Trans-membrane protein and colocalizes with the tight junction Vasopressin proteins ZO-1 [1,4,5] and occluding [1]. Immunostaining on human embryos showed that CLMP was expressed in many tissues including the gut. Knock down experiments of the orthologue in zebrafish resulted in general developmental defects including an affected intestine. Goblet cells are normally present in the mid intestine in zebrafish (which resembles the small intestine in humans), and can therefore be used as a marker for this epithelial tissue. Since the goblet cells were absent in the morphant zebrafish, knock down of the orthologue of CLMP in zebrafish would probably result in the absence of the small intestine. All these findings suggest that CLMP has an important role in intestinal development, although its function is still largely unclear [4]. However, it is known that transient transfection of human CLMP into human intestinal epithelial T84 cells showed CLMP localization at the cell-cell membrane contacts [4]. It is also known that CLMP co-localizes with tight junction proteins, and is therefore claimed as a tight junction-associated protein. Because tight junction proteins play an important role in proliferation [7,8], we have suggested that loss-of-function of CLMP might affect proliferation [4]. Moreover, it was shown that transfection of human CLMP into MDCK cells increases transepithelial resistance [1]. Whether it is proliferation,or transepithelial resistance, or indeed another process in which CLMP plays a crucial role and that has impact on the pathophysiology of CSBS, is still unknown. To elucidate the function of CLMP we performed several functional assays using T84 cells as a model. As previously reported, transient transfection of human CLMP into T84 cells showed localization of WT-CLMP at the cell membrane and mislocalization of Pentagastrin mutant-CLMP (V124D) in the cytoplasm [4]. Although others have shown that transfection of human CLMP into MDCK cells increases transepithelial electrical resistance [1], we did not observe any differences in the transepithelial electrical resistance in T84 cells. We cannot say whether this discrepancy is due to the use of distinct cell types (MDCK versus T84) or to the fact that CLMP is simply not involved in this process. MDCK cells are kidney cells derived from a seemingly normal adult female cocker spaniel. Many different strains of the MDCK cell line are available and the transepithelial resistance in these different strains differs depending on the tight junction proteins that are expressed [12]. This illustrates that even in the same cell line, different results can be obtained. Unfortunately, Raschperger et al did not mention which strain of the MDCK cell line they used [1]. T84 cells are human colon carcinoma cells that have been widely used to study intestinal epithelial function [9?1]. We would therefore like to argue that T84 cells form a more representative model for studying the function of CLMP than MDCK cells. We reported earlier that the missense mutation in CLMP, which was identified in one of the CSBS patients, leads to CLMP cytoplasmatic mislocalization [4]. To study whether this missense mutation would also affect the adhesion properties of CLMP, we performed a cell aggregation assay using a widely accepted protocol implemented by Boterberg et al (Metastasis Research Protocols) [13]. As a positive control we transfected CHO cells with cadherin 1, also called epithelial or E-cadherin, a cell-cellNo Role for.Trans-membrane protein and colocalizes with the tight junction proteins ZO-1 [1,4,5] and occluding [1]. Immunostaining on human embryos showed that CLMP was expressed in many tissues including the gut. Knock down experiments of the orthologue in zebrafish resulted in general developmental defects including an affected intestine. Goblet cells are normally present in the mid intestine in zebrafish (which resembles the small intestine in humans), and can therefore be used as a marker for this epithelial tissue. Since the goblet cells were absent in the morphant zebrafish, knock down of the orthologue of CLMP in zebrafish would probably result in the absence of the small intestine. All these findings suggest that CLMP has an important role in intestinal development, although its function is still largely unclear [4]. However, it is known that transient transfection of human CLMP into human intestinal epithelial T84 cells showed CLMP localization at the cell-cell membrane contacts [4]. It is also known that CLMP co-localizes with tight junction proteins, and is therefore claimed as a tight junction-associated protein. Because tight junction proteins play an important role in proliferation [7,8], we have suggested that loss-of-function of CLMP might affect proliferation [4]. Moreover, it was shown that transfection of human CLMP into MDCK cells increases transepithelial resistance [1]. Whether it is proliferation,or transepithelial resistance, or indeed another process in which CLMP plays a crucial role and that has impact on the pathophysiology of CSBS, is still unknown. To elucidate the function of CLMP we performed several functional assays using T84 cells as a model. As previously reported, transient transfection of human CLMP into T84 cells showed localization of WT-CLMP at the cell membrane and mislocalization of mutant-CLMP (V124D) in the cytoplasm [4]. Although others have shown that transfection of human CLMP into MDCK cells increases transepithelial electrical resistance [1], we did not observe any differences in the transepithelial electrical resistance in T84 cells. We cannot say whether this discrepancy is due to the use of distinct cell types (MDCK versus T84) or to the fact that CLMP is simply not involved in this process. MDCK cells are kidney cells derived from a seemingly normal adult female cocker spaniel. Many different strains of the MDCK cell line are available and the transepithelial resistance in these different strains differs depending on the tight junction proteins that are expressed [12]. This illustrates that even in the same cell line, different results can be obtained. Unfortunately, Raschperger et al did not mention which strain of the MDCK cell line they used [1]. T84 cells are human colon carcinoma cells that have been widely used to study intestinal epithelial function [9?1]. We would therefore like to argue that T84 cells form a more representative model for studying the function of CLMP than MDCK cells. We reported earlier that the missense mutation in CLMP, which was identified in one of the CSBS patients, leads to CLMP cytoplasmatic mislocalization [4]. To study whether this missense mutation would also affect the adhesion properties of CLMP, we performed a cell aggregation assay using a widely accepted protocol implemented by Boterberg et al (Metastasis Research Protocols) [13]. As a positive control we transfected CHO cells with cadherin 1, also called epithelial or E-cadherin, a cell-cellNo Role for.
