Figure 6.

Intracellular pathogens exploit PI4KIIs and BLOC-1 to achieve their life cycle. (A) Viral production assessed by plaque assay was plotted as plaque forming units per milliliter (PFU/ml). A549 cells treated with siCTRL or siPI4KIIs were infected with PR8 at MOI of 3 for 8 h. A single experiment composed of six samples is shown and is representative of three independent experiments. (B) Cells treated with siCTRL or siPI4KIIs were infected or mock-infected with PR8 at MOI of 3 for 8 h and processed by IFM. Cytosolic viral inclusions were identified using antibodies against RAB11 (green) and viral NP (magenta) proteins. Insets show RAB11 and NP co-localization. Nuclei (blue) and cell periphery were delimited by yellow-dashed lines. (C) Frequency distribution of NP viral inclusions within the three area categories (in µm²) was plotted for each condition. (D) HeLa cells treated with siCTRL or siPI4KIIs were infected 12 h with C. trachomatis serovar D (Ctr D) expressing mCherry (top panels), then fixed and analyzed by IFM using antibodies against the bacterial inclusion protein Cap1 (bottom panels). Arrowheads point to Cap1⁺ tubules elongating from the inclusion membrane. Cell periphery is delimited by yellow lines. (E and F) Ctr D-infected cells were analyzed for the average number/cell (E) and the mean length (F) of Cap1⁺ tubules observed in D. (G) Working model of the role of PI4P, PI4KIIs, and BLOC-1 during recycling endosomal tubule formation from the early sorting endosomal membrane. (1) At PI3P⁺ early endosomal membrane subdomains, PI4P locally produced by PI4KIIs is quickly depleted by SAC2 phosphatase activity. (2) In PI3P-deficient domains, endosomal PI4P locally synthesized by PI4KIIs accumulates. (3) BLOC-1 concentration on PI4P⁺ domains facilitates the formation of nascent tubules with diameter and curvature compatible with BLOC-1 association/stabilization to membrane. (4) Nascent tubules accumulate PI4P, produced by local PI4KIIs, that sustains elongation through continuous BLOC-1 concentration to the PI4P⁺ tubular membrane and concomitant pulling by KIF13 motors along microtubules (Delevoye et al., 2016). (5) Eventually, nascent-extended RE tubules are severed at their neck through BLOC-1 cooperation with actin-polymerizing machinery (Delevoye et al., 2016) to release PI4P⁺ tubules required for endosomal cargo (e.g., Tf) recycling. The machineries required for the biogenesis of RE tubules can be exploited by pathogens to either use RE membranes (e.g., IAV) or tubulate parasitophorous vacuoles (e.g., Chlamydia). (B) Non-parametric two-way ANOVA test, followed by Sidak’s multiple comparisons test (***, P < 0.001); ∼45 cells/condition from three independent experiments. (A, E, and F) Data are the average of three independent experiments presented as the mean ± SEM; two-tailed unpaired t test (**, P < 0.01; ****, P < 0.0001). Scale bars: (main panels) 10 μm; (insets) 2.5 μm.