Inversin forms a complex with catenins and N-cadherin in polarized epithelial cells

Abstract

Nephrogenesis starts with the reciprocal induction of two embryonically distinct analages, metanephric mesenchyme and ureteric bud. This complex process requires the refined and coordinated expression of numerous developmental genes, such as inv. Mice that are homozygous for a mutation in the inv gene (inv/inv) develop renal cysts resembling autosomal-recessive polycystic kidney disease. The gene locus containing inv has been proposed to serve as a common modifier for some human and rodent polycystic kidney disease phenotypes. We generated polyclonal antibodies to inversin to study its subcellular distribution, potential binding partners, and functional aspects in cultured murine proximal tubule cells. A 125-kDa inversin protein isoform was found at cell-cell junctions. Two inversin isoforms, 140- and 90-kDa, were identified in the nuclear and perinuclear compartments. Plasma membrane allocation of inversin is dependent upon cell-cell contacts and was redistributed when cell adhesion was disrupted after incubation of the cell monolayer with low-calcium/EGTA medium. We further show that the membrane-associated 125-kDa inversin forms a complex with N-cadherin and the catenins. The 90-kDa nuclear inversin complexes with beta-catenin. These findings indicate that the inv gene product functions in several cellular compartments, including the nucleus and cell-cell adhesion sites.

Figure 1

Characterization of inversin antibody. (A) Total protein extracted from confluent S1 cells (1% Triton X-100 buffer) was separated by 7.5% SDS-PAGE, transferred to membranes, and incubated with affinity-purified inversin antibody. Three bands were detected at 140, 125, and 90 kDa with anti-inversin alone, but no bands were detected when inversin antibody was preincubated with the immunizing recombinant protein. (B) Postnuclear homogenate from confluent S1 cells was immunoprecipitated with affinity-purified inversin antibody and was resolved by 7.5% SDS-PAGE. The 140-kDa band was excised, trypsin digested in situ, and analyzed by mass spectrometry. Table shows peak values measured from mass spectrum as compared with calculated fragment masses after trypsin digestion of inversin. Differences between these values ranged in the expected variability.

Figure 2

Confocal microscopy of subconfluent (A, C, and E) and confluent (B, D, and F) S1 cells fixed in paraformaldehyde and triple-labeled with inversin antibody (A and B), DAPI (C and D), and phalloidin-rhodamine (E and F). In both subconfluent (A) and confluent (B) S1 cells, the inversin antibody stained nuclei and less intensely the perinuclear compartment. Cell membranes appeared uniformly stained in confluent cells (B), but were focally stained in subconfluent cells at early cell-cell contacts (A, arrowheads). Staining seen with inversin antibody (A and B) was absent in cells stained with inversin antibody preincubated with immunizing protein, and secondary antibody alone (A, insets).

Figure 3

Vertical sections (X-Z axis) of inversin staining. Confluent S1 cells were grown on filters, fixed in paraformaldehyde, and triple-labeled with DAPI (A), inversin antibody (B), and phalloidin-rhodamine (C). The vertical sections (X-Z axis) demonstrate distribution of inversin to membranes of cell-cell contacts, but not to apical or basal plasma membranes. (D) overlay of all three channels.

Figure 4

Subcellular distribution of inversin. (A) Iodixinol subcellular fractionations were collected from the lightest (top fraction 1) to the heaviest (bottom fraction 12) of the gradient. Equivalent fraction volumes were resolved by 7.5% SDS-PAGE and immunoblotted with antibodies to inversin, pan-cadherin, or β-catenin. Bands (140- and 90-kDa) were detected with anti-inversin in all fractions, but a 125-kDa band was detected only in membrane fraction 1. The bands detected by anti-pan-cadherin and anti-β-catenin were restricted to the lightest fractions. The accompanying graph expresses phosphorimager values for each band as a percentage of the total volume of all fractions measured for each antibody. (B) Nuclear and membrane protein extracts from confluent S1 cells were separated by 7.5% SDS-PAGE and were immunoblotted with anti-inversin. The inversin antibody detected only one band of 125 kDa in the membrane protein extract and two bands of 140- and 90- kDa in the nuclear protein extracts. (C) Total protein was extracted from confluent S1 cells with and without 1% Triton X-100 followed by inversin immunoblot analysis. Anti-inversin detected bands at 140 and 90 kDa in both extracts, but only in the presence of Triton X-100 did the inversin antibody detect a band at 125 kDa.

Figure 5

(A) Homogenates from confluent S1 cells were immunoprecipitated with inversin antibody and precipitates were immunoblotted with a panel of antibodies. Arrowheads indicate expected molecular weights. Bands were detected for α-, β-, and γ-catenin, and pan-, and N-cadherin, but no bands were detected for E-cadherin, B1-integrin, and vinculin despite long film exposure. (B) Confluent S1 cell homogenates were immunoprecipitated with anti-β-catenin (lane 1) and anti-N-cadherin (lanes 2 and 3), and precipitates were immunoblotted with anti-inversin or anti-β-catenin. The inversin antibody detected 125- and 90-kDa bands in both the β-catenin precipitate (left panel) and the N-cadherin precipitate (middle panel). The N-cadherin precipitate that was immunoblotted for β-catenin detected a 92-kDa band (right panel). (C) Iodixanol fractions 1 and 12 were collected as for Figure 3A, immunoprecipitated with inversin antibody, and immunoblotted with antibodies to β-catenin and N-cadherin. Inversin precipitates from fraction 1 contained β-catenin and N-cadherin (lanes 1 and 2), but no bands were detected in fraction 12 (lanes 3 and 4). Protein extracts in A, B, and C were separated by 7.5% SDS-PAGE and transferred to nitrocellulose membranes.

Figure 6

Colabeling of inversin with β-catenin or N-cadherin. Confluent S1 cells were double-labeled with antibodies to inversin (A) and β-catenin (B) or inversin (D) and N-cadherin (E) and were analyzed by confocal microscopy. Inversin colocalized with β-catenin at cell membranes, but there was partial overlap in nuclei (C, yellow overlap). Inversin colocalized with N-cadherin only at cell membranes.

Figure 7

Calcium depletion in confluent S1 cells by confocal microscopy. Confluent S1 cells were calcium depleted by incubating with medium containing 4 mM EGTA. At time 0, 45, and 90 min, cells were triple labeled with anti-inversin (A-C), anti-β-catenin (D-F), and phalloidin (G-I) or anti-inversin (J-L), anti-N-cadherin (M-O), and phalloidin (P-R). At 45 and 90 min of calcium depletion, cells progressively lost cell-cell contacts, as displayed by staining of the F-actin cytoskeleton (H, I, Q, and R). Calcium depletion also led to diminished staining of inversin (B, C, K and L), β-catenin (E and F), and N-cadherin (N and O) from cell membranes. Nuclear staining of inversin and β-catenin remained unchanged under low calcium conditions (C, F, and L).