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GTP avoidance in Tetrahymena thermophila requires tyrosine kinase activity, intracellular calcium, NOS, and guanylyl cyclase.

Bartholomew J, Reichart J, Mundy R, Recktenwald J, Keyser S, Riddle M, Kuruvilla H - Purinergic Signal. (2007)

Bottom Line: However, the intracellular mechanisms involved in GTP avoidance have not been previously documented.Similarly, pharmacological inhibitors of phospholipase C, NOS, and guanylyl cyclase all eliminated Tetrahymena avoidance to GTP.Immunofluorescence data shows evidence of tyrosine kinase activity in the cilia, suggesting that this enzyme activity could be directly involved in ciliary reversal.

View Article: PubMed Central - PubMed

Affiliation: Department of Science and Mathematics, Cedarville University, Cedarville, OH, 45314, USA.

ABSTRACT
Guanosine 5'-triphosphate (GTP) is a chemorepellent in Tetrahymena thermophila that has been shown to stimulate cell division as well as ciliary reversal. Previous studies have proposed that GTP avoidance is linked to a receptor-mediated, calcium-based depolarization. However, the intracellular mechanisms involved in GTP avoidance have not been previously documented. In this study, we examine the hypothesis that GTP signals through a tyrosine kinase pathway in T. thermophila. Using behavioral assays, enzyme immunosorbent assays, Western blotting, and immunofluorescence, we present data that implicate a tyrosine kinase, phospholipase C, intracellular calcium, nitric oxide synthase (NOS) and guanylyl cyclase in GTP signaling. The tyrosine kinase inhibitor genistein eliminates GTP avoidance in Tetrahymena in behavioral assays. Similarly, pharmacological inhibitors of phospholipase C, NOS, and guanylyl cyclase all eliminated Tetrahymena avoidance to GTP. Immunofluorescence data shows evidence of tyrosine kinase activity in the cilia, suggesting that this enzyme activity could be directly involved in ciliary reversal.

No MeSH data available.


Related in: MedlinePlus

Guanosine 5'-triphosphate (GTP) exposure increases tyrosine kinase activity in Tetrahymena. a–c Immunofluorescence labeling of phosphotyrosines in Tetrahymena is evidence of tyrosine kinase activity. Control (a), GTP-exposed (b), and genistein-treated GTP-exposed cells (c) were fixed and labeled with a polyclonal antiphosphotyrosine antibody. GTP-exposed cells showed higher fluorescence intensity than did control cells and cells treated with the tyrosine kinase inhibitor genistein. In addition, GTP-exposed cells showed ciliary staining in addition to punctuate staining of the cytosol. Total magnification 400×. Cell length ∼ 50 μm. d Western blot of whole-cell extract obtained from control and GTP-exposed cells using a polyclonal antiphosphotyrosine antibody shows increased phosphorylation levels in extract taken from GTP-exposed cells (lane 2) relative to extract from control cells (lane 3). A 66-kDa phosphoprotein stained similarly in both the GTP and control lanes, whereas bands of 42, 35, and 21 kDa were more heavily stained in the GTP lane relative to the control lane. Molecular weight markers are shown in the first lane (molecular weights in kDa). A Coomassie-stained sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) run as a loading control showed equivalent staining of proteins in all lanes (not shown)
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Fig3: Guanosine 5'-triphosphate (GTP) exposure increases tyrosine kinase activity in Tetrahymena. a–c Immunofluorescence labeling of phosphotyrosines in Tetrahymena is evidence of tyrosine kinase activity. Control (a), GTP-exposed (b), and genistein-treated GTP-exposed cells (c) were fixed and labeled with a polyclonal antiphosphotyrosine antibody. GTP-exposed cells showed higher fluorescence intensity than did control cells and cells treated with the tyrosine kinase inhibitor genistein. In addition, GTP-exposed cells showed ciliary staining in addition to punctuate staining of the cytosol. Total magnification 400×. Cell length ∼ 50 μm. d Western blot of whole-cell extract obtained from control and GTP-exposed cells using a polyclonal antiphosphotyrosine antibody shows increased phosphorylation levels in extract taken from GTP-exposed cells (lane 2) relative to extract from control cells (lane 3). A 66-kDa phosphoprotein stained similarly in both the GTP and control lanes, whereas bands of 42, 35, and 21 kDa were more heavily stained in the GTP lane relative to the control lane. Molecular weight markers are shown in the first lane (molecular weights in kDa). A Coomassie-stained sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) run as a loading control showed equivalent staining of proteins in all lanes (not shown)

Mentions: In an attempt to localize the phosphotyrosines within the cell, immunofluorescence experiments were performed using a polyclonal antiphosphotyrosine antibody. Three separate trials were done with this antibody, and representative photographs are compiled in Fig. 3a–c. GTP-exposed cells (Fig. 3b) showed higher overall fluorescence intensity than did the controls and genistein-treated GTP-exposed cells (Fig. 3a,c). Ciliary staining was seen in the GTP-exposed cells, along with punctuate staining of the cytosol. The control and genistein-treated GTP-exposed cells showed the same cytosolic staining pattern, but ciliary staining was not detected in these control groups, perhaps because the overall fluorescence level of these cells was so low.Fig. 3


GTP avoidance in Tetrahymena thermophila requires tyrosine kinase activity, intracellular calcium, NOS, and guanylyl cyclase.

Bartholomew J, Reichart J, Mundy R, Recktenwald J, Keyser S, Riddle M, Kuruvilla H - Purinergic Signal. (2007)

Guanosine 5'-triphosphate (GTP) exposure increases tyrosine kinase activity in Tetrahymena. a–c Immunofluorescence labeling of phosphotyrosines in Tetrahymena is evidence of tyrosine kinase activity. Control (a), GTP-exposed (b), and genistein-treated GTP-exposed cells (c) were fixed and labeled with a polyclonal antiphosphotyrosine antibody. GTP-exposed cells showed higher fluorescence intensity than did control cells and cells treated with the tyrosine kinase inhibitor genistein. In addition, GTP-exposed cells showed ciliary staining in addition to punctuate staining of the cytosol. Total magnification 400×. Cell length ∼ 50 μm. d Western blot of whole-cell extract obtained from control and GTP-exposed cells using a polyclonal antiphosphotyrosine antibody shows increased phosphorylation levels in extract taken from GTP-exposed cells (lane 2) relative to extract from control cells (lane 3). A 66-kDa phosphoprotein stained similarly in both the GTP and control lanes, whereas bands of 42, 35, and 21 kDa were more heavily stained in the GTP lane relative to the control lane. Molecular weight markers are shown in the first lane (molecular weights in kDa). A Coomassie-stained sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) run as a loading control showed equivalent staining of proteins in all lanes (not shown)
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2377316&req=5

Fig3: Guanosine 5'-triphosphate (GTP) exposure increases tyrosine kinase activity in Tetrahymena. a–c Immunofluorescence labeling of phosphotyrosines in Tetrahymena is evidence of tyrosine kinase activity. Control (a), GTP-exposed (b), and genistein-treated GTP-exposed cells (c) were fixed and labeled with a polyclonal antiphosphotyrosine antibody. GTP-exposed cells showed higher fluorescence intensity than did control cells and cells treated with the tyrosine kinase inhibitor genistein. In addition, GTP-exposed cells showed ciliary staining in addition to punctuate staining of the cytosol. Total magnification 400×. Cell length ∼ 50 μm. d Western blot of whole-cell extract obtained from control and GTP-exposed cells using a polyclonal antiphosphotyrosine antibody shows increased phosphorylation levels in extract taken from GTP-exposed cells (lane 2) relative to extract from control cells (lane 3). A 66-kDa phosphoprotein stained similarly in both the GTP and control lanes, whereas bands of 42, 35, and 21 kDa were more heavily stained in the GTP lane relative to the control lane. Molecular weight markers are shown in the first lane (molecular weights in kDa). A Coomassie-stained sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) run as a loading control showed equivalent staining of proteins in all lanes (not shown)
Mentions: In an attempt to localize the phosphotyrosines within the cell, immunofluorescence experiments were performed using a polyclonal antiphosphotyrosine antibody. Three separate trials were done with this antibody, and representative photographs are compiled in Fig. 3a–c. GTP-exposed cells (Fig. 3b) showed higher overall fluorescence intensity than did the controls and genistein-treated GTP-exposed cells (Fig. 3a,c). Ciliary staining was seen in the GTP-exposed cells, along with punctuate staining of the cytosol. The control and genistein-treated GTP-exposed cells showed the same cytosolic staining pattern, but ciliary staining was not detected in these control groups, perhaps because the overall fluorescence level of these cells was so low.Fig. 3

Bottom Line: However, the intracellular mechanisms involved in GTP avoidance have not been previously documented.Similarly, pharmacological inhibitors of phospholipase C, NOS, and guanylyl cyclase all eliminated Tetrahymena avoidance to GTP.Immunofluorescence data shows evidence of tyrosine kinase activity in the cilia, suggesting that this enzyme activity could be directly involved in ciliary reversal.

View Article: PubMed Central - PubMed

Affiliation: Department of Science and Mathematics, Cedarville University, Cedarville, OH, 45314, USA.

ABSTRACT
Guanosine 5'-triphosphate (GTP) is a chemorepellent in Tetrahymena thermophila that has been shown to stimulate cell division as well as ciliary reversal. Previous studies have proposed that GTP avoidance is linked to a receptor-mediated, calcium-based depolarization. However, the intracellular mechanisms involved in GTP avoidance have not been previously documented. In this study, we examine the hypothesis that GTP signals through a tyrosine kinase pathway in T. thermophila. Using behavioral assays, enzyme immunosorbent assays, Western blotting, and immunofluorescence, we present data that implicate a tyrosine kinase, phospholipase C, intracellular calcium, nitric oxide synthase (NOS) and guanylyl cyclase in GTP signaling. The tyrosine kinase inhibitor genistein eliminates GTP avoidance in Tetrahymena in behavioral assays. Similarly, pharmacological inhibitors of phospholipase C, NOS, and guanylyl cyclase all eliminated Tetrahymena avoidance to GTP. Immunofluorescence data shows evidence of tyrosine kinase activity in the cilia, suggesting that this enzyme activity could be directly involved in ciliary reversal.

No MeSH data available.


Related in: MedlinePlus