Figure 1.
Expression of AQP2 and various phosphorylation mutants in LLCPK1 cells.
Cell lysates were prepared from stable cells expressing WT AQP2 or AQP2 phosphorylation mutants, including S256A, S256D, S261A, S269A and S269D. Using an antibody recognizing the myc tag that is attached to the C-terminus of AQP2, AQP2 appears as an approximately 25 kDa band on western blot.
Figure 2.
Formation of AQP2 perinuclear patch after cold block at 20°C in cells expressing various AQP2 phosphorylation mutants.
After pretreatment with cycloheximide for one hour, cells underwent cold block at 20°C for up to 150 minutes. Cells were fixed and subjected to staining with anti-c-myc antibody to detect AQP2. During 120 minutes of cold block the development of the perinuclear AQP2 patch is evident in all cells expressing AQP2 variants except for AQP2-S256D, which remains mainly on the cell membrane (LLC 256D). Scale bar = 20 µm.
Figure 3.
Quantified formation of AQP2 perinuclear patch after cold block at 20°C in cells expressing various AQP2 phosphorylation mutants.
The time course of the development of the AQP2 perinuclear patch was quantified by measuring mean pixel intensity of the patch using IPlab software. The results are presented as the increase in mean patch fluorescence value starting from 0 minutes of cold block. The overall accumulation of the perinuclear patch reached a maximal density after cold block for 150 minutes with all mutants. The experiment was repeated in triplicate, N for each mean is ≥24. Bars represent standard error.
Figure 4.
Dissolution of the AQP2 perinuclear patch after cold block release.
Cells were cold blocked for 120 minutes to form the perinuclear patch after which the cold block was released by returning the cells to 37°C. The perinuclear patch rapidly disintegrated over 30 minutes of cold block release as the AQP2 mutants (except for AQP2-S256D which remained on the membrane) redistributed throughout the cytosol and membrane. Scale bar = 20 µm.
Figure 5.
Quantified dissolution of the AQP2 perinuclear patch after cold block release.
To quantify the rate of AQP2 redistribution a time course analysis of the dissolution of the patch was assayed by immunofluorescence staining and images were quantified using the IPlab software. The results are presented as the decrease from the maximum mean patch fluorescence value after release of cold block. The experiment was repeated in triplicate N for each mean is > = 24. Bars represent standard error.
Figure 6.
Association of AQP2 and cellular compartment markers during the dynamic process of cold block and cold block release.
The association of subcellular compartment markers with AQP2 during the cold block and subsequent release was investigated. GM130, clathrin, EEA1, and HSP/HSC70, were co-stained with AQP2 (A, B, C, D). Panel A, GM130 staining also overlaps with AQP2 in the perinuclear patch but not after release of the cold block. In panel B, clathrin appeared to colocalize mostly with AQP2 during the development of the perinuclear patch and redistribution after cold block release. Panel C, EEA staining was colocalized with AQP2 at the early stage of endocytosis, and was partially associated with the perinuclear patch. After CBR at 37°C, a few large EEA/AQP2 positive structures appeared, but most AQP2 was not associated with EEA. Panel D, HSP/HSC70 partially colocalized with AQP2 during the formation of the perinuclear patch, but not during AQP2 redistribution after cold block release. Scale bar = 20 µm.