13 research outputs found
Inositol hexakisphosphate and Gle1P direct mRNA export by spatially regulating the dead-box protein Dbp5P at the nuclear pore complex
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A role for eisosomes in maintenance of plasma membrane phosphoinositide levels
The plasma membrane delineates the cell and mediates its communication and material exchange with the environment. Many processes of the plasma membrane occur through interactions of proteins with phosphatidylinositol(4,5)-bisphosphate (PI(4,5)P2), which is highly enriched in this membrane and is a key determinant of its identity. Eisosomes function in lateral organization of the plasma membrane, but the molecular function of their major protein subunits, the BAR domain–containing proteins Pil1 and Lsp1, is poorly understood. Here we show that eisosomes interact with the PI(4,5)P2 phosphatase Inp51/Sjl1, thereby recruiting it to the plasma membrane. Pil1 is essential for plasma membrane localization and function of Inp51 but not for the homologous phosphatidylinositol bisphosphate phosphatases Inp52/Sjl2 and Inp53/Sjl3. Consistent with this, absence of Pil1 increases total and available PI(4,5)P2 levels at the plasma membrane. On the basis of these findings, we propose a model in which the eisosomes function in maintaining PI(4,5)P2 levels by Inp51/Sjl1 recruitment
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Regulation of Mammalian Autophagy by Class II and III PI 3-Kinases through PI3P Synthesis
Synthesis of phosphatidylinositol-3-phosphate (PI3P) by Vps34, a class III phosphatidylinositol 3-kinase (PI3K), is critical for the initial steps of autophagosome (AP) biogenesis. Although Vps34 is the sole source of PI3P in budding yeast, mammalian cells can produce PI3P through alternate pathways, including direct synthesis by the class II PI3Ks; however, the physiological relevance of these alternate pathways in the context of autophagy is unknown. Here we generated Vps34 knockout mouse embryonic fibroblasts (MEFs) and using a higher affinity 4x-FYVE finger PI3P-binding probe found a Vps34-independent pool of PI3P accounting for ~35% of the total amount of this lipid species by biochemical analysis. Importantly, WIPI-1, an autophagy-relevant PI3P probe, still formed some puncta upon starvation-induced autophagy in Vps34 knockout MEFs. Additional characterization of autophagy by electron microscopy as well as protein degradation assays showed that while Vps34 is important for starvation-induced autophagy there is a significant component of functional autophagy occurring in the absence of Vps34. Given these findings, class II PI3Ks (α and β isoforms) were examined as potential positive regulators of autophagy. Depletion of class II PI3Ks reduced recruitment of WIPI-1 and LC3 to AP nucleation sites and caused an accumulation of the autophagy substrate, p62, which was exacerbated upon the concomitant ablation of Vps34. Our studies indicate that while Vps34 is the main PI3P source during autophagy, class II PI3Ks also significantly contribute to PI3P generation and regulate AP biogenesis
Inositol hexakisphosphate and Gle1P direct mRNA export by spatially regulating the dead-box protein Dbp5P at the nuclear pore complex
<i>Vps34</i> KO MEFs show a decrease in LC3 conjugation and LC3 puncta formation upon starvation.
<p>(<b>A</b>) Control and <i>Vps34</i> KO MEFs were cultured in normal medium (N) or HBSS (St) in the presence or absence of 50nM Bafilomycin (Nm+B or St+B, respectively) for 90 min. Right: Lysates were analyzed by immunoblotting using the indicated antibodies. Left: Relative LC3-II levels normalized to actin (n=4). (<b>B</b>) Control and <i>Vps34</i> KO MEFs were cultured in normal media (Nm) or HBSS (St) for 30 and 90 min, fixed and immunostained. Confocal analysis of LC3, p62 and GFP-Cre fluorescence, which is artificially shown in green, red and blue colors, respectively. Arrowheads indicate LC3 and p62 colocalization (yellow). Scale bar: 10 µm. (<b>C</b>) Quantification of the number of LC3 (left panel) and p62 puncta (middle panel) per cell. Colocalization of p62 with LC3 puncta is also shown (right panel) (colocalization was defined as the number of pixels overlapping in the p62 and LC3 channels normalized per cell) (n=35-45 cells).</p
A higher affinity PI3P-binding probe, 4x-FYVE<sup>Hrs</sup>, reveals a larger pool of intracellular PI3P in the absence of Vps34 compared to the conventional 2x-FYVE<sup>Hrs</sup> probe.
<p>Control and <i>Vps34</i> KO MEFs were transiently transfected with both RFP-2x-FYVE<sup>Hrs</sup> and GFP-4x-FYVE<sup>Hrs</sup> PI3P-binding constructs for 24hr, grown in normal media and fixed. Confocal microscopy analysis of RFP-2x-FYVE<sup>Hrs</sup> (red) and GFP-4x-FYVE<sup>Hrs</sup> (green) is shown. Scale bar: 10 µm.</p
Silencing class II PI3Ks decreases autophagy in both control and <i>Vps34</i> null MEFs.
<p>(<b>A</b>) Control MEFs were transfected with mock or PI3K-C2α/β siRNA as well as GFP-WIPI-1 for 48 hrs, cultured in normal media or HBSS for 90 min and fixed. Right: Confocal microscopy images of GFP-WIPI-1 fluorescence in mock or PI3K-C2α/β siRNA-treated control cells after HBSS starvation for 90 min. Scale bar: 10 µm. Left: Quantification of the number and size (arbitrary units) of GFP-WIPI-1 puncta observed after 90 min HBSS starvation (n=11-15 cells). (<b>B</b>) Cells prepared as in (A) were fixed and immunostained. Right: Confocal microscopy images showing endogenous LC3 (green) in cells cultured in HBSS in the presence of 50 nM Bafilomycin (St+B) for 30 min. DAPI is shown in blue. Scale bar: 10 µm. Left: Quantification of the number and size (arbitrary units) of LC3 puncta observed under normal media (Nm), HBSS (St) and HBSS in the presence of Bafilomycin (St+B) conditions (n=12-19, 23-40 and 26-53 cells for Nm, St and St+B conditions, respectively). Scale bars: 10 µm.(<b>C</b>) Control and <i>Vps34</i> KO MEFs were transfected for 48 hrs with mock or PI3K-C2α/β siRNA, cultured in normal medium (N), HBSS (St) or HBSS with 50 nM Bafilomycin (St+B) for 30 min, lysed and analyzed by immunoblotting using the indicated antibodies (n=3).</p
Macroautophagy-mediated protein degradation is partially impaired in <i>Vps34</i> null MEFs.
<p>(<b>A</b>) Quantification of total [<sup>14</sup>C]-valine long-lived protein degradation induced by nutrient deprivation (HBSS) in control and <i>Vps34</i> KO MEFs. (<b>B</b>) Assessment of autophagy efficiency in control and <i>Vps34</i> KO MEFs by <sup>14</sup>C-valine long-lived protein degradation under starvation (HBSS), starvation with 3MA or starvation with NH<sub>4</sub>Cl conditions (see Methods) (n=8 for both A and B).</p
Recruitment of WIPI-1, a PI3P-binding protein, to sites of AP biogenesis occurs in <i>Vps34</i> KO MEFs but at diminished levels.
<p>Control and <i>Vps34</i> KO MEFs were transiently transfected with GFP-WIPI-1 for 24 hrs, cultured in HBSS for 90 min (90 min St) and fixed. Left: Confocal microscopy analysis of GFP-WIPI-1 fluorescence (green). Nuclear inactive and active Tomato-Cre is shown in red. The contrast was enhanced to reveal the WIPI puncta over the cytosolic background fluorescence. Right: Quantification of the number of GFP-WIPI-1 puncta (n=13-15 cells). Scale bar: 10 µm.</p
Ablation of Vps34 alters levels of Vps34 complex proteins.
<p>(<b>A</b>) Western blot analysis showing a time-course of Vps34 protein levels in <i>Vps34</i><sup>Flox/Flox</sup> MEFs infected with lentiviruses expressing either an inactive Cre (Δ) or active (CRE) full-length Cre recombinase for 7-13 days. (<b>B</b>) Western blot using an antibody directed to the NH<sub>2</sub>-terminus of Vps34 in control and <i>Vps34</i> KO cell extracts. (<b>C</b>) Western blot analysis of Vps34 complex components and endosomal protein levels in control and <i>Vps34</i> KO MEF lysates. Quantification of protein levels after normalization to tubulin (n=3).</p