scholarly journals Regulation of organelle transport in melanophores by calcineurin.

1990 ◽  
Vol 111 (5) ◽  
pp. 1939-1948 ◽  
Author(s):  
C D Thaler ◽  
L T Haimo
Keyword(s):  
Protein Phosphorylation ◽  
Phosphatase Activity ◽  
Okadaic Acid ◽  
Pigment Granule ◽  
Retrograde Transport ◽  
Organelle Transport ◽  
Calmodulin Antagonists ◽  
Activity Inhibition ◽  
Pigment Aggregation ◽  
Calcineurin Activity

Previous studies have shown that pigment granule dispersion and aggregation in melanophores of the African cichlid, Tilapia mossambica, are regulated by protein phosphorylation and dephosphorylation, respectively (Rozdzial, M. M., and L. T. Haimo. 1986. Cell. 47:1061-1070). The present studies suggest that calcineurin, a Ca2+/calmodulin-stimulated phosphatase, is the endogenous phosphatase that mediates pigment aggregation in melanophores. Aggregation, but not dispersion, is inhibited by okadaic acid at concentrations consistent with an inhibition of calcineurin activity. Inhibition of aggregation in melanophores that have been BAPTA loaded or treated with calmodulin antagonists implicate Ca2+ and calmodulin, respectively, in this process. Moreover, addition of calcineurin rescues aggregation in lysed melanophores which are otherwise incapable of aggregating pigment. Immunoblotting with an anticalcineurin IgG reveals that calcineurin is a component of the dermis, which contains the melanophores, and indirect immunofluorescence localizes calcineurin specifically to the melanophores. Finally, this antibody, which inhibits calcineurin's phosphatase activity (Tash, J. S., M. Krinks, J. Patel, R. L. Means, C. B. Klee, and A. R. Means. 1988. J. Cell Biol. 106:1625-1633), inhibits aggregation but has no effect on pigment granule dispersion. Together these studies indicate that retrograde transport of pigment granules to the melanophore cell center depends upon the participation of calcineurin.

scholarly journals CK1 activates minus-end–directed transport of membrane organelles along microtubules

2011 ◽  
Vol 22 (8) ◽  
pp. 1321-1329 ◽  
Author(s):  
Kazuho Ikeda ◽  
Olga Zhapparova ◽  
Ilya Brodsky ◽  
Irina Semenova ◽  
Jennifer S. Tirnauer ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Pigment Granule ◽  
Organelle Transport ◽  
Signaling Mechanism ◽  
Pigment Granules ◽  
Dynein Motor ◽  
Dynein Intermediate Chain ◽  
Phosphatase 2A ◽  
Pigment Aggregation ◽  
Rapid Activation

Microtubule (MT)-based organelle transport is driven by MT motor proteins that move cargoes toward MT minus-ends clustered in the cell center (dyneins) or plus-ends extended to the periphery (kinesins). Cells are able to rapidly switch the direction of transport in response to external cues, but the signaling events that control switching remain poorly understood. Here, we examined the signaling mechanism responsible for the rapid activation of dynein-dependent MT minus-end–directed pigment granule movement in Xenopus melanophores (pigment aggregation). We found that, along with the previously identified protein phosphatase 2A (PP2A), pigment aggregation signaling also involved casein kinase 1ε (CK1ε), that both enzymes were bound to pigment granules, and that their activities were increased during pigment aggregation. Furthermore we found that CK1ε functioned downstream of PP2A in the pigment aggregation signaling pathway. Finally, we discovered that stimulation of pigment aggregation increased phosphorylation of dynein intermediate chain (DIC) and that this increase was partially suppressed by CK1ε inhibition. We propose that signal transduction during pigment aggregation involves successive activation of PP2A and CK1ε and CK1ε-dependent phosphorylation of DIC, which stimulates dynein motor activity and increases minus-end–directed runs of pigment granules.

Ca2+ and calmodulin-dependent protein phosphatase from Leishmania donovani

Parasitology ◽  
1999 ◽  
Vol 118 (6) ◽  
pp. 567-573 ◽  
Author(s):  
C. BANERJEE ◽  
D. SARKAR ◽  
A. BHADURI
Keyword(s):  
Cyclosporin A ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Leishmania Donovani ◽  
Protein Phosphatases ◽  
Cross Reactivity ◽  
Calmodulin Antagonists ◽  
Type Protein ◽  
Complete Obliteration ◽  
Dependent Protein

A protein phosphatase exclusively dependent upon micromolar amounts of Ca2+ and calmodulin has been identified and partially purified from Leishmania spp. Complete obliteration of its activity is observed in the presence of calmodulin antagonists such as trifluoperazine, fluphenazine and calmidazolium. Relative insensitivity to okadaic acid and lack of activation in the absence of Ca2+ and calmodulin distinguishes this enzyme from PP1, PP2A and PP2C-type protein phosphatases. Cross-reactivity of the enzyme was observed with antibodies that recognize both the A and B chains of calcineurin, a PP2B type Ca2+ and calmodulin-dependent phosphatase from brain. FK506, an immunosuppresive drug that inhibits the enzyme from other sources inhibited the enzyme only in the presence of exogenous FK binding protein, whereas Cyclosporin A inhibited the enzyme in crude preparations. Taken together these results reveal the presence of a Ca2+ and calmodulin-dependent phosphatase from Leishmania. This is the first report of the presence of a PP2B-type protein phosphatase from a pathogenic protozoa.

scholarly journals Protein kinase C activation antagonizes melatonin-induced pigment aggregation in Xenopus laevis melanophores.

1992 ◽  
Vol 119 (6) ◽  
pp. 1515-1521 ◽  
Author(s):  
D Sugden ◽  
S J Rowe
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Xenopus Laevis ◽  
Pigment Granule ◽  
Melatonin Receptors ◽  
Kinase C ◽  
Pigment Granules ◽  
Slow Aggregation ◽  
Pigment Aggregation ◽  
Induced Aggregation

The pineal hormone, melatonin (5-methoxy N-acetyltryptamine) induces a rapid aggregation of melanin-containing pigment granules in isolated melanophores of Xenopus laevis. Treatment of melanophores with activators of protein kinase C (PKC), including phorbol esters, mezerein and a synthetic diacylglycerol, did not affect pigment granule distribution but did prevent and reverse melatonin-induced pigment aggregation. This effect was blocked by an inhibitor of PKC, Ro 31-8220. The inhibitory effect was not a direct effect on melatonin receptors, per se, as the slow aggregation induced by a high concentration of an inhibitor of cyclic AMP-dependent protein kinase (PKA), adenosine 3',5'-cyclic monophosphothioate, Rp-diastereomer (Rp-cAMPS), was also reversed by PKC activation. Presumably activation of PKC, like PKA activation, stimulates the intracellular machinery involved in the centrifugal translocation of pigment granules along microtubules. alpha-Melanocyte stimulating hormone (alpha-MSH), like PKC activators, overcame melatonin-induced aggregation but this response was not blocked by the PKC inhibitor, Ro 31-8220. This data indicates that centrifugal translocation (dispersion) of pigment granules in Xenopus melanophores can be triggered by activation of either PKA, as occurs after alpha-MSH treatment, or PKC. The very slow aggregation in response to inhibition of PKA with high concentrations of Rp-cAMPS, suggests that the rapid aggregation in response to melatonin may involve multiple intracellular signals in addition to the documented Gi-mediated inhibition of adenylate cyclase.

Organelle motility and metabolism in axons vs dendrites of cultured hippocampal neurons

1996 ◽  
Vol 109 (5) ◽  
pp. 971-980 ◽  
Author(s):  
C.C. Overly ◽  
H.I. Rieff ◽  
P.J. Hollenbeck
Keyword(s):  
Regional Variation ◽  
Hippocampal Neurons ◽  
Retrograde Transport ◽  
Organelle Transport ◽  
Microtubule Polarity ◽  
Large Phase ◽  
Organelle Motility ◽  
The Difference ◽  
Metabolic Properties ◽  
Cultured Hippocampal Neurons

Regional regulation of organelle transport seems likely to play an important role in establishing and maintaining distinct axonal and dendritic domains in neurons, and in managing differences in local metabolic demands. In addition, known differences in microtubule polarity and organization between axons and dendrites along with the directional selectivity of microtubule-based motor proteins suggest that patterns of organelle transport may differ in these two process types. To test this hypothesis, we compared the patterns of movement of different organelle classes in axons and different dendritic regions of cultured embryonic rat hippocampal neurons. We first examined the net direction of organelle transport in axons, proximal dendrites and distal dendrites by video-enhanced phase-contrast microscopy. We found significant regional variation in the net transport of large phase-dense vesicular organelles: they exhibited net retrograde transport in axons and distal dendrites, whereas they moved equally in both directions in proximal dendrites. No significant regional variation was found in the net transport of mitochondria or macropinosomes. Analysis of individual organelle motility revealed three additional differences in organelle transport between the two process types. First, in addition to the difference in net transport direction, the large phase-dense organelles exhibited more persistent changes in direction in proximal dendrites where microtubule polarity is mixed than in axons where microtubule polarity is uniform. Second, while the net direction of mitochondrial transport was similar in both processes, twice as many mitochondria were motile in axons than in dendrites. Third, the mean excursion length of moving mitochondria was significantly longer in axons than in dendrites. To determine whether there were regional differences in metabolic activity that might account for these motility differences, we labeled mitochondria with the vital dye, JC-1, which reveals differences in mitochondrial transmembrane potential. Staining of neurons with this dye revealed a greater proportion of highly charged, more metabolically active, mitochondria in dendrites than in axons. Together, our data reveal differences in organelle motility and metabolic properties in axons and dendrites of cultured hippocampal neurons.

Okadaic acid, a phosphatase inhibitor, decreases macrophage motility

1991 ◽  
Vol 260 (2) ◽  
pp. L105-L112 ◽  
Author(s):  
A. K. Wilson ◽  
A. Takai ◽  
J. C. Ruegg ◽  
P. de Lanerolle
Keyword(s):  
Protein Phosphorylation ◽  
Okadaic Acid ◽  
Light Chain ◽  
Intracellular Signaling ◽  
Mammalian Cells ◽  
Morphological Changes ◽  
Phosphoprotein Phosphatase ◽  
Phosphatase Inhibitor ◽  
Signaling Mechanisms ◽  
Dose Dependent

Cellular locomotion results from a series of spatially and temporally integrated reactions. The coordinated regulation of these reactions requires sensitive intracellular signaling mechanisms. Because protein phosphorylation reactions represent important signaling mechanisms in mammalian cells, we investigated the effect of okadaic acid, a phosphoprotein phosphatase inhibitor, on protein phosphorylation and macrophage motility. Okadaic acid was applied to rat alveolar macrophages, and motility was quantitated by a directed chemotaxis assay. Okadaic acid inhibits macrophage motility in a dose-dependent fashion; the concentrations for 50 and 100% inhibition were 3 and 25 microM, respectively. Protein phosphorylation studies demonstrated a 2.5-fold increase in total protein phosphorylation in macrophages treated with 25 microM okadaic acid. These experiments also demonstrated a dose-dependent increase in the phosphorylation of the 20-kDa light chain of myosin. Moreover, 25 microM okadaic acid 1) maximally increased myosin light chain phosphorylation by 6.6-fold, 2) raised the level of myosin associated with the cytoskeleton from a basal level of 47.0 to 96.7% of the total myosin, and 3) induced profound morphological changes as visualized by scanning electron microscopy. These data correlate an increase in protein phosphorylation with a decrease in macrophage motility. Furthermore, they suggest that phosphoprotein phosphatase inhibition may prevent motility by uncoupling coordinated processes, such as cytoskeletal reorganization, that are essential for macrophage motility.

Extended Release Tacrolimus (LCP-TAC) Prolongs Calcineurin Activity Inhibition During Drug Doses Intervals

2018 ◽  
Vol 102 ◽  
pp. S592-S593
Author(s):  
Pere Fontova ◽  
Raul Rigo-Bonnin ◽  
Anna Vidal-Alabró ◽  
Gema Cerezo ◽  
Oriol Bestard ◽  
...  
Keyword(s):  
Extended Release ◽  
Activity Inhibition ◽  
Drug Doses ◽  
Calcineurin Activity

scholarly journals Axonal transport of mitochondria along microtubules and F-actin in living vertebrate neurons.

1995 ◽  
Vol 131 (5) ◽  
pp. 1315-1326 ◽  
Author(s):  
R L Morris ◽  
P J Hollenbeck
Keyword(s):  
Sympathetic Neurons ◽  
Large Body ◽  
Retrograde Transport ◽  
Organelle Transport ◽  
Neuronal Cultures ◽  
Essentially Normal ◽  
Cytochalasin E ◽  
Mitochondrial Motility ◽  
Continuous Presence

A large body of evidence indicates that microtubules (MTs) conduct organelle transport in axons, but recent studies on extruded squid axoplasm have suggested that actin microfilaments (MFs) may also play a role in this process. To investigate the separate contributions to transport of each class of cytoskeletal element in intact vertebrate axons, we have monitored mitochondrial movements in chick sympathetic neurons experimentally manipulated to eliminate MTs, MFs, or both. First, we grew neurons in the continuous presence of: (a) cytochalasin E to create neurites which had never contained MFs; or (b) nocodazole or vinblastine to produce neurites which had never contained MTs. Mitochondria moved bidirectionally at normal velocities along the length of neurites which contained MTs and lacked MFs, but did not even enter neurites grown without MTs but containing MFs. In a second approach, we treated established neuronal cultures with cytoskeletal drugs to disrupt either MTs or MFs in axons already containing mitochondria. In cytochalasin-treated cells, which retained MTs but lacked MFs, average mitochondrial velocity increased in both directions, but net directional transport decreased. In vinblastine-treated cells, which lacked MTs but retained essentially normal levels of MFs, mitochondria continued to move bidirectionally but the average mitochondrial velocity and excursion length were reduced for both directions of movement, and the mitochondria spent threefold as much time moving in the retrograde as in the anterograde direction, resulting in net retrograde transport. Treatment of established cultures with both drugs produced neurites lacking MTs and MFs but still rich in neurofilaments; these showed a striking absence of any mitochondrial motility. These data indicate that axonal organelle transport can occur along both MTs and MFs in vivo, but with different velocities and net transport properties.

scholarly journals Inhibitory effect of okadaic acid on the p-nitrophenyl phosphate phosphatase activity of protein phosphatases

1991 ◽  
Vol 275 (1) ◽  
pp. 233-239 ◽  
Author(s):  
A Takai ◽  
G Mieskes
Keyword(s):  
Phosphatase Activity ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Inhibitory Effect ◽  
Enzyme Concentration ◽  
Alkaline Phosphatases ◽  
Nitrophenyl Phosphate ◽  
Type 1 Phosphatase

The phosphatase activities of type 2A, type 1 and type 2C protein phosphatase preparations were measured against p-nitrophenyl phosphate (pNPP), a commonly used substrate for alkaline phosphatases. Of the three types of phosphatase examined, the type 2A phosphatase exhibited an especially high pNPP phosphatase activity (119 +/- 8 mumol/min per mg of protein; n = 4). This activity was strongly inhibited by pico- to nano-molar concentrations of okadaic acid, a potent inhibitor of type 2A and type 1 protein phosphatases that has been shown to have no effect on alkaline phosphatases. The dose-inhibition relationship was markedly shifted to the right and became steeper by increasing the concentration of the enzyme, as predicted by the kinetic theory for tightly binding inhibitors. The enzyme concentration estimated by titration with okadaic acid agreed well with that calculated from the protein content and the molecular mass for type 2A phosphatase. These results strongly support the idea that the pNPP phosphatase activity is intrinsic to type 2A protein phosphatase and is not due to contamination by alkaline phosphatases. pNPP was also dephosphorylated, but at much lower rates, by type 1 phosphatase (6.4 +/- 8 nmol/min per mg of protein; n = 4) and type 2C phosphatase (1.2 +/- 3 nmol/min per mg of protein; n = 4). The pNPP phosphatase activity of the type 1 phosphatase preparation shows a susceptibility to okadaic acid similar to that of its protein phosphatase activity, whereas it was interestingly very resistant to inhibitor 2, an endogenous inhibitory factor of type 1 protein phosphatase. The pNPP phosphatase activity of type 2C phosphatase preparation was not affected by up to 10 microM-okadaic acid.

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