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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

CSPP-L localizes to the Desmosome.IF of CSPP-L (a-CSPP-L, green) in the basal-like breast cancer cell line HCC1937 showed co-localization with the desmosomal protein Desmoplakin (A, a-DSP, red; upper panel) but not with the AJ associated protein β-catenin (B, a-CTNNB1, red; lower panel), as tested by co-localization analysis (linear dependence, Pearson’s coefficient). (C-E) 3D-super-resolution microscopy refined the localization of CSPP-L to single patches that frame Desmoplakin at the cell junction (C) and do not co-localize with β-catenin (D). Higher magnification image and line plots of signal intensities across individual desmosomes at cell junctions (E). Dashed line in overview image connects peaks in Desomplakin signals. Line plots show signal intensities along an 800nm line across desmosomes depicted below (dashed line in magnifications, scale bars magnifications = 200nm). Average distances (with standard deviation) of peak intensities of CSPP-L patches and Desmoplakin (26 cells with non-separated and eleven cells with separated Desmoplakin signals).
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pone.0134789.g001: CSPP-L localizes to the Desmosome.IF of CSPP-L (a-CSPP-L, green) in the basal-like breast cancer cell line HCC1937 showed co-localization with the desmosomal protein Desmoplakin (A, a-DSP, red; upper panel) but not with the AJ associated protein β-catenin (B, a-CTNNB1, red; lower panel), as tested by co-localization analysis (linear dependence, Pearson’s coefficient). (C-E) 3D-super-resolution microscopy refined the localization of CSPP-L to single patches that frame Desmoplakin at the cell junction (C) and do not co-localize with β-catenin (D). Higher magnification image and line plots of signal intensities across individual desmosomes at cell junctions (E). Dashed line in overview image connects peaks in Desomplakin signals. Line plots show signal intensities along an 800nm line across desmosomes depicted below (dashed line in magnifications, scale bars magnifications = 200nm). Average distances (with standard deviation) of peak intensities of CSPP-L patches and Desmoplakin (26 cells with non-separated and eleven cells with separated Desmoplakin signals).

Mentions: We recently reported the localization of CSPP-L at motile cilia of mouse trachea epithelial cells by immunogold and immunofluorescence analysis (IF) [37]. During the investigation of murine epithelial cells CSPP-L labelling at desmosomal junctions was noticed (S1 Fig). The staining of CSPP-L at undetermined apical cell junctions was seen earlier in apical-basal polarized Madin-Darbey canine kidney cells (MDCK, S1 Fig), but yet not characterized in human cell line models. We therefore examined the localization of CSPP-L in confluent, apical-basal polarized cells of the human basal-like breast cancer cell line HCC1937 (Fig 1). In congruence with the staining pattern of CSPP-L in MDCK cells, CSPP-L localized to apical cell junctions barely overlapping with the AJ protein β-catenin (Fig 1A; Pearson’s co-localization coefficient = -0.07) but co-localizing with the desmosomal protein Desmoplakin (Fig 1B; Pearson’s colocalization coefficient = 0.85) as determined by structured illumination microscopy (SIM, ApoTome [38]). The Desmosome is a highly organized, electron dense structure of less than 1 μm in diameter, which bridges cytoplasmic plaques of opposing cells via membrane transpassing desmosomal cadherins that bind within an approximately 35nm wide intercellular space ([4] and S1 Fig). Desmoplakin connects the membrane adjacent outer dense plaque with the intermediate filament organizing inner dense plaque. We applied three-dimensional super-resolution microscopy (3D-SIM, [39,40]) to resolve the localization of CSPP-L in HCC1937 cells at a lateral resolution of about 125nm (Fig 1C–1E). 3D-SIM revealed that CSPP-L is localized in paired patches that frame Desmoplakin labelled plaques at the cytolasmic site (Fig 1C). These paired patches are well separated from membrane associated β-catenin (Fig 1D). In most desmosomes the desmoplakin label of individual plaques was below the resolution limit (Fig 1E), while Desmoplakin framing CSPP-L patches were separated by 180±30nm. Interestingly, CSPP-L associated with the cytoplasmic side of desmosmal plaques prior to merging of Desmoplakin signals (Fig 1E).


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)

CSPP-L localizes to the Desmosome.IF of CSPP-L (a-CSPP-L, green) in the basal-like breast cancer cell line HCC1937 showed co-localization with the desmosomal protein Desmoplakin (A, a-DSP, red; upper panel) but not with the AJ associated protein β-catenin (B, a-CTNNB1, red; lower panel), as tested by co-localization analysis (linear dependence, Pearson’s coefficient). (C-E) 3D-super-resolution microscopy refined the localization of CSPP-L to single patches that frame Desmoplakin at the cell junction (C) and do not co-localize with β-catenin (D). Higher magnification image and line plots of signal intensities across individual desmosomes at cell junctions (E). Dashed line in overview image connects peaks in Desomplakin signals. Line plots show signal intensities along an 800nm line across desmosomes depicted below (dashed line in magnifications, scale bars magnifications = 200nm). Average distances (with standard deviation) of peak intensities of CSPP-L patches and Desmoplakin (26 cells with non-separated and eleven cells with separated Desmoplakin signals).
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Related In: Results  -  Collection

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pone.0134789.g001: CSPP-L localizes to the Desmosome.IF of CSPP-L (a-CSPP-L, green) in the basal-like breast cancer cell line HCC1937 showed co-localization with the desmosomal protein Desmoplakin (A, a-DSP, red; upper panel) but not with the AJ associated protein β-catenin (B, a-CTNNB1, red; lower panel), as tested by co-localization analysis (linear dependence, Pearson’s coefficient). (C-E) 3D-super-resolution microscopy refined the localization of CSPP-L to single patches that frame Desmoplakin at the cell junction (C) and do not co-localize with β-catenin (D). Higher magnification image and line plots of signal intensities across individual desmosomes at cell junctions (E). Dashed line in overview image connects peaks in Desomplakin signals. Line plots show signal intensities along an 800nm line across desmosomes depicted below (dashed line in magnifications, scale bars magnifications = 200nm). Average distances (with standard deviation) of peak intensities of CSPP-L patches and Desmoplakin (26 cells with non-separated and eleven cells with separated Desmoplakin signals).
Mentions: We recently reported the localization of CSPP-L at motile cilia of mouse trachea epithelial cells by immunogold and immunofluorescence analysis (IF) [37]. During the investigation of murine epithelial cells CSPP-L labelling at desmosomal junctions was noticed (S1 Fig). The staining of CSPP-L at undetermined apical cell junctions was seen earlier in apical-basal polarized Madin-Darbey canine kidney cells (MDCK, S1 Fig), but yet not characterized in human cell line models. We therefore examined the localization of CSPP-L in confluent, apical-basal polarized cells of the human basal-like breast cancer cell line HCC1937 (Fig 1). In congruence with the staining pattern of CSPP-L in MDCK cells, CSPP-L localized to apical cell junctions barely overlapping with the AJ protein β-catenin (Fig 1A; Pearson’s co-localization coefficient = -0.07) but co-localizing with the desmosomal protein Desmoplakin (Fig 1B; Pearson’s colocalization coefficient = 0.85) as determined by structured illumination microscopy (SIM, ApoTome [38]). The Desmosome is a highly organized, electron dense structure of less than 1 μm in diameter, which bridges cytoplasmic plaques of opposing cells via membrane transpassing desmosomal cadherins that bind within an approximately 35nm wide intercellular space ([4] and S1 Fig). Desmoplakin connects the membrane adjacent outer dense plaque with the intermediate filament organizing inner dense plaque. We applied three-dimensional super-resolution microscopy (3D-SIM, [39,40]) to resolve the localization of CSPP-L in HCC1937 cells at a lateral resolution of about 125nm (Fig 1C–1E). 3D-SIM revealed that CSPP-L is localized in paired patches that frame Desmoplakin labelled plaques at the cytolasmic site (Fig 1C). These paired patches are well separated from membrane associated β-catenin (Fig 1D). In most desmosomes the desmoplakin label of individual plaques was below the resolution limit (Fig 1E), while Desmoplakin framing CSPP-L patches were separated by 180±30nm. Interestingly, CSPP-L associated with the cytoplasmic side of desmosmal plaques prior to merging of Desmoplakin signals (Fig 1E).

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