scholarly journals Alternative splicing isoforms of synaptotagmin VII in the mouse, rat and human

2002 ◽  
Vol 365 (1) ◽  
pp. 173-180 ◽  
Author(s):  
Mitsunori FUKUDA ◽  
Yukie OGATA ◽  
Chika SAEGUSA ◽  
Eiko KANNO ◽  
Katsuhiko MIKOSHIBA
Keyword(s):  
Plasma Membrane ◽  
Alternative Splicing ◽  
Brefeldin A ◽  
Green Fluorescence Protein ◽  
Peptide Sequence ◽  
Actin Binding ◽  
Pcr Analysis ◽  
Protein Fusion ◽  
Synaptotagmin Vii ◽  
Splicing Isoforms

Synaptotagmin VII (Syt VII) has been proposed to regulate several different types of Ca2+-dependent exocytosis, but its subcellular localization (lysosome or plasma membrane) and the number of alternative splicing isoforms of Syt VII (single or multiple forms) are matters of controversy. In the present study, we show by reverse transcriptase-PCR analysis that mouse Syt VII has one major isoform (Syt VIIα), the original Syt VII, and two minor isoforms (Syt VIIβ and Syt VIIγ), which contain unique insertions (of 44 and 116 amino acids respectively) in the spacer domain between the transmembrane and C2 domains of Syt VIIα. Similar results were obtained with respect to rat and human Syt VII mRNA expression. An antibody against the N-terminal domain of mouse Syt VII [anti-(Syt VII-N)], which specifically recognized recombinant Syt VII but not other Syt isoforms expressed in COS-7 cells, recognized two major, closely co-migrating bands (p58 and p60) and minor bands of approx. 65kDa in mouse brain. Immunoaffinity purification of proteins that bind the anti-(Syt VII-N) antibody, and peptide sequence analysis revealed that: (i) the major p58 and p60 bands are identified as adenylate cyclase-associated protein 2; (ii) actin-binding protein is localized at the plasma membrane; and (iii) Syt VIIα (65kDa) is the major Syt VII isoform, but with a much lower expression level than previously thought. It was also shown that FLAG-Syt VII—green-fluorescence-protein fusion protein stably expressed in PC12 cells is localized in the perinuclear region (co-localization with TGN38 protein, even after brefeldin A treatment) and in the tips of neurites (co-localization with Syt I), and not in the plasma membrane.

scholarly journals Exploration of Alternative Splicing Events in Mesenchymal Stem Cells from Human Induced Pluripotent Stem Cells

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 737
Author(s):  
Ji-Eun Jeong ◽  
Binna Seol ◽  
Han-Seop Kim ◽  
Jae-Yun Kim ◽  
Yee-Sook Cho
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Alternative Splicing ◽  
Functional Enrichment ◽  
Actin Binding ◽  
Pcr Analysis ◽  
Valuable Insight ◽  
Sequencing Analysis ◽  
Induced Pluripotent ◽  
Insight Into

Although comparative genome-wide transcriptomic analysis has provided insight into the biology of human induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs), the distinct alternative splicing (AS) signatures of iMSCs remain elusive. Here, we performed Illumina RNA sequencing analysis to characterize AS events in iMSCs compared with tissue-derived MSCs. A total of 4586 differentially expressed genes (|FC| > 2) were identified between iMSCs and umbilical cord blood-derived MSCs (UCB-MSCs), including 2169 upregulated and 2417 downregulated genes. Of these, 164 differentially spliced events (BF > 20) in 112 genes were identified between iMSCs and UCB-MSCs. The predominant type of AS found in iMSCs was skipped exons (43.3%), followed by retained introns (19.5%), alternative 3′ (15.2%) and 5′ (12.8%) splice sites, and mutually exclusive exons (9.1%). Functional enrichment analysis showed that the differentially spliced genes (|FC| > 2 and BF > 20) were mainly enriched in functions associated with focal adhesion, extracellular exosomes, extracellular matrix organization, cell adhesion, and actin binding. Splice isoforms of selected genes including TRPT1, CNN2, and AP1G2, identified in sashimi plots, were further validated by RT-PCR analysis. This study provides valuable insight into the biology of iMSCs and the translation of mechanistic understanding of iMSCs into therapeutic applications.

scholarly journals Primary structure of rat plasma membrane Ca2+-ATPase isoform 4 and analysis of alternative splicing patterns at splice site A

1995 ◽  
Vol 306 (3) ◽  
pp. 779-785 ◽  
Author(s):  
T P Keeton ◽  
G E Shull
Keyword(s):  
Plasma Membrane ◽  
Alternative Splicing ◽  
Splice Site ◽  
Primary Structure ◽  
Sequence Divergence ◽  
Rat Plasma ◽  
Pcr Analysis ◽  
Rat Tissues ◽  
Mrna Tissue Distribution ◽  
Splicing Patterns

We have determined the primary structure of the rat plasma membrane Ca(2+)-ATPase isoform 4 (PMCA4), and have analysed its mRNA tissue distribution and alternative splicing patterns at splice site A. Rat PMCA4 (rPMCA4) genomic clones were isolated and used to determine the coding sequences and intron/exon organization of the 5′-end of the gene, and the remaining coding sequence was determined from PCR-amplified cDNA fragments. Pairwise comparisons reveal that the amino acid sequence of rPMCA4 has diverged substantially from those of rPMCA isoforms 1, 2 and 3 (73-76% identity) and from that of human PMCA4 (87%). Despite the high degree of sequence divergence between the two species, comparisons of intron and untranslated mRNA sequences with the corresponding human sequences confirm the identity of this rat isoform as PMCA4. Northern blot studies demonstrate that the PMCA4 mRNA is expressed in all rat tissues examined except liver, with the highest levels in uterus and stomach. A combination of PCR analysis of alternative splicing patterns and sequence analysis of the gene demonstrate that a 36 nt exon at site A is included in PMCA4 mRNAs of most tissues but is largely excluded in heart and testis. Alternative splicing of both the 36 nt exon and a previously characterized 175 nt exon at splice site C, each of which can be either included or excluded in a highly tissue-specific manner, leads to the production of four different PMCA4 variants ranging in size from 1157 to 1203 amino acids.

scholarly journals A Cysteine-Rich Receptor-Like Protein Kinase CaCKR5 Contributes to Immune Defense Against Ralstonia Solanacearum in Pepper

2020 ◽  
Author(s):  
Shaoliang Mou ◽  
Feng Gao ◽  
Tingting Zhang ◽  
Weihong He ◽  
Qianqian Meng ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescence Protein ◽  
Immune Defense ◽  
Bimolecular Fluorescence Complementation ◽  
Pcr Analysis ◽  
Virus Induced Gene Silencing ◽  
Mobility Shift ◽  
Positive Role ◽  
Physiological Processes ◽  
Mobility Shift Assay

Abstract Background: Cysteine-rich receptor-like kinases (CRKs) represent a large subfamily of receptor-like kinases and have important roles in numerous different physiological processes in plants. Results: CaCRK5 transcripts were observed to accumulate after the inoculation of R. solanacearum and treatment with salicylic acid. The fusion between CaCRK5 and the green fluorescence protein was targeted to the plasma membrane. Suppression of CaCRK5 via virus-induced gene silencing made pepper plants significantly susceptible to R. solanacearum infection, which was accompanied by decreased expression of defense related genes CaPR1, CaSAR8.2, CaDEF1 and CaACO1. Overexpression of CaCRK5 in tobacco conferred increased resistance against R. solanacearum. Furthermore, electrophoretic mobility shift assay and chromatin immunoprecipitation with quantitative real-time PCR analysis verified that a homeodomain zipper I protein CaHDZ27 can bind to the CaCRK5 promoter, and directly active its expression. Yeast two-hybrid and bimolecular fluorescence complementation analyses suggested that CaCRK5 could heterodimerize with the homologous member CaCRK6 in the plasma membrane. Conclusions: Our data indicated that CaCRK5 played a positive role in pepper resistance against R. solanacearum infection.

scholarly journals A cysteine-rich receptor-like protein kinase CaCKR5 modulates immune response against Ralstonia solanacearum infection in pepper

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shaoliang Mou ◽  
Qianqian Meng ◽  
Feng Gao ◽  
Tingting Zhang ◽  
Weihong He ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Ralstonia Solanacearum ◽  
Green Fluorescence Protein ◽  
Bimolecular Fluorescence Complementation ◽  
Pcr Analysis ◽  
Virus Induced Gene Silencing ◽  
Mobility Shift ◽  
Positive Role ◽  
Physiological Processes ◽  
Mobility Shift Assay

Abstract Background Cysteine-rich receptor-like kinases (CRKs) represent a large subfamily of receptor-like kinases and play vital roles in diverse physiological processes in regulating plant growth and development. Results CaCRK5 transcripts were induced in pepper upon the infection of Ralstonia solanacearum and treatment with salicylic acid. The fusions between CaCRK5 and green fluorescence protein were targeted to the plasma membrane. Suppression of CaCRK5 via virus-induced gene silencing (VIGS) made pepper plants significantly susceptible to R. solanacearum infection, which was accompanied with decreased expression of defense related genes CaPR1, CaSAR8.2, CaDEF1 and CaACO1. Overexpression of CaCRK5 increased resistance against R. solanacearum in Nicotiana benthamiana. Furthermore, electrophoretic mobility shift assay and chromatin immunoprecipitation coupled with quantitative real-time PCR analysis revealed that a homeodomain zipper I protein CaHDZ27 can active the expression of CaCRK5 through directly binding to its promoter. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) analyses suggested that CaCRK5 heterodimerized with the homologous member CaCRK6 on the plasma membrane. Conclusions Our data revealed that CaCRK5 played a positive role in regulating immune responses against R. solanacearum infection in pepper.

scholarly journals Natural Antisense Transcript PEBP1P3 Regulates the RNA Expression, DNA Methylation and Histone Modification of CD45 Gene

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 759
Author(s):  
Zhongjing Su ◽  
Guangyu Liu ◽  
Bin Zhang ◽  
Ze Lin ◽  
Dongyang Huang
Keyword(s):  
Dna Methylation ◽  
Alternative Splicing ◽  
Histone Modification ◽  
Antisense Rna ◽  
Noncoding Rna ◽  
Antisense Transcript ◽  
Natural Antisense Transcript ◽  
Regulation Of Expression ◽  
Splicing Isoforms ◽  
Intron 2

The leukocyte common antigen CD45 is a transmembrane phosphatase expressed on all nucleated hemopoietic cells, and the expression levels of its splicing isoforms are closely related to the development and function of lymphocytes. PEBP1P3 is a natural antisense transcript from the opposite strand of CD45 intron 2 and is predicted to be a noncoding RNA. The genotype-tissue expression and quantitative PCR data suggested that PEBP1P3 might be involved in the regulation of expression of CD45 splicing isoforms. To explore the regulatory mechanism of PEBP1P3 in CD45 expression, DNA methylation and histone modification were detected by bisulfate sequencing PCR and chromatin immunoprecipitation assays, respectively. The results showed that after the antisense RNA PEBP1P3 was knocked down by RNA interference, the DNA methylation of CD45 intron 2 was decreased and histone H3K9 and H3K36 trimethylation at the alternative splicing exons of CD45 DNA was increased. Knockdown of PEBP1P3 also increased the binding levels of chromatin conformation organizer CTCF at intron 2 and the alternative splicing exons of CD45. The present results indicate that the natural antisense RNA PEBP1P3 regulated the alternative splicing of CD45 RNA, and that might be correlated with the regulation of histone modification and DNA methylation.

scholarly journals Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane.

1994 ◽  
Vol 125 (2) ◽  
pp. 381-391 ◽  
Author(s):  
J Mulholland ◽  
D Preuss ◽  
A Moon ◽  
A Wong ◽  
D Drubin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Binding Proteins ◽  
Ultrastructural Level ◽  
Actin Binding ◽  
Cortical Actin ◽  
Actin Binding Proteins ◽  
Double Labeling ◽  
Wall Growth ◽  
Cortical Cytoskeleton

We characterized the yeast actin cytoskeleton at the ultrastructural level using immunoelectron microscopy. Anti-actin antibodies primarily labeled dense, patchlike cortical structures and cytoplasmic cables. This localization recapitulates results obtained with immunofluorescence light microscopy, but at much higher resolution. Immuno-EM double-labeling experiments were conducted with antibodies to actin together with antibodies to the actin binding proteins Abp1p and cofilin. As expected from immunofluorescence experiments, Abp1p, cofilin, and actin colocalized in immuno-EM to the dense patchlike structures but not to the cables. In this way, we can unambiguously identify the patches as the cortical actin cytoskeleton. The cortical actin patches were observed to be associated with the cell surface via an invagination of plasma membrane. This novel cortical cytoskeleton-plasma membrane interface appears to consist of a fingerlike invagination of plasma membrane around which actin filaments and actin binding proteins are organized. We propose a possible role for this unique cortical structure in wall growth and osmotic regulation.

Barrier role of actin filaments in regulated mucin secretion from airway goblet cells

2005 ◽  
Vol 288 (1) ◽  
pp. C46-C56 ◽  
Author(s):  
Camille Ehre ◽  
Andrea H. Rossi ◽  
Lubna H. Abdullah ◽  
Kathleen De Pestel ◽  
Sandra Hill ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Yellow Fluorescent Protein ◽  
Goblet Cells ◽  
Actin Binding ◽  
Secretory Cells ◽  
Cortical Actin ◽  
Mucin Secretion

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/Gq-coupled P2Y2receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of β- and γ-actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of β- or γ-actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca2+-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis.

Abstract 273: P2Y2 Receptor-Mediated Lymphotoxin-α Secretion Regulates ICAM-1 Expression in Smooth Muscle Cells

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Cheikh Seye ◽  
Wilbert Derbigny ◽  
Shaomin Qian
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Protein Transport ◽  
Brefeldin A ◽  
Muscle Cells ◽  
Secretory Vesicle ◽  
Actin Binding ◽  
Filamin A ◽  
Artery Disease ◽  
Lymphotoxin Α

Rationale: Single nucleotide polymorphism (SNP) in the LGASL2 galectin-2 (Gal-2) gene leads to altered secretion of lymphotoxin-α (LT-α) and is associated with coronary artery disease. Objective:Our aim was to determine whether factors other than genetic variations in LGASL2 regulate LT-α release and to define the role of this pro-inflammatory in vascular smooth muscle cells (SMC). Methods and results: The proinflammatory cytokine lymphotoxin-alpha (LTA) is thought to contribute to the pathogenesis of atherosclerosis. However, the mechanisms that regulate its expression in VSMC are poorly understood. The ability of exogenous nucleotides to stimulate LTA production was evaluated in VSMC by ELISA. The P2Y 2 nucleotide receptor (P2Y 2 R) agonist UTP stimulates a strong and sustained release of LTA from wild-type but not P2Y 2 R -/- SMC. Assessment of LTA gene transcription by LTA promoter-luciferase construct indicated that LTA levels are controlled at the level of transcription. We show using RNAi techniques that knockdown of the actin-binding protein filamin-A (FLNa) severely impaired nucleotide-induced Rho activation and consequent Rho-mediated LTA secretion. Re-introduction of FLNa in FLNa RNAi SMC rescued UTP-induced LTA expression. In addition, we found UTP-stimulated LTA secretion is not sensitive to brefeldin A (BFA), which blocks the formation of vesicles involved in protein transport from the ER to the Golgi apparatus, suggesting that P2Y 2 R/filamin-mediated secretion of LTA is independent of the ER/Golgi secretory vesicle route. Furthermore, UTP selectively induces ICAM-1 expression in WT but not SMC expressing a truncated P2Y 2 R deficient in LTA secretion. Conclusion: These data suggest that P2Y 2 R recruits FLNa to provide a cytoskeletal scaffold necessary for Rho signaling pathway upstream of LTA release and subsequent stimulation of ICAM-1 expression on VSMC.

scholarly journals Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes

1992 ◽  
Vol 103 (4) ◽  
pp. 1139-1152
Author(s):  
J.W. Kok ◽  
K. Hoekstra ◽  
S. Eskelinen ◽  
D. Hoekstra
Keyword(s):  
Plasma Membrane ◽  
Intracellular Ph ◽  
Brefeldin A ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Recycling Pathway ◽  
Golgi Network ◽  
Extensive Involvement

Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide, 6(-)[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosyl sphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membrane occurs, as could be shown after treating the cells with the microtubule-disrupting agent nocodazole, which causes inhibition of the glycolipid's trafficking from peripheral early endosomes to centrally located late endosomes. When the microtubuli are intact, at least part of the glucosylceramide is transported from early to late endosomes together with ricin. Interestingly, also N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a membrane marker of the fluid-phase endocytic pathway, is transported to this endosomal compartment. However, in contrast to both ricin and N-Rh-PE, the glucosylceramide can escape from this organelle and recycle to the plasma membrane. Monensin and brefeldin A have little effect on this recycling pathway, which would exclude extensive involvement of early Golgi compartments in recycling. Hence, the small fraction of the glycolipid that colocalizes with transferrin (Tf) in the Golgi area might directly recycle via the trans-Golgi network. When the intracellular pH was lowered to 5.5, recycling was drastically reduced, in accordance with the impeding effect of low intracellular pH on vesicular transport during endocytosis and in the biosynthetic pathway. Our results thus demonstrate the existence of at least two recycling pathways for glucosylceramide and indicate the relevance of early endosomes in recycling of both proteins and lipids.

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