scholarly journals The C. elegans TspanC8 tetraspanin TSP-14 exhibits isoform-specific localization and function

2021 ◽  
Author(s):  
Zhiyu Liu ◽  
Herong Shi ◽  
Jun Liu
Keyword(s):  
Amino Acids ◽  
Expression Patterns ◽  
Basolateral Membrane ◽  
Living Organism ◽  
Distribution Patterns ◽  
Biochemical Properties ◽  
Bmp Signaling ◽  
Mesoderm Development ◽  
C Elegans

Tetraspanin proteins are a unique family of highly conserved four-pass transmembrane proteins in metazoans. While much is known about their biochemical properties, the in vivo functions and distribution patterns of different tetraspanin proteins are less understood. Previous studies have shown that two paralogous tetraspanins that belong to the TspanC8 subfamily, TSP12 and TSP-14, function redundantly to promote both Notch signaling and bon morphogenetic protein (BMP) signaling in C. elegans. TSP-14 has two isoforms, TSP-14A and TSP-14B, where TSP-14B has an additional 24 amino acids at its N-terminus compared to TSP-14A. By generating isoform specific knock-ins and knock-outs using CRISPR, we found that TSP-14A and TSP-14B share distinct as well as overlapping expression patterns and functions. While TSP14A functions redundantly with TSP-12 to regulate body size and embryonic and vulva development, TSP-14B primarily functions redundantly with TSP-12 to regulate postembryonic mesoderm development. Importantly, TSP-14A and TSP-14B exhibit distinct subcellular localization patterns. TSP-14A is localized apically and on early and late endosomes. TSP-14B is localized to the basolateral cell membrane. We further identified a di-leucine motif within the Nterminal 24 amino acids of TSP-14B that serves as a basolateral membrane targeting sequence, and showed that the basolateral membrane localization of TSP-14B is important for its function. Our work highlights the diverse and intricate functions of TspanC8 tetraspanins in C. elegans, and demonstrates the importance of dissecting the functions of these important proteins in an intact living organism.

scholarly journals Tetraspanins TSP-12 and TSP-14 function redundantly to regulate the trafficking of the type II BMP receptor in Caenorhabditis elegans

2020 ◽  
Vol 117 (6) ◽  
pp. 2968-2977
Author(s):  
Zhiyu Liu ◽  
Herong Shi ◽  
Anthony K. Nzessi ◽  
Anne Norris ◽  
Barth D. Grant ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Cell Surface ◽  
Molecular Mechanisms ◽  
Transmembrane Protein ◽  
Expression Patterns ◽  
Bmp Signaling ◽  
Type I ◽  
Type Ii ◽  
Bmp Receptors

Tetraspanins are a unique family of 4-pass transmembrane proteins that play important roles in a variety of cell biological processes. We have previously shown that 2 paralogous tetraspanins in Caenorhabditis elegans, TSP-12 and TSP-14, function redundantly to promote bone morphogenetic protein (BMP) signaling. The underlying molecular mechanisms, however, are not fully understood. In this study, we examined the expression and subcellular localization patterns of endogenously tagged TSP-12 and TSP-14 proteins. We found that TSP-12 and TSP-14 share overlapping expression patterns in multiple cell types, and that both proteins are localized on the cell surface and in various types of endosomes, including early, late, and recycling endosomes. Animals lacking both TSP-12 and TSP-14 exhibit reduced cell-surface levels of the BMP type II receptor DAF-4/BMPRII, along with impaired endosome morphology and mislocalization of DAF-4/BMPRII to late endosomes and lysosomes. These findings indicate that TSP-12 and TSP-14 are required for the recycling of DAF-4/BMPRII. Together with previous findings that the type I receptor SMA-6 is recycled via the retromer complex, our work demonstrates the involvement of distinct recycling pathways for the type I and type II BMP receptors and highlights the importance of tetraspanin-mediated intracellular trafficking in the regulation of BMP signaling in vivo. As TSP-12 and TSP-14 are conserved in mammals, our findings suggest that the mammalian TSP-12 and TSP-14 homologs may also function in regulating transmembrane protein recycling and BMP signaling.

scholarly journals Mouse embryo geometry drives formation of robust signaling gradients through receptor localization

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Zhechun Zhang ◽  
Steven Zwick ◽  
Ethan Loew ◽  
Joshua S. Grimley ◽  
Sharad Ramanathan
Keyword(s):  
Cell Fate ◽  
Mouse Embryo ◽  
Basolateral Membrane ◽  
Embryonic Stem ◽  
Bmp Signaling ◽  
Receptor Localization ◽  
Bmp Receptors ◽  
Early Mouse

Abstract Morphogen signals are essential for cell fate specification during embryogenesis. Some receptors that sense these morphogens are known to localize to only the apical or basolateral membrane of polarized cell lines in vitro. How such localization affects morphogen sensing and patterning in the developing embryo remains unknown. Here, we show that the formation of a robust BMP signaling gradient in the early mouse embryo depends on the restricted, basolateral localization of BMP receptors. The mis-localization of receptors to the apical membrane results in ectopic BMP signaling in the mouse epiblast in vivo. With evidence from mathematical modeling, human embryonic stem cells in vitro, and mouse embryos in vivo, we find that the geometric compartmentalization of BMP receptors and ligands creates a signaling gradient that is buffered against fluctuations. Our results demonstrate the importance of receptor localization and embryo geometry in shaping morphogen signaling during embryogenesis.

The zebrafish homologs of SET/I2PP2A oncoprotein: expression patterns and insights into their physiological roles during development

2016 ◽  
Vol 473 (24) ◽  
pp. 4609-4627 ◽  
Author(s):  
Iliana Serifi ◽  
Eleni Tzima ◽  
Katerina Soupsana ◽  
Zoe Karetsou ◽  
Dimitris Beis ◽  
...  
Keyword(s):  
Amino Acids ◽  
Lateral Line ◽  
Gene Networks ◽  
Expression Patterns ◽  
Otic Vesicle ◽  
Lateral Line System ◽  
Loss Of Function ◽  
Line System ◽  
Eye Retina

The oncoprotein SET/I2PP2A (protein phosphatase 2A inhibitor 2) participates in various cellular mechanisms such as transcription, cell cycle regulation and cell migration. SET is also an inhibitor of the serine/threonine phosphatase PP2A, which is involved in the regulation of cell homeostasis. In zebrafish, there are two paralogous set genes that encode Seta (269 amino acids) and Setb (275 amino acids) proteins which share 94% identity. We show here that seta and setb are similarly expressed in the eye, the otic vesicle, the brain and the lateral line system, as indicated by in situ hybridization labeling. Whole-mount immunofluorescence analysis revealed the expression of Seta/b proteins in the eye retina, the olfactory pit and the lateral line neuromasts. Loss-of-function studies using antisense morpholino oligonucleotides targeting both seta and setb genes (MOab) resulted in increased apoptosis, reduced cell proliferation and morphological defects. The morphant phenotypes were partially rescued when MOab was co-injected with human SET mRNA. Knockdown of setb with a transcription-blocking morpholino oligonucleotide (MOb) resulted in phenotypic defects comparable with those induced by setb gRNA (guide RNA)/Cas9 [CRISPR (clustered regularly interspaced short palindromic repeats)-associated 9] injections. In vivo labeling of hair cells showed a significantly decreased number of neuromasts in MOab-, MOb- and gRNA/Cas9-injected embryos. Microarray analysis of MOab morphant transcriptome revealed differential expression in gene networks controlling transcription in the sensory organs, including the eye retina, the ear and the lateral line. Collectively, our results suggest that seta and setb are required during embryogenesis and play roles in the zebrafish sensory system development.

Increased Resistance of Peptides to Serum Proteases by Modification of their Amino Groups

2003 ◽  
Vol 58 (7-8) ◽  
pp. 558-561 ◽  
Author(s):  
Rossella Galati ◽  
Alessandra Verdina ◽  
Giuliana Falasca ◽  
Alberto Chersi
Keyword(s):  
Amino Acids ◽  
Proteolytic Enzymes ◽  
Living Organism ◽  
Amino Group ◽  
Amino Groups ◽  
Protein Fragments ◽  
Beta Alanine ◽  
Synthetic Protein ◽  
In Vivo Tests

Abstract The ability of synthetic protein fragments to survive the degradative action of aminopeptidases and serum proteolytic enzymes can be remarkably enhanced by slight modifications at their N-terminal alpha-amino group. This can be achieved by addition of beta-alanine or amino acids of the d-configuration, amino acids which are seldom found in a living organism. These modifications do scarcely modify the chemical and physical properties of the peptides, and should be preferrred, especially for in vivo tests, to drastic alterations of peptides as produced by dinitrophenylation or dansylation of the amino groups.

scholarly journals Tissue specific targeting of DNA nanodevices in a multicellular living organism

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Kasturi Chakraborty ◽  
Palapuravan Anees ◽  
Sunaina Surana ◽  
Simona Martin ◽  
Jihad Aburas ◽  
...  
Keyword(s):  
Living Organism ◽  
Cell Types ◽  
Specific Cell ◽  
Full Potential ◽  
Tissue Specific ◽  
C Elegans ◽  
Specific Targeting ◽  
Synthetic Receptor ◽  
Ligand Interactions

Nucleic acid nanodevices present great potential as agents for logic-based therapeutic intervention as well as in basic biology. Often, however, the disease targets that need corrective action are localized in specific organs and thus realizing the full potential of DNA nanodevices also requires ways to target them to specific cell-types in vivo. Here we show that by exploiting either endogenous or synthetic receptor-ligand interactions and by leveraging the biological barriers presented by the organism, we can target extraneously introduced DNA nanodevices to specific cell types in C. elegans, with sub-cellular precision. The amenability of DNA nanostructures to tissue-specific targeting in vivo significantly expands their utility in biomedical applications and discovery biology.

scholarly journals Crystal Structure of CRN-4: Implications for Domain Function in Apoptotic DNA Degradation

2008 ◽  
Vol 29 (2) ◽  
pp. 448-457 ◽  
Author(s):  
Yu-Yuan Hsiao ◽  
Akihisa Nakagawa ◽  
Zhonghao Shi ◽  
Shohei Mitani ◽  
Ding Xue ◽  
...  
Keyword(s):  
Cell Death ◽  
Active Site ◽  
Structural Model ◽  
Active Form ◽  
Dna Degradation ◽  
Biochemical Properties ◽  
Chromosomal Dna ◽  
C Elegans ◽  
Cell Functions

ABSTRACT Cell death related nuclease 4 (CRN-4) is one of the apoptotic nucleases involved in DNA degradation in Caenorhabditis elegans. To understand how CRN-4 is involved in apoptotic DNA fragmentation, we analyzed CRN-4's biochemical properties, in vivo cell functions, and the crystal structures of CRN-4 in apo-form, Mn2+-bound active form, and Er3+-bound inactive form. CRN-4 is a dimeric nuclease with the optimal enzyme activity in cleaving double-stranded DNA in apoptotic salt conditions. Both mutational studies and the structures of the Mn2+-bound CRN-4 revealed the geometry of the functional nuclease active site in the N-terminal DEDDh domain. The C-terminal domain, termed the Zn-domain, contains basic surface residues ideal for nucleic acid recognition and is involved in DNA binding, as confirmed by deletion assays. Cell death analysis in C. elegans further demonstrated that both the nuclease active site and the Zn-domain are required for crn-4's function in apoptosis. Combining all of the data, we suggest a structural model where chromosomal DNA is bound at the Zn-domain and cleaved at the DEDDh nuclease domain in CRN-4 when the cell is undergoing apoptosis.

Identification of the differential distribution patterns of mRNAs and consensus binding sequences for mouse DAF-16 homologues

2000 ◽  
Vol 349 (2) ◽  
pp. 629-634 ◽  
Author(s):  
Tatsuo FURUYAMA ◽  
Toru NAKAZAWA ◽  
Itsuko NAKANO ◽  
Nozomu MORI
Keyword(s):  
Target Genes ◽  
Binding Protein ◽  
Expression Patterns ◽  
Distribution Patterns ◽  
Mobility Shift ◽  
Differential Distribution ◽  
C Elegans ◽  
Core Sequence ◽  
Mobility Shift Assay ◽  
Responsive Element

daf-16 is a forkhead-type transcription factor, functioning downstream of insulin-like signals, and is known to be critical to the regulation of life span in Caenorhabditis elegans. Mammalian DAF-16 homologues include AFX, FKHR and FKHRL1, which contain a conserved forkhead domain and three putative phosphorylation sites for the Ser/Thr kinase Akt/protein kinase B (PKB), as well as for DAF-16. To assess the function of the homologues, we examined tissue distribution patterns of mRNAs for DAF-16 homologues in mice. In the embryos, expressions of AFX, FKHR and FKHRL1 mRNAs were complementary to each other and were highest in muscle, adipose tissue and embryonic liver. The characteristic expression pattern remained in the adult, except that signals of FKHRL1 became evident in more tissues, including the brain. In order to clarify whether each DAF-16 homologue had different target genes, we determined the consensus sequences for the binding of DAF-16 and the mouse homologues. The binding sequences for all four proteins shared a core sequence, TTGTTTAC, daf-16 family protein-binding element (DBE) binding protein. However, electrophoretic mobility shift assay showed that the binding affinity of DAF-16 homologues to the core sequence was stronger than that to the insulin-responsive element in the insulin-like growth factor binding protein-1 promoter region, which has been identified as a binding sequence for them. We identified one copy of the DBE upstream of the first exon of sod-3 by searching the genomic database of C. elegans. Taken together, DAF-16 homologues can fundamentally regulate the common target genes in insulin-responsive tissues and the specificity to target genes of each protein is partially determined by the differences in their expression patterns.

scholarly journals Tissue specific targeting of DNA nanodevices in a multicellular living organism

2021 ◽  
Author(s):  
Kasturi Chakraborty ◽  
Sunaina Surana ◽  
Simona Martin ◽  
Jihad Aburas ◽  
Sandrine Moutel ◽  
...  
Keyword(s):  
Living Organism ◽  
Cell Types ◽  
Specific Cell ◽  
Full Potential ◽  
Tissue Specific ◽  
C Elegans ◽  
Specific Targeting ◽  
Synthetic Receptor ◽  
Ligand Interactions

AbstractNucleic acid nanodevices present great potential as agents for logic-based therapeutic intervention as well as in basic biology. Often, however, the disease targets that need corrective action are localized in specific organs and thus realizing the full potential of DNA nanodevices also requires ways to target them to specific cell-types in vivo. Here we show that by exploiting either endogenous or synthetic receptor-ligand interactions and by leveraging the biological barriers presented by the organism, we can target extraneously introduced DNA nanodevices to specific cell types in C. elegans, with sub-cellular precision. The amenability of DNA nanostructures to tissue-specific targeting in vivo significantly expands their utility in biomedical applications and discovery biology.

scholarly journals Golgi localization of the LIN-2/7/10 complex points to a role in basolateral secretion of LET-23 EGFR in the C. elegans vulval precursor cells

Development ◽  
2021 ◽  
pp. dev.194167
Author(s):  
Kimberley D. Gauthier ◽  
Christian E. Rocheleau
Keyword(s):  
Cell Fate ◽  
Spatial Organization ◽  
Basolateral Membrane ◽  
Growth Factor Receptor ◽  
Precursor Cells ◽  
C Elegans ◽  
Receptor Localization ◽  
Epidermal Growth ◽  
Complex Forms

The evolutionarily conserved LIN-2 (CASK)/LIN-7 (Lin7A-C)/LIN-10 (APBA1) complex plays an important role in regulating spatial organization of membrane proteins and signaling components. In C. elegans, the complex is essential for development of the vulva by promoting the localization of the sole Epidermal Growth Factor Receptor (EGFR) orthologue, LET-23, to the basolateral membrane of the vulva precursor cells (VPCs) where it can specify the vulval cell fate. To understand how the LIN-2/7/10 complex regulates receptor localization, we determined its expression and localization during vulva development. We found that LIN-7 colocalizes with LET-23 EGFR at the basolateral membrane, whereas the LIN-2/7/10 complex colocalizes with LET-23 EGFR at cytoplasmic punctae, that mostly overlap with the Golgi. Furthermore, LIN-10 recruits LIN-2, which in turn recruits LIN-7. We demonstrate that the complex forms in vivo with particularly strong interaction and colocalization between LIN-2 and LIN-7 consistent with their forming a subcomplex. Thus, the LIN-2/7/10 complex forms on the Golgi where it likely targets LET-23 EGFR trafficking to the basolateral membrane rather than functioning as a tether.

scholarly journals Characterization of mannitol metabolism genes in Saccharina explains its key role in mannitol biosynthesis and evolutionary significance in Laminariales

2018 ◽  
Author(s):  
Shan Chi ◽  
Tao Liu ◽  
Cui Liu ◽  
Yuemei Jin ◽  
Hongxin Yin ◽  
...  
Keyword(s):  
Brown Algae ◽  
Molecular Mechanisms ◽  
Specific Activity ◽  
Expression Patterns ◽  
Dominant Gene ◽  
Biochemical Properties ◽  
Transcriptional Analysis ◽  
Evolutionary Significance ◽  
Mannitol Metabolism

AbstractAs a unique photosynthetic product in brown algae, mannitol exhibits high synthesis and accumulation in Saccharina japonica. Mannitol acts as a carbon-storage compound and is an osmoprotectant, imparting increased tolerance to osmotic stress. However, the underlying biochemical and molecular mechanisms in macroalgae have not been studied. Analysis of genomic and transcriptomic data has shown that mannitol metabolism in S. japonica is a circular pathway composed of four steps. In this study, one S. japonica mannitol-1-phosphate dehydrogenase (M1PDH2) and two mannitol-1-phosphatase (M1Pase) proteins were recombinantly expressed to analysis enzyme biochemical properties. RNA sequencing and droplet digital polymerase chain reaction were used to analyze the gene expression patterns of mannitol metabolism in different generations, tissues, sexes, and abiotic stresses. Our findings revealed insights into the mannitol synthesis pathways in brown algae. All genes were constitutively expressed in all samples, allowing maintenance of basic mannitol anabolism and dynamic maintenance of the “saccharide pool” in vivo as the main storage and antistress mechanism. Enzyme assays confirmed that the recombinant proteins produced mannitol, with the specific activity of SjaM1Pase1 being 1.8–4831 times that of other algal enzymes. Combined with the transcriptional analysis, SjaM1Pase1 was shown to be the dominant gene of mannitol metabolism. Mannitol metabolism genes in multicellular filamentous (gametophyte) and large parenchyma thallus (sporophyte) generations had different expression levels and responded differently under environmental stresses (hyposaline and hyperthermia) in gametophytes and sporophytes. The considerable variation in enzyme characteristics and expression of mannitol synthesis genes suggest their important ecophysiological significance in the evolution of complex systems (filamentous and thallus) and environmental adaptation of Laminariales.

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