The Vac14^{β-geo/β-geo} mouse has no detectable Vac14 protein and has a decrease in PI(3,5)P₂ and PI(5)P levels. (a) PI(3,5)P₂ and PI(5)P levels decrease, whereas PI(3)P levels increase in Vac14^{β-geo/β-geo} mice. Primary fibroblasts were starved in inositol-free DMEM for 12 h and labeled with myo-[2-³H]inositol for 24 h. Lipids were extracted, deacylated, and analyzed by anion-exchange HPLC chromatography. To assess whether differences in the lipid levels were statistically significant, we applied a two-tailed, paired Student's t test. Significance was set at P = 0.15. PI(3)P increased (P = 0.11), whereas PI(3,5)P₂ and PI(5)P decreased in Vac14^{β-geo/β-geo} cells (P = 0.0070 and 0.0038, respectively). PI(4)P and PI(4,5)P₂ levels did not change (P = 0.36 and 0.87, respectively). For all measurements, n = 3. Error bars represent the standard deviations. (b and c) Regions of HPLC profiles showing PI(3,5)P₂ (b) and PI(5)P (c) from Vac14^+/+ (Left) and Vac14^{β-geo/β-geo} (Right).

Lesions in neural tissue from Vac14^{β-geo/β-geo} mutant mice. (a) Sagittal sections of the whole brain of P0 mice. Examples of lesions (holes) in affected regions, midbrain (Upper) and pons (Lower), are shown on the right with higher magnification. (Scale bars, 100 μm.) Insets are also indicated by the squares in the whole brain. (b) Lesions composed of large intracellular vacuoles (arrows) or acellular areas were found in olfactory bulb, trigeminal ganglion, and dorsal root ganglion. In some cases, whole cell bodies appeared to be either missing or filled with one giant vacuole (arrowheads). (Scale bars, 100 μm.)

Expression of Vac14-mCit and overexpression of mCit-Fab1/PIKfyve in Vac14^{β-geo/β-geo} fibroblasts leads to the suppression of vacuole formation. (a) Vacuolation occurs in neuronal primary cultures. Cortices were dissected from E16.5 embryos and cultured for 2 weeks. Images of live cells were taken on a Nikon TE-2000 inverted microscope with a ×40 phase-contrast objective. (Scale bars, 20 μm.) (b) Cells were transfected with the indicated plasmids. Fluorescent cells were scored for the presence of vacuoles. Each data point is a mean of four independent experiments. For each experiment >100 cells were counted. Error bars represent SD. (c) Representative cells for 6 and 24 h after transfection are shown. Arrows indicate transfected cells.

Vacuoles appear to form from late endosomes/lysosomes. (a) Primary fibroblasts were labeled with GM130 (Golgi marker), EEA1 (early endosome marker), or Lamp2 (late endosome and lysosome marker). Although the cis-Golgi appears normal in Vac14^{β-geo/β-geo} cells, early endosomes are frequently enlarged. The limiting membrane of the vacuoles in Vac14^{β-geo/β-geo} cells is labeled with Lamp2. (b) Vacuoles in Vac14^{β-geo/β-geo} cells are incapable of receiving fluid phase marker dextran. Vac14^+/+ and Vac14^{β-geo/β-geo} fibroblasts were incubated with dextran Cascade blue for 16 h and chased for 2 h. (Scale bars, 10 μm.)

Endosome-to-TGN trafficking is defective in Vac14^{β-geo/β-geo} fibroblasts. (a) CI-MPR is trapped in a mixed population of endosomes. Vac14^+/+ and Vac14^{β-geo/β-geo} fibroblasts were fixed as above and double labeled with CI-MPR (green) and EEA1 or Lamp2 (red). Images were taken on a Zeiss confocal microscope. Peripheral regions of cells marked by squares are shown in an amplified view. (b) Pro- and intermediate cathepsin D accumulate in Vac14^{β-geo/β-geo} fibroblasts. Steady-state levels of cathepsin D in whole-cell lysates from Vac14^+/+ and Vac14^{β-geo/β-geo} fibroblasts were analyzed by Western blot.