Fig 1.
SORBS2 isoforms expressed in cultured human and canine epithelial cells.
(A) Immunoblotted lysates from SKco15 cells reveal three distinct SORBS2 protein bands with sizes between approximately 65–85 kDa. After CRISPR KO SORBS2 protein is no longer detectable by immunoblot. (B) One distinct SORBS2 band, approximately 75 kDa in size, is visible in immunoblotted lysates from MDCKII cells and after CRISPR KO SORBS2 protein is no longer detectable. (C) DNA gel from PCR products generated by using six primer pairs to identify various isoforms of human SORBS2. Primer pair 1 is showing a 216 bp product, confirming isoform 9. Primer pair 2 is showing a distinct band at 185 bp consistent with isoform 9 and a weak band at 401 bp (could be isoforms 3, 4, 5, 12). Isoforms 2 and 8 can be excluded due to a lack of bands at 326 and 242 bp respectively. Isoform 2 can again be excluded due to the lack of a 176 bp product with primer pair 3. Primer pair 4 shows a 457 bp, confirming presence of isoforms 9 and 12. Primer pairs 5 and 6 both show the presence of isoform 3 at 303 bp and 540 bp respectively. (D) PCR products generated by using specific primers to identify various isoforms of canine SORBS2. Primer pair 1 should give a 900 bp product for isoform X23, which is confirmed. Isoform X25 would have been 878 bp, isoform X26 1109 bp and isoform X27 1177 bp respectively, which means these isoforms could be excluded by size. Isoform X25 has a very similar expected size to isoform X23, but we could exclude it based on the negative result obtained with primer pair 3. Primer pair 2 should give a 900 bp product for isoforms X23 and X26 which further confirms the presence of isoform X23. Primer pair 3 should identify isoforms X25 and X26 at 900 bp, but there is no evidence of a distinct product that size. We already excluded isoforms X25 and X27 and therefore the 850 bp product is thus isoform X23. Primer pairs 5 and 6 should recognize all potential SORBS2 isoforms (NCBI mRNA isoforms X23, X25, X26, X27) at 850 bp and 500 bp respectively. (E) Schematic figure of SORBS2 isoforms identified in human SKco15 cells and canine MDCKII cells. These are the only splice forms compatible with PCR results and the band sizes on the immunoblots.
Fig 2.
SORBS2 partially colocalizes with ZO-1 in murine bile canaliculi and with ZO-1 and E-cadherin in polarized MDCKII cells.
(A) Confocal immunofluorescence analysis reveals that SORBS2 is localized to bile canalicular TJs in murine liver and is partially colocalized with ZO-1 in X, Y and Z planes (63x oil objective was used, scale bar: 10 μm). Images are maximum intensity projections of Z-stacks (total depth 4.5 μm). Merged images show that they overlap, but that ZO-1 also extends more apically. (B) SORBS2 is colocalized with ZO-1 and the apical portion of E-cadherin in polarized MDCKII cells cultured on Transwell filters for nine days (63x oil objective used, scale bar: 10 μm). Images are maximum intensity projections of Z-stacks (total depth for X-Y images ca 4.5 μm (to avoid the strong signal from basal actin stress fibers). SORBS2 is also faintly visible as green dots at the bottom of the cells (cross sections of actin stress fibers) in Z projections, especially in panel B. (full stack:10 μm).
Fig 3.
STED super resolution microscopy reveals that SORBS2 colocalizes with apical junctional actin and afadin, and partially overlaps with ZO-1, occludin and E-cadherin in polarized MDCKII cells.
(A) Super resolution microscopy shows that SORBS2 in fact only partially colocalizes with ZO-1 in a discontinuous pattern along cell-cell contacts. Line scans of fluorescence intensity at 90 degrees to the cell contacts confirmed the visual observations, graph to right (N = 20, mean ± SEM). (B) As with ZO-1, SORBS2 is decorating occludin in a discontinuous pattern at cell-cell junctions and we noticed that SORBS2, at this level, is absent from tricellular junctions. (C) SORBS2 is colocalized with junctional actin, as confirmed both by visual appearance and line scan results. (D) Myosin IIB is localized in a discontinuous pattern at approximately the same distance from the cell-cell junction as SORBS2, but to note is that myosin IIB is present where SORBS2 is absent. (E) The very apical portion of E-cadherin is in the same X/Y plane as SORBS2, but SORBS2 localized farther from the membrane than E-cadherin. Scale bar: 0.5 μm. Images are maximum intensity projections of Z-stacks, total depth 2 μm.
Fig 4.
SORBS2 colocalizes with the thick contractile perijunctional actin, alpha-actinin and myosin structure induced by ZO-1, ZO-2 double knock-down in MDCKII cells.
Super resolution Airyscan immunofluorescent images confirms that actin is redistributed in ZO-1, ZO-2 dKD cells (C, lower panel, middle image) and SORBS2 which is normally colocalized with apical junctional actin in MDCKII cells (C, upper panel) is now redistributed with actin (C, lower panel, left and right images). Myosin IIB and alpha-actinin are also redistributed in ZO-1, ZO-2 dKD cells (Myosin IIB: A, lower panel center and D, lower panel center; alpha-actinin: B, lower panel center and D, lower panel left) as compared to cells rescued with ZO-1 (Myosin IIB: A and D, top panel center; alpha-actinin: B and D, top panel center). SORBS2 appears to co-localize with alpha-actinin and mostly colocalize with actin in both ZO-1 rescue cells and ZO-1, ZO-2 dKD cells (alpha-actinin: B, right images in upper and lower panel; actin: C, right images in upper and lower panel). Myosin IIB in ZO-1, ZO-2 dKD cells decorate the distal part of both SORBS2 (A: right image in lower panel) and alpha-actinin (D: right image in lower panel). (63x oil objective used, scale bar: 20 μm). Images are maximum intensity projection of Z-stacks (total depth range: 2–3.3 μm).
Fig 5.
Tight junction, adherens junction and cytoskeletal proteins are not changed either at cell-cell contacts or at actin stress fibers in SORBS2 knock-out cells.
(A) Localization of the TJ proteins ZO-1 and cingulin is not changed in SORBS2 KO cells (right panel) as compared to WT cells (left panel). SORBS2 is present in a discontinuous pattern at apical cell-cell contacts in WT cells (left panel) and is not visible in SORBS2 KO cells (right panel). The adherens junction proteins E-cadherin (panel A) and afadin (panel B) are both unchanged by SORBS2 KO as compared to WT cells (right and left panels respectively). (B) the cytoskeletal proteins actin, vinculin and myosin IIB are also not affected by SORBS2 KO (right panel) as compared to WT cells (left panel). (C) SORBS2 is also localized at actin stress fibers (WT, left panel) and is not visible in SORBS2 KO cells (right panel). Actin, vinculin and myosin IIB are not changed at actin stress fibers in SORBS2 KO cells (right panel) as compared to WT cells (left panel). 63x objective was used, apical images are maximum intensity projections (depth range: 1.7–3 μm depending on the basolateral distribution of each protein, scale bar: 20 μm). Basal images: SORBS2 and actin are maximum intensity projections (1.26 μm), vinculin (0.82 μm) and Myosin IIB (0.42 μm). Scale bar: 20 μm.
Fig 6.
Knock-out of SORBS2 does not affect TJ recovery after calcium switch.
(A) SORBS2 colocalizes with ZO-1 in WT SKco15 cells under normal culture conditions. (B) After overnight culture in low-calcium both WT and SORBS2 KO cells are rounded up and no normal cell-cell junctions are visible. Already one hour after normal calcium is restored cell-cell junctions are starting to recover (C) and the recovery progresses after 2 hours (D) and after 7 hours (E) the cell-cell contacts look normal by immunofluorescent microscopy. Scale bar: 20 μm. Images are maximum intensity projections of Z-stacks (total depth:10 μm). (F) TER is high in both WT and SORBS2 KO SKco15cells before calcium removal. However, after overnight low calcium TER is almost undetectable. TER recovers as the cell-cell contacts are reassembled and is almost fully recovered 24 hours after normal calcium is restored. Data shown are mean ±SEM of duplicate samples.
Fig 7.
HGF-induced cell scattering and accelerated wound healing is the same in SORBS2 knock-out and control MDCKII cells.
(A) SORBS2 colocalizes with ZO-1 in WT MDCKII cells. (B) Twenty-four hours after HGF treatment cells scatter and ZO-1 becomes discontinuous at cell-cell contacts in both WT and SORBS2 KO cells, as does SORBS2 in WT cells. (C) Forty-eight hours after HGF treatment cell scattering patterns again looks the same in both WT and SORBS2 KO cells. (D) HGF speeds up wound healing in both SORBS2 KO and WT cells (12 hours) as compared to normal complete media (28 hours), but there is no difference between SORBS2 KO and WT MDCKII cells (63 x oil objective, scale bar: 20 μm).
Fig 8.
GFP-SORBS2 expression in MDCKII cells recruits actin, alpha-actinin, vinculin, N-WASP and possibly CIP4.
Overexpression of GFP-SORBS2 is strongly associated with accumulation of actin, alpha-actinin and vinculin (A, B, C) and weakly associated with N-WASP and possibly CIP4 (D, A) as shown by confocal immunofluorescence imaging. Afadin accumulation was not induced by expression of gGFP-SORBS2 (E). 20x objective was used, scale bar: 40 μm. Images shown are maximum intensity projection of Z-stacks (total depth: 10 μm).