Modulation of Rab5 and Rab7 recruitment to phagosomes by phosphatidylinositol 3-kinase - PubMed (original) (raw)
Modulation of Rab5 and Rab7 recruitment to phagosomes by phosphatidylinositol 3-kinase
Otilia V Vieira et al. Mol Cell Biol. 2003 Apr.
Abstract
Phagosomal biogenesis is central for microbial killing and antigen presentation by leukocytes. However, the molecular mechanisms governing phagosome maturation are poorly understood. We analyzed the role and site of action of phosphatidylinositol 3-kinases (PI3K) and of Rab GTPases in maturation using both professional and engineered phagocytes. Rab5, which is recruited rapidly and transiently to the phagosome, was found to be essential for the recruitment of Rab7 and for progression to phagolysosomes. Similarly, functional PI3K is required for successful maturation. Remarkably, inhibition of PI3K did not preclude Rab5 recruitment to phagosomes but instead enhanced and prolonged it. Moreover, in the presence of PI3K inhibitors Rab5 was found to be active, as deduced from measurements of early endosome antigen 1 binding and by photobleaching recovery determinations. Though their ability to fuse with late endosomes and lysosomes was virtually eliminated by wortmannin, phagosomes nevertheless recruited a sizable amount of Rab7. Moreover, Rab7 recruited to phagosomes in the presence of PI3K antagonists retained the ability to bind its effector, Rab7-interacting lysosomal protein, suggesting that it is functionally active. These findings imply that (i) dissociation of Rab5 from phagosomes requires products of PI3K, (ii) PI3K-dependent effectors of Rab5 are not essential for the recruitment of Rab7 by phagosomes, and (iii) recruitment and activation of Rab7 are insufficient to induce fusion of phagosomes with late endosomes and lysosomes. Accordingly, transfection of constitutively active Rab7 did not bypass the block of phagolysosome formation exerted by wortmannin. We propose that Rab5 activates both PI3K-dependent and PI3K-independent effectors that act in parallel to promote phagosome maturation.
Figures
FIG. 1.
Effect of wortmannin on Rab5 acquisition by phagosomes. (A to F) Time-lapse recordings of the distribution of Rab5-GFP during phagosome maturation. RAW cells were transiently transfected with Rab5-GFP. Untreated (A to C) or wortmannin-pretreated (D to F) cells were allowed to internalize IgG-opsonized beads while fluorescence images were acquired at regular intervals. The arrows in panels B and E point to phagosomes that had formed within 1 min of acquisition of the image. (A and D) Corresponding differential interference contrast images. (C and F) Images of the corresponding fields acquired after an additional 3 min. Scale bars = 10 μm. (G and H) Quantification of the fraction of Rab5-positive phagosomes in control (open bars) and wortmannin-treated (black bars) cells as a function of time after phagosome sealing in RAW (10-min phagocytosis pulse [G]) and CHO-IIA cells (20-min phagocytosis pulse [H]). Note that the time scales are different in these panels. Data are means + SE (error bars) of four separate experiments each with at least 100 individual phagosomes from ∼30 different cells expressing low levels of Rab5-GFP counted per condition.
FIG. 2.
Effect of dominant-negative Rab5 on fluid phase uptake and EEA1 acquisition. CHO-IIA cells were transiently transfected with dominant-negative Rab5-GFP (DN-Rab5). (A and B) The cells were incubated for 30 min in medium containing rhodamine-dextran (100 μg/ml) to assess fluid phase intake. (C and D) The cells were allowed to internalize IgG-opsonized beads for 20 min, and this was followed by fixation and EEA1 immunostaining. (A and C) GFP fluorescence; (B) fluorescence of rhodamine-dextran; (D) distribution of EEA1. Asterisks identify transfected cells. Arrows point to phagosomes. In panel B, transfected cells are outlined to facilitate identification of their boundaries. Scale bar = 10 μm. (E) Quantification of the effects of DN-Rab5 and wortmannin (Wort) on EEA1 acquisition by phagosomes in CHO-IIA cells. Cells were allowed to internalize beads for 20 min, fixed, and immunostained for EEA1. Where indicated, cells were pretreated with 100 nM wortmannin for 30 min. The cells were analyzed microscopically, and phagosomes lined by clearly observable EEA1 were scored. Data are means + SE (error bars) of four separate experiments, each with at least 100 individual phagosomes from 30 to 40 different cells counted per condition.
FIG. 3.
Effect of wortmannin on fusion of nascent phagosomes with early endosomes. (A to F) CHO-IIA cells that were untreated (A to C) or pretreated with wortmannin (D to F) were allowed to internalize IgG-opsonized latex beads for 20 min. After washing excess beads, the cells were incubated briefly on ice with anti-human antibody conjugated with fluorescein isothiocyanate to identify adherent, incompletely internalized beads (B and E). The cells were then incubated for an additional 10 min at 37°C with FM4-64.The red fluorescence of FM4-64 is shown in A and D (arrows). C and F are the differential interference contrast images corresponding to A and D, respectively. (G to I) CHO-IIA cells transiently transfected with syntaxin 13-GFP were treated with wortmannin (H and black columns in I) or left untreated (G and empty columns in I) and allowed to internalize particles for 20 min. Adherent, incompletely internalized beads were then stained as above using Texas red-conjugated antibodies. Lastly, the cells were chased for the indicated times and fixed, and confocal fluorescence microscopy was used to visualize the distribution of syntaxin 13. (G and H) representative fluorescence images, with corresponding differential interference contrast insets. Arrows point to phagosomes. Scale bars =10 μm. (I) Quantitation of the effect of wortmannin on syntaxin 13 acquisition by phagosomes. Data are means + SE (error bars) of three separate experiments, each with at least 100 individual phagosomes from 30 to 50 different cells counted.
FIG. 4.
Rab5-GFP FRAP. CHO-IIA cells were transfected with either wild-type (WT) or constitutively active (CA) Rab5-GFP. Where indicated, the cells had been pretreated with wortmannin (Wort). (A) Labeled endosomes were identified and their initial fluorescence quantified and defined as 100%. Bleaching was then performed and fluorescence recovery monitored as described in Materials and Methods. (B) Cells transfected with WT-Rab5 were treated with wortmannin, while cells transfected with CA-Rab5 were left untreated. The cells were then allowed to ingest opsonized beads, fluorescent phagosomes were identified, and FRAP was measured as above. Representative experiments are shown in the main panels. The inset tables show the corresponding times for 50% recovery (T1/2) and the fractional recovery (R). Data in the tables are means + SE (error bars) of 8 to 10 determinations, each involving 10 different cells.
FIG. 5.
Role of Rab5 in fusion of phagosomes with late endocytic compartments. CHO-IIA cells were transfected with DN-Rab5-GFP (A, B and black column in E) or with RN-tre-GFP (C, D, and gray column in E). Transfected cells (labeled by asterisks) are identifiable by their green fluorescence (A and D). The cells were allowed to internalize opsonized beads for 20 min. After washing unbound beads, incubation at 37°C proceeded for 30 more min. The cells were then fixed, permeabilized with methanol at −20°C, and stained for LAMP-1 (B, D, and E). Arrows and arrowheads point to LAMP-1-positive and -negative phagosomes, respectively. Scale bars = 10 μm. (E) Quantification of the effect of DN-Rab5 (black column) and RN-tre (gray column) overexpression on LAMP-1 acquisition by phagosomes, compared to untransfected control cells (open bar). Results shown are means + SE (error bars) of three separate experiments, each with at least 100 phagosomes from 20 to 40 different cells counted.
FIG. 6.
Effect of dominant-negative Rab5 on Rab7 acquisition. CHO-IIA cells were cotransfected with wild-type Rab7-myc and either GFP (A to C and empty columns in G) or with DN-Rab5-GFP (D to F). The cells were incubated for 20 min with IgG-opsonized beads, chased for the indicated times, and then fixed, permeabilized, and immunostained. (A and D) Green fluorescence; (B and E) distribution of Rab7, revealed by immunostaining the myc epitope; (C and F) corresponding differential interference contrast images. Arrows and arrowheads point to Rab7-myc positive and -negative phagosomes, respectively. Scale bars = 10 μm. (G) quantification of the effect of DN-Rab5 on Rab7 recruitment by phagosomes, from experiments like those in panels A to F. Empty columns, control; black columns, cells transfected with DN-Rab5-GFP. Results shown are means + SE (error bars) of four separate experiments, each with at least 100 individual phagosomes counted per condition in cells with high expression levels of DN-Rab5.
FIG. 7.
Effect of wortmannin on Rab7 acquisition by phagosomes. CHO-IIA cells (A to E) or RAW cells (F) were transfected with wild-type Rab7-GFP. The cells were either left untreated (A, B, and empty columns in E and F) or were treated with 100 nM wortmannin for 30 min (C, D, and black columns in E and F). The cells were allowed to internalize IgG-opsonized beads and the distribution of Rab7 was monitored by confocal microscopy at the indicated times. (A and C) Phagocytosis was for 20 min, without chase. The arrows and arrowheads point to Rab7-GFP-positive and -negative phagosomes, respectively. (B and D) Corresponding differential interference contrast images. Scale bars = 10 μm. (E and F) Quantification of the effect of wortmannin on Rab7-GFP acquisition by phagosomes in CHO-IIA (E) and RAW cells (F). Empty columns, control; black columns, cells treated with wortmannin. Data are means + SE (error bars) of four separate experiments, each with at least 100 individual phagosomes from 30 to 40 different cells expressing low levels of Rab7-GFP counted per condition.
FIG. 8.
RILP recruitment to phagosomes. CHO-IIA cells were cotransfected with wild-type Rab7-GFP and RILP-HA. (D to F) Cells were treated with wortmannin 30 min before phagocytosis. Phagocytosis was allowed to proceed for 20 min, and this was followed by a 30-min chase. The cells were then fixed and immunostained for the HA epitope. (A and D) Distribution of Rab7-GFP; (B and E) the distribution of RILP-HA; (C and F) corresponding differential interference contrast images. The arrows point to phagosomes positive for both Rab7 and RILP. Asterisks identify transfected cells. Bars = 10 μm. Results are representative of four similar experiments, each with ∼30 cells counted.
FIG. 9.
Effect of constitutively active Rab7 on LAMP-1 acquisition by phagosomes. CHO-IIA cells were transfected with CA-Rab7-GFP and either left untreated (A to C and empty columns in G) or treated with wortmannin (D to F and black columns in G) prior to phagocytosis. After 20 min of phagocytosis the unbound particles were removed and the cells were incubated for 30 more min to allow phagosome maturation. The cells were then fixed, permeabilized, and immunostained with anti-LAMP-1 antibodies. (A and D) Distribution of CA-Rab7-GFP; (B and E) distribution of LAMP-1. Arrows and arrowheads point to phagosomes that were positive or negative, respectively, for Rab7 or LAMP-1, as appropriate. Asterisks identify transfected cell. Scale bars = 10 μm. (C and F) Corresponding differential interference contrast images. (G) Quantitation of LAMP-1 acquisition by phagosomes in cells treated with wortmannin. The graph compares cells that were untransfected (black bars) with CA-Rab7-GFP-transfected cells (open bars). Data are means + SE (error bars) of four separate experiments, each with at least 100 individual phagosomes from 20 to 40 different cells counted per condition.
FIG. 10.
Schematic representation of phagosome maturation, highlighting putative sites of action of PI3K. Nascent phagosomes formed upon fission from the plasma membrane contain small amounts of Rab5. Additional Rab5 is acquired by fusion with early endosomes. Inhibition of PI3K prevents the dissociation of active Rab5 from early phagosomes and presumably also from early endosomes (dotted line). Recruitment or activation of (some) Rab5 effectors also requires products of PI3K. Intermediary phagosomes are formed upon acquisition of Rab7 from either the soluble pool or from a PI3K-independent vesicular compartment. Late phagosomes form by fusion with late endosomes, which themselves bear Rab7. This step is blocked by wortmannin. Late phagosomes become phagolysosomes upon fusion with lysosomes. Wortmannin also prevents this step. A putative Rab7-bearing organelle that can still fuse with phagosomes despite the presence of PI3K inhibitors is indicated by the purple oval (lower left).
References
- Aderem, A., and D. M. Underhill. 1999. Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 17:593-623. - PubMed
- Alvarez-Dominguez, C., A. M. Barbieri, W. Beron, A. Wandinger-Ness, and P. D. Stahl. 1996. Phagocytosed live. Listeria monocytogenes influences Rab5-regulated in vitro phagosome-endosome fusion. J. Biol. Chem. 271:13834-13843. - PubMed
- Alvarez-Dominguez, C., and P. D. Stahl. 1998. Interferon-gamma selectively induces Rab5a synthesis and processing in mononuclear cells. J. Biol. Chem. 273:33901-33904. - PubMed
- Bucci, C., R. G. Parton, I. H. Mather, H. Stunnenberg, K. Simons, B. Hoflack, and M. Zerial. 1992. The small, GTPase rab5 functions as a regulatory factor in the early endocytic pathway. Cell 70:715-728. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources