Characterization of the enterocyte-like brush border cytoskeleton of the C2BBe clones of the human intestinal cell line, Caco-2

1992 ◽  
Vol 102 (3) ◽  
pp. 581-600 ◽  
Author(s):  
M.D. Peterson ◽  
M.S. Mooseker
Keyword(s):  
Molecular Mass ◽  
Cell Line ◽  
Heavy Chain ◽  
Brush Border ◽  
Laser Scanning ◽  
Human Colon ◽  
Cell Types ◽  
Intestinal Cell ◽  
Myosin I

The brush border (BB) of the enterocyte is a well-studied example of the actin-based cytoskeleton. We describe here a cell culture model that expresses a faithful representation of the in vivo structure. Two clones (C2BBe 1 and 2) isolated from the cell line Caco-2 (derived from a human colonic adenocarcinoma) formed a polarized monolayer with an apical BB morphologically comparable to that of the human colon. BBs could be isolated by standard methods and contained the microvillar proteins villin, fimbrin, sucrase-isomaltase and BB myosin I, and the terminal web proteins fodrin and myosin II. The immunolocalization of these proteins in confluent, filter-grown monolayers was determined by laser scanning confocal microscopy; patterns of distribution comparable to those in human enterocytes were observed. Sedimentation analysis of cell homogenates derived from C2BBe cells and human colonic epithelial cells demonstrated similar patterns of fractionation of BB proteins; the physical association of those proteins, as determined by extraction from the BB, was also comparable between the two cell types. Like enterocytes of the human intestine, C2BBe cells expressed multiple myosin I immunogens reactive with a head domain-specific monoclonal antibody raised against avian BB myosin I, one of which co-migrated with the approximately 110 kilodalton (kDa) heavy chain of human BB myosin I. In addition, the C2BBe cells express a pair of higher molecular mass immunogens (130 and 140 kDa). These myosin I immunogens all exhibit ATP-dependent association with the C2BBe cytoskeleton. Although the higher molecular mass immunogens were detected in several other human intestinal lines examined, including the parent Caco-2 line, none of these other lines expressed detectable levels of the 110 kDa immunogen, which is presumed to be the heavy chain of human BB myosin I.

Ultrastructural Study of a differentiating human colon carcinoma cell line

1990 ◽  
Vol 48 (3) ◽  
pp. 208-209
Author(s):  
Sylvie Polak-Charcon ◽  
Mehrdad Hekmati ◽  
Yehuda Ben Shaul
Keyword(s):  
Cell Line ◽  
Morphological Changes ◽  
Secretory Granules ◽  
Human Colon ◽  
Cell Types ◽  
Culture Conditions ◽  
Morphological Evidence ◽  
Human Colon Carcinoma Cell ◽  
Colon Cell

The epithelium of normal human colon mucosa “in vivo” exhibits a gradual pattern of differentiation as undifferentiated stem cells from the base of the crypt of “lieberkuhn” rapidly divide, differentiate and migrate toward the free surface. The major differentiated cell type of the intestine observed are: absorptive cells displaying brush border, goblet cells containing mucous granules, Paneth and endocrine cells containing dense secretory granules. These different cell types are also found in the intestine of the 13-14 week old embryo.We present here morphological evidence showing that HT29, an adenocarcinoma of the human colon cell line, can differentiate into various cell types by changing the growth and culture conditions and mimic morphological changes found during development of the intestine in the human embryo.HT29 cells grown in tissue-culture dishes in DMEM and 10% FCS form at late confluence a multilayer of morphologically undifferentiated cell culture covered with irregular microvilli, and devoid of tight junctions (Figs 1-3).

Localization of the rat myosin I molecules myr 1 and myr 2 and in vivo targeting of their tail domains

1995 ◽  
Vol 108 (12) ◽  
pp. 3775-3786 ◽  
Author(s):  
C. Ruppert ◽  
J. Godel ◽  
R.T. Muller ◽  
R. Kroschewski ◽  
J. Reinhard ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Brush Border ◽  
Subcellular Fractionation ◽  
Actin Binding ◽  
Resting Cells ◽  
Medium Density ◽  
Subcellular Targeting ◽  
Myosin I ◽  
Punctate Pattern

Myr 1 is a widely distributed mammalian myosin I molecule related to brush border myosin 1. A second widely distributed myosin I molecule similar to myr 1 and brush border myosin I, called myr 2, has now been identified. Specific antibodies and expression of epitope-tagged molecules were used to determine the subcellular localization of myr 1 and myr 2 in NRK cells. Myr 1 was detected at the plasma membrane and was particularly enriched in cell protrusions like lamellipodia, membrane ruffles and filopodia. In dividing cells myr 1 localized to the cleavage furrow. Myr 2 was localized in a discrete punctate pattern in resting cells and in cells undergoing cytokinesis. In subcellular fractionation experiments myr 1 and myr 2 were both partly soluble and partly associated with smooth membranes of medium density. The tail domains of myosin I molecules have been proposed to interact with a receptor and thereby determine the subcellular localization. To test this hypothesis we expressed the tail domains of myr 1 and myr 2 that lack the F-actin-binding myosin head domain in NRK cells. These tail domains also partly copurified with smooth membranes of medium density and immunolocalized similar to the respective endogenous myosin I; however, they exhibited a lower affinity for membranes and an increased diffuse cytosolic localization. These results suggest that the tail domains of myr 1 and myr 2 are sufficient for subcellular targeting but that their head domains also contribute significantly to maintaining a proper subcellular localization.

Isolation of a cDNA corresponding to a developmentally regulated transcript in rat intestine

1988 ◽  
Vol 8 (10) ◽  
pp. 4243-4249
Author(s):  
J Filmus ◽  
J G Church ◽  
R N Buick
Keyword(s):  
Cell Line ◽  
Sequence Data ◽  
Human Colon ◽  
Intestinal Cell ◽  
Homologous Gene ◽  
Rat Intestine ◽  
Human Colon Carcinoma Cell ◽  
Reading Frame ◽  
Intestinal Cell Line ◽  
High Level

We report the isolation of a cDNA clone corresponding to a transcript that is accumulated differentially in rat intestine during development. Clone OCI-5 was selected from the rat intestinal cell line IEC-18, which represents primitive intestinal epithelial crypt cells. Expression was high in rat fetal intestine between 15 and 19 days of development and thereafter was progressively down regulated, becoming undetectable after weaning. Clone OCI-5 detected homologous sequences in human and murine cells. In particular, a high level of expression was detected in CaCo-2, a human colon carcinoma cell line, which is known to express molecules characteristic of fetal small intestinal cells. Expression of a homologous gene was also detected in F9 murine teratocarcinoma cells when they were induced to differentiate into parietal or visceral endodermlike cells. When IEC-18 cells were transformed by activated H-ras or v-src genes, expression of clone OCI-5 was suppressed; the degree of down-regulation correlated with the extent of morphological change induced in the transformed IEC-18 cells. The sequence of clone OCI-5 showed an open reading frame that was capable of encoding a protein of 597 amino acids, but no strong homology was found with any of the proteins registered in the protein sequence data base.

scholarly journals Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor μ2 kinase

2003 ◽  
Vol 163 (2) ◽  
pp. 231-236 ◽  
Author(s):  
Antony P. Jackson ◽  
Alexander Flett ◽  
Carl Smythe ◽  
Lindsay Hufton ◽  
Frank R. Wettey ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Heavy Chain ◽  
Transferrin Receptor ◽  
Coated Pits ◽  
Permeabilized Cells ◽  
Clathrin Heavy Chain

Endocytic cargo such as the transferrin receptor is incorporated into clathrin-coated pits by associating, via tyrosine-based motifs, with the AP2 complex. Cargo–AP2 interactions occur via the μ2 subunit of AP2, which needs to be phosphorylated for endocytosis to occur. The most likely role for μ2 phosphorylation is in cargo recruitment because μ2 phosphorylation enhances its binding to internalization motifs. Here, we investigate the control of μ2 phosphorylation. We identify clathrin as a specific activator of the μ2 kinase and, in permeabilized cells, we show that ligand sequestration, driven by exogenous clathrin, results in elevated levels of μ2 phosphorylation. Furthermore, we show that AP2 containing phospho-μ2 is mainly associated with assembled clathrin in vivo, and that the level of phospho-μ2 is strongly reduced in a chicken B cell line depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via the modulation of phospho-μ2 levels.

scholarly journals SipA Activation of Caspase-3 Is a Decisive Mediator of Host Cell Survival at Early Stages of Salmonella enterica Serovar Typhimurium Infection

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Anne McIntosh ◽  
Lynsey M. Meikle ◽  
Michael J. Ormsby ◽  
Beth A. McCormick ◽  
John M. Christie ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Actin Polymerization ◽  
Caspase 3 ◽  
Ex Vivo ◽  
Cell Types ◽  
Laser Scanning Microscopy ◽  
Limiting Factor ◽  
Dual Function ◽  
Content Type

ABSTRACT Salmonella invasion protein A (SipA) is a dual-function effector protein that plays roles in both actin polymerization and caspase-3 activation in intestinal epithelial cells. To date its function in other cell types has remained largely unknown despite its expression in multiple cell types and its extracellular secretion during infection. Here we show that in macrophages SipA induces increased caspase-3 activation early in infection. This activation required a threshold level of SipA linked to multiplicity of infection and may be a limiting factor controlling bacterial numbers in infected macrophages. In polymorphonuclear leukocytes, SipA or other Salmonella pathogenicity island 1 effectors had no effect on induction of caspase-3 activation either alone or in the presence of whole bacteria. Tagging of SipA with the small fluorescent phiLOV tag, which can pass through the type three secretion system, allowed visualization and quantification of caspase-3 activation by SipA-phiLOV in macrophages. Additionally, SipA-phiLOV activation of caspase-3 could be tracked in the intestine through multiphoton laser scanning microscopy in an ex vivo intestinal model. This allowed visualization of areas where the intestinal epithelium had been compromised and demonstrated the potential use of this fluorescent tag for in vivo tracking of individual effectors.

scholarly journals Aberrant Epithelial Differentiation Contributes to Pathogenesis in a Murine Model of Congenital Tufting Enteropathy

2020 ◽  
Author(s):  
Barun Das ◽  
Kevin Okamoto ◽  
John Rabalais ◽  
Jocelyn Young ◽  
Kim E. Barrett ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Epithelial Cell ◽  
Murine Model ◽  
Brush Border ◽  
Diarrheal Disease ◽  
Cell Types ◽  
Intestinal Cell ◽  
Secretory Cells ◽  
Absorptive Cells ◽  
Absorptive Enterocytes

AbstractBackground & AimsCongenital Tufting Enteropathy (CTE) is an intractable diarrheal disease of infancy caused by mutation of Epithelial Cell Adhesion Molecule (EpCAM). The cellular and molecular basis of CTE pathology has been elusive. We hypothesized that the loss of EpCAM in CTE results in altered lineage differentiation and defects in absorptive enterocytes thereby contributing to CTE pathogenesis.MethodsIntestine from CTE mice was evaluated for specific markers by RT-qPCR, western blotting and immunostaining. Body weight, blood glucose and intestinal enzyme activity were also investigated. A CTE enteroid model was used to assess whether the decreased census of secretory cells could be rescued.ResultsCTE mice exhibited alterations in brush-border function, disaccharidase activity and glucose absorption, potentially contributing to nutrient malabsorption and impaired weight gain. Altered cell differentiation in CTE mice led to decreased secretory cells and increased numbers of absorptive cells, though the absorptive enterocytes lacked key features, causing brush border malfunction. Further, treatment with Notch signaling inhibitor, DAPT, increased the numbers of major secretory cell types in CTE enteroids (Graphical abstract 1).ConclusionsAlterations in intestinal epithelial cell differentiation in CTE mice favor an increase in absorptive cells at the expense of secretory cells. Although the proportion of absorptive enterocytes is increased, they lack key functional properties. We conclude that these effects underlie pathogenic features of CTE such as malabsorption and diarrhea, and ultimately the failure to thrive seen in patients. The ability of DAPT to reverse aberrant differentiation suggests a possible therapeutic strategy.SynopsisA murine model of Congenital Tufting Enteropathy exhibits altered intestinal cell differentiation, leading to increased absorptive and decreased secretory cells, which can be reversed with DAPT. Absorptive enterocytes in these mice are also dysfunctional, contributing to disease pathogenesis.Graphical Abstract

scholarly journals The Expression And The Tumor Suppressor Role of CLDN6 In Colon Cancer

2021 ◽  
Author(s):  
Huinan Qu ◽  
Min Wang ◽  
Miaomiao Wang ◽  
Yuanyuan Liu ◽  
Chengshi Quan
Keyword(s):  
Colon Cancer ◽  
Tumor Suppressor ◽  
Cell Line ◽  
Cancer Cell Line ◽  
Human Colon ◽  
Colon Cancer Cell Line ◽  
Migration And Invasion ◽  
Epithelial Cell Line

Abstract As a member of the tight junction family, CLDN6 is a tumor suppressor gene in breast cancer, but its role in colon cancer is unknown. In this research, we aimed at revealing the function of CLDN6 in colon cancer. We found that CLDN6 expressed lower in colon cancer tissues compared with adjacent normal tissues and the low expression of CLDN6 was correlated with lymph node metastasis. Similarly, CLDN6 expressed lower in the colon cancer cell line SW1116 compared with the normal human colon epithelial cell line NCM460. Upon CLDN6 overexpression in SW1116 cells, the proliferation of cells was suppressed in vitro and in vivo. Consistently, the migration and invasion abilities of cells were significantly inhibited after CLDN6 overexpression. Furthermore, the TYK2/STAT3 pathway was activated in SW1116/CLDN6 cells, and inhibition of this pathway with AG490 reversed the inhibition of migration and invasion of SW1116 cells by CLDN6. Therefore, our data indicated that CLDN6 acted as a tumor suppressor and had the potential to be regarded as a biomarker for the progression of colon cancer.

scholarly journals Isolation of a cDNA corresponding to a developmentally regulated transcript in rat intestine.

1988 ◽  
Vol 8 (10) ◽  
pp. 4243-4249 ◽  
Author(s):  
J Filmus ◽  
J G Church ◽  
R N Buick
Keyword(s):  
Cell Line ◽  
Sequence Data ◽  
Human Colon ◽  
Intestinal Cell ◽  
Homologous Gene ◽  
Rat Intestine ◽  
Human Colon Carcinoma Cell ◽  
Reading Frame ◽  
Intestinal Cell Line ◽  
High Level

We report the isolation of a cDNA clone corresponding to a transcript that is accumulated differentially in rat intestine during development. Clone OCI-5 was selected from the rat intestinal cell line IEC-18, which represents primitive intestinal epithelial crypt cells. Expression was high in rat fetal intestine between 15 and 19 days of development and thereafter was progressively down regulated, becoming undetectable after weaning. Clone OCI-5 detected homologous sequences in human and murine cells. In particular, a high level of expression was detected in CaCo-2, a human colon carcinoma cell line, which is known to express molecules characteristic of fetal small intestinal cells. Expression of a homologous gene was also detected in F9 murine teratocarcinoma cells when they were induced to differentiate into parietal or visceral endodermlike cells. When IEC-18 cells were transformed by activated H-ras or v-src genes, expression of clone OCI-5 was suppressed; the degree of down-regulation correlated with the extent of morphological change induced in the transformed IEC-18 cells. The sequence of clone OCI-5 showed an open reading frame that was capable of encoding a protein of 597 amino acids, but no strong homology was found with any of the proteins registered in the protein sequence data base.

Flexible-substratum technique for viewing cells from the side: some in vivo properties of primary (9+0) cilia in cultured kidney epithelia

1988 ◽  
Vol 89 (4) ◽  
pp. 457-466 ◽  
Author(s):  
K.E. Roth ◽  
C.L. Rieder ◽  
S.S. Bowser
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Kidney Cell ◽  
Primary Cilia ◽  
Length Distribution ◽  
Cell Types ◽  
Phenotypic Marker ◽  
Kidney Epithelial Cell

Cells cultured on thin plastic (e.g. Formvar, Teflon, polycarbonate) membranes can be clearly imaged from the side in vivo by video microscopy. We have used this flexible-substratum technique to examine the behaviour and properties of primary cilia in confluent cultures of the kidney epithelial cell lines PtK1, PtK2, LLC-PK1, MDCK and BSC-40. In these cells primary cilia appear as rigid rods, up to 55 micron long, which project at various angles from the dorsal cell surface. The length distribution of primary cilia in confluent cultures is a distinct characteristic of each established kidney cell line examined, with LLC-PK1 exhibiting three distinct length populations. Primary cilia of kidney cell lines bend passively in response to flow but do not display propagated bending or vortical motions. Up to 26% of the cilia in the cell types examined possess one or more conspicuous swellings along the ciliary shaft. Treatment with 0.05% trypsin, which is sufficient to cause cell rounding, does not induce the resorption or shedding of the cilium. These direct observations demonstrate that kidney epithelial-cell primary cilia are non-motile and longer than previously thought, and suggest that their length represents a phenotypic marker for each cell line.

scholarly journals Partial deduced sequence of the 110-kD-calmodulin complex of the avian intestinal microvillus shows that this mechanoenzyme is a member of the myosin I family.

1989 ◽  
Vol 109 (6) ◽  
pp. 2895-2903 ◽  
Author(s):  
A Garcia ◽  
E Coudrier ◽  
J Carboni ◽  
J Anderson ◽  
J Vandekerkhove ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Heavy Chain ◽  
Brush Border ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Head Domain ◽  
Myosin I ◽  
Chicken Tissue ◽  
Carboxy Terminal

The actin bundle within each microvillus of the intestinal brush border is laterally tethered to the membrane by bridges composed of the protein complex, 110-kD-calmodulin. Previous studies have shown that avian 110-kD-calmodulin shares many properties with myosins including mechanochemical activity. In the present study, a cDNA molecule encoding 1,000 amino acids of the 110-kD protein has been sequenced, providing direct evidence that this protein is a vertebrate homologue of the tail-less, single-headed myosin I first described in amoeboid cells. The primary structure of the 110-kD protein (or brush border myosin I heavy chain) consists of two domains, an amino-terminal "head" domain and a 35-kD carboxy-terminal "tail" domain. The head domain is homologous to the S1 domain of other known myosins, with highest homology observed between that of Acanthamoeba myosin IB and the S1 domain of the protein encoded by bovine myosin I heavy chain gene (MIHC; Hoshimaru, M., and S. Nakanishi. 1987. J. Biol. Chem. 262:14625-14632). The carboxy-terminal domain shows no significant homology with any other known myosins except that of the bovine MIHC. This demonstrates that the bovine MIHC gene most probably encodes the heavy chain of bovine brush border myosin I (BBMI). A bacterially expressed fusion protein encoded by the brush border 110-kD cDNA binds calmodulin. Proteolytic removal of the carboxy-terminal domain of the fusion protein results in loss of calmodulin binding activity, a result consistent with previous studies on the domain structure of the 110-kD protein. No hydrophobic sequence is present in the molecule indicating that chicken BBMI heavy chain is probably not an integral membrane protein. Northern blot analysis of various chicken tissue indicates that BBMI heavy chain is preferentially expressed in the intestine.

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