Ocadaic acid treatment alters the intracellular localization of caveolin-1 and caveolin-2 in HepG2 cells

In this paper we provide evidences that protein phosphatases could regulate the intracellular localization of caveolin isoforms in a hepatoma cell line (HepG2). Ocadaic acid (OA) - a serine/threonine phosphatase inhibitor – was used in various concentrations (4nM and 100nM) to study the localization of caveolin-1 and caveolin-2 in HepG2 cells. Using fluorescent and confocal immunocytochemistry we have found that OA in both concentrations has significantly altered the intracellular localization and distribution of the caveolin-1 and caveolin-2 as well. In control (-OA treatment) the caveolin-1 was present in discrete punctate structures in the cytoplasm and also on the cell membrane. Caveolin-2 has partly overlapped with caveolin-1, but a significant amount caveolin-2 was detected around the nucleus. After OA (4 and 100 nM) treatment caveolin-1 has disappeared from the cell membrane, it was present mainly in the cytoplasm in larger vesicle or vacuole-like structures that were arranged along the cables of the cytoskeleton. In many cases caveolin-2 was found to colocalize with caveolin-1, but there was always a significant amount of caveolin-2 present around the nucleus. Immunoprecipitation and Western blot analysis revealed that in OA-treated cells a ~24 kDa protein identified as caveolin-2 was strongly phosphorylated on tyrosine residues. The effect of OA was not reversible, since the removal of OA has not resulted in the dephosphorylation of caveolin-2 and the perinuclear localization of caveolin-2 remained. Our data indicate that phophorylation of caveolin-2 can alter not only the intracellular localization of caveolin isoforms but also the distribution of caveolae. The cytoskeleton seems to play an important role in the normal and altered distribution of caveolae, and the tyrosine phosphorylation or the absence of dephosphorylation of caveolin-2 isoform can inhibit the recycling of caveolae

Localization of Caveolin-1 and C-SRC in Mature and Differentiating Photoreceptors: Raft Proteins Co-Distribute With Rhodopsin During Development

Numerous biochemical and morphological studies have provided insight into the distribution pattern of caveolin-1 and the presence of membrane rafts in the vertebrate retina. To date however, studies have not addressed the localization profile of raft specific proteins during development. Therefore the purpose of our studies was to follow the localization pattern of caveolin-1, phosphocaveolin-1 and c-src in the developing retina and compare it to that observed in adults. Specific antibodies were used to visualize the distribution of caveolin-1, c-src, a kinase phosphorylating caveolin-1, and phospho-caveolin-1. The labeling pattern of this scaffolded complex was compared to those of rhodopsin and rhodopsin kinase. Samples were analyzed at various time points during postnatal development and compared to adult retinas. The immunocytochemical studies were complemented with immunoblots and immunoprecipitation studies. In the mature retina caveolin-1 and c-src localized mainly to the cell body and IS of photoreceptors, with only very weakly labeled OS. In contrast, phospho-caveolin-1 was only detectable in the OS of photoreceptors. During development we followed the expression and distribution profile of these proteins in a temporal sequence with special attention to the period when OS formation is most robust. Double labeling immunocytochemistry and immunoprecipitation showed rhodopsin to colocalize and co-immunoprecipitate with caveolin-1 and c-src. Individual punctate structures between the outer limiting membrane and the outer plexiform layer were seen at P10 to be labeled by both rhodopsin and caveolin-1 as well as by rhodopsin and c-src, respectively. These studies suggest that membrane raft specific proteins are co-distributed during development, thereby pointing to a role for such complexes in OS formation. In addition, the presence of small punctate structures containing caveolin-1, c-src and rhodopsin raise the possibility that these proteins may transport together to OS during development and that caveolin-1 exists predominantly in a phosphorylated form in the OS

A fotoreceptor-fejlődés sejt- és molekuláris biológiája = Cell and molecular biology of photoreceptor development

Az opszinváltás szabályozása fajonként eltérő, tiroxin és TRbéta2 nélkülözhetetlen a zöld csapfejlődéshez. A receptormegoszlás időbeli és térbeli mintázatával igazoltuk a tiroxint mediáló TRbéta2 szerepét. Tenyésztő módszerünkkel a fotoreceptor-differenciálódás szérummentes környezetben is végbemegy, a világon először definitív médiumban vizsgálható. Más faktorok (A- és E-vitamin, BDNF) is hatnak a pigment-expresszióra. Szelektív neurotrophin antagonisták az opszin és a TrkB receptor kapcsoltságára utalnak. Az erythropoietin retinális expressziója HIF1-α szabályozás alatt áll és időben változik a fejlődés korai szakaszában. Szemnyitás után a retinában lecsökken a transzdukciós molekulák szintje, kivéve a fotoreceptorokat. A sejttestben szintetizálódnak, közös lipid rafton kerülnek a kültagba és játszanak szerepet a fejlődésben. Az STK38L gén homozigóta mutációja befolyásolja a fotoreceptor-fejlődést. Sejthalál és proliferáció egyaránt jelen van. Hibrid sejtek jelennek meg pálcika-, kisebb mértékben S-opszin termeléssel. A differenciált, mutáns sejtek a degenerációs gén hatására megőrzik osztódóképességüket. A melatonin éjszaka termelődik, éjjeli világítás a hormontermelés gátlásával patológiás folyamatokat (emlő és colorectalis carcinoma) okozhat. A pineális szerv emlősben elvesztette fotoreceptor működését és szimpatikus rostokon a retinából kap információt. Mivel a pineális melatoninképzést rövidhullámú fény gátolja, éjjeli műszakban hosszúhullámú megvilágítást kell használni. | Regulation of opsin-switch varies across species. Thyroxin and TRß2 receptor is essential for green cone development. Spatial and temporal receptor distribution proved the role of TRß2 mediating thyroxin. Photoreceptor differentiation is completed in our culture method in serum-free medium, allowing its examination in a definitive paradigm. Other factors such as Vitamins A and E, BDNF) also influence visual pigment expression. Selective neurotrophin antagonists reveal the close connection of opsin and Trkß. Retinal expression of erythropoietin is under the regulation of HIF1-α, and changes during development. Transduction molecule levels decrease after eye opening, except for photoreceptors. They are synthesized in the cell body, located and transported in the outer segment on common lipid rafts and play a developmental role. Homozygotic mutation of STK38L influences photoreceptor development. Cell death and proliferation are equally present. Hybrid photoreceptors appear that express rod and, to a lesser extent, S-opsin. The degenerated, hybrid mutants retain their capacity to divide. Melatonin is produced at night. Nocturnal light exposition inhibiting hormone production may generate pathological processes (e.g.: carcinomas). The pineal organ in mammals has lost its photoreceptor function and receives information from the retina via sympathetic fibers. Since short wave light inhibits melatonin production, long-wave illumination is recommended during night-shift

Localization of Caveolin-1 and C-Src in Mature and Differentiating Photoreceptors: Raft Proteins Co-Distribute with Rhodopsin During Development

Numerous biochemical and morphological studies have provided insight into the distribution pattern of caveolin-1 and the presence of membrane rafts in the vertebrate retina. To date however, studies have not addressed the localization profile of raft specific proteins during development. Therefore the purpose of our studies was to follow the localization pattern of caveolin-1, phospho-caveolin-1 and c-src in the developing retina and compare it to that observed in adults. Specific antibodies were used to visualize the distribution of caveolin-1, c-src, a kinase phosphorylating caveolin-1, and phospho-caveolin-1. The labeling pattern of this scaffolded complex was compared to those of rhodopsin and rhodopsin kinase. Samples were analyzed at various time points during postnatal development and compared to adult retinas. The immunocytochemical studies were complemented with immunoblots and immunoprecipitation studies. In the mature retina caveolin-1 and c-src localized mainly to the cell body and IS of photoreceptors, with only very weakly labeled OS. In contrast, phospho-caveolin-1 was only detectable in the OS of photoreceptors. During development we followed the expression and distribution profile of these proteins in a temporal sequence with special attention to the period when OS formation is most robust. Double labeling immunocytochemistry and immunoprecipitation showed rhodopsin to colocalize and co-immunoprecipitate with caveolin-1 and c-src. Individual punctate structures between the outer limiting membrane and the outer plexiform layer were seen at P10 to be labeled by both rhodopsin and caveolin-1 as well as by rhodopsin and c-src, respectively. These studies suggest that membrane raft specific proteins are co-distributed during development, thereby pointing to a role for such complexes in OS formation. In addition, the presence of small punctate structures containing caveolin-1, c-src and rhodopsin raise the possibility that these proteins may transport together to OS during development and that caveolin-1 exists predominantly in a phosphorylated form in the OS. © 2011 Springer Science+Business Media B.V

Fényérzékeny molekulák és fotoreceptorok a gerincesek retinájában és tobozmirigyében = Photosensitive molecules and photoreceptors in the vertebrate retina and pineal gland

Míg az emlősök legtöbbjében, amelyekben legalább kétféle színérzékenységű fotoreceptor található, kimutatható a transzdifferenciáció, vagyis a rövidhullám-érzékeny (kék) csapok korai megjelenése, és ezek egy részében a középhullám-érzékeny (zöld) pigment későbbi expressziója. Az eredeti kék pigment eltűnése eredményezi a definitív zöld csapokat, míg azok, amelyekben a zöld pigment egyáltalán nem jelenik meg, adják a kék csap-populációt. Elvetettük a hipotézist, hogy a monokromatikus fajokan is a kék-pigment expresszióján keresztül vezet az út. Felnőttben is előfordulnak két pigmentet tartalmazó csapok. A transzdifferenciáció (csap-fejlődés) nem áll meg újszülött korban, hanem folytatódik felnőttben is. Ezek idegi őssejtek, amelyek a retina regenerációjában játszhatnak szerepet (anti-PCNA). A csapok differenciálódásában közrejátszó tényezők közül a BDNF, az NT-3 és a TrkB szerepet játszanak a kék-zöld átalakulásban. Tenyésztés és immuncitokémia bizonyítja, hogy a kék csapok fejlődéséhez elegendő a megfelelő feltételek fennállása. A zöld csapok megjelenéséhez azonban az említett tényezők is szükségesek. A nem-vizuális fotoreceptorok feladata a diurnális ritmus beállítása. A bennük lévő cryptochromok kék-érzékenyek. Az ékszakai megvilágítás patológiás hatásait (emlőrák, colorectalis tumorok, stb.) a pineális melatonin gátlása okozza. Ezek redukálhatók kék fény-mentes megvilágítással, azaz megfelelő színszűrők használatával. | In mammals that possess two color-specific photoreceptors, transdifferentiation is present. That means the early appearance of short wave (blue) cones and the later expression of middle wave (green) pigment in some of them. The disappearance of the original blue pigment results in the development of definitive green cones. Those in which no green pigment appears at all will make the blue cone population. The hypothesis that in monochromatic species green cones come about through the transitory appearance of blue pigment has been rejected. Dual cones expressing two visual pigments occur also in adults. Obviously, the transdifferentiation does not come to an end in the early postnatal period, rather it goes on in the adulthood. These might be neuronal stem cells playing a role in the regeneration of the retina (anti-PCNA). Of the factors having a role in the cone differentiation, BDNF, NT-3 and TrkB seem to be effective in the blue-green transition. Immunocytochemistry and tissue culture prove that proper culture technique is enough for the development of blue cones, however the above factors are indispensable for the green cones. Non-visual photoreceptors play a role in the entrainment of the diurnal clock. Their cryptochromes proved to be blue-sensitive. The pathologic effects of night work are attributed to the inhibition of pineal melatonin. The adverse reactions might be reduced with blue light-free illumination, with proper color filters

Localization of Caveolin-1 and C-Src in Mature and Differentiating Photoreceptors: Raft Proteins Co-Distribute With Rhodopsin During Development

Numerous biochemical and morphological studies have provided insight into the distribution pattern of caveolin-1 and the presence of membrane rafts in the vertebrate retina. To date however, studies have not addressed the localization profile of raft specific proteins during development. Therefore the purpose of our studies was to follow the localization pattern of caveolin-1, phospho-caveolin-1 and c-src in the developing retina and compare it to that observed in adults. Specific antibodies were used to visualize the distribution of caveolin-1, c-src, a kinase phosphorylating caveolin-1, and phospho-caveolin-1. The labeling pattern of this scaffolded complex was compared to those of rhodopsin and rhodopsin kinase. Samples were analyzed at various time points during postnatal development and compared to adult retinas. The immunocytochemical studies were complemented with immunoblots and immunoprecipitation studies. In the mature retina caveolin-1 and c-src localized mainly to the cell body and IS of photoreceptors, with only very weakly labeled OS. In contrast, phospho-caveolin-1 was only detectable in the OS of photoreceptors. During development we followed the expression and distribution profile of these proteins in a temporal sequence with special attention to the period when OS formation is most robust. Double labeling immunocytochemistry and immunoprecipitation showed rhodopsin to colocalize and co-immunoprecipitate with caveolin-1 and c-src. Individual punctate structures between the outer limiting membrane and the outer plexiform layer were seen at P10 to be labeled by both rhodopsin and caveolin-1 as well as by rhodopsin and c-src, respectively. These studies suggest that membrane raft specific proteins are co-distributed during development, thereby pointing to a role for such complexes in OS formation. In addition, the presence of small punctate structures containing caveolin-1, c-src and rhodopsin raise the possibility that these proteins may transport together to OS during development and that caveolin-1 exists predominantly in a phosphorylated form in the OS. © 2011 Springer Science+Business Media B.V

Distribution of caveolin isoforms in the lemur retina

The distribution of caveolin isoforms was previously evaluated in the retinas of different species, but has not yet been described in the primate retina. In this study, the distribution of caveolins was assessed via immunochemistry using isoform-specific antibodies in the retina of the black-and-white ruffed lemur. Here, we report the presence of a variety of caveolin isoforms in many layers of the lemur retina. As normal human retinas were not available for research and the retinas of primates are fairly similar to those of humans, the lemur retina can be utilized as a model for caveolin distribution in normal humans

Análise dos antígenos de histocompatibilidade leucocitária de classe II-DR em crianças e adolescentes brasileiros com lúpus eritematoso sistêmico

OBJECTIVE: To analyze the frequency of human leukocyte antigens class II-DR in children and adolescents with systemic lupus erythematosus. PATIENTS AND METHODS: Fifty-fiveBrazilian systemic lupus erythematosus children and adolescents and 308 healthy individuals were studied. Gender, race, and age of onset of systemic lupus erythematosus were recorded. The human leukocyte antigens typing of class II-DR was carried out by polymerase chain reaction amplification with sequence-specific primers (PCR-SSP). Data were analyzed statistically using the chi square test with Yates' correction, Fisher's exact test, and Bonferroni's correction. RESULTS: Human leukocyte antigen-DR 15 was the most frequently detected antigen in this group of children and adolescents, and it also occurred more frequently in the female group, in children with onset of systemic lupus erythematosus between 0 and 9 years and between 10 to 14 years, and in the Black race group, but these associations were not statistically significants. CONCLUSION: In this group of children and adolescents with a high degree of racial admixture, we could not verify a significant association between human leukocyte antigens class II-DR and systemic lupus erythematosus.OBJETIVO: Analisar a freqüência dos antígenos de histocompatibilidade leucocitária de classe II-DR em crianças e adolescentes com o lúpus eritematoso sistêmico. PACIENTES E MÉTODOS: Cinqüenta e cinco crianças e adolescentes lúpicos brasileiros e 308 indivíduos sadios foram estudados. Os sexos, os grupos étnicos e as idades de início da doença foram anotados. A tipagem de histocompatibilidade leucocitária de classe II-DR foi realizada pela reação de polimerase em cadeia com amplificação de sondas de seqüência específica (PCR-SSP). Na análise estatística foram utilizados o teste de qui-quadrado com correção de Yates, o teste exato de Fisher e a correção de Bonferroni. RESULTADOS: A histocompatibilidade leucocitária-DR15 foi a mais freqüente neste grupo de crianças e adolescentes, sendo também mais freqüente nas mulheres, nas crianças com idade de início da doença entre zero e nove anos e entre 10 e 14 anos e nas crianças de raça negra, mas estas correlações não foram estatisticamente significativas. CONCLUSÃO: Neste grupo de crianças e adolescentes com alto grau de miscigenação não pudemos observar associação significativa entre os antígenos de histocompatibilidade leucocitária de classe II-DR e o lúpus eritematoso sistêmico