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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

Apical-basal polarity is not disrupted in CSPP-L and Desmoplakin depleted multi-lumen Caco-2 spheroids.(A) Quantification of the multi-lumen phenotype in Caco-2 siRNA transfectants (siGFP, siCSPP1, siCSPP1 smart pool, siDSP). The bar diagram shows average of two experiments, error bars depict standard deviation. Statistical significance was tested by paired t-test. (B) CSPP-L and Desmoplakin depletion in Caco-2 spheroids was validated by immunoblotting (right panel). (C) Multi-lumen spheroids in CSPP1 and Desmoplakin depleted cells depict filamentous actin stabilization indicated by solid arrow heads (Phalloidin, white) and PKCζ enrichment (a-PKCζ, red) at the lumen facing apical membrane. Occasional weak PKCζ staining at the basal-side of outer-rim cells is seen in siCSPP1 and siDSP transfectants (open arrowheads). (D) Centrosomes (a-Pericentrin, green) positioned in the lumen oriented cytoplasm (see also S1, S2, and S3 Videos).
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pone.0134789.g006: Apical-basal polarity is not disrupted in CSPP-L and Desmoplakin depleted multi-lumen Caco-2 spheroids.(A) Quantification of the multi-lumen phenotype in Caco-2 siRNA transfectants (siGFP, siCSPP1, siCSPP1 smart pool, siDSP). The bar diagram shows average of two experiments, error bars depict standard deviation. Statistical significance was tested by paired t-test. (B) CSPP-L and Desmoplakin depletion in Caco-2 spheroids was validated by immunoblotting (right panel). (C) Multi-lumen spheroids in CSPP1 and Desmoplakin depleted cells depict filamentous actin stabilization indicated by solid arrow heads (Phalloidin, white) and PKCζ enrichment (a-PKCζ, red) at the lumen facing apical membrane. Occasional weak PKCζ staining at the basal-side of outer-rim cells is seen in siCSPP1 and siDSP transfectants (open arrowheads). (D) Centrosomes (a-Pericentrin, green) positioned in the lumen oriented cytoplasm (see also S1, S2, and S3 Videos).

Mentions: Desmosomal organization of intermediate filaments and columnar MTs integrity is an important factor in epithelial tissue morphogenesis and homeostasis. We therefore investigated the expression of CSPP-L in apical-basal polarized layers or spheres of intestinal epithelial Caco-2 cells and studied the effects of CSPP-L depletion on spheroid formation. CSPP-L and Desmoplakin co-localized at apical cell junctions of Caco-2 cell layers (S2 Fig), similar to the localization pattern in HCC1937 cells (Fig 1). To study the localization of CSPP-L in spheroids Caco-2 cells were seeded in a Matrigel-matrix, which promotes apical-basal polarization, spheroid growth and lumen formation [2]. IF of cells in Matrigel-matrix requires para-formaldehyde fixation for preservation of spheroid morphology. Unfortunately, para-formaldehyde fixation abrogated staining of CSPP-L and Desmoplakin at the desmosome in Caco-2 cell spheroids and monolayers (data not shown) and could hence not be evaluated at this compartment. However, at two-cell stage detectable CSPP-L prominently localized in a spotted pattern proximal to the central filamentous actin layer at the site of forming apical membrane and apical end of E-cadherin staining (Fig 5A). This localization pattern along the apical filamentous actin layer was observed throughout all stages of spheroid formation. The specificity of the cytoplasmic CSPP-L staining pattern was validated by transfection with CSPP1 targeting siRNA (Fig 5B and Fig 6). Interestingly, CSPP-L depleted Caco-2 spheroids developed multiple lumen or multiple central filamentous actin structures. Furthermore, multi-lumen spheroids formed by Desmoplakin or CSPP-L depleted Caco-2 cells showed aberrant MT networks and depicted similar morphology (Fig 5B and Fig 6).


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)

Apical-basal polarity is not disrupted in CSPP-L and Desmoplakin depleted multi-lumen Caco-2 spheroids.(A) Quantification of the multi-lumen phenotype in Caco-2 siRNA transfectants (siGFP, siCSPP1, siCSPP1 smart pool, siDSP). The bar diagram shows average of two experiments, error bars depict standard deviation. Statistical significance was tested by paired t-test. (B) CSPP-L and Desmoplakin depletion in Caco-2 spheroids was validated by immunoblotting (right panel). (C) Multi-lumen spheroids in CSPP1 and Desmoplakin depleted cells depict filamentous actin stabilization indicated by solid arrow heads (Phalloidin, white) and PKCζ enrichment (a-PKCζ, red) at the lumen facing apical membrane. Occasional weak PKCζ staining at the basal-side of outer-rim cells is seen in siCSPP1 and siDSP transfectants (open arrowheads). (D) Centrosomes (a-Pericentrin, green) positioned in the lumen oriented cytoplasm (see also S1, S2, and S3 Videos).
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Related In: Results  -  Collection

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pone.0134789.g006: Apical-basal polarity is not disrupted in CSPP-L and Desmoplakin depleted multi-lumen Caco-2 spheroids.(A) Quantification of the multi-lumen phenotype in Caco-2 siRNA transfectants (siGFP, siCSPP1, siCSPP1 smart pool, siDSP). The bar diagram shows average of two experiments, error bars depict standard deviation. Statistical significance was tested by paired t-test. (B) CSPP-L and Desmoplakin depletion in Caco-2 spheroids was validated by immunoblotting (right panel). (C) Multi-lumen spheroids in CSPP1 and Desmoplakin depleted cells depict filamentous actin stabilization indicated by solid arrow heads (Phalloidin, white) and PKCζ enrichment (a-PKCζ, red) at the lumen facing apical membrane. Occasional weak PKCζ staining at the basal-side of outer-rim cells is seen in siCSPP1 and siDSP transfectants (open arrowheads). (D) Centrosomes (a-Pericentrin, green) positioned in the lumen oriented cytoplasm (see also S1, S2, and S3 Videos).
Mentions: Desmosomal organization of intermediate filaments and columnar MTs integrity is an important factor in epithelial tissue morphogenesis and homeostasis. We therefore investigated the expression of CSPP-L in apical-basal polarized layers or spheres of intestinal epithelial Caco-2 cells and studied the effects of CSPP-L depletion on spheroid formation. CSPP-L and Desmoplakin co-localized at apical cell junctions of Caco-2 cell layers (S2 Fig), similar to the localization pattern in HCC1937 cells (Fig 1). To study the localization of CSPP-L in spheroids Caco-2 cells were seeded in a Matrigel-matrix, which promotes apical-basal polarization, spheroid growth and lumen formation [2]. IF of cells in Matrigel-matrix requires para-formaldehyde fixation for preservation of spheroid morphology. Unfortunately, para-formaldehyde fixation abrogated staining of CSPP-L and Desmoplakin at the desmosome in Caco-2 cell spheroids and monolayers (data not shown) and could hence not be evaluated at this compartment. However, at two-cell stage detectable CSPP-L prominently localized in a spotted pattern proximal to the central filamentous actin layer at the site of forming apical membrane and apical end of E-cadherin staining (Fig 5A). This localization pattern along the apical filamentous actin layer was observed throughout all stages of spheroid formation. The specificity of the cytoplasmic CSPP-L staining pattern was validated by transfection with CSPP1 targeting siRNA (Fig 5B and Fig 6). Interestingly, CSPP-L depleted Caco-2 spheroids developed multiple lumen or multiple central filamentous actin structures. Furthermore, multi-lumen spheroids formed by Desmoplakin or CSPP-L depleted Caco-2 cells showed aberrant MT networks and depicted similar morphology (Fig 5B and Fig 6).

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