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First published online August 24, 2006
doi: 10.1242/10.1242/jcs.03091

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cAMP synthesis and degradation by phagosomes regulate actin assembly and fusion events: consequences for mycobacteria

Stefanos A. Kalamidas^1,2,*, Mark P. Kuehnel^1,*,, Pascale Peyron^1,3, Vladimir Rybin¹, Susanne Rauch¹, Othon B. Kotoulas², Miles Houslay⁴, Brian A. Hemmings⁵, Maximiliano G. Gutierrez⁶, Elsa Anes⁷ and Gareth Griffiths¹

¹ EMBL, Postfach 102209, 69117 Heidelberg, Germany
² Department of Anatomy, Histology and Embryology, Medical School, University of Ioannina, Ioannina 45 110, Greece
³ Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, Toulouse 31077, France
⁴ Division Biochemistry and Molecular Biology, IBLS, Wolfson Building, University of Glasgow, Glasgow, Scotland, UK
⁵ Friedrich Miescher Institute for Biomedical Research, Basel 4058, Switzerland
⁶ Laboratorio de Biología Celular y Molecular, IHEM-CONICET, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza 5500, Argentina
⁷ Molecular Pathogenesis Centre, Faculty of Pharmacy, University of Lisbon, Av. Forcas Armadas, 1600-083 Lisbon, Portugal

View larger version (23K): [in a new window]   Fig. 1. Cyclic AMP inhibits LBP actin assembly. Effects of cAMP (A) and SQ 22536 (B), on the in vitro de novo actin assembly of latex bead phagosomes isolated by flotation (after 1 hour pulse and 1 hour chase) from J774A.1 mouse macrophages, at low (0.2 mM) and high (5 mM) ATP concentrations. (C) Effect of cAMP-modulating drugs on actin nucleation. Results represent means ± s.d. from three independent experiments. Asterisks indicate significance as determined by the Student's t-test: *P<0.05; **P<0.01 compared with levels in the control.

View larger version (12K): [in a new window]   Fig. 2. LBPs can synthesize and degrade cAMP. HPLC analysis of cAMP on phagosomes treated as indicated. Red color indicates high (5 mM) ATP, blue, intermediate ATP conditions and green low ATP conditions (200 µM ATP). The inset shows a calibration of the system with pure cAMP. The absorbance values represent the peak areas of cAMP. SQ, SQ22356.

View larger version (62K): [in a new window]   Fig. 3. Effects of cAMP on actin (A), PKA RII (B), fusion (C) and acidification (D) in J774 macrophages. (A) Effects of indicated treatments on LBP-associated actin. Micrograph shows the effect of actin on intracellular LBPs visualized by Rhodamine-phalloidin labeling with a magnification of the LBP area in untreated cells. (Left) DIC-image; (right) Rhodamine-phalloidin image. The histogram shows percentage of LBPs positive for actin under indicated conditions. (B) Analysis of the co-localization of PKA RII. Micrograph shows DIC image of LBPs in macrophages (left), immunofluorescent labeling of PKA RII on LBPs (middle) and the overlay (right). The histogram shows the percentage of LBPs co-localizing with PKA RII under indicated conditions. The blot shows identification of PKA RII on LBPs. (C) Analysis of fusion of LBPs with Rhodamine-gold-filled late endosomes and lysosomes. Micrograph shows an example of lysosomal Rhodamine-gold particles co-localizing with LBPs with a magnification of the LBP area in untreated cells: DIC image (left), Rhodamine-gold image (right). The histogram shows the percentage of LBPs co-localizing with lysosomal Rhodamine-gold under indicated conditions. (D) Analysis of phagosomal acidification. The histogram shows the percentage of LBPs that co-localize with LysoTracker Red DND-99 under indicated conditions. All histograms represent mean ± s.d. of three individual experiments. Asterisks indicate significance as determined by the Student's t-test: *P<0.01 compared with levels in the control.

View larger version (18K): [in a new window]   Fig. 4. Effects of cAMP on the destruction of mycobacteria as viewed by CFU plating assay. (A) M. smegmatis survival. Results represent mean ± s.d. from three independent experiments. (B) M. tuberculosis H37Rv survival. Reagents were added as follows: 8-Br-cAMP (50 µM) added at 3 hours and 2 days post infection. SQ22536 (50 µM) added at 3 hours and 2 day post infection. H-89 (10 µM) added at 3 hours post infection. Asterisks indicate significance as determined by the Student's t-test: *P<0.01 compared with levels in the control.