Figure 4
Vps34 deletion impairs acute endocytosis of injected ovalbumin-texasRed. WT or Vps34∆/∆ mice were injected at P14 with 180 µg TexasRed-labelled ovalbumin (TR-Ova) in the orbital plexus and euthanized at 20 min. Kidneys were fixed by immersion and whole cryostat sagittal sections were collected. (a) Low-power view of TR-Ova signal by stitched images of the two upper thirds of the sagittal sections recorded by Zeiss Mirax Midi and examined for red emission. Dashed white and yellow lines define the kidney contours and the cortico-medullary junctions, respectively. Boxes are enlarged below. Notice in WT mice the homogenous cortical signal indicating extensive recapture by all PTCs, with abrupt arrest at the cortico-medullary boundary (between cortex and outer stripe of outer medulla, OSOM). In Vps34∆/∆, much fewer PTCs are labeled and red signal extends deeper into the OSOM. (b) High magnification analysis by confocal microscopy with triple PTC imaging for TR-ovalbumin as endocytic cargo (red), megalin as endocytic receptor (white) and LAMP-1 as membrane marker of lysosomes where undigested tracer accumulates and free Texas Red is trapped (green). PTCs are circumscribed by broken white lines. Fields in the boxed rectangles are shown enlarged below with separate emission channels in white then merged channels in colors. Preserved PTC due to mosaicism in cKO is indicated by two radial broken yellow lines. In WT PTCs, megalin signal is restricted to a continuous apical layer. Intensely red dots indicating endocytosed TR-ovalbumin are mostly surrounded by LAMP-1, still in the subapical layer, indicating extensive endocytic uptake and transfer to lysosomes. In Vps34∆/∆ PTCs, LAMP-1 objects are much more numerous and become mislocalized to the deeper cytoplasm. Megalin has mostly disappeared from the apical layer and is instead redistributed into cytoplasmic dots colocalizing with LAMP-1. TR-Ova is not detected, except in the few preserved PTCs at the bottom of the section, where megalin and LAMP-1 show normal localization and intensity.
