Reactive oxygen species prevent lysosome coalescence during PIKfyve inhibition

Lysosomes are terminal, degradative organelles of the endosomal pathway that undergo repeated fusion-fission cycles with themselves, endosomes, phagosomes, and autophagosomes. Lysosome number and size depends on balanced fusion and fission rates. Thus, conditions that favour fusion over fission can reduce lysosome numbers while enlarging their size. Conversely, favouring fission over fusion may cause lysosome fragmentation and increase their numbers. PIKfyve is a phosphoinositide kinase that generates phosphatidylinositol-3,5-bisphosphate to modulate lysosomal functions. PIKfyve inhibition causes an increase in lysosome size and reduction in lysosome number, consistent with lysosome coalescence. This is thought to proceed through reduced lysosome reformation and/or fission after fusion with endosomes or other lysosomes. Previously, we observed that photo-damage during live-cell imaging prevented lysosome coalescence during PIKfyve inhibition. Thus, we postulated that lysosome fusion and/or fission dynamics are affected by reactive oxygen species (ROS). Here, we show that ROS generated by various independent mechanisms all impaired lysosome coalescence during PIKfyve inhibition and promoted lysosome fragmentation during PIKfyve re-activation. However, depending on the ROS species or mode of production, lysosome dynamics were affected distinctly. H2O2 impaired lysosome motility and reduced lysosome fusion with phagosomes, suggesting that H2O2 reduces lysosome fusogenecity. In comparison, inhibitors of oxidative phosphorylation, thiol groups, glutathione, or thioredoxin, did not impair lysosome motility but instead promoted clearance of actin puncta on lysosomes formed during PIKfyve inhibition. Additionally, actin depolymerizing agents prevented lysosome coalescence during PIKfyve inhibition. Thus, we discovered that ROS can generally prevent lysosome coalescence during PIKfyve inhibition using distinct mechanisms depending on the type of ROS.

Dynamic remodeling of host membranes by self-organizing bacterial effectors

During infection Legionella bacteria translocate a variety of effectors into host cells that modify host cell membrane trafficking for the benefit of the intracellular pathogen. Here we found a self-organizing system consisting of a bacterial phosphoinositide kinase and its opposing phosphatase that formed spatiotemporal patterns, including traveling waves, to remodel host cellular membranes. The Legionella effector MavQ, a phosphatidylinositol (PI) 3-kinase, was targeted to the endoplasmic reticulum (ER). MavQ and the Legionella PI 3-phosphatase SidP, even in the absence of other bacterial components, drove rapid PI 3-phosphate turnover on the ER, spontaneously forming traveling waves that spread along ER subdomains inducing vesicle/tubule budding. Thus, bacteria can exploit a self-organizing membrane-targeting mechanism to hijack host cellular structures for survival.

Gambogic Acid Affects Ribosomal Occurrence in Glioma Cells by Downregulating the Phosphoinositide Kinase-3/Protein Kinase B/Mammalian Target of Rapamycin Signaling Pathway

Gambogic acid (GA) is a natural compound with a polyprenylated xanthone structure that has antiinflammatory, antioxidant, and neuroprotective properties and acts as a chemopreventive agent. GA exhibits anti-tumor, antimicrobial, and anti-proliferative effects on cancer cells. In the current study, the effect of GA on phosphoinositide kinase-3 (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway was examined in human U251 glioma cells. Cell viability and apoptosis were evaluated by MTT and Annexin V/PI Double Staining. The expressions of P38, AKT, and mTOR were evaluated by western blot and qRT-PCR, respectively. MagBeads Total RNA Extraction Kit was used to isolate cell tissue RNA. GA decreased the phosphorylation of P38, AKT, and mTOR. Inhibitors of PI3K (LY294002) enhanced the phosphorylation of P38, AKT, and mTOR. GA reduced the phosphorylation of ribosomal protein precursors (Pre) and upstream binding factor (UBF), and insulin-like growth factor I (IGF-1) further enhanced the cell proliferation and expression of Pre and UBF. These results suggested that downregulation of PI3K/AKT/mTOR signaling pathway may be an important mediator in GA-affected ribosomal occurrence in glioma cells.

Reactive oxygen species rescue lysosome coalescence during PIKfyve inhibition

Lysosomes are terminal, degradative organelles of the endosomal pathway that undergo repeated fusion-fission cycles with themselves and other organelles like endosomes, phagosomes, and autophagosomes. Lysosome number, size and degradative flux depends on the equilibrium between fusion and fission rates. Thus, conditions that favour fusion over fission will reduce lysosome numbers while enlarging remaining lysosomes. Conversely, conditions that favour fission over fusion will cause lysosome fragmentation and increase their numbers. PIKfyve is a phosphoinositide kinase that generates phosphatidylinositol-3,5-bisphosphate to modulate several lysosomal functions. PIKfyve inhibition causes a dramatic increase in lysosome size and reduction in lysosome number, consistent with lysosome coalescence. This is thought to proceed through reduced lysosome reformation and/or fission after fusion with endosomes or other lysosomes. Previously, we observed that photo-damage during live-cell imaging prevented lysosome coalescence during acute PIKfyve inhibition. Thus, we postulated that lysosome fusion and/or fission dynamics are affected by reactive oxygen species (ROS). Here, we show that ROS generated by four independent mechanisms all arrested lysosome coalescence during PIKfyve inhibition and accelerated lysosome fragmentation during PIKfyve re-activation. However, depending on the ROS species and/or mode of production, lysosome dynamics were affected distinctly. H2O2 impaired lysosome motility and reduced lysosome fusion with phagosomes, suggesting that H2O2 prevents lysosome coalescence in PIKfyve-impaired cells by reducing lysosome fusogenecity. In comparison, inhibitors of oxidative phosphorylation, glutathione, and thioredoxin that produce superoxide, did not impair lysosome motility but instead promoted clearance of actin puncta on lysosomes formed during PIKfyve inhibition. Additionally, actin depolymerizing agents prevented lysosome coalescence during PIKfyve inhibition. Thus, we discovered that ROS can generally prevent lysosome coalescence during PIKfyve inhibition using distinct mechanisms.