Expression and distribution of Trophoblast Glycoprotein in the mouse retina

AbstractWe recently identified the leucine-rich repeat adhesion protein, trophoblast glycoprotein (TPBG), as a novel PKCα-dependent phosphoprotein in retinal rod bipolar cells (RBCs). Since TPBG has not been thoroughly examined in the retina, this study characterizes the localization and expression patterns of TPBG in the developing and adult mouse retina using two antibodies, one against the N-terminal, leucine-rich domain and the other against the C-terminal PDZ-interacting motif. Both antibodies labeled dendrites and synaptic terminals of RBCs, as well as the cell bodies and dendrites of an uncharacterized class of amacrine cell. In transfected HEK293 cells, TPBG was localized to the plasma membrane and intracellular membranes and was associated with the tips of thin filopodia-like membrane projections. TPBG immunofluorescence in RBCs detected with the C-terminal antibody was strongly dependent on the activity state of the adult retina, with less labeling in dark-adapted compared to light-adapted retina, and less labeling in light-adapted PKCα knockout and TRPM1 knockout retinas compared to wild type, despite no change in total TPBG detected by immunoblotting. These results suggest that the C-terminal epitope is blocked in the dark-adapted and knockout retinas compared to light-adapted wild type retinas, possibly through interaction with a PDZ domain protein. During development, TPBG expression increases dramatically just prior to eye opening with a time course closely correlated with that of TRPM1 expression. In the retina, leucine-rich repeat proteins like TPBG have been implicated in the development and maintenance of functional bipolar cell synapses, and TPBG may play a similar role in RBCs.

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Modulation of Excitatory Synaptic Transmission by GABAC Receptor-Mediated Feedback in the Mouse Inner Retina

Journal of Neurophysiology ◽

10.1152/jn.2001.86.5.2285 ◽

2001 ◽

Vol 86 (5) ◽

pp. 2285-2298 ◽

Cited By ~ 36

Author(s):

Ko Matsui ◽

Jun Hasegawa ◽

Masao Tachibana

Keyword(s):

Nmda Receptors ◽

Time Course ◽

Feedback Inhibition ◽

Plexiform Layer ◽

Amacrine Cells ◽

Bipolar Cells ◽

Mouse Retina ◽

Receptor Subtypes ◽

Inner Plexiform Layer ◽

Cone Bipolar Cells

In many vertebrate CNS synapses, the neurotransmitter glutamate activates postsynaptic non- N-methyl-d-aspartate (NMDA) and NMDA receptors. Since their biophysical properties are quite different, the time course of excitatory postsynaptic currents (EPSCs) depends largely on the relative contribution of their activation. To investigate whether the activation of the two receptor subtypes is affected by the synaptic interaction in the inner plexiform layer (IPL) of the mouse retina, we analyzed the properties of the light-evoked responses ofon-cone bipolar cells and on-transient amacrine cells in a retinal slice preparation. on-transient amacrine cells were whole cell voltage-clamped, and the glutamatergic synaptic input from bipolar cells was isolated by a cocktail of pharmacological agents (bicuculline, strychnine, curare, and atropine). Direct puff application of NMDA revealed the presence of functional NMDA receptors. However, the light-evoked EPSC was not significantly affected byd(−)-2-amino-5-phosphonopentanoic acid (d-AP5), but suppressed by 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) or 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466). These results indicate that the light-evoked EPSC is mediated mainly by AMPA receptors under this condition. Since bipolar cells have GABACreceptors at their terminals, it has been suggested that bipolar cells receive feedback inhibition from amacrine cells. Application of (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), a specific blocker of GABAC receptors, suppressed both the GABA-induced current and the light-evoked feedback inhibition observed in on-cone bipolar cells and enhanced the light-evoked EPSC of on-transient amacrine cells. In the presence of TPMPA, the light-evoked EPSC of amacrine cells was composed of AMPA and NMDA receptor-mediated components. Our results suggest that photoresponses of on-transient amacrine cells in the mouse retina are modified by the activation of presynaptic GABAC receptors, which may control the extent of glutamate spillover.

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Identification of PKCα-dependent phosphoproteins in mouse retina

10.1101/589184 ◽

2019 ◽

Cited By ~ 1

Author(s):

Colin M. Wakeham ◽

Phillip A. Wilmarth ◽

Jennifer M. Cunliffe ◽

John E. Klimek ◽

Gaoying Ren ◽

...

Keyword(s):

Transcriptional Regulation ◽

Functional Groups ◽

Phorbol Ester ◽

Bipolar Cells ◽

Mouse Retina ◽

Wild Type ◽

Light Intensities ◽

Retinal Rod ◽

Wide Range ◽

Rod Bipolar Cells

AbstractAdjusting to a wide range of light intensities is an essential feature of retinal rod bipolar cell (RBC) function. While persuasive evidence suggests this modulation involves phosphorylation by protein kinase C-alpha (PKCα), the targets of PKCα phosphorylation in the retina have not been identified. PKCα activity and phosphorylation in RBCs was examined by immunofluorescence confocal microscopy using a conformation-specific PKCα antibody and antibodies to phosphorylated PKC motifs. PKCα activity was dependent on light and expression of TRPM1, and RBC dendrites were the primary sites of light-dependent phosphorylation. PKCα-dependent retinal phosphoproteins were identified using a phosphoproteomics approach to compare total protein and phosphopeptide abundance between phorbol ester-treated wild type and PKCα knockout (PKCα-KO) mouse retinas. Phosphopeptide mass spectrometry identified over 1100 phosphopeptides in mouse retina, with 12 displaying significantly greater phosphorylation in WT compared to PKCα-KO samples. The differentially phosphorylated proteins fall into the following functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein). Two strongly differentially expressed phosphoproteins, BORG4 and TPBG, were localized to the synaptic layers of the retina, and may play a role in PKCα-dependent modulation of RBC physiology. Data are available via ProteomeXchange with identifier PXD012906.SignificanceRetinal rod bipolar cells (RBCs), the second-order neurons of the mammalian rod visual pathway, are able to modulate their sensitivity to remain functional across a wide range of light intensities, from starlight to daylight. Evidence suggests that this modulation requires the serine/threonine kinase, PKCα, though the specific mechanism by which PKCα modulates RBC physiology is unknown. This study examined PKCα phosophorylation patterns in mouse rod bipolar cells and then used a phosphoproteomics approach to identify PKCα-dependent phosphoproteins in the mouse retina. A small number of retinal proteins showed significant PKCα-dependent phosphorylation, including BORG4 and TPBG, suggesting a potential contribution to PKCα-dependent modulation of RBC physiology.HighlightsPKCα is a major source of phosphorylation in retinal RBC dendrites and its activity in RBCs is light dependent.Proteins showing differential phosphorylation between phorbol ester-treated wild type and PKCα-KO retinas belong to the following major functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein).The PKCα-dependent phosphoproteins, BORG4 and TPBG, are present in the synaptic layers of the retina and may be involved in PKCα-dependent modulation of RBC physiology.

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Postsynaptic calcium feedback between rods and rod bipolar cells in the mouse retina

Visual Neuroscience ◽

10.1017/s095252380421611x ◽

2004 ◽

Vol 21 (6) ◽

pp. 913-924 ◽

Cited By ~ 17

Author(s):

AMY BERNTSON ◽

ROBERT G. SMITH ◽

W. ROWLAND TAYLOR

Keyword(s):

Intracellular Calcium ◽

Time Constant ◽

Time Course ◽

Dynamic Range ◽

Amacrine Cells ◽

Bipolar Cells ◽

Mouse Retina ◽

Inward Current ◽

Rod Bipolar Cells ◽

In Cells

Light-evoked currents were recorded from rod bipolar cells in a dark-adapted mouse retinal slice preparation. Low-intensity light steps evoked a sustained inward current. Saturating light steps evoked an inward current with an initial peak that inactivated, with a time constant of about 60–70 ms, to a steady plateau level that was maintained for the duration of the step. The inactivation was strongest at hyperpolarized potentials, and absent at positive potentials. Inactivation was mediated by an increase in the intracellular calcium concentration, as it was abolished in cells dialyzed with 10 mM BAPTA, but was present in cells dialyzed with 1 mM EGTA. Moreover, responses to brief flashes of light were broader in the presence of intracellular BAPTA indicating that the calcium feedback actively shapes the time course of the light responses. Recovery from inactivation observed for paired-pulse stimuli occurred with a time constant of about 375 ms. Calcium feedback could act to increase the dynamic range of the bipolar cells, and to reduce variability in the amplitude and duration of the single-photon signal. This may be important for nonlinear processing at downstream sites of convergence from rod bipolar cells to AII amacrine cells. A model in which intracellular calcium rapidly binds to the light-gated channel and reduces the conductance can account for the results.

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Arabidopsis 4-COUMAROYL-COA LIGASE 8 contributes to the biosynthesis of the benzenoid ring of coenzyme Q in peroxisomes

Biochemical Journal ◽

10.1042/bcj20190688 ◽

2019 ◽

Vol 476 (22) ◽

pp. 3521-3532

Author(s):

Eric Soubeyrand ◽

Megan Kelly ◽

Shea A. Keene ◽

Ann C. Bernert ◽

Scott Latimer ◽

...

Keyword(s):

Oxidative Metabolism ◽

Time Course ◽

Reverse Genetics ◽

De Novo ◽

Coenzyme Q ◽

Control Level ◽

Wild Type ◽

Fluorescent Reporters ◽

Coa Ligase ◽

Peroxisomal Targeting

Plants have evolved the ability to derive the benzenoid moiety of the respiratory cofactor and antioxidant, ubiquinone (coenzyme Q), either from the β-oxidative metabolism of p-coumarate or from the peroxidative cleavage of kaempferol. Here, isotopic feeding assays, gene co-expression analysis and reverse genetics identified Arabidopsis 4-COUMARATE-COA LIGASE 8 (4-CL8; At5g38120) as a contributor to the β-oxidation of p-coumarate for ubiquinone biosynthesis. The enzyme is part of the same clade (V) of acyl-activating enzymes than At4g19010, a p-coumarate CoA ligase known to play a central role in the conversion of p-coumarate into 4-hydroxybenzoate. A 4-cl8 T-DNA knockout displayed a 20% decrease in ubiquinone content compared with wild-type plants, while 4-CL8 overexpression boosted ubiquinone content up to 150% of the control level. Similarly, the isotopic enrichment of ubiquinone's ring was decreased by 28% in the 4-cl8 knockout as compared with wild-type controls when Phe-[Ring-13C6] was fed to the plants. This metabolic blockage could be bypassed via the exogenous supply of 4-hydroxybenzoate, the product of p-coumarate β-oxidation. Arabidopsis 4-CL8 displays a canonical peroxisomal targeting sequence type 1, and confocal microscopy experiments using fused fluorescent reporters demonstrated that this enzyme is imported into peroxisomes. Time course feeding assays using Phe-[Ring-13C6] in a series of Arabidopsis single and double knockouts blocked in the β-oxidative metabolism of p-coumarate (4-cl8; at4g19010; at4g19010 × 4-cl8), flavonol biosynthesis (flavanone-3-hydroxylase), or both (at4g19010 × flavanone-3-hydroxylase) indicated that continuous high light treatments (500 µE m−2 s−1; 24 h) markedly stimulated the de novo biosynthesis of ubiquinone independently of kaempferol catabolism.

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Postnatal growth rate and gonadal development in circadian tau mutant hamsters reared in constant dim red light

Reproduction ◽

10.1530/reprod/118.2.327 ◽

2000 ◽

pp. 327-330 ◽

Cited By ~ 7

Author(s):

RJ Lucas ◽

JA Stirland ◽

YN Mohammad ◽

AS Loudon

Keyword(s):

Time Course ◽

Red Light ◽

Somatic Growth ◽

Postnatal Growth ◽

Gonadal Development ◽

Testicular Development ◽

Wild Type ◽

Similar Time ◽

Syrian Hamsters ◽

Body Weights

The role of the circadian clock in the reproductive development of Syrian hamsters (Mesocricetus auratus was examined in wild type and circadian tau mutant hamsters reared from birth to 26 weeks of age under constant dim red light. Testis diameter and body weights were determined at weekly intervals in male hamsters from 4 weeks of age. In both genotypes, testicular development, subsequent regression and recrudescence exhibited a similar time course. The age at which animals displayed reproductive photosensitivity, as exhibited by testicular regression, was unrelated to circadian genotype (mean +/- SEM: 54 +/- 3 days for wild type and 59 +/- 5 days for tau mutants). In contrast, our studies revealed a significant impact of the mutation on somatic growth, such that tau mutants weighed 18% less than wild types at the end of the experiment. Our study reveals that the juvenile onset of reproductive photoperiodism in Syrian hamsters is not timed by the circadian system.

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Analysis of maxillopedia Expression Pattern and Larval Cuticular Phenotype in Wild-Type and Mutant Tribolium

Genetics ◽

10.1093/genetics/155.2.721 ◽

2000 ◽

Vol 155 (2) ◽

pp. 721-731 ◽

Cited By ~ 7

Author(s):

Teresa D Shippy ◽

Jianhua Guo ◽

Susan J Brown ◽

Richard W Beeman ◽

Robin E Denell

Keyword(s):

Rna Interference ◽

Expression Pattern ◽

Tribolium Castaneum ◽

Expression Patterns ◽

Ectopic Expression ◽

Homeotic Gene ◽

Wild Type ◽

Double Stranded Rna ◽

Similar Expression ◽

Mutant Phenotypes

Abstract The Tribolium castaneum homeotic gene maxillopedia (mxp) is the ortholog of Drosophila proboscipedia (pb). Here we describe and classify available mxp alleles. Larvae lacking all mxp function die soon after hatching, exhibiting strong transformations of maxillary and labial palps to legs. Hypomorphic mxp alleles produce less severe transformations to leg. RNA interference with maxillopedia double-stranded RNA results in phenocopies of mxp mutant phenotypes ranging from partial to complete transformations. A number of gain-of-function (GOF) mxp alleles have been isolated based on transformations of adult antennae and/or legs toward palps. Finally, we have characterized the mxp expression pattern in wild-type and mutant embryos. In normal embryos, mxp is expressed in the maxillary and labial segments, whereas ectopic expression is observed in some GOF variants. Although mxp and Pb display very similar expression patterns, pb null embryos develop normally. The mxp mutant larval phenotype in Tribolium is consistent with the hypothesis that an ancestral pb-like gene had an embryonic function that was lost in the lineage leading to Drosophila.

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Time course analysis of ABA and non-ionic osmotic stress-induced changes in water status, chlorophyll fluorescence and osmotic adjustment in Arabidopsis thaliana wild-type (Columbia) and ABA-deficient mutant (aba2)

Environmental and Experimental Botany ◽

10.1016/j.envexpbot.2010.09.008 ◽

2013 ◽

Vol 86 ◽

pp. 44-51 ◽

Cited By ~ 19

Author(s):

Ceyda Ozfidan ◽

Ismail Turkan ◽

Askim Hediye Sekmen ◽

Burcu Seckin

Keyword(s):

Arabidopsis Thaliana ◽

Chlorophyll Fluorescence ◽

Osmotic Stress ◽

Osmotic Adjustment ◽

Time Course ◽

Water Status ◽

Deficient Mutant ◽

Wild Type ◽

Induced Changes

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Modulation by Zn2+ of GABA responses in bipolar cells of the mouse retina

Visual Neuroscience ◽

10.1017/s0952523800172098 ◽

2000 ◽

Vol 17 (2) ◽

pp. 273-281 ◽

Cited By ~ 20

Author(s):

M. KANEDA ◽

B. ANDRÁSFALVY ◽

A. KANEKO

Keyword(s):

Axon Terminal ◽

Inhibitory Action ◽

Phosphinic Acid ◽

Amacrine Cells ◽

Bipolar Cells ◽

Mouse Retina ◽

Induced Responses ◽

Inhibitory Interaction ◽

Cell Recording ◽

Recording Conditions

The localization of endogenous Zn2+ in the mouse retina was examined histochemically and the inhibitory action of Zn2+ on GABA-induced responses was studied in bipolar cells isolated from the mouse retina. Accumulation of endogenous Zn2+ was detected in photoreceptors, bipolar, and/or amacrine cells by either the bromopyridylazo-diethylaminophenol method or the dithizone method. Under whole-cell recording conditions, GABA induced a Cl− current in isolated bipolar cells. The current consisted of two components. The first component was inhibited completely by application of 100 μM bicuculline, suggesting that this is a GABAA-receptor mediated current. The second component was inhibited completely by 100 μM 3-aminopropyl-(methyl)-phosphinic acid, suggesting that this is a GABAC-receptor mediated current. GABAC receptors were present at a higher density on the axon terminal than on dendrites. Zn2+ inhibited both GABAA and GABAC receptors. GABAC receptors were more susceptible to Zn2+; the IC50 for the GABAA receptor was 67.4 μM and that for the GABAC receptor was 1.9 μM. These results suggest that Zn2+ modulates the inhibitory interaction between amacrine and bipolar cells, particularly that mediated by the GABAC receptor.

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Isolation of a chitinase overproducing mutant of Streptomyces peucetius defective in daunorubicin biosynthesis

Canadian Journal of Microbiology ◽

10.1139/w00-079 ◽

2000 ◽

Vol 46 (10) ◽

pp. 956-960 ◽

Cited By ~ 2

Author(s):

Kuzhandhaivel S Vetrivel ◽

Kuppamuthu Dharmalingam

Keyword(s):

Biosynthetic Pathway ◽

Time Course ◽

Polyketide Synthase ◽

Protein Profile ◽

Restriction Enzymes ◽

Wild Type ◽

Anthracycline Antibiotic ◽

Streptomyces Peucetius ◽

Chitinase Production ◽

Major Alteration

Streptomyces peucetius, producer of the antitumor anthracycline antibiotic daunorubicin, was mutagenized, and mutants defective in daunorubicin biosynthesis were screened. One mutant (SPVI), which failed to produce daunorubicin, was found to overproduce an extracellular chitinase. Time course analyses of chitinase production and of the extracellular protein profile showed that the increase in activity is due to increased synthesis of the enzyme protein. The production of chitinase in SPVI was repressed by glucose as in the case of wild-type S. peucetius. PFGE analysis of VspI restriction fragments of S. peucetius and SPVI showed that there was no major alteration in the mutant genome. The hybridization pattern of S. peucetius and SPVI genomic DNA digested with various restriction enzymes was identical when probed with dnrUVJI genes of the S. peucetius daunorubicin cluster and chiA of Streptomyces lividans 66. The possible step affected in the daunorubicin biosynthetic pathway could be a polyketide synthase, since aklanonic acid, the earliest detectable intermediate in the daunorubicin pathway, was not synthesized in SPVI.Key words: Streptomyces peucetius, chitinase, daunorubicin, NTG mutagenesis.

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