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CSPP-L Associates with the Desmosome of Polarized Epithelial Cells and Is Required for Normal Spheroid Formation.

Sternemalm J, Geimer S, Frikstad KA, Schink KO, Stokke T, Patzke S - PLoS ONE (2015)

Bottom Line: Importantly, siRNA conferred depletion of CSPP-L or Desmoplakin promoted multi-lumen spheroid formation in 3D-cultures of non-ciliated human colon carcinoma Caco-2 cells.Multi-lumen spheroids of CSPP1 siRNA transfectants showed disrupted apical cell junction localization of the cytoskeleton organizing RhoGEF ECT2.Our results hence identify a novel, non-ciliary role for CSPP-L in epithelial morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Biology, Division of Cancer Medicine, Surgery and Transplantation, Institute for Cancer Research, Oslo University Hospitals-Norwegian Radium Hospital, Oslo, Norway.

ABSTRACT
Deleterious mutations of the Centrosome/Spindle Pole associated Protein 1 gene, CSPP1, are causative for Joubert-syndrome and Joubert-related developmental disorders. These disorders are defined by a characteristic mal-development of the brain, but frequently involve renal and hepatic cyst formation. CSPP-L, the large protein isoform of CSPP1 localizes to microtubule ends of the mitotic mid-spindle and the ciliary axoneme, and is required for ciliogenesis. We here report the microtubule independent but Desmoplakin dependent localization of CSPP-L to Desmosomes in apical-basal polarized epithelial cells. Importantly, siRNA conferred depletion of CSPP-L or Desmoplakin promoted multi-lumen spheroid formation in 3D-cultures of non-ciliated human colon carcinoma Caco-2 cells. Multi-lumen spheroids of CSPP1 siRNA transfectants showed disrupted apical cell junction localization of the cytoskeleton organizing RhoGEF ECT2. Our results hence identify a novel, non-ciliary role for CSPP-L in epithelial morphogenesis.

No MeSH data available.


Related in: MedlinePlus

Desmoplakin is required for CSPP-L localization to the Desmosome.IF (A-C) and immunoblotting of total cell lysates (D) of HCC1937 cells treated with indicated siRNAs. Cells were transfected in low calcium medium. 72hrs post-transfection cells were allowed to form cell-cell junctions for 40 min by change to pre-warmed, normal calcium medium. Cell-cell junction staining of CSPP-L (green in overlay image) is siCSPP1 sensitive (A). Depletion of CSPP-L does not impair cell-cell junction localization of Desmoplakin (A, red) or β-catenin (B, red). Depletion of Desmoplakin (C, red) results in loss of CSPP-L (green) staining at cell-cell junctions. Knockdown efficacy was monitored by immunoblotting for Desmoplakin, CSPP-L and compared to the loading control γ-tubulin (D).
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pone.0134789.g002: Desmoplakin is required for CSPP-L localization to the Desmosome.IF (A-C) and immunoblotting of total cell lysates (D) of HCC1937 cells treated with indicated siRNAs. Cells were transfected in low calcium medium. 72hrs post-transfection cells were allowed to form cell-cell junctions for 40 min by change to pre-warmed, normal calcium medium. Cell-cell junction staining of CSPP-L (green in overlay image) is siCSPP1 sensitive (A). Depletion of CSPP-L does not impair cell-cell junction localization of Desmoplakin (A, red) or β-catenin (B, red). Depletion of Desmoplakin (C, red) results in loss of CSPP-L (green) staining at cell-cell junctions. Knockdown efficacy was monitored by immunoblotting for Desmoplakin, CSPP-L and compared to the loading control γ-tubulin (D).

Mentions: We next investigated if CSPP-L and Desmoplakin are interdependent for their sub-cellular localization to cell junctions. Initial experiments using transfection of confluent HCC1937 cells with siRNAs targeting CSPP1 or DSP mRNAs did not result in any marked decrease in cell-cell contact staining (data not shown). We hence speculated that siRNA mediated depletion of these proteins at desmosomes might be impaired by slow turn-over of CSPP-L within cell-cell contact protein complexes. Calcium is an essential co-factor for cadherin based cell-cell contact formation, which is inhibited at low calcium concentration and inducible by restoring normal calcium levels through re-addition of normal growth medium (calcium-switch). We therefore transfected HCC1937 cells at sub-confluence with CSPP1 or DSP targeting siRNAs in medium containing low calcium concentration. 72 hours post siRNA transfection calcium levels were restored and cell-cell contacts allowed to form for 40 minutes. Cell-cell contact formation was monitored by IF (Fig 2A–2C) and knock-down efficacy also analyzed by immunoblotting for Desmoplakin and CSPP-L in total cell lysates (Fig 2D). GFP targeting control siRNA transfectants readily formed CSPP-L and Desmoplakin comprising patches at cell junctions. siRNA mediated knock-down of CSPP-L slightly decreased, but did not abolish Desmoplakin staining at cell-cell contacts (Fig 2A). β-catenin staining at cell junctions in siCSPP1 transfectants was indistinguishable from siGFP control transfected HCC1937 cells (Fig 2B). In contrast, siRNA mediated depletion of Desmoplakin strongly decreased CSPP-L staining at cell junctions (Fig 2C).


CSPP-L Associates with the Desmosome of Polarized Epithelial Cells and Is Required for Normal Spheroid Formation.

Sternemalm J, Geimer S, Frikstad KA, Schink KO, Stokke T, Patzke S - PLoS ONE (2015)

Desmoplakin is required for CSPP-L localization to the Desmosome.IF (A-C) and immunoblotting of total cell lysates (D) of HCC1937 cells treated with indicated siRNAs. Cells were transfected in low calcium medium. 72hrs post-transfection cells were allowed to form cell-cell junctions for 40 min by change to pre-warmed, normal calcium medium. Cell-cell junction staining of CSPP-L (green in overlay image) is siCSPP1 sensitive (A). Depletion of CSPP-L does not impair cell-cell junction localization of Desmoplakin (A, red) or β-catenin (B, red). Depletion of Desmoplakin (C, red) results in loss of CSPP-L (green) staining at cell-cell junctions. Knockdown efficacy was monitored by immunoblotting for Desmoplakin, CSPP-L and compared to the loading control γ-tubulin (D).
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Related In: Results  -  Collection

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pone.0134789.g002: Desmoplakin is required for CSPP-L localization to the Desmosome.IF (A-C) and immunoblotting of total cell lysates (D) of HCC1937 cells treated with indicated siRNAs. Cells were transfected in low calcium medium. 72hrs post-transfection cells were allowed to form cell-cell junctions for 40 min by change to pre-warmed, normal calcium medium. Cell-cell junction staining of CSPP-L (green in overlay image) is siCSPP1 sensitive (A). Depletion of CSPP-L does not impair cell-cell junction localization of Desmoplakin (A, red) or β-catenin (B, red). Depletion of Desmoplakin (C, red) results in loss of CSPP-L (green) staining at cell-cell junctions. Knockdown efficacy was monitored by immunoblotting for Desmoplakin, CSPP-L and compared to the loading control γ-tubulin (D).
Mentions: We next investigated if CSPP-L and Desmoplakin are interdependent for their sub-cellular localization to cell junctions. Initial experiments using transfection of confluent HCC1937 cells with siRNAs targeting CSPP1 or DSP mRNAs did not result in any marked decrease in cell-cell contact staining (data not shown). We hence speculated that siRNA mediated depletion of these proteins at desmosomes might be impaired by slow turn-over of CSPP-L within cell-cell contact protein complexes. Calcium is an essential co-factor for cadherin based cell-cell contact formation, which is inhibited at low calcium concentration and inducible by restoring normal calcium levels through re-addition of normal growth medium (calcium-switch). We therefore transfected HCC1937 cells at sub-confluence with CSPP1 or DSP targeting siRNAs in medium containing low calcium concentration. 72 hours post siRNA transfection calcium levels were restored and cell-cell contacts allowed to form for 40 minutes. Cell-cell contact formation was monitored by IF (Fig 2A–2C) and knock-down efficacy also analyzed by immunoblotting for Desmoplakin and CSPP-L in total cell lysates (Fig 2D). GFP targeting control siRNA transfectants readily formed CSPP-L and Desmoplakin comprising patches at cell junctions. siRNA mediated knock-down of CSPP-L slightly decreased, but did not abolish Desmoplakin staining at cell-cell contacts (Fig 2A). β-catenin staining at cell junctions in siCSPP1 transfectants was indistinguishable from siGFP control transfected HCC1937 cells (Fig 2B). In contrast, siRNA mediated depletion of Desmoplakin strongly decreased CSPP-L staining at cell junctions (Fig 2C).

Bottom Line: Importantly, siRNA conferred depletion of CSPP-L or Desmoplakin promoted multi-lumen spheroid formation in 3D-cultures of non-ciliated human colon carcinoma Caco-2 cells.Multi-lumen spheroids of CSPP1 siRNA transfectants showed disrupted apical cell junction localization of the cytoskeleton organizing RhoGEF ECT2.Our results hence identify a novel, non-ciliary role for CSPP-L in epithelial morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Biology, Division of Cancer Medicine, Surgery and Transplantation, Institute for Cancer Research, Oslo University Hospitals-Norwegian Radium Hospital, Oslo, Norway.

ABSTRACT
Deleterious mutations of the Centrosome/Spindle Pole associated Protein 1 gene, CSPP1, are causative for Joubert-syndrome and Joubert-related developmental disorders. These disorders are defined by a characteristic mal-development of the brain, but frequently involve renal and hepatic cyst formation. CSPP-L, the large protein isoform of CSPP1 localizes to microtubule ends of the mitotic mid-spindle and the ciliary axoneme, and is required for ciliogenesis. We here report the microtubule independent but Desmoplakin dependent localization of CSPP-L to Desmosomes in apical-basal polarized epithelial cells. Importantly, siRNA conferred depletion of CSPP-L or Desmoplakin promoted multi-lumen spheroid formation in 3D-cultures of non-ciliated human colon carcinoma Caco-2 cells. Multi-lumen spheroids of CSPP1 siRNA transfectants showed disrupted apical cell junction localization of the cytoskeleton organizing RhoGEF ECT2. Our results hence identify a novel, non-ciliary role for CSPP-L in epithelial morphogenesis.

No MeSH data available.


Related in: MedlinePlus