Fig 1.
Validation of MT-3 protein interactions by MT-3 mediated pulldown and V5-mediated co-immunoprecipitation.
(A). Zn7-MT-3 cross-linked agarose beads pulldown proteins isolated from cultured human proximal tubule cells (HK-2) include β-actin (a), tropomyosin 3 (b), enolase 1 (c), and aldolase A (d). (B). MT-3 interacts with myosin-9 (a), β-actin* (b) and enolase 1 (c) in V5-tagged MT-3 expressing proximal tubule cells. To verify the binding of MT-3 to myosin-9, β-actin, and enolase 1 in vitro, the V5 antibody was cross-linked to agarose beads through aldehyde linkage and incubated with HK-2 MT-3-V5 cell lysates. Bound proteins were eluted and detected by western blotting. The corresponding protein was not detected in non-transfected control (no V5 tagged MT-3), no antibody control, and no lysate control. *Note: Heat causes Protein A/G to come off the magnetic beads, and the reducing agent dissociates the heavy and light chains of the antibody, resulting in detection via secondary antibody reactivity towards protein A/G.
Table 1.
Identification of MT-3-binding proteins by MT-3 affinity chromatography.
Fig 2.
Intracellular localization of MT-3 in a doming population of HK-2(MT-3) cells.
MT-3 (green) is seen in a non-doming monolayer (A-E), a moderately tall dome (F-J), and in a taller dome structure (K-O). Nuclei are stained with DAPI (blue). The z-section level (0.35 μm depth) relative to the total number of z-sections through that region is indicated as a ratio in the upper right corner of each image. The scale bar at the bottom right of each image shows increments of 5 μm up to 25 μm.
Fig 3.
Orthogonal images of MT-3 localization.
MT-3 (green) and nuclei (blue) are seen in orthogonal sections along the x-axis (A, C and E) and y-axis (B, D and F) for same 3 fields shown in Fig 1. The non-doming monolayer (A and B), the moderately tall dome (C and D) and the taller dome structure (E and F) are all shown.
Fig 4.
Intracellular localization of F-actin in a doming population of HK-2(MT-3) cells.
F-actin (red) is seen in a non-doming monolayer (A-E), a moderately tall dome (F-J), and in a taller dome structure (K-O). Nuclei are stained with DAPI (blue). The z-section level (0.35 μm depth) relative to the total number of z-sections through that region are indicated as a ratio in the upper right corner of each image. The scale bar at the bottom right of each image shows increments of 5 μm up to 25 μm.
Fig 5.
Orthogonal images of F-actin localization.
F-actin (red) and nuclei (blue) are seen in orthogonal sections along the x-axis (A, C and E) and y-axis (B, D and F) for same 3 fields shown in Fig 4. The non-doming monolayer (A and B), the moderately tall dome (C and D) and the taller dome structure (E and F) are all shown.
Fig 6.
Intracellular co-localization of MT-3 and its binding partners.
Images of MT-3 co-localization with the identified binding partners are shown in individual z-sections (0.35 μm depth) for the non-doming monolayer (A-E) and a doming region (F-J). Larger panels have the 25 μm scale bar indicated on the bottom left, and there is an inset indicated with a higher magnification image that contains a 5 μm scale bar. Respective orthogonal views are shown for each image and are indicated (’) for scanning along the x-axis and (") for scanning along the Y-axis. In each panel MT-3 staining is shown in green and the binding partners in red: F-actin (A and F); myosin-9 (B and G); tropomyosin 3 (C and H); enolase 1 (D and I); aldolase A (E and J). The z-section level is shown as a ratio relative to the total z-images taken for that panel.
Fig 7.
Three dimensional projection of the intracellular localization for the MT-3 binding partners.
Each of the binding partners are shown in red: Myosin-9 (A, E and I); tropomyosin 3 (B, F and J); enolase 1 (C, G and K); and aldolase A (D, H and L) and nuclei are shown in blue. The 3 different 3D views shown are side angle view (A-D), apical surface from the top down (E-H), and a portion of the field cut away in a 3D slice (cut in the X, Y, and Z planes) are shown in (I-K). All panels shown are for a representative dome structure.
Table 2.
Protein-protein molecular docking scores obtained for the best two conformations of MT-3 and its binding partners.
Fig 8.
Inter-biomolecular interactions.
The surface residues that are taking part in the interactions and making contacts between MT-3 (green) and the proteins (A) tropomyosin 3, B) enolase 1 (C) aldolase A, and (D) β-actin. All residues are colored and labeled.