A CEP104-CSPP1 Complex Is Required for Formation of Primary Cilia Competent in Hedgehog Signaling

CEP104 is an evolutionarily conserved centrosomal and ciliary tip protein. CEP104 loss-of-function mutations are reported in patients with Joubert syndrome, but their function in the etiology of ciliopathies is poorly understood. Here, we show that cep104 silencing in zebrafish causes cilia-related manifestations: shortened cilia in Kupffer's vesicle, heart laterality, and cranial nerve development defects. We show that another Joubert syndrome-associated cilia tip protein, CSPP1, interacts with CEP104 at microtubules for the regulation of axoneme length. We demonstrate in human telomerase reverse transcriptase-immortalized retinal pigmented epithelium (hTERT-RPE1) cells that ciliary translocation of Smoothened in response to Hedgehog pathway stimulation is both CEP104 and CSPP1 dependent. However, CEP104 is not required for the ciliary recruitment of CSPP1, indicating that an intra-ciliary CEP104-CSPP1 complex controls axoneme length and Hedgehog signaling competence. Our in vivo and in vitro analyses of CEP104 define its interaction with CSPP1 as a requirement for the formation of Hedgehog signaling-competent cilia, defects that underlie Joubert syndrome

3D-Structured Illumination Microscopy of Centrosomes in Human Cell Lines

The centrosome is the main microtubule-organizing center of animal cells, and is composed of two barrel-shaped microtubule-based centrioles embedded in protein dense pericentriolar material. Compositional and architectural re-organization of the centrosome drives its duplication, and enables its microtubule-organizing activity and capability to form the primary cilium, which extends from the mature (mother) centriole, as the cell exits the cell cycle. Centrosomes and primary cilia are essential to human health, signified by the causal role of centrosome- and cilia-aberrations in numerous congenic disorders, as well as in the etiology and progression of cancer. The list of disease-associated centrosomal proteins and their proximitomes is steadily expanding, emphasizing the need for high resolution mapping of such proteins to specific substructures of the organelle. Here, we provide a detailed 3D-structured illumination microscopy (3D-SIM) protocol for comparative localization analysis of fluorescently labeled proteins at the centrosome in fixed human cell lines, at approximately 120 nm lateral and 300 nm axial resolution. The procedure was optimized to work with primary antibodies previously known to depend on more disruptive fixation reagents, yet largely preserves centriole and centrosome architecture, as shown by transposing acquired images of landmark proteins on previously published transmission electron microscopy (TEM) images of centrosomes. Even more advantageously, it is compatible with fluorescent protein tags. Finally, we introduce an internal reference to ensure correct 3D channel alignment. This protocol hence enables flexible, swift, and information-rich localization and interdependence analyses of centrosomal proteins, as well as their disorder-associated mutations

Prognostic significance of S100A4 expression in stage II and III colorectal cancer: results from a population-based series and a randomized phase III study on adjuvant chemotherapy

Published version. Source at <a href=http://dx.doi.org/10.1002/cam4.766> http://dx.doi.org/10.1002/cam4.766 </a>Current clinical algorithms are unable to precisely predict which colorectal cancer patients would benefit from adjuvant chemotherapy, and there is a need for novel biomarkers to improve the selection of patients. The metastasis-promoting protein S100A4 predicts poor outcome in colorectal cancer, but whether it could be used to guide clinical decision making remains to be resolved. S100A4 expression was analyzed by immunohistochemistry in primary colorectal carcinomas from a consecutively collected, population-representative cohort and a randomized phase III study on adjuvant 5-fluorouracil/ levamisole. Sensitivity to treatment with 5-fluorouracil in S100A4 knockdown cells was investigated using 2D and 3D cell culture assays. Strong nuclear expression of S100A4 was detected in 19% and 23% of the tumors in the two study cohorts, respectively. In both cohorts, nuclear immunoreactivity was associated with reduced relapse-free (P < 0.001 and P = 0.010) and overall survival (P = 0.046 and P = 0.006) in univariate analysis. In multivariate analysis, nuclear S100A4 was a predictor of poor relapse-free survival in the consecutive series (P = 0.002; HR 1.9), but not in the randomized study. Sensitivity to treatment with 5-fluorouracil was not affected by S100A4 expression in in vitro cell culture assays, and there was no indication from subgroup analyses in the randomized study that S100A4 expression was associated with increased benefit of adjuvant treatment with 5-fluorouracil/ levamisole. The present study confirms that nuclear S100A4 expression is a negative prognostic biomarker in colorectal cancer, but the clinical utility in selection of patients for adjuvant fluoropyrimidine-based chemotherapy is limited

Prognostic significance of S100A4 expression in stage II and III colorectal cancer: results from a population-based series and a randomized phase III study on adjuvant chemotherapy

Current clinical algorithms are unable to precisely predict which colorectal cancer patients would benefit from adjuvant chemotherapy, and there is a need for novel biomarkers to improve the selection of patients. The metastasis-promoting protein S100A4 predicts poor outcome in colorectal cancer, but whether it could be used to guide clinical decision making remains to be resolved. S100A4 expression was analyzed by immunohistochemistry in primary colorectal carcinomas from a consecutively collected, population-representative cohort and a randomized phase III study on adjuvant 5-fluorouracil/levamisole. Sensitivity to treatment with 5-fluorouracil in S100A4 knockdown cells was investigated using 2D and 3D cell culture assays. Strong nuclear expression of S100A4 was detected in 19% and 23% of the tumors in the two study cohorts, respectively. In both cohorts, nuclear immunoreactivity was associated with reduced relapse-free (P < 0.001 and P = 0.010) and overall survival (P = 0.046 and P = 0.006) in univariate analysis. In multivariate analysis, nuclear S100A4 was a predictor of poor relapse-free survival in the consecutive series (P = 0.002; HR 1.9), but not in the randomized study. Sensitivity to treatment with 5-fluorouracil was not affected by S100A4 expression in in vitro cell culture assays, and there was no indication from subgroup analyses in the randomized study that S100A4 expression was associated with increased benefit of adjuvant treatment with 5-fluorouracil/levamisole. The present study confirms that nuclear S100A4 expression is a negative prognostic biomarker in colorectal cancer, but the clinical utility in selection of patients for adjuvant fluoropyrimidine-based chemotherapy is limited

Depletion of CSPP-L and Desmoplakin cause multi-lumen spheroid formation in Caco-2 spheroids.

<p>(A) Localization of CSPP-L (a-CSPP-L, green), filamentous actin (Phalloidin, white), E-cadherin (a-E-cadherin, red), and DNA (blue) during different stages of spheroid development of Caco-2 cells. Cells were grown in 3D-Matrigel culture and formalin fixed for IF. Images show projections of z-sections enclosing the entire lumen volume. CSPP-L shows prominent enrichment juxtapose to the apical filamentous actin throughout all stages of spheroid development (B) The apical CSPP-L staining pattern (a-CSPP-L, green) is CSPP1 siRNA sensitive and not altered by Desmoplakin depletion (a-α-tubulin, red; phalloidin, white). CSPP1 and Desmoplakin siRNA Caco-2 transfectants develop disorganized cell aggregates with multiple lumen.</p

Apical-basal polarity is not disrupted in CSPP-L and Desmoplakin depleted multi-lumen Caco-2 spheroids.

<p>(A) Quantification of the multi-lumen phenotype in Caco-2 siRNA transfectants (siGFP, siCSPP1, siCSPP1 <i>s</i>mart <i>p</i>ool, 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 <i>siCSPP1</i> and <i>siDSP</i> transfectants (open arrowheads). (D) Centrosomes (a-Pericentrin, green) positioned in the lumen oriented cytoplasm (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134789#pone.0134789.s003" target="_blank">S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134789#pone.0134789.s004" target="_blank">S2</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134789#pone.0134789.s005" target="_blank">S3</a> Videos).</p

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

<div><p>Deleterious mutations of the Centrosome/Spindle Pole associated Protein 1 gene, <i>CSPP1</i>, 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 <i>CSPP1</i> 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 <i>CSPP1</i> 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.</p></div

A CEP104-CSPP1 Complex Is Required for Formation of Primary Cilia Competent in Hedgehog Signaling

Contains fulltext : 206876.pdf (publisher's version ) (Open Access

CSPP1 stabilizes growing microtubule ends and damaged lattices from the luminal side

Microtubules are dynamic cytoskeletal polymers, and their organization and stability are tightly regulated by numerous cellular factors. While regulatory proteins controlling the formation of interphase microtubule arrays and mitotic spindles have been extensively studied, the biochemical mechanisms responsible for generating stable microtubule cores of centrioles and cilia are poorly understood. Here, we used in vitro reconstitution assays to investigate microtubule-stabilizing properties of CSPP1, a centrosome and cilia-associated protein mutated in the neurodevelopmental ciliopathy Joubert syndrome. We found that CSPP1 preferentially binds to polymerizing microtubule ends that grow slowly or undergo growth perturbations and, in this way, resembles microtubule-stabilizing compounds such as taxanes. Fluorescence microscopy and cryo-electron tomography showed that CSPP1 is deposited in the microtubule lumen and inhibits microtubule growth and shortening through two separate domains. CSPP1 also specifically recognizes and stabilizes damaged microtubule lattices. These data help to explain how CSPP1 regulates the elongation and stability of ciliary axonemes and other microtubule-based structures.</p

Mis-localization of ECT2 in CSPP1 depleted multi-lumen Caco-2 spheroids.

<p>Spheroids of Caco-2 cells transfected with indicated siRNAs were stained for ECT2 (a-ECT2, green), the filamentous actin (Phalloidin, white) and α-tubulin (a-α-tubulin,red). ECT2 localized to apical cell-cell junctions in single-lumen spheroids of siGFP control transfectants and mal-organized spheroids of Desmoplakin depleted cells. The apical cell-cell junction ECT2 staining pattern is lost in multi-lumen spheroids of CSPP-L depleted cells. Bar diagram shows frequency of spheroids with lost or strongly reduced junctional ECT2 staining (100 spheroids per treatment scored in two experiments, error bars depict SEM, statistical significance was tested in paired t-test).</p