A novel mutation of adenomatous polyposis coli (APC) gene results in the formation of supernumerary teeth

Supernumerary teeth are teeth that are present in addition to normal teeth. Although several hypotheses and some molecular signalling pathways explain the formation of supernumerary teeth, but their exact disease pathogenesis is unknown. To study the molecular mechanisms of supernumerary tooth‐related syndrome (Gardner syndrome), a deeper understanding of the aetiology of supernumerary teeth and the associated syndrome is needed, with the goal of inhibiting disease inheritance via prenatal diagnosis. We recruited a Chinese family with Gardner syndrome. Haematoxylin and eosin staining of supernumerary teeth and colonic polyp lesion biopsies revealed that these patients exhibited significant pathological characteristics. APC gene mutations were detected by PCR and direct sequencing. We revealed the pathological pathway involved in human supernumerary tooth development and the mouse tooth germ development expression profile by RNA sequencing (RNA‐seq). Sequencing analysis revealed that an APC gene mutation in exon 15, namely 4292‐4293‐Del GA, caused Gardner syndrome in this family. This mutation not only initiated the various manifestations typical of Gardner syndrome but also resulted in odontoma and supernumerary teeth in this case. Furthermore, RNA‐seq analysis of human supernumerary teeth suggests that the APC gene is the key gene involved in the development of supernumerary teeth in humans. The mouse tooth germ development expression profile shows that the APC gene plays an important role in tooth germ development. We identified a new mutation in the APC gene that results in supernumerary teeth in association with Gardner syndrome. This information may shed light on the molecular pathogenesis of supernumerary teeth. Gene‐based diagnosis and gene therapy for supernumerary teeth may become available in the future, and our study provides a high‐resolution reference for treating other syndromes associated with supernumerary teeth.     

APC nuclear membrane association and microtubule polarity

Background information. Directional cell migration is a fundamental feature of embryonic development, the inflammatory response and the metastatic spread of cancer. Migrating cells have a polarized morphology with an asymmetric distribution of signalling molecules and of the actin and microtubule cytoskeletons. The dynamic reorganization of the actin cytoskeleton provides the major driving force for migration in all mammalian cell types, but microtubules also play an important role in many cells, most notably neuronal precursors. Results. We previously showed, using primary fibroblasts and astrocytes in in vitro scratch‐induced migration assays, that the accumulation of APC (adenomatous polyposis coli; the APC tumour suppressor protein) at microtubule plus‐ends promotes their association with the plasma membrane at the leading edge. This is required for polarization of the microtubule cytoskeleton during directional migration. Here, we have examined the organization of microtubules in the soma of migrating neurons and fibroblasts. Conclusions. We find that APC, through a direct interaction with the NPC (nuclear pore complex) protein Nup153 (nucleoporin 153), promotes the association of microtubules with the nuclear membrane.     

Adenomatous polyposis coli (APC) protein moves along microtubules and concentrates at their growing ends in epithelial cells

Adenomatous polyposis coli (APC) tumor suppressor protein has been shown to be localized near the distal ends of microtubules (MTs) at the edges of migrating cells. We expressed green fluorescent protein (GFP)-fusion proteins with full-length and deletion mutants of Xenopus APC in Xenopus epithelial cells, and observed their dynamic behavior in live cells. During cell spreading and wound healing, GFP-tagged full-length APC was concentrated as granules at the tip regions of cellular extensions. At higher magnification, APC appeared to move along MTs and concentrate as granules at the growing plus ends. When MTs began to shorten, the APC granules dropped off from the MT ends. Immunoelectron microscopy revealed that fuzzy structures surrounding MTs were the ultrastructural counterparts for these GFP signals. The COOH-terminal region of APC was targeted to the growing MT ends without forming granular aggregates, and abruptly disappeared when MTs began to shorten. The APC lacking the COOH-terminal region formed granular aggregates that moved along MTs toward their plus ends in an ATP-dependent manner. These findings indicated that APC is a unique MT-associated protein that moves along selected MTs and concentrates at their growing plus ends through their multiple functional domains.     

应用蛋白截短技术检测APC基因胚系突变

建立蛋白截短检测技术,分析家族性腺瘤样息肉病（FAP）发病相关基因APC基因的胚系突变,研究基因突变型与FAP疾病表型的关系。经临床诊断的22例FAP患者及43例散发性大肠癌患者,分别取外周静脉血和正常结肠粘膜组织,常规提取基因组DNA。应用蛋白截短检测技术,分段分析APC基因巨大的第15外显子,对检出截短蛋白的样本进行测序,以确定突变位点及突变性质。22例FAP患者中,5例患者存在APC基因第15外显子的胚系突变,均为碱基缺失造成的移码突变,导致截短蛋白的产生;43例散发性大肠癌患者中未检测到APC基因第15外显子的胚系突变。蛋白截短检测技术是一种高效的基因突变分析技术,适用于大片段基因（如APC基因第15外显子）截短型突变的检测,可作为FAP症状出现前的常规基因诊断技术的一项有效补充。     

Single-nucleotide polymorphisms (SNPs) of the IRF6 and TFAP2A in non-syndromic cleft lip with or without cleft palate (NSCLP) in a northern Chinese population

The conserved armadillo repeat (ARM) domain of adenomatous polyposis coli (APC) protein plays an important role in the recognition of its binding partners. In this study, we report the crystal structure of APC-ARM (residues 407-775), which was determined to 2.9 Å resolution. Our structure shows that the seven armadillo repeats of APC-ARM fold together into a compact domain, with Arm2 and Arm5 presenting some deviations from canonical armadillo repeats. There is a positively charged groove on the surface of APC-ARM, which might be the recognition site for APC-binding partners. Comparison of this structure with our previously reported structure of APC (407-751), together with normal mode analysis, reveals that the APC-ARM domain possesses a limited intrinsic flexibility. We propose that this intrinsic flexibility might be an inherent property of ARM domains in general.     

Reconstitution of the destruction complex defines roles of AXIN polymers and APC in β-catenin capture,phosphorylation, and ubiquitylation

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Microtubules regulate the organization of actin filaments at the cortical region in root hair cells ofHydrocharis

Summary We studied the mechanism controlling the organization of actin filaments (AFs) inHydrocharis root hair cells, in which reverse fountain streaming occurs. The distribution of AFs and microtubules (MTs) in root hair cells were analyzed by fluorescence microscopy and electron microscopy. AFs and MTs were found running in the longitudinal direction of the cell at the cortical region. AFs were observed in the transvacuolar strand, but not MTs. Ultrastructural studies revealed that AFs and MTs were colocalized and that MTs were closer to the plasma membrane than AFs. To examine if MTs regulate the organization of AFs, we carried out a double inhibitor experiment using cytochalasin B (CB) and propyzamide, which are inhibitors of AFs and MTs, respectively. CB reversibly inhibited cytoplasmic streaming while propyzamide alone had no effect on it. However, after treatment with both CB and propyzamide, removal of CB alone did not lead to recovery of cytoplasmic streaming. In these cells, AFs showed a meshwork structure. When propyzamide was also removed, cytoplasmic streaming and the original organization of AFs were recovered. These results strongly suggest that MTs are responsible for the organization of AFs inHydrocharis root hair cells.     

Effects of altered gravity on the actin and microtubule cytoskeleton of human SH-SY5Y neuroblastoma cells

Summary. Human SH-SY5Y neuroblastoma cells were used to study the effects of altered gravity on the actin and microtubule cytoskeleton dynamics. A cholinergic stimulation of the cells during a 6 min period of changing gravity (3 parabolas) resulted in an enhanced actin-driven protrusion of evoked lamellipodia. Likewise, the spontaneous protrusive activity of nonactivated cells was promoted during exposure to changing gravity (6 up to 31 parabolas). Ground-based experiments revealed a similar enhancement of the spontaneous and evoked lamellar protrusive activity when the cells were kept at 2 g hypergravity for at least 6 min. This gravity response was independent of the direction of the acceleration vector in respect to the cells. Exposure of the cells to “simulated weightlessness” (clinorotation) had no obvious influence on this type of lamellar actin cytoskeleton dynamics. A 20 min exposure of the cells to simulated weightlessness or to changing gravity (6 to 31 parabolas) – but not to 2 g (hypergravity, centrifugation) – resulted in an altered arrangement of microtubules indicated by bending, turning, and loop formation. A similar altered arrangement was shown by microtubules which had polymerized into lamellipodia after release from a taxol block at simulated weightlessness (clinorotation) or during changing gravity (5 parabolas). Our data suggest that in human SH-SY5Y neuroblastoma cells, microgravity affects the dynamics and spatial arrangement of microtubules but has no influence on the Rac-controlled lamellar actin cytoskeleton dynamics and cell spreading. The latter, however, seems to be promoted at hypergravity. Correspondence and reprints: Cell and Developmental Neurobiology, Institute of Zoology, University of Hohenheim, Garbenstrasse 30, 70593 Stuttgart, Federal Republic of Germany.     

Regulatory role of CK2 during the progression of cell cycle

The protein kinase casein kinase 2 (CK2) is a ubiquitous eukaryotic serine/threonine protein kinase that plays an important role in cell cycle progression. We find that (1) CK2 interacts with a tumor suppressor protein, adenomatous polyposis coli (APC) that occurs at the highest level in G₂/M, and (2) the C-terminal region of APC, between amino acid residues 2086–2394, has the strongest activity to suppress CK2. Over-expression of this fragment in HEK293 cells or colorectal carcinoma cells that have truncated mutant APC proteins down-regulates cell proliferation rates as well as colony formation on soft agar. These results indicate that the complex formation between CK2 and full-length APC regulates CK2 activity that, in turn, regulates cell cycle progression, whereas truncated APC in colorectal carcinomas are unable to regulate the cell cycle. In the process to look for the downstream target for CK2, we found that eukaryotic translation initiation factor 5 (eIF5) is phosphorylated by CK2 in vivo as well as in vitro These results suggest an important role of CK2 on promotion of cell growth through eIF5.     

Modulation of the epithelial barrier by dexamethasone and prolactin in cultured Madin-Darby canine kidney (MDCK) cells

Glucocorticoids and prolactin (PRL) have a direct effect on the formation and maintenance of tight junctions (TJs) in cultured endothelial and mammary gland epithelial cells. In this work, we investigated the effect of a synthetic glucocorticoid dexamethasone (DEX) and PRL on the paracellular barrier function in MDCK renal epithelial cells. DEX (4 microM)+PRL (2 microg/ml) and DEX alone increased significantly the transepithelial electrical resistance after chronic treatment (4 days) of confluent MDCK monolayers or after 24 h treatment of subconfluent monolayers. Immunoblotting and immunocytochemistry revealed no changes in the expression and distribution of TJ-associated proteins occludin, ZO-1 and claudin-1 in confluent monolayers after hormone addition. However, a marked increase in junctional content for occludin and ZO-1 with no changes in their total expression was observed in subconfluent MDCK monolayers 24 h exposed to DEX or DEX+PRL. No change in cell proliferation/growth was detected at subconfluent conditions following hormone treatment. An increase in the total number of viable cells was observed only in confluent MDCK monolayers after exposure to DEX+PRL suggesting that the main effect of these hormones on already established barrier may be associated with the inhibition of cell death. In conclusion, our data suggest that these hormones (specially dexamethasone) have an effect on TJ structure and function only during the formation of MDCK epithelial barrier by probably modulating the localization, stability or assembly of TJ proteins to membrane sites of intercellular contact.     

The cyclic reorientation of cortical microtubules in epidermal cells of azuki bean epicotyls: the role of actin filaments in the progression of the cycle

The orientation of microtubules (MTs) was examined in epidermal cells of azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls. The orientation of MTs adjacent to the outer tangential wall of the cells, which has a crossed polylamellate structure with lamellae of longitudinal cellulose microfibrils alternating with lamellae of transverse cellulose microfibrils, differed from one cell to another. Treatment with an auxin-free solution caused the accumulation of cells with longitudinal MTs and subsequent treatment with a solution that contained auxin resulted in the accumulation of cells with transverse MTs, showing that sequential treatments with auxin-free and auxin-containing solutions can synchronize the reorientation of MTs. The MTs, once reoriented from longitudinal to transverse, returned to longitudinal and then back to transverse once again, the duration of the cycle being about 6 h. Gibberellic acid, known to increase the percentage of cells with transverse MTs, promoted reorientation of MTs from longitudinal to transverse and inhibited that from transverse to longitudinal. Cytochalasin D, an agent that disrupts actin filaments, speeded up the reorientation from transverse to longitudinal and slowed down that from longitudinal to transverse. It caused an increase in the percentage of cells with MTs in mixed orientation, and the percentage of such cells was highest when the percentage of cells with longitudinal MTs was decreasing and that of cells with transverse MTs was increasing. Received: 27 November 1997 / Accepted: 7 January 1998     

Changes in the cellular glycosaminoglycans of cultured mastocytoma cells induced by sodium butyrate

The effect of sodium butyrate on the cellular glycosaminoglycans of cultured mastocytoma p-815-4 cells was investigated using enzymic digestion, electrophoresis, nitrous acid degradation, and sequential partition fractionation. The average cellular glycosaminoglycan content of mastocytoma p-815-4 cells grown in the presence of 2 mM sodium butyrate was ten times as much as that of the control p-815-4 cells. Approximately 90% of the glycosaminoglycans isolated from the control cells and 70% from the butyrate-treated cells were found to be chondroitin 4-sulfate by enzymic digestion. The remainders were chondroitinase ABC-resistant. Hyaluronic acid and dermatan sulfate were not detected in either control cells or butyrate-treated cells. The chondroitinase ABC-resistant fraction of glycosaminoglycans from butyrate-treated cells showed a molar ratio of sulfate to uronic acid of more than 2.0, and provided some physicochemical properties characteristics to reference bovine lung heparin.     

Stabilization of cortical microtubules by the cell wall in cultured tobacco cells

Cortical microtubules (MTs) in protoplasts prepared from tobacco (Nicotiana tabacum L.) BY-2 cells were found to be sensitive to cold. However, as the protoplasts regenerated cell walls they became resistant to cold, indicating that the cell wall stabilizes cortical MTs against the effects of cold. Since poly-l-lysine was found to stabilize MTs in protoplasts, we examined extensin, an important polycationic component of the cell wall, and found it also to be effective in stabilizing the MTs of protoplasts. Both extensin isolated from culture filtrates of tobacco BY-2 cells and extensin isolated in a similar way from cultures of tobacco XD-6S cells rendered the cortical MTs in protoplasts resistant to cold. Extensin at 0.1 mg·ml⁻¹ was as effective as the cell wall in this respect. It is probable that extensin in the cell wall plays an important role in stabilizing cortical MTs in tobacco BY-2 cells.     

Apical membrane localization of the adenomatous polyposis coli tumor suppressor protein and subcellular distribution of the beta-catenin destruction complex in polarized epithelial cells

The adenomatous polyposis coli (APC) protein is implicated in the majority of hereditary and sporadic colon cancers. APC is known to function as a tumor suppressor through downregulation of beta-catenin as part of a high molecular weight complex known as the beta-catenin destruction complex. The molecular composition of the intact complex and its site of action in the cell are still not well understood. Reports on the subcellular localization of APC in various cell systems have differed significantly and have been consistent with an association with a cytosolic complex, with microtubules, with the nucleus, or with the cortical actin cytoskeleton. To better understand the role of APC and the destruction complex in colorectal cancer, we have begun to characterize and isolate these complexes from confluent polarized human colon epithelial cell monolayers and other epithelial cell types. Subcellular fractionation and immunofluorescence microscopy reveal that a predominant fraction of APC associates tightly with the apical plasma membrane in a variety of epithelial cell types. This apical membrane association is not dependent on the mutational status of either APC or beta-catenin. An additional pool of APC is cytosolic and fractionates into two distinct high molecular weight complexes, 20S and 60S in size. Only the 20S fraction contains an appreciable portion of the cellular axin and small but detectable amounts of glycogen synthase kinase 3beta and beta-catenin. Therefore, it is likely to correspond to the previously characterized beta-catenin destruction complex. Dishevelled is almost entirely cytosolic, but does not significantly cofractionate with the 20S complex. The disproportionate amount of APC in the apical membrane and the lack of other destruction complex components in the 60S fraction of APC raise questions about whether these pools of APC take part in the degradation of beta-catenin, or alternatively, whether they could be involved in other functions of the protein that still must be determined.     

Asymmetric localization of Notch2 on the microvillous surface in choroid plexus epithelial cells

Notch family molecules are transmembrane receptors that play various roles in contact-dependent cell–cell interactions in a wide range of organs. In the brain, Notch2, but not the other members of Notch, is expressed in the choroid plexus at an exceptionally high level. We immunohistochemically examined the cellular and subcellular localization of Notch2 protein in the choroid plexus using confocal and electron microscopy. Unexpectedly, Notch2 was asymmetrically localized on the microvillous surface of epithelial cells in the choroid plexus of both postnatal and adult rats. This localization pattern of Notch2 suggests its novel and unknown role independent of contact with adjacent cells in the choroid plexus. In organotypic cultures of the choroid plexus, the addition of anti-Notch2 antibody resulted in deformation of microvilli in epithelial cells, which suggests a role of Notch2 in the maintenance of the microvillous structure in choroid plexus epithelial cells.     

Association of glycoproteins and pepsinogen in the secretory granules of fundic epithelial cells isolated from guinea pig stomach

Epithelial cells were isolated from the fundic portion of the guinea pig stomach. Cells were separated by velocity sedimentation at unit gravity in a Ficoll 70 gradient and pooled in three fractions. By morphological and biochemical criteria, each fraction was characterized as a population highly enriched in one of the three main functional types: oxyntic cell; chief cell and mucus-secreting cell. Measure of the pepsinogen content and specific stainings of the secretory granules for light and electron microscopy led to the definition of two types of mucus-secreting cells in nearly equal quantity; mucous cells with smaller secretory granules entirely glycoproteic in nature and muco-peptic cells containing larger heterogeneous secretory granules. These granules were made of a proteic core containing pepsinogen surrounded by a thin membrane and a voluminous cap, both containing carbohydrates. The cap appeared as if built of orderly packed layers of glycoproteins. Secretory granules of chief cells were also surrounded by a membrane containing glycoproteins and occasionally a small glycoproteic cap. Pepsinogen content was estimated to be three times higher in a single chief cell than in a muco-peptic cell.     

Morphometry of cellular protrusions of mesodermal cells and fibrous extracellular matrix in the primitive streak stage chick embryo

Summary Chick mesodermal cells, having become invaginated and beginning to locomote prior to the formation of the mesodermal cell layer at an early primitive streak stage, extend many filopodia and flatten themselves against the basal surface of the epiblast. Morphometry on scanning electron micrographs of chick mesodermal cells revealed two statistically significant tendencies. Each cell took an extended form and protruded filopodia, preferably along its major axis, suggesting that the force extending the cell body was generated by both ends rich in filopodia. The cells also tended to protrude filopodia most frequently in a direction away from Hensen's node. The orientation of the fibrous extracellular matrix (fECM), running on the basal surface of the epiblast, was assessed quantitatively, and it was proved statistically that the orientation of the fECM was radial around the primitive streak: With an immunogold staining technique, fECM, to which the filopodia of the mesodermal cells attached frequently and closely, was confirmed to be rich in fibronectin (FN). These results lead us to conclude that the mesodermal cells in chick gastrula were guided to locomote towards the periphery of the area pellucida by FN-rich fECM laid on the basal surface of the epiblast, and that this movement was due to an in vivo locomotive mechanism using filopodia. Offprint requests to: R. Toyoizumi