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Insect Stage-Specific Adenylate Cyclases Regulate Social Motility in African Trypanosomes

Miguel A. Lopez, Edwin A. Saada, Kent L. Hill
Miguel A. Lopez
aDepartment of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA
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Edwin A. Saada
aDepartment of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA
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Kent L. Hill
aDepartment of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, USA
bMolecular Biology Institute, University of California, Los Angeles, Los Angeles, California, USA
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DOI: 10.1128/EC.00217-14
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    FIG 1

    Social motility is regulated by a subset of procyclic stage-specific adenylate cyclases. (A) Gene-specific knockdowns targeting the indicated ACs were assayed for social motility in comparison with 2913 control cells (ctrl) on plates containing tetracycline to induce RNAi. (B) Social motility assay results are shown for the indicated knockdowns, maintained in the absence or presence of tetracycline (Tet) to induce RNAi. (C) Quantitation of the number of projections formed for wild-type cells (WT) or the indicated knockdown lines, maintained in the absence or presence of tetracycline. Mean values and standard errors are shown. P values (unpaired t test) are shown. Sample sizes: WT –Tet, n = 12; WT +Tet, n = 7; AC1 and -2KD –Tet, n = 24; AC1 and -2KD +Tet, n = 20; AC6KD –Tet, n = 16; AC6KD +Tet, n = 23; AC5KD –Tet, n = 36; AC5KD +Tet, n = 36.

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    Social motility is modulated by AC6 catalytic activity. (A) Social motility assays on AC6-UTR-knockdown cells (AC6 uKD), AC6-RNAi-immune cells (AC6-Ri), or the RNAi-immune catalytic domain mutant cells (AC6**-Ri). Cells were maintained in the absence or presence of tetracycline (Tet) to induce RNAi. Schematics at the top of the panel illustrate the region targeted for RNAi. RNAi targets the 3′ UTR of the endogenous AC6 gene, while transgenes are immune to RNAi, owing to the presence of an alternate UTR. (B) Northern blot probed with an AC6-specific probe (top). RNA was prepared from bloodstream cells (BSF), procyclic 2913 control cells (PCF), AC6-uKD cells, AC6-Ri cells, or AC6**-Ri cells maintained with or without Tet as indicated. The position of the endogenous AC6 mRNA (open arrowhead) and transgene mRNA (closed arrowhead) are indicated. Total RNA was visualized by UV illumination of the blot (bottom). (C) Quantitation of the number of projections formed for the indicated cell lines maintained in the absence or presence of Tet. Mean values and standard errors are shown. P values (unpaired t test) are indicated. Sample sizes: AC6uKD –Tet, n = 90; AC6uKD +Tet, n = 90; AC6-Ri –Tet, n = 34; AC6-Ri +Tet, n = 34; AC6**-Ri –Tet, n = 30; AC6**-Ri +Tet, n = 30.

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    AC6 is localized to the tip of the trypanosome flagellum. (A) Western blot of protein extracts from 2913 cells and from cells expressing wild-type HA-tagged AC6 (AC6HA) or catalytically inactive AC6 (AC6**HA). Blots were probed with anti-HA antibody, or anti-tubulin antibody as a loading control. (B) Control cells (2913) or cells expressing HA-tagged wild-type AC6 (AC6HA) or the catalytic domain mutant (AC6**HA) were subjected to immunofluorescence with anti-HA antibody (green) and DAPI (blue). Bar, 2 μm.

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    FIG 4

    Model for cAMP microdomain regulation of social motility. (A) Schematic model for AC6-dependent control of social motility in wild-type cells (WT) and AC6 knockdown or catalytic mutant cells [AC6(−)]. AC6 is one of several ACs in the trypanosome flagellum. The model posits that ACs recognize different ligands, depending on their divergent extracellular domains, and that ligand binding regulates AC activity. cAMP produced specifically by AC6 acts to inhibit social motility, and when AC6 activity is reduced through ligand-mediated regulation (WT), this results in reduced cAMP and activation of social motility. Constitutive inactivation of AC6, e.g., through knockdown or expression of a catalytic mutant [AC6(−)], causes a signal-independent decrease in local cAMP, resulting in a precocious, hypersocial phenotype. (B) Reciprocal relationship between social motility (SoMo) and cAMP concentration.

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Insect Stage-Specific Adenylate Cyclases Regulate Social Motility in African Trypanosomes
Miguel A. Lopez, Edwin A. Saada, Kent L. Hill
Eukaryotic Cell Dec 2014, 14 (1) 104-112; DOI: 10.1128/EC.00217-14

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Insect Stage-Specific Adenylate Cyclases Regulate Social Motility in African Trypanosomes
Miguel A. Lopez, Edwin A. Saada, Kent L. Hill
Eukaryotic Cell Dec 2014, 14 (1) 104-112; DOI: 10.1128/EC.00217-14
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