scholarly journals Immunocytochemical Labeling of Rhabdomeric Proteins inDrosophilaPhotoreceptor Cells Is Compromised by a Light-dependent Technical Artifact

2019 ◽  
Vol 67 (10) ◽  
pp. 745-757 ◽  
Author(s):  
Krystina Schopf ◽  
Thomas K. Smylla ◽  
Armin Huber
Keyword(s):  
Structural Changes ◽  
Protein Transport ◽  
Light Exposure ◽  
Protein Localization ◽  
Photoreceptor Cells ◽  
Antibody Staining ◽  
Technical Artifact ◽  
Stage Process ◽  
Stage 1 ◽  
Antibody Labeling

Drosophila photoreceptor cells are employed as a model system for studying membrane protein transport. Phototransduction proteins like rhodopsin and the light-activated TRPL ion channel are transported within the photoreceptor cell, and they change their subcellular distribution in a light-dependent way. Investigating the transport mechanisms for rhodopsin and ion channels requires accurate histochemical methods for protein localization. By using immunocytochemistry the light-triggered translocation of TRPL has been described as a two-stage process. In stage 1, TRPL accumulates at the rhabdomere base and the adjacent stalk membrane a few minutes after onset of illumination and is internalized in stage 2 by endocytosis after prolonged light exposure. Here, we show that a commonly observed crescent shaped antibody labeling pattern suggesting a fast translocation of rhodopsin, TRP, and TRPL to the rhabdomere base is a light-dependent antibody staining artifact. This artifact is most probably caused by the profound structural changes in the microvillar membranes of rhabdomeres that result from activation of the signaling cascade. By using alternative labeling methods, either eGFP-tags or the self-labeling SNAP-tag, we show that light activation of TRPL transport indeed results in fast changes of the TRPL distribution in the rhabdomere but not in the way described previously.

Day-To-Day Variability of Protein Transport Used as a Method for Analyzing Peritoneal Permeability in CAPD

1991 ◽  
Vol 11 (3) ◽  
pp. 217-223 ◽  
Author(s):  
Désirée Zemel ◽  
Raymond T. Krediet ◽  
Gerardus C.M. Koomen ◽  
Dirk G. Struijk ◽  
Lambertus Arisz
Keyword(s):  
Surface Area ◽  
Structural Changes ◽  
Protein Transport ◽  
Intrinsic Permeability ◽  
Effective Surface Area ◽  
Intraindividual Variation ◽  
Effective Surface ◽  
Peritoneal Surface ◽  
Beta 2 ◽  
Beta 2 Microglobulin

The transperitoneal transport of macromolecules is dependent on both effective peritoneal surface area and intrinsic permeability of the peritoneum. For passage of small solutes, the effective surface area is the main determinant. We hypothesized that day-to-day variations in peritoneal clearances are caused by changes in the effective surface area and not in the intrinsic permeability. Four CAPD {continuous ambulatory peritoneal dialysis) patients without peritonitis were investigated on 28 consecutive days. Concentrations of beta-2-microglobulin, albumin, IgG, and alpha-2-macroglobulin were determined daily in dialysate {night bags) and weekly in serum. Clearances and their coefficients of variation were calculated. Mean coefficients of the intraindividual variation of protein clearances increased, the higher the molecular weight: they ranged from 12% for beta-2microglobulin clearance to 22% for alpha-2-macroglobulin clearance. Correlations were present between the clearances of albumin, IgG, and alpha-2-macroglobulin, but not between any of these and beta-2-microglobulin clearance. In all patients, protein clearance {C) was a power function of the free diffusion coefficient in water {D) according to the equation: C=a. Db in which b represents the restriction coefficient of the peritoneum, and thus intrinsic permeability. The coefficient of variation of the restriction coefficient was low (range 4–6%). This supports our assumption that the intrinsic permeability is fairly constant on the short term. Day-to-day variations in protein clearances are thus mainly caused by alterations in the effective peritoneal surface area. Longterm follow-up of the restriction coefficient in individual patients might identify those at risk for the development of structural changes in the peritoneal membrane.

scholarly journals The rhodopsin-retinochrome system for retinal re-isomerization predates the origin of cephalopod eyes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Oliver Vöcking ◽  
Lucas Leclère ◽  
Harald Hausen
Keyword(s):  
In Situ Hybridization ◽  
Photoreceptor Cells ◽  
Visual Cycle ◽  
Phylogenetic Distribution ◽  
Enzymatic Process ◽  
Antibody Staining ◽  
The Individual ◽  
Functional Components ◽  
Visual Rhodopsin

Abstract Background The process of photoreception in most animals depends on the light induced isomerization of the chromophore retinal, bound to rhodopsin. To re-use retinal, the all-trans-retinal form needs to be re-isomerized to 11-cis-retinal, which can be achieved in different ways. In vertebrates, this mostly includes a stepwise enzymatic process called the visual cycle. The best studied re-isomerization system in protostomes is the rhodopsin-retinochrome system of cephalopods, which consists of rhodopsin, the photoisomerase retinochrome and the protein RALBP functioning as shuttle for retinal. In this study we investigate the expression of the rhodopsin-retinochrome system and functional components of the vertebrate visual cycle in a polyplacophoran mollusk, Leptochiton asellus, and examine the phylogenetic distribution of the individual components in other protostome animals. Results Tree-based orthology assignments revealed that orthologs of the cephalopod retinochrome and RALBP are present in mollusks outside of cephalopods. By mining our dataset for vertebrate visual cycle components, we also found orthologs of the retinoid binding protein RLBP1, in polyplacophoran mollusks, cephalopods and a phoronid. In situ hybridization and antibody staining revealed that L. asellus retinochrome is co-expressed in the larval chiton photoreceptor cells (PRCs) with the visual rhodopsin, RALBP and RLBP1. In addition, multiple retinal dehydrogenases are expressed in the PRCs, which might also contribute to the rhodopsin-retinochrome system. Conclusions We conclude that the rhodopsin-retinochrome system is a common feature of mollusk PRCs and predates the origin of cephalopod eyes. Our results show that this system has to be extended by adding further components, which surprisingly, are shared with vertebrates.

A role for Pax-1 as a mediator of notochordal signals during the dorsoventral specification of vertebrae

Development ◽  
1993 ◽  
Vol 119 (3) ◽  
pp. 649-660 ◽  
Author(s):  
H. Koseki ◽  
J. Wallin ◽  
J. Wilting ◽  
Y. Mizutani ◽  
A. Kispert ◽  
...  
Keyword(s):  
Structural Changes ◽  
Genetic Interaction ◽  
Paraxial Mesoderm ◽  
Lumbar Region ◽  
Subsequent Formation ◽  
Developmental Abnormalities ◽  
Short Tail ◽  
Antibody Staining ◽  
Skeletal Phenotype ◽  
Subsequent Loss

The notochord plays an important role in the differentiation of the paraxial mesoderm and the neural tube. We have analyzed the role of the notochord in somite differentiation and subsequent formation of the vertebral column using a mouse mutant, Danforth's short-tail (Sd). In this mutant, the skeletal phenotype is most probably a result of degeneration and subsequent loss of the notochord. The Sd gene is known to interact with undulated (un), a sclerotome mutant. Double mutants between Sd and un alleles show an increase in the severity of the defects, mainly in the ventral parts of the vertebrae. We also show that part of the Sd phenotype is strikingly similar to that of the un alleles. As un is known to be caused by a mutation in the Pax-1 gene, we analyzed Pax-1 expression in Sd embryos. In Sd embryos, Pax-1 expression is reduced, providing a potential molecular basis for the genetic interaction observed. A complete loss of Pax-1 expression in morphologically intact mesenchyme was found in the lower thoracic-lumbar region, which is phenotypically very similar to the corresponding region in a Pax-1 null mutant, Undulated short-tail. The sclerotome developmental abnormalities in Sd coincide closely, both in time and space, with notochordal changes, as determined by whole-mount T antibody staining. These findings indicate that an intact notochord is necessary for normal Pax-1 expression in sclerotome cells, which is in turn required for the formation of the ventral parts of the vertebrae. The observed correlation among structural changes of the notochord, Pax-1 expression levels and skeletal phenotypes, suggests that Pax-1 might be an intrinsic mediator of notochordal signals during the dorsoventral specification of vertebrae.

scholarly journals LIM kinase1 regulates mitotic centrosome integrity via its activity on dynein light intermediate chains

Open Biology ◽  
2018 ◽  
Vol 8 (6) ◽  
pp. 170202 ◽  
Author(s):  
Sirong Ou ◽  
Mei-Hua Tan ◽  
Ting Weng ◽  
HoiYeung Li ◽  
Cheng-Gee Koh
Keyword(s):  
Protein Transport ◽  
Protein Localization ◽  
Cytoplasmic Dynein ◽  
Spindle Pole ◽  
Centrosomal Protein ◽  
And Function ◽  
Cytoskeleton Dynamics ◽  
Dynein Light Intermediate Chains ◽  
Intermediate Chains ◽  
Mitotic Spindle Pole

Abnormal centrosome number and function have been implicated in tumour development. LIM kinase1 (LIMK1), a regulator of actin cytoskeleton dynamics, is found to localize at the mitotic centrosome. However, its role at the centrosome is not fully explored. Here, we report that LIMK1 depletion resulted in multi-polar spindles and defocusing of centrosomes, implicating its involvement in the regulation of mitotic centrosome integrity. LIMK1 could influence centrosome integrity by modulating centrosomal protein localization at the spindle pole. Interestingly, dynein light intermediate chains (LICs) are able to rescue the defects observed in LIMK1-depleted cells. We found that LICs are potential novel interacting partners and substrates of LIMK1 and that LIMK1 phosphorylation regulates cytoplasmic dynein function in centrosomal protein transport, which in turn impacts mitotic spindle pole integrity.

scholarly journals Protective Effect of Astaxanthin on Blue Light Light-Emitting Diode-Induced Retinal Cell Damage via Free Radical Scavenging and Activation of PI3K/Akt/Nrf2 Pathway in 661W Cell Model

Marine Drugs ◽  
2020 ◽  
Vol 18 (8) ◽  
pp. 387 ◽  
Author(s):  
Chao-Wen Lin ◽  
Chung-May Yang ◽  
Chang-Hao Yang
Keyword(s):  
Antioxidant Enzymes ◽  
Free Radical ◽  
Protective Effect ◽  
Blue Light ◽  
Nuclear Translocation ◽  
Light Exposure ◽  
Cell Damage ◽  
Radical Scavenging ◽  
Photoreceptor Cells ◽  
Light Emitting

Light-emitting diodes (LEDs) are widely used and energy-efficient light sources in modern life that emit higher levels of short-wavelength blue light. Excessive blue light exposure may damage the photoreceptor cells in our eyes. Astaxanthin, a xanthophyll that is abundantly available in seafood, is a potent free radical scavenger and anti-inflammatory agent. We used a 661W photoreceptor cell line to investigate the protective effect of astaxanthin on blue light LED-induced retinal injury. The cells were treated with various concentrations of astaxanthin and then exposed to blue light LED. Our results showed that pretreatment with astaxanthin inhibited blue light LED-induced cell apoptosis and prevented cell death. Moreover, the protective effect was concentration dependent. Astaxanthin suppressed the production of reactive oxygen species and oxidative stress biomarkers and diminished mitochondrial damage induced by blue light exposure. Western blot analysis confirmed that astaxanthin activated the PI3K/Akt pathway, induced the nuclear translocation of Nrf2, and increased the expression of phase II antioxidant enzymes. The expression of antioxidant enzymes and the suppression of apoptosis-related proteins eventually protected the 661W cells against blue light LED-induced cell damage. Thus, our results demonstrated that astaxanthin exerted a dose-dependent protective effect on photoreceptor cells against damage mediated by blue light LED exposure.

scholarly journals Exploring the potential role of community engagement in evaluating clinical and translational science grant proposals

2018 ◽  
Vol 2 (3) ◽  
pp. 139-146 ◽  
Author(s):  
Jeffrey W. Treem ◽  
Margaret Schneider ◽  
Robynn L. Zender ◽  
Dara H. Sorkin
Keyword(s):  
Community Engagement ◽  
Potential Role ◽  
Review Process ◽  
Translational Science ◽  
Community Members ◽  
Grant Proposals ◽  
Stage Process ◽  
Stage 1 ◽  
The University

IntroductionThis study explored the effects of integrating community members into the evaluation of clinical and translational science grants.MethodsThe University of California, Irvine Institute for Clinical and Translational Sciences (ICTS) engaged 21 community reviewers alongside scientific reviewers in a 2-stage process of evaluating research proposals. In Stage 1 reviewers scored proposals, and during Stage 2 two study sections convened: one a mix of community reviewers and scientific reviewers, and one only engaging scientific reviewers. In total, 4 studies were discussed by both study sections.ResultsComparisons of reviews revealed little difference between ratings of community reviewers and those of scientific reviewers, and that community reviewers largely refrained from critiquing scientific or technical aspects of proposals.ConclusionsThe findings suggest that involving community reviewers early in the grant cycle, and exposing them to the entirety of the review process, can bolster community engagement without compromising the rigor of grant evaluations.

Molecular Structural Changes of Plasticized PVC after UV Light Exposure

2013 ◽  
Vol 117 (50) ◽  
pp. 16336-16344 ◽  
Author(s):  
Jeanne M. Hankett ◽  
William R. Collin ◽  
Zhan Chen
Keyword(s):  
Structural Changes ◽  
Uv Light ◽  
Light Exposure ◽  
Plasticized Pvc

scholarly journals Crumbs limits oxidase-dependent signaling to maintain epithelial integrity and prevent photoreceptor cell death

2012 ◽  
Vol 198 (6) ◽  
pp. 991-998 ◽  
Author(s):  
François J.-M. Chartier ◽  
Émilie J.-L. Hardy ◽  
Patrick Laprise
Keyword(s):  
Nadph Oxidase ◽  
Molecular Mechanisms ◽  
Light Exposure ◽  
Photoreceptor Cells ◽  
Protective Role ◽  
Superoxide Production ◽  
Epithelial Tissue ◽  
Complex Activity ◽  
Tissue Organization ◽  
Nadph Oxidase Complex

Drosophila melanogaster Crumbs (Crb) and its mammalian orthologues (CRB1–3) share evolutionarily conserved but poorly defined roles in regulating epithelial polarity and, in photoreceptor cells, morphogenesis and stability. Elucidating the molecular mechanisms of Crb function is vital, as mutations in the human CRB1 gene cause retinal dystrophies. Here, we report that Crb restricts Rac1–NADPH oxidase-dependent superoxide production in epithelia and photoreceptor cells. Reduction of superoxide levels rescued epithelial defects in crb mutant embryos, demonstrating that limitation of superoxide production is a crucial function of Crb and that NADPH oxidase and superoxide contribute to the molecular network regulating epithelial tissue organization. We further show that reduction of Rac1 or NADPH oxidase activity or quenching of reactive oxygen species prevented degeneration of Crb-deficient retinas. Thus, Crb fulfills a protective role during light exposure by limiting oxidative damage resulting from Rac1–NADPH oxidase complex activity. Collectively, our results elucidate an important mechanism by which Crb functions in epithelial organization and the prevention of retinal degeneration.

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

2005 ◽  
Vol 169 (4) ◽  
pp. 635-646 ◽  
Author(s):  
Slobodan Beronja ◽  
Patrick Laprise ◽  
Ophelia Papoulas ◽  
Milena Pellikka ◽  
John Sisson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Transport ◽  
Basolateral Membrane ◽  
Photoreceptor Cells ◽  
Secretory Vesicles ◽  
Membrane Domains ◽  
Animal Cells ◽  
Light Sensing ◽  
Membrane Growth ◽  
Essential Function

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.

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