spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 27 January 2009
doi: 10.1242/jcs.034199

Journal of Cell Science 122, 499-504 (2009)
Published by The Company of Biologists 2009
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kuehnel, Mark. P.
Right arrow Articles by Griffiths, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kuehnel, Mark. P.
Right arrow Articles by Griffiths, G.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Lipids regulate P2X7-receptor-dependent actin assembly by phagosomes via ADP translocation and ATP synthesis in the phagosome lumen

Mark. P. Kuehnel1, Vladimir Rybin1, Paras K. Anand1, Elsa Anes2 and Gareth Griffiths1,*

1 EMBL, Meyerhofstr. 1, 69117 Heidelberg, Germany
2 Molecular Pathogenesis Centre, Unit of Retrovirus and Associated Infections, Faculty of Pharmacy, University of Lisbon, Av. Forcas Armadas, 1600-083 Lisbon, Portugal


Figure 1
View larger version (34K):
[in this window]
[in a new window]

  		Fig. 1. (A) Procedure for quantification of luminal phagosome ATP. (B) Comparison of LBP luminal ATP concentrations estimated by luciferin fluorescence; red line indicates the average basal ATP level in unstimulated LBPs. (C) Luminal ATP levels from LBPs treated with different lipids in the presence of 1 mM ADP and ATP. Lower panel summarizes actin assembly data (Anes et al., 2003Go). Inset shows the effects of ceramide-1-phosphate (C1P) on actin assembly at 0.2 mM (low) and 5 mM (high) [ATP]. (D) LBP actin assembly with 1 mM ATP (ATP), 1 mM ATP plus 1 mM ADP (ATP + ADP) and the phagosomes containing apyrase-coated beads and control avidin-coated beads incubated with 1 mM ATP and 1 mM ADP or ATP alone. (E) Luminal ATP levels of LBP of different maturation ages, with and without PIP stimulation. Ctr, control containing solvents but no lipids. Ctr 0, LBP analysed immediately after thawing from frozen stocks. Ctr 30, incubation for 30 minutes at 37°C. TX, Triton X-100 treatment. Statistical significance was determined by the Student's t-test. P values given for the effects of lipids relative to the average values seen with unstimulated LBP. In all figures, mean ± s.d. is shown.

 

Figure 2
View larger version (26K):
[in this window]
[in a new window]

  		Fig. 2. (A) HPLC analysis of 3H-labeled nucleotide compositions [ATP (T), ADP (D) and AMP (M)] within the lumen of control phagosomes (C0, no incubation;C30, 30 minute incubation at 37°C) compared with phagosomes stimulated by S1P or PIP for 30 minutes. All control and lysed conditions had 1 mM ADP and 1 mM ATP; one set of conditions contained 10 µCi [3H]ADP, the other 10 µCi [3H]ATP. (B) Testing of adenylate kinase activity in extracts of phagosomes. LBPs were treated with Triton X-100 and then incubated with either tritium-labeled ADP (left) or AMP plus ATP (right) and nucleotide levels were analyzed by HPLC after 30 minutes of incubation. The mass action concentrations at the beginning and end of the 30 minute reaction are given for a representative experiment. (C) Estimation of [ATP] by luciferase fluorescence of phagosomal extracts incubated with 1 mM ADP and sphingosine kinase inhibitor (DHS), phosphoinositide 4-kinase inhibitor (adenosine) or adenylate kinase inhibitor (Ad2P5).

 

Figure 3
View larger version (40K):
[in this window]
[in a new window]

  		Fig. 3. (A) Actin nucleation assay performed with phagosomes from control (WT) mice and P2X7R-knockout (–/–) mice. The mean fraction of phagosomes nucleating actin filaments is given, along with s.d. Asterisks indicate significance below P<0.05 for the indicated comparisons. The equivalent comparisons for the P2X7R-knockout phagosomes were not statistically significant. (B) Immunofluorescence microscopy of P2X7R on control (WT), P2X7R-knockout (ko) and J774 LBPs in vitro. Green, P2X7; Red, LBP (false coloration of the phase-contrast images). (C) Western blot for P2X7R in spleen tissue from wild-type and P2X7R-knockout mice. Inset shows a western blot for P2X7R in 2 hour LBPs from J774 macrophages.

 

Figure 4
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 4. Model showing the role of P2X7R in phagosome actin assembly. Phosphorylated lipids, such as S1P, which is made by sphingosine kinase (SP kinase), are proposed to open a channel or transporter that allows ADP to enter the phagosome lumen. There, adenylate kinase (AK) converts ADP to ATP, which can bind to the luminal domain of P2X7R. This receptor then transmits a signal across the membrane to activate the actin-assembly machinery on the cytoplasmic side of the phagosome.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2009