Figure 5. α-cat proximity partner,... | Rockefeller University Press

Figure 5.

$α-cat proximity partner, LZTS2, localizes to apical junctions, base of primary cilia and midbody and is required for successful cell division in MDCK cells. (A) Confocal en face image (maximum z-projection; inverted grayscale) and orthogonal view images of MDCK cells on polycarbonate filters showing LZTS2 localizes to apical junctions and basal body (primary cilium), with schematic illustration. α-cat (green) and LZTS2 (magenta). Scale bar, en face 10 μm; orthogonal scale, 3 μm. (B) Confocal images of MDCK cells on coverslips showing LZTS2 is enriched at midbodies during cell abscission (magenta arrows) and multivertex junctions in mitosis (magenta arrowhead; see also Fig. 8). α-cat (green), LZTS2 (magenta), acetylated tubulin (cyan). Scale bar, 10 μm. (C) Confocal images showing transient knockdown of LZTS2 expression using shRNA in MDCK cells promotes binucleation (colored arrows). LZTS2 (magenta), Acetylated tubulin (cyan). Scale bar, 50 μm. Representative images from two independent experiments (n = 2) are shown. (D) Immunoblots of total cell extracts showing partial knockdown of LZTS2 expression using siRNA (48 or 72 h after transfection). Asterisk (*) denotes non-specific band refractory to knock-down and possibly responsible for strong detection of perinuclear/Golgi structures by immunofluorescence (evident in C and Fig. S3). (E) Percentage of multinucleated MDCK cells upon transient knockdown of LZTS2 expression using either shRNA (three biological replicates) or siRNA (two biological replicates). Two-tailed unpaired t test, *P < 0.05. ***P < 0.001. Graph indicates mean ± SD. Source data are available for this figure: SourceData F5. Refer to the image caption for details.$

α-cat proximity partner, LZTS2, localizes to apical junctions, base of primary cilia and midbody and is required for successful cell division in MDCK cells. (A) Confocal en face image (maximum z-projection; inverted grayscale) and orthogonal view images of MDCK cells on polycarbonate filters showing LZTS2 localizes to apical junctions and basal body (primary cilium), with schematic illustration. α-cat (green) and LZTS2 (magenta). Scale bar, en face 10 μm; orthogonal scale, 3 μm. (B) Confocal images of MDCK cells on coverslips showing LZTS2 is enriched at midbodies during cell abscission (magenta arrows) and multivertex junctions in mitosis (magenta arrowhead; see also Fig. 8). α-cat (green), LZTS2 (magenta), acetylated tubulin (cyan). Scale bar, 10 μm. (C) Confocal images showing transient knockdown of LZTS2 expression using shRNA in MDCK cells promotes binucleation (colored arrows). LZTS2 (magenta), Acetylated tubulin (cyan). Scale bar, 50 μm. Representative images from two independent experiments (n = 2) are shown. (D) Immunoblots of total cell extracts showing partial knockdown of LZTS2 expression using siRNA (48 or 72 h after transfection). Asterisk (*) denotes non-specific band refractory to knock-down and possibly responsible for strong detection of perinuclear/Golgi structures by immunofluorescence (evident in C and Fig. S3). (E) Percentage of multinucleated MDCK cells upon transient knockdown of LZTS2 expression using either shRNA (three biological replicates) or siRNA (two biological replicates). Two-tailed unpaired t test, *P < 0.05. ***P < 0.001. Graph indicates mean ± SD. Source data are available for this figure: SourceData F5.