Phylogenetic, expression, and functional analyses of anoctamin homologs inCaenorhabditis elegans
Ying WangTashrique AlamKatherine L Hill-HarfeAlejandro J. LopezChi K. LeungDaniela IribarneBradley J. BruggemanMichael M. MiyamotoBrian D. HarfeKeith Choe
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Ca(2+)-activated Cl(-) channels (CaCCs) are critical to processes such as epithelial transport, membrane excitability, and signal transduction. Anoctamin, or TMEM16, is a family of 10 mammalian transmembrane proteins, 2 of which were recently shown to function as CaCCs. The functions of other family members have not been firmly established, and almost nothing is known about anoctamins in invertebrates. Therefore, we performed a phylogenetic analysis of anoctamins across the animal kingdom and examined the expression and function of anoctamins in the genetically tractable nematode Caenorhabditis elegans. Phylogenetic analyses support five anoctamin clades that are at least as old as the deuterostome/protosome ancestor. This includes a branch containing two Drosophila paralogs that group with mammalian ANO1 and ANO2, the two best characterized CaCCs. We identify two anoctamins in C. elegans (ANOH-1 and ANOH-2) that are also present in basal metazoans. The anoh-1 promoter is active in amphid sensory neurons that detect external chemical and nociceptive cues. Within amphid neurons, ANOH-1::GFP fusion protein is enriched within sensory cilia. RNA interference silencing of anoh-1 reduced avoidance of steep osmotic gradients without disrupting amphid cilia development, chemotaxis, or withdrawal from noxious stimuli, suggesting that ANOH-1 functions in a sensory mode-specific manner. The anoh-2 promoter is active in mechanoreceptive neurons and the spermatheca, but loss of anoh-2 had no effect on motility or brood size. Our study indicates that at least five anoctamin duplicates are evolutionarily ancient and suggests that sensory signaling may be a basal function of the anoctamin protein family.Ciliopathies
Motile cilium
Primary (astronomy)
Intraflagellar Transport
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Ciliogenesis
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In response to DNA damage, p53 undergoes post-translational modifications (including acetylation) that are critical for its transcriptional activity. However, the mechanism by which p53 acetylation is regulated is still unclear. Here, we describe an essential role for HLA-B-associated transcript 3 (Bat3)/Scythe in controlling the acetylation of p53 required for DNA damage responses. Depletion of Bat3 from human and mouse cells markedly impairs p53-mediated transactivation of its target genes Puma and p21 . Although DNA damage-induced phosphorylation, stabilization, and nuclear accumulation of p53 are not significantly affected by Bat3 depletion, p53 acetylation is almost completely abolished. Bat3 forms a complex with p300, and an increased amount of Bat3 enhances the recruitment of p53 to p300 and facilitates subsequent p53 acetylation. In contrast, Bat3-depleted cells show reduced p53–p300 complex formation and decreased p53 acetylation. Furthermore, consistent with our in vitro findings, thymocytes from Bat3-deficient mice exhibit reduced induction of puma and p21, and are resistant to DNA damage-induced apoptosis in vivo. Our data indicate that Bat3 is a novel and essential regulator of p53-mediated responses to genotoxic stress, and that Bat3 controls DNA damage-induced acetylation of p53.
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The primary cilium is a microtubule-based structure found in most cell types in mammals. Disruption of cilium function causes a diverse set of human diseases collectively known as ciliopathies. We report that Rab effector-related proteins Rab-interacting lysosomal protein-like 1 (Rilpl1) and Rilpl2 regulate protein localization in the primary cilium. Rilpl2 was initially identified as up-regulated in ciliating mouse tracheal epithelial cells. Rilpl1 and Rilpl2 both localize to the primary cilium and centrosome, Rilpl1 specifically to the distal end of the mother centriole. Live-cell microscopy reveals that Rilpl2 primary cilium localization is dynamic and that it is associated with tubulovesicular structures at the base of the cilium. Depletion of Rilpl1 and Rilpl2 results in accumulation of signaling proteins in the ciliary membrane and prevents proper epithelial cell organization in three-dimensional culture. These data suggest that Rilp-like proteins function in regulation of ciliary membrane protein concentration by promoting protein removal from the primary cilium.
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Centriole
Ciliogenesis
Rab
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Intraflagellar Transport
Motile cilium
Exocyst
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Purpose of review Primary (immotile) cilia are specialized organelles present on most cell types. Almost all of proteins associated with a broad spectrum of human cystic kidney diseases have been localized to the region in or around the cilia. Abnormal cilia structure and function have both been reported in animal models and human cystic kidneys. The goal of this review is to discuss current understanding of the mechanisms by which abnormal genes/proteins and cilia interact to potentially influence renal cystogenesis. Recent findings Novel direct recording of cilia calcium levels/channel activity suggests that cilia form a calcium-mediated signaling microenvironment separate from the cytoplasm, which could provide a mechanism for cilia-specific downstream signaling. Genetic-based studies confirm that cilia are not required for cystogenesis, but modulate cystic kidney disease severity through a novel, undefined mechanism. Mechanisms by which both cilia-associated and noncilia-associated proteins can alter cilia structure/function have also been identified. Summary Considerable progress has been made in defining the mechanisms by which abnormal genes and proteins affect cilia structure and function. However, the exact mechanisms by which these interactions cause renal cyst formation and progression of cystic kidney disease are still unknown.
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Cystic kidney disease
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Abstract Proteins specialized in the detection, generation, or stabilization of membrane curvature play important roles in establishing various morphologies of cells and cellular organelles. Primary cilia are cellular organelles that protrude from the cell surface using a microtubule‐based cytoskeleton called the axoneme as a structural support. It is unclear whether the integrity of the high curvature of the ciliary membrane depends on membrane curvature‐related proteins. Charged Multivesicular Body Protein 4B (CHMP4B), a subunit of the endosomal sorting complexes required for transport (ESCRT), can stabilize membrane curvature. Here we show that CHMP4B is involved in the assembly and maintenance of primary cilia. CHMP4B was localized to primary cilia in mammalian cells. Knockdown of CHMP4B interfered with cilium assembly and also caused fragmentation of preexisting cilia. By contrast, cilium formation was unaffected by the interruption of the ESCRT‐dependent endocytic degradation pathway. Morpholino (MO)‐mediated CHMP4B depletion in zebrafish embryos induced characteristic phenotypes of ciliary defects such as curved body axis, hydrocephalus, otolith malformation, and kidney cyst. Our study reveals a new role for the multifunctional protein CHMP4B as a key factor in maintaining the structural integrity of primary cilia.
ESCRT
Structural integrity
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Membrane integrity
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SUMMARY A hitherto unrecorded virus having flexible rod‐shaped particles about 740–760 × 13 nm was isolated from Anthoxanthum odoratwn L. It was transmitted by sap inoculation, but not by several species of insect, seed or soil to 18 species of Gramineae including wheat, oats and barley. In susceptible species the virus normally produced a mosaic mottling of the leaves which was sometimes followed by a necrotic streaking or striping.
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HLA-B-associated transcript 3 (BAT3) was originally identified as one of the genes located within human major histocompatibility complex. It encodes a large proline-rich protein with unknown function. In this study, we found that a fragment of the BAT3 gene product interacts with a candidate tumor suppressor, DAN, in the yeast-based two-hybrid system. We cloned the full-length rat BAT3 cDNA from a fibroblast 3Y1 cDNA library. Our sequence analysis has demonstrated that rat BAT3 cDNA is 3617 nucleotides in length and encodes a full-length BAT3 (1098 amino acids) with an estimated molecular mass of 114,801 daltons, which displays an 87.4% identity with human BAT3. The deletion experiment revealed that the N-terminal region (amino acid residues 1-80) of DAN was required for the interaction with BAT3. Green fluorescent protein-tagged BAT3 was largely localized in the cytoplasm of COS cells. Northern hybridization showed that BAT3 mRNA was expressed in all the adult rat tissues examined but predominantly in testis. In addition, the level of BAT3 mRNA expression was more downregulated in some of the transformed cells, including v-mos- and v-Ha-ras-transformed 3Y1 cells, than in the parental cells.
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