pH of endophagosomes controls association of their membranes with Vps34 and PtdIns(3)P levels

Phagocytosis of filamentous bacteria occurs through tubular phagocytic cups (tPCs) and takes many minutes to engulf these filaments into phagosomes. Contravening the canonical phagocytic pathway, tPCs mature by fusing with endosomes. Using this model, we observed the sequential recruitment of early and late endolysosomal markers to the elongating tPCs. Surprisingly, the regulatory early endosomal lipid phosphatidylinositol-3-phosphate (PtdIns(3)P) Persists on tPCs as long as their luminal pH remains neutral. Interestingly, by manipulating cellular pH, we determined that PtdIns(3) P behaves similarly in canonical phagosomes as well as endosomes. We found that this is the product of a pH-based mechanism that induces the dissociation of the Vps34 class III phosphatidylinositol-3-kinase from these organelles as they acidify. The detachment of Vps34 stops the production of PtdIns(3)P, allowing for the turnover of this lipid by PIKfyve. Given that PtdIns(3)P-dependent signaling is important for multiple cellular pathways, this mechanism for pH-dependent regulation of Vps34 could be at the center of many PtdIns(3)P-dependent cellular processes. </p

Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes

Phagocytes engulf unwanted particles into phagosomes that then fuse with lysosomes to degrade the enclosed particles. Ultimately, phagosomes must be recycled to help recover membrane resources that were consumed during phagocytosis and phagosome maturation, a process referred to as “phagosome resolution.” Little is known about phagosome resolution, which may proceed through exocytosis or membrane fission. Here, we show that bacteria-containing phagolysosomes in macrophages undergo fragmentation through vesicle budding, tubulation, and constriction. Phagosome fragmentation requires cargo degradation, the actin and microtubule cytoskeletons, and clathrin. We provide evidence that lysosome reformation occurs during phagosome resolution since the majority of phagosome-derived vesicles displayed lysosomal properties. Importantly, we show that clathrin-dependent phagosome resolution is important to maintain the degradative capacity of macrophages challenged with two waves of phagocytosis. Overall, our work suggests that phagosome resolution contributes to lysosome recovery and to maintaining the degradative power of macrophages to handle multiple waves of phagocytosis. </p

(A) Cells were cotransfected with Rab5A-GFP and PH(PLCδ)-RFP or synaptojanin-2–CAAX, PH(PLCδ)-RFP, and Rab5A-GFP as indicated

Cells were then infected with either WT or mutant expressing RFP, fixed at 10-min after infection, and stained for extracellular bacteria. Intracellular bacteria colocalizing with Rab5A-GFP was then determined by microscopic analysis. Data are means ± SEM of three separate experiments (>100 bacteria analyzed per experiment). (B) Cells were transfected with the indicated construct and then infected with either WT or mutant labeled covalently with Alexa Fluor 647. Infected cells were analyzed using a spinning disk confocal microscope. Images were acquired at 1-min intervals for ∼1 h. Localization of PI(3)P to SCVs (colocalization with 2FYVE-GFP or PX-mCherry) during the course of infection is shown. Data are means ± SEM of three separate experiments for WT- (94 SCVs analyzed) or mutant– (68 SCVs analyzed) infected cells expressing 2FYVE-GFP. Five separate experiments were analyzed for mutant–infected cells expressing synaptojanin 2-CAAX, PH(PLCδ)-RFP, and 2FYVE-GFP (121 SCVs analyzed), and three separate experiments were analyzed for mutant–infected cells expressing 2PH(PLCδ)-GFP and PX-mCherry-GFP (120 SCVs analyzed). The p-values are shown.<p><b>Copyright information:</b></p><p>Taken from "SopB promotes phosphatidylinositol 3-phosphate formation on vacuoles by recruiting Rab5 and Vps34"</p><p></p><p>The Journal of Cell Biology 2008;182(4):741-752.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518712.</p><p></p

(A and B) Cells were transfected with 2FYVE-GFP and infected with WT (A) or a deletion mutant (B)

Left panels indicate 2FYVE-GFP relative to the RFP-expressing bacteria (red) in the merged images in the right panels. Insets are enlarged from the indicated areas (dashed boxes). Cells were analyzed using confocal microscopy. These images correspond to <p><b>Copyright information:</b></p><p>Taken from "SopB promotes phosphatidylinositol 3-phosphate formation on vacuoles by recruiting Rab5 and Vps34"</p><p></p><p>The Journal of Cell Biology 2008;182(4):741-752.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518712.</p><p></p

(A) Cells were transfected with GFP-tagged Rab5A, Rab5B, or Rab5C as indicated

Cell lysates were then immunoblotted for either Rab5A or Rab5B. (B) Cells were treated with siRNA to the indicated Rab5 isoform, all three Rab5 isoforms (Pool), or control siRNA that does not affect Rab5 expression. Cell lysates were immunoblotted for Rab5A or tubulin (loading control). (C) Samples from B were immunoblotted for Rab5B or tubulin. (D) Cells were treated with either control siRNA or Rab5 Pool siRNA and transfected with 2FYVE-GFP. Cells were then infected with either WT or mutant bacteria, fixed at 10-min after infection, and stained for bacteria. Intracellular bacteria colocalizing with 2FYVE-GFP was then quantified. Data are means ± SEM of three separate experiments (>100 bacteria analyzed per experiment). The p-value is shown.<p><b>Copyright information:</b></p><p>Taken from "SopB promotes phosphatidylinositol 3-phosphate formation on vacuoles by recruiting Rab5 and Vps34"</p><p></p><p>The Journal of Cell Biology 2008;182(4):741-752.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518712.</p><p></p

(A and B) Localization of PH(PLCδ)-RFP (A) and synaptojanin-2–CAAX construct (B) in control cells

Arrowheads indicate plasma membrane localization of synaptojanin-2–CAAX construct. (C–F) Cells were cotransfected with synaptojanin-2–CAAX, PH(PLCδ)-RFP (C), and PH(Akt)-GFP (D). (E) Merged image showing localization of bacteria (labeled with Alexa Fluor 647) relative to signal for PH(Akt)-GFP. (F) Cells were cotransfected as in C–E, infected with WT . , and analyzed by confocal microscopy. Images were acquired at 1-min intervals for ∼1 h. Colocalization of the bacteria with PH(Akt)-GFP during infection is shown. As a control, cells were cotransfected with PH(PLCδ)-RFP and PH(Akt)-GFP but not synaptojanin-2–CAAX. Data are means ± SEM of three separate experiments for synaptojanin-2–CAAX-expressing cells (20 ruffles analyzed) and two separate experiments for control cells (13 ruffles analyzed). The p-value is shown. (G–J) Cells were cotransfected with synaptojanin-2–CAAX, PH(PLCδ)-RFP (G), and 2FYVE-GFP (H). (I) Merged image showing localization of bacteria (labeled with Alexa Fluor 647) relative to signal for 2FYVE-GFP. Insets are enlarged from dashed boxes. (J) Cells were cotransfected as in G–I, infected with WT bacteria, and analyzed by confocal microscopy. Images were acquired at 1-min intervals for at least 1 h. Colocalization of the bacteria with 2FYVE-GFP during infection is shown. As a control, cells were cotransfected with PH(PLCδ)-RFP and 2FYVE-GFP but not synaptojanin-2–CAAX. Data are means ± SEM of three separate experiments for synaptojanin-2–CAAX- expressing cells (111 SCVs analyzed) and two separate experiments for control cells (44 SCVs analyzed). The p-value is shown. Bars, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "SopB promotes phosphatidylinositol 3-phosphate formation on vacuoles by recruiting Rab5 and Vps34"</p><p></p><p>The Journal of Cell Biology 2008;182(4):741-752.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518712.</p><p></p

Data_Sheet_1_Small Rho GTPases and the Effector VipA Mediate the Invasion of Epithelial Cells by Filamentous Legionella pneumophila.pdf

<p>Legionella pneumophila (Lp) exhibits different morphologies with varying degrees of virulence. Despite their detection in environmental sources of outbreaks and in respiratory tract secretions and lung autopsies from patients, the filamentous morphotype of Lp remains poorly studied. We previously demonstrated that filamentous Lp invades lung epithelial cells (LECs) and replicates intracellularly in a Legionella containing vacuole. Filamentous Lp activates β1integrin and E-cadherin receptors at the surface of LECs leading to the formation of actin-rich cell membrane structures we termed hooks and membrane wraps. These structures entrap segments of an Lp filament on host cell surface and mediate bacterial internalization. Here we investigated the molecular mechanisms responsible for the actin rearrangements needed for the formation and elongation of these membrane wraps and bacterial internalization. We combined genetic and pharmacological approaches to assess the contribution of signaling downstream of β1integrin and E-cadherin receptors, and Lp Dot/Icm secretion system- translocated effectors toward the invasion process. Our studies demonstrate a multi-stage mechanism of LEC invasion by filamentous Lp. Bacterial attachment to host cells depends on signaling downstream of β1integrin and E-cadherin activation, leading to Rho GTPases-dependent activation of cellular actin nucleating proteins, Arp2/3 and mDia. This mediates the formation of primordial membrane wraps that entrap the filamentous bacteria on the cell surface. Following this, in a second phase of the invasion process the Dot/Icm translocated effector VipA mediates rapid membrane wrap elongation, leading to the engulfment of the filamentous bacteria by the LECs. Our findings provide the first description of Rho GTPases and a Dot/Icm effector VipA regulating the actin dynamics needed for the invasion of epithelial cells by Lp.</p