism of the vid gene in the optic lobe. The complete or partial lethality generated by different mutations at the vid locus suggests that this gene's role may not be limited to the visual system, but may also affect a vital function during Drosophila development.     

Conserved and divergent aspects of Robo receptor signaling and regulation between Drosophila Robo1 and C. elegans SAX-3

The evolutionarily conserved Roundabout (Robo) family of axon guidance receptors control midline crossing of axons in response to the midline repellant ligand Slit in bilaterian animals including insects, nematodes, and vertebrates. Despite this strong evolutionary conservation, it is unclear whether the signaling mechanism(s) downstream of Robo receptors are similarly conserved. To directly compare midline repulsive signaling in Robo family members from different species, here we use a transgenic approach to express the Robo family receptor SAX-3 from the nematode Caenorhabditis elegans in neurons of the fruit fly, Drosophila melanogaster. We examine SAX-3’s ability to repel Drosophila axons from the Slit-expressing midline in gain of function assays, and test SAX-3’s ability to substitute for Drosophila Robo1 during fly embryonic development in genetic rescue experiments. We show that C. elegans SAX-3 is properly translated and localized to neuronal axons when expressed in the Drosophila embryonic CNS, and that SAX-3 can signal midline repulsion in Drosophila embryonic neurons, although not as efficiently as Drosophila Robo1. Using a series of Robo1/SAX-3 chimeras, we show that the SAX-3 cytoplasmic domain can signal midline repulsion to the same extent as Robo1 when combined with the Robo1 ectodomain. We show that SAX-3 is not subject to endosomal sorting by the negative regulator Commissureless (Comm) in Drosophila neurons in vivo, and that peri-membrane and ectodomain sequences are both required for Comm sorting of Drosophila Robo1.