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Biomedical subjects

A I McClatchey

Publications and source records attributed to A I McClatchey.

At least 19 recordsLinked to original sources
PubMed2025

Distinct phenotypic consequences of cholangiocarcinoma-associated FGFR2 alterations depend on biliary epithelial cell state.

Epithelial cancers disrupt tissue architecture and are often driven by mutations in genes that play important roles in normal epithelial morphogenesis. The intrahepatic biliary system is an epithelial tubular network that forms within the developing liver via the de novo initiation and expansion of apical lumens. Intrahepatic biliary tumors (intrahepatic cholangiocarcinoma) commonly harbor activating genomic alterations in the FGFR2 receptor tyrosine kinase, which plays important roles in epithelial morphogenesis in other developmental settings. Using a physiologic and quantitative 3D model we demonstrate that FGFR signaling is important for biliary morphogenesis and that oncogenic FGFR2 fusions and in-frame deletions disrupt biliary architecture. Importantly, we show that the trafficking of and signaling from the FGFR2 mutants, as well as their phenotypic impacts, are governed by the epithelial state of the cell. Unexpectedly, we also found that distinct tumor-driving FGFR2 mutants disrupt biliary morphogenesis in completely different and clinically relevant ways, informing our understanding of morphogenesis and tumorigenesis and highlighting the importance of convergent studies of both.

Journal Article↗
PubMed2001

The Nf2 tumor suppressor, merlin, functions in Rac-dependent signaling.

Mutations in the neurofibromatosis type II (NF2) tumor suppressor predispose humans and mice to tumor development. The study of Nf2+/- mice has demonstrated an additional effect of Nf2 loss on tumor metastasis. The NF2-encoded protein, merlin, belongs to the ERM (ezrin, radixin, and moesin) family of cytoskeleton:membrane linkers. However, the molecular basis for the tumor- and metastasis- suppressing activity of merlin is unknown. We have now placed merlin in a signaling pathway downstream of the small GTPase Rac. Expression of activated Rac induces phosphorylation and decreased association of merlin with the cytoskeleton. Furthermore, merlin overexpression inhibits Rac-induced signaling in a phosphorylation-dependent manner. Finally, Nf2-/- cells exhibit characteristics of cells expressing activated alleles of Rac. These studies provide insight into the normal cellular function of merlin and how Nf2 mutation contributes to tumor initiation and progression.

3T3 Cells↗
PubMed1998

Mice heterozygous for a mutation at the Nf2 tumor suppressor locus develop a range of highly metastatic tumors.

A role for the membrane/cytoskeleton interface in the development and progression of cancer is established, yet poorly understood. The neurofibromatosis type II (NF2) tumor suppressor gene encodes a member of the ezrin/radixin/moesin (ERM) family of membrane/cytoskeleton linker proteins thought to be important for cell adhesion and motility. We report that in contrast to the narrow spectrum of benign tumors in human NF2 patients, Nf2 heterozygous mice develop a variety of malignant tumors. Using the fact that Nf2 is linked to the p53 tumor suppressor locus in the mouse we have also investigated the effects of genetic linkage of cancer-predisposing mutations on tumorigenesis and examined the genetic pathway to tumor formation involving Nf2 loss. Importantly, we observed a very high rate of metastasis associated with Nf2 deficiency, with or without loss of p53 function, and we provide experimental evidence supporting a role for Nf2 loss in metastatic potential. Together, our results suggest an important role for the NF2 tumor suppressor, and perhaps the ERM family in tumor formation and metastasis.

Animals↗
PubMed1998

Regulation of the neurofibromatosis type 2 tumor suppressor protein, merlin, by adhesion and growth arrest stimuli.

The neurofibromatosis type 2 tumor suppressor gene is inactivated in the development of familial and sporadic schwannomas and meningiomas. The encoded protein, Merlin, is closely related to the Ezrin, Radixin, and Moesin family of membrane/cytoskeletal linker proteins. Examination of Merlin in several cell lines revealed that the protein migrates as two distinct species near 70 kDa. Phosphatase treatment and orthophosphate labeling demonstrated that the species with decreased mobility is phosphorylated. Given Merlin's localization to cortical actin structures, we examined the effect of cell-cell contact or other forms of growth arrest on Merlin expression and post-translational modification. Under conditions of confluency or serum deprivation, the levels of phosphorylated and unphosphorylated Merlin species increased significantly. Cells arrested in G1 by other methods or other phases of the cell cycle did not show changes in Merlin levels. Furthermore, loss of adhesion resulted in a nearly complete dephosphorylation of Merlin, which was reversed upon re-plating of cells, suggesting Merlin phosphorylation may be responsive to cell spreading or changes in cell shape. Thus, the tumor suppressor function of Merlin may involve the regulation of cellular responses to cues such as cell-cell contact, growth factor microenvironment, or changes in cell shape.

3T3 Cells↗
PubMed1998

Tumor suppressor mutations in mice: the next generation.

Mouse strains carrying tumor suppressor mutations genetically mimic familial forms of human cancer. New tumor suppressors have and will be identified and mutated in the mouse; however, it is clear that future investigation will focus on a new generation of experiments aimed at improving existing models, and using them to delineate the molecular pathways to tumorigenesis and to test the value of rationally designed drug therapies.

Animals↗
PubMed1998

Localization and functional domains of the neurofibromatosis type II tumor suppressor, merlin.

The neurofibromatosis type II (NF2) tumor suppressor gene is inactivated in the development of familial and sporadic schwannomas and meningiomas. The encoded protein, merlin, is closely related to ezrin, radixin, and moesin, which are members of the band 4.1 family of membrane/cytoskeletal linker proteins. We have examined the localization and effects of overexpressing epitope-tagged full-length isoforms of merlin as well as amino- and carboxyl-terminal truncations. The full-length and the amino-terminal domain of merlin localize to cortical actin, particularly areas of dynamic actin rearrangements such as membrane ruffles. Furthermore, overexpression of the carboxyl half of merlin induces cell death in NIH3T3 cells. The effect is splice-form specific and is not observed in the context of the full-length molecule. Thus, as has been described for the erzin, radixin, and moesin proteins, the activities of the carboxyl-terminal domain of merlin may be suppressed by the amino-terminal domain.

3T3 Cells↗
PubMed1997

The Nf2 tumor suppressor gene product is essential for extraembryonic development immediately prior to gastrulation.

The neurofibromatosis type II (NF2) tumor suppressor encodes a putative cytoskeletal associated protein, the loss of which leads to the development of Schwann cell tumors associated with NF2 in humans. The NF2 protein merlin belongs to the band 4.1 family of proteins that link membrane proteins to the cytoskeleton and are thought to be involved in dynamic cytoskeletal reorganization. Beyond its membership in this family, however, the function of merlin remains poorly understood. In order to analyze the function of merlin during embryogenesis and to develop a system to study merlin function in detail, we have disrupted the mouse Nf2 gene by homologous recombination in embryonic stem cells. Most embryos homozygous for a mutation at the Nf2 locus fail between embryonic days 6.5 and 7.0, exhibiting a collapsed extraembryonic region and the absence of organized extraembryonic ectoderm. The embryo proper continues to develop, but fails to initiate gastrulation. These observations are supported by the expression patterns of markers of the extraembryonic lineage and the lack of expression of mesodermal markers in the mutant embryos. Mosaic studies demonstrate that merlin function is not required cell autonomously in mesoderm, and support the proposition that merlin function is essential for the development of extraembryonic structures during early mouse development.

Animals↗
PubMed1993

Modification of the Na+ current conducted by the rat skeletal muscle alpha subunit by coexpression with a human brain beta subunit.

Sodium channels are multimeric structures composed of alpha and beta subunits. Oocytes injected with RNA encoding only the alpha subunit express voltage-gated Na+ currents. The kinetics of inactivation, however, are abnormal. Co-injection of rat brain alpha and beta subunits modifies inactivation of INa such that it closely resembles endogenous currents. Here we show that a beta subunit derived from human brain directs the same functional modification of INa expressed by a rat skeletal muscle alpha subunit. This implies that functional domains for the interaction of alpha and beta subunits are highly conserved across both tissues and species.

Animals↗
PubMed1993

The cloning and expression of a sodium channel beta 1-subunit cDNA from human brain.

Electrical excitability of neurons and muscle cells is mediated largely through the actions of the voltage-gated sodium channel. Initiation and propagation of the action potential is a direct result of the precisely controlled inward flux of sodium through these channels. Much attention has been paid to the sodium channel alpha-subunit, the major, pore-forming component. However, alpha-subunits are associated with one or more smaller beta-subunits, which have been implicated in the critical fine tuning of the gating properties of the channel. To investigate the properties of the beta-subunit, we have isolated a cDNA encoding the human brain beta 1-subunit and assigned the corresponding gene to chromosome 19. We have also examined the effects of expressing the brain beta 1-subunit on the kinetics of a coexpressed muscle sodium channel alpha-subunit. Our results underscore the functional importance of the beta 1-subunit and imply a conserved mechanism for the interaction of the beta 1-subunit with different alpha-subunits.

Amino Acid Sequence↗
PubMed1992

Temperature-sensitive mutations in the III-IV cytoplasmic loop region of the skeletal muscle sodium channel gene in paramyotonia congenita.

Paramyotonia congenita (PMC), a dominant disorder featuring cold-induced myotonia (muscle stiffness), has recently been genetically linked to a candidate gene, the skeletal muscle sodium channel gene SCN4A. We have now established that SCN4A is the disease gene in PMC by identifying two different single-base coding sequence alterations in PMC families. Both mutations affect highly conserved residues in the III-IV cytoplasmic loop, a portion of the sodium channel thought to pivot in response to membrane depolarization, thereby blocking and inactivating the channel. Abnormal function of this cytoplasmic loop therefore appears to produce the Na+ current abnormality and the unique temperature-sensitive clinical phenotype in this disorder.

Amino Acid Sequence↗
PubMed1992

Sequence-tagged sites (STSs) spanning 4p16.3 and the Huntington disease candidate region.

The generation of sequence-tagged sites (STSs) has been proposed as a unifying approach to correlating the disparate results generated by genetic and various physical techniques being used to map the human genome. We have developed an STS map to complement the existing physical and genetic maps of 4p16.3, the region containing the Huntington disease gene. A total of 18 STSs span over 4 Mb of 4p16.3, with an average spacing of about 250 kb. Eleven of the STSs are located within the primary candidate HD region of 2.5 Mb between D4S126 and D4S168. The availability of STSs makes the corresponding loci accessibility to the general community without the need for distribution of cloned DNA. These STSs should also provide the means to isolate yeast artificial chromosome clones spanning the HD candidate region.

Base Sequence↗
PubMed1992

Assay by polymerase chain reaction (PCR) of multi-allele polymorphisms in the Huntington's disease region of chromosome 4.

The Huntington's disease-linked D4S115 marker has been converted from a DNA blot assay to a more sensitive and rapid polymerase chain reaction (PCR) assay. PCR amplification of a tandem repeat at D4S115 revealed 7 allelic fragments, ranging in size from approximately 610 to 915 bp, differing in their apparent copy number of a approximately 55 bp core repeat. This repeat unit differs strikingly in sequence from the repeat units of other multi-allele markers from chromosome region 4p 16.3, arguing that the VNTR (Variable Number of Tandem Repeats) loci clustered in this region did not arise from a common ancestral sequence. The D4S115 marker can be assayed simultaneously with PCR products from D4S125, D4S95 and D4S43 on a single agarose gel, providing a rapid scan for successful amplification of these difficult-to-assay VNTRs, and for inheritance of the entire candidate Huntington's disease region. This approach should help to increase the speed, informativeness and accuracy of presymptomatic and prenatal linkage testing in this devastating disorder.

Alleles↗
PubMed1992

The telomeric 60 kb of chromosome arm 4p is homologous to telomeric regions on 13p, 15p, 21p, and 22p.

A telomere YAC clone containing the most distal 115 kb of chromosome arm 4p has been previously isolated. This clone is of particular interest as it spans a potential candidate region for the Huntington disease gene. The YAC was subcloned into a phage vector, and a high-resolution restriction map extending to within 13 kb of the telomere was constructed. In situ hybridization of the YAC to human metaphase spreads gives a peak of hybridization on 4pter but also an increase in the number of signals close to several other telomeres. Where possible, these results were investigated further by the hybridization of probes from the YAC to somatic cell hybrids containing single human chromosomes. This analysis indicates that the most telomeric 60 kb of chromosome arm 4p is homologous to telomeric regions on 13p, 15p, 21p, and 22p. The extent of this homology makes it less likely that the mutation for Huntington's disease is located within the telomere YAC clone.

Animals↗
PubMed1992

Novel mutations in families with unusual and variable disorders of the skeletal muscle sodium channel.

Mutations in the skeletal muscle sodium channel gene (SCN4A) have been described in paramyotonia congenita (PMC) and hyperkalaemic periodic paralysis (HPP). We have found two mutations in SCN4A which affect regions of the sodium channel not previously associated with a disease phenotype. Furthermore, affected family members display an unusual mixture of clinical features reminiscent of PMC, HPP and of a third disorder, myotonia congenita (MC). The highly variable individual expression of these symptoms, including in some cases apparent non-penetrance, implies the existence of modifying factors. Mutations in SCN4A can produce a broad range of phenotypes in muscle diseases characterized by episodic abnormalities of membrane excitability.

Adult↗