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First published online 3 April 2007
doi: 10.1242/jcs.03439

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The control of tissue architecture over nuclear organization is crucial for epithelial cell fate

¹ Department of Basic Medical Sciences and Cancer Center, Purdue University, 625 Harrison Street, West Lafayette, IN 47907-2026, USA
² Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA

View larger version (55K): [in this window] [in a new window]   Fig. 1. Tumor nodules display altered tissue polarity and nuclear organization compared with acini formed by non-neoplastic cells. S1 and T4-2 cells were cultured in 3D in the presence of MatrigelTM for 10 days and labeled for differentiation markers collagen IV, 6-integrin, -catenin, mucin-1 and Ki-67, and markers of nuclear organization H3K9m, H4K20m, NuMA, SC35, PML and lamin B. (A) Immunostaining for basal polarity markers collagen IV (green) and 6-integrin (green), lateroapical polarity marker -catenin (red), and apical polarity marker mucin-1 (red) in acinar S1 cells (top panel) and malignant T4-2 cells (bottom panel). Drawings show the organization of S1 and T4-2 cells after 10 days of 3D culture; nuclei are represented in black. (B) Immunostaining for cell cycle marker Ki67 (green). Nuclei are counterstained with DAPI (blue). Bar graph shows the percentage of cells in the cell cycle based on Ki67 expression. (C) Organization of heterochromatin markers H3K9m (red) and H4K20m (red) in the nuclei of acinar (S1) and malignant (T4-2) cells. Arrows indicate the concentration of H3K9m and H4K20m around the nucleolus (*) in differentiated cells. (D) Organization of coiled-coil protein NuMA (green), splicing factor speckle marker SC35 (green), PML (red, with DAPI-counterstained nuclei in blue) and lamin B (red) in the nuclei of acinar (S1) and malignant (T4-2) cells. The arrowhead indicates the formation of enlarged NuMA foci in mid-nucleus in S1 cells. SC35 is organized into large domains in the nucleus of S1 cells (dotted line delineates the cell nucleus). The arrow points to the twisted shape of the malignant nucleus as shown by lamin B staining. Bars, 5 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 2. AG1478-induced phenotypic reversion of malignant cells is accompanied by partial tissue polarity and the re-establishment of the organization of markers of higher order nuclear structure. Malignant T4-2 cells were treated either with the reverting agent AG1478 or vehicle DMSO for 10 days in 3D culture in the presence of MatrigelTM and labeled for differentiation markers collagen IV, 6-integrin, -catenin, mucin-1, and Ki67, and for nuclear proteins H3K9m, H4K20m, NuMA, SC35 and lamin B. (A) Immunostaining for basal polarity markers collagen IV (green) and 6-integrin (green), lateroapical polarity marker -catenin (red), and apical polarity marker mucin-1 (red) in malignant T4-2 cells (top panel) and reverted RT4-2 cells (bottom panel). Drawings represent the organization of T4-2 and RT4-2 cells after 10 days of 3D culture. Nuclei are represented in black. (B) Immunostaining for cell cycle marker Ki67 (green). Nuclei are counterstained with DAPI (blue). Bar graph shows the percentage of cells in the cell cycle based on Ki67 expression. (C) Organization of heterochromatin markers H3K9m (red) and H4K20m (red) in the nuclei of malignant (T4-2) and reverted (RT4-2) cells. Arrows indicate the concentration of H3K9m and H4K20m around the nucleolus (*) in differentiated cells. (D) Organization of coiled-coil protein NuMA (green), splicing factor speckle marker SC35 (green), and lamin B (red) in the nuclei of malignant (T4-2) and reverted (RT4-2) cells. The arrowhead indicates the formation of enlarged NuMA foci in mid-nucleus in RT4-2 cells. SC35 is organized into large domains in the nucleus of RT4-2 cells (a dotted line delineates the cell nucleus). The arrows point to the twisted shape, as shown by lamin B staining, of the nucleus in malignant and reverted cells. (E) Left histogram: Percentage of nuclei in S1 acini, T4-2 tumor nodules, and RT4-2 spheroids with nuclear proteins displaying a distribution characteristic of acinar differentiation. Drawings show the distributions of H4K20m, H3K9m and SC35 representative of acinar differentiation. Right histogram: ratios of cells with differentiation-like distribution of NuMA over cells with nondifferentiation-like distribution of NuMA in the S1, RT4-2 and T4-2 populations.*P<0.01. Bars, 5 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 3. LY294002 induced-phenotypic reversion of malignant cells is accompanied with the re-establishment of basal polarity and higher order nuclear structure. Malignant T4-2 cells were treated either with the reverting agent LY294002 or vehicle DMSO for 10 days in 3D culture in the presence of MatrigelTM and labeled for differentiation markers collagen IV, 6-integrin, mucin-1 and Ki67, and nuclear proteins H4K20m, NuMA and SC35. (A) Immunostaining for basal polarity markers collagen IV (green) and 6-integrin (green), apical polarity marker mucin-1 (red), and proliferation marker Ki67 (green) in malignant (T4-2) cells (small tumor nodules are shown) and reverted (RT4-2) cells. Nuclei are counterstained with DAPI (blue). (B) Organization of heterochromatin marker H4K20m (red), coiled-coil protein NuMA (green; arrowheads indicate the presence of enlarged foci in mid-nucleus) and splicing factor speckle marker SC35 (green) in the nuclei of malignant (T4-2) and reverted (RT4-2) cells. The dotted line delineates the cell nucleus. Bars, 5 µm.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Alteration of NuMA induces apoptosis in non-neoplastic S1 cells and entry into the cell cycle in reverted T4-2 cells. Non-neoplastic S1 cells and malignant T4-2 cells treated with or without the reverting agent AG1478 were cultured in 3D MatrigelTM for 10 days, followed by digitonin permeabilization and incubation with antibodies against NuMA (NuMA mAb) or nonspecific mouse immunoglobulins (IgGs) for 3 days. (A) Immunostaining with a FITC-tagged antibody against mouse IgG reveals the location of IgGs in the cytoplasm and NuMA mAb in the cell nucleus of permeabilized RT4-2 and/or T4-2 cells. Arrowheads indicate the location of NuMA at the poles of mitotic spindles in malignant cells. (B) Immunostaining for collagen IV (green, see arrowhead) in RT4-2 and T4-2 multicellular structures. Arrows indicate the lack of continuous collagen IV staining in RT4-2 cells treated with NuMA mAb. (Note that fluorescent staining for NuMA mAb can be seen in the nuclei; both collagen IV and NuMA antibodies are mouse IgG1, thus they are recognized by the same secondary antibody.) Nuclei are counterstained with DAPI (blue). (C) Histograms of the percentage of apoptotic cells. (D) Histograms of the percentage of cells in the cell cycle (Ki67-positive). Immunostaining for NuMA (green) shows evidence of mitotic spindle formation in NuMA mAb-treated RT4-2 cells (see image and arrowhead). A minimum of 300 cells were scored per replicate in three independent experiments. *P<0.05. Bars, 5 µm.

View larger version (48K): [in this window] [in a new window]   Fig. 5. S1 cells that form multicellular structures with incomplete tissue polarity are induced to enter the cell cycle upon incubation with NuMA antibodies. (A) Ten day 3D MatrigelTM culture of non-neoplastic S1, malignant T4-2 and reverted RT4-2 cells immunostained for tight junction marker ZO-1 (green). Apical polarization of S1 acini is indicated by the apical location of ZO-1. Arrows indicate the presence of ZO-1 in the nucleus of RT4-2 cells. (B) Immunostaining for collagen IV (green), 6-integrin (green), -catenin (red) and ZO-1 (red) in non-neoplastic S1 cells cultured in collagen I for 10 days (Coll I) or cultured in collagen I for 9 days and replated in Matrigel for 24 hours (Coll I + MG). Multicellular structures in collagen I display alterations in all markers tested, as shown by the absence of continuous peripheral staining of collagen IV, the overlap of 6-integrin and -catenin staining at cell-cell junctions – see yellow – and the diffuse location of ZO-1. By contrast, only ZO-1 staining remains altered in multicellular structures replated in MatrigelTM, as shown by the presence of staining at the basal side of cells (see arrows). (C) Non-neoplastic S1 cells were cultured in MatrigelTM for 10 days to produce basoapically polarized acini (MG) or in collagen I for 9 days before replating in MatrigelTM for 24 hours (Coll I + MG) to produce a population enriched with only basally polarized multicellular structures. Acini and multicellular structures were then permeabilized with digitonin, incubated with NuMA antibodies (NuMA mAb) or nonspecific immunoglobulins (IgGs) for 3 days and immunostained for Ki67 (red) and mouse immunoglobulin (green). Immunostaining for mouse immunoglobulins highlights the nuclear location of NuMA mAb and the cytoplasmic location of nonspecific mouse IgGs. Arrows indicate Ki67-positive cells. Nuclei are counterstained with DAPI (blue). Bars, 10 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 6. Partially differentiated acini enter the cell cycle upon NuMA alteration regardless of the method used to induce incomplete polarity. (A) Histogram of the percentage of S1 multicellular structures obtained following 9 days in collagen I and 4 days in MatrigelTM (black bars) that possess intact collagen IV and 6-integrin staining, and apical location of ZO-1 compared with S1 cells cultured in MatrigelTM (shaded bars) for 13 days. (B) Non-neoplastic S1 cells were cultured in MatrigelTM for 10 days to produce basoapically polarized acini (shaded bars) or in collagen I for 9 days before replating in MatrigelTM for 24 hours (black bars) to produce a population enriched with only basally polarized multicellular structures. Acini and multicellular structures were then permeabilized with digitonin, incubated with NuMA antibodies (NuMA mAb) or nonspecific immunoglobulins (IgGs) for 3 days. Shown is the percentage of cells positive for cell cycle marker Ki67. 300 cells were scored in each replicate in three independent experiments. (C) Histogram of the percentage of S1 multicellular structures with ZO-1 apically located after treatment with EGTA for 24 hours. (D) Acini and multicellular structures were treated with EGTA for 1 hour, then briefly permeabilized with digitonin and incubated with NuMA antibodies (NuMA mAb) or nonspecific immunoglobulins (IgGs) for 3 days. EGTA was only left in the culture medium during the first 24 hours of antiNuMA antibody treatment. Shown is the percentage of cells positive for cell cycle marker Ki67. 300 cells were scored in each replicate in three independent experiments. *P<0.05. (E) Dual staining for NuMA (red) and Ki67 (green) in acini formed by cells transfected with 50 nM siRNA NuMA or 50 nM nontargeting siRNA and cultured in 3D for 8 days. Nuclei are counterstained with DAPI (blue). Arrowheads indicate strong Ki67 staining in nuclei that lack NuMA staining.

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