Apoptotic Fragmentation of Tricellulin

Apoptotic extrusion of cells from epithelial cell layers is of central importance for epithelial homeostasis. As a prerequisite cell–cell contacts between apoptotic cells and their neighbors have to be dissociated. Tricellular tight junctions (tTJs) represent specialized structures that seal polarized epithelial cells at sites where three cells meet and are characterized by the specific expression of tricellulin and angulins. Here, we specifically addressed the fate of tricellulin in apoptotic cells. Methods: Apoptosis was induced by staurosporine or camptothecin in MDCKII and RT-112 cells. The fate of tricellulin was analyzed by Western blotting and immunofluorescence microscopy. Caspase activity was inhibited by Z-VAD-FMK or Z-DEVD-FMK. Results: Induction of apoptosis induces the degradation of tricellulin with time. Aspartate residues 487 and 441 were identified as caspase cleavage-sites in the C-terminal coiled-coil domain of human tricellulin. Fragmentation of tricellulin was inhibited in the presence of caspase inhibitors or when Asp487 or Asp441 were mutated to asparagine. Deletion of the tricellulin C-terminal amino acids prevented binding to lipolysis-stimulated lipoprotein receptor (LSR)/angulin-1 and thus should impair specific localization of tricellulin to tTJs. Conclusions: Tricellulin is a substrate of caspases and its cleavage in consequence contributes to the dissolution of tTJs during apoptosis.

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Stage-specific Localization and Expression of c-kit in the Adult Human Testis

Journal of Histochemistry & Cytochemistry ◽

10.1369/jhc.2009.953737 ◽

2009 ◽

Vol 57 (9) ◽

pp. 861-869 ◽

Cited By ~ 41

Author(s):

Sreepoorna K. Unni ◽

Deepak N. Modi ◽

Shilpa G. Pathak ◽

Jayesh V. Dhabalia ◽

Deepa Bhartiya

Keyword(s):

Current Knowledge ◽

Protein Localization ◽

Immunohistochemical Analysis ◽

Human Testis ◽

Specific Expression ◽

Adult Human ◽

Kit Receptor ◽

Specific Localization ◽

Human Spermatogenesis ◽

And Function

The c-kit receptor (KIT) and its ligand, stem cell factor (SCF), represent one of the key regulators of testicular formation, development, and function and have been extensively studied in various animal models. The present study was undertaken to characterize the pattern of localization and expression of c-kit in normal adult human testis. Immunohistochemical analysis showed that KIT is expressed in the cytoplasm of spermatogonia, acrosomal granules of spermatids, and Leydig cells. Interestingly, a rather heterogenous pattern of expression of the protein along the basement membrane was observed. Intense protein localization in spermatogonia was detected in stages I–III, whereas low expression was observed in stages IV–VI of the seminiferous epithelium, indicating that the expression of the molecule was stage specific. In situ hybridization studies revealed that the transcripts of the gene were also localized in a similar non-uniform pattern. To the best of our knowledge, such a stage-specific expression of KIT has not been reported previously in the human testis. The results of the present study may expand current knowledge about the c-kit/SCF system in human spermatogenesis.

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A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation

Science ◽

10.1126/science.aaw4912 ◽

2019 ◽

Vol 365 (6448) ◽

pp. eaaw4912 ◽

Cited By ~ 27

Author(s):

Richard T. Timms ◽

Zhiqian Zhang ◽

David Y. Rhee ◽

J. Wade Harper ◽

Itay Koren ◽

...

Keyword(s):

Quality Control ◽

Protein Stability ◽

Protein Degradation ◽

E3 Ligase ◽

Cleavage Sites ◽

Caspase Cleavage ◽

E3 Ligases ◽

Ring E3 Ligase ◽

The Stability ◽

Ring E3

The N-terminal residue influences protein stability through N-degron pathways. We used stability profiling of the human N-terminome to uncover multiple additional features of N-degron pathways. In addition to uncovering extended specificities of UBR E3 ligases, we characterized two related Cullin-RING E3 ligase complexes, Cul2ZYG11B and Cul2ZER1, that act redundantly to target N-terminal glycine. N-terminal glycine degrons are depleted at native N-termini but strongly enriched at caspase cleavage sites, suggesting roles for the substrate adaptors ZYG11B and ZER1 in protein degradation during apoptosis. Furthermore, ZYG11B and ZER1 were found to participate in the quality control of N-myristoylated proteins, in which N-terminal glycine degrons are conditionally exposed after a failure of N-myristoylation. Thus, an additional N-degron pathway specific for glycine regulates the stability of metazoan proteomes.

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Increased seed cytokinin levels in transgenic tobacco influence embryo and seedling development

Functional Plant Biology ◽

10.1071/pp01211 ◽

2002 ◽

Vol 29 (9) ◽

pp. 1107 ◽

Cited By ~ 19

Author(s):

Qing-Hu Ma ◽

Zhan-Bing Lin ◽

De-Zhi Fu

Keyword(s):

Transgenic Tobacco ◽

Dry Weight ◽

Polymerase Chain Reaction Analysis ◽

Single Copy ◽

Ipt Gene ◽

Specific Expression ◽

Tobacco Seeds ◽

Cell Layers ◽

Polymerase Chain ◽

Transgenic Embryos

Seed-specific expression of a heterologous vicilin-isopentenyl transferase (ipt) gene has been determined in transgenic tobacco lines 7 and 16. Genetic analysis of self-fertilized progeny showed that a single copy of the vicilin-ipt gene had been integrated, and T2 progeny had become homozygous for the transgene. Stable inheritance of the vicilin-ipt gene in T3 progeny was verified by polymerase chain reaction analysis. Seed cytokinin levels were 2-3-fold higher than control levels. Cytological analyses revealed a significant increase in the number of plerome cell layers and enlargement of diameter in transgenic embryos compared with controls. Dry weight of mature seeds and subsequent seedling growth was increased significantly. This correlated with the level of vicilin-ipt overexpression and increased cytokinin levels in transgenic tobacco seeds. The results suggest a crucial role for cytokinins in regulation of tobacco embryo development.

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Death Receptor-induced Apoptosis Reveals a Novel Interplay between the Chromosomal Passenger Complex and CENP-C during Interphase

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0409 ◽

2007 ◽

Vol 18 (4) ◽

pp. 1337-1347 ◽

Cited By ~ 12

Author(s):

Alison J. Faragher ◽

Xiao-Ming Sun ◽

Michael Butterworth ◽

Nick Harper ◽

Mike Mulheran ◽

...

Keyword(s):

Death Receptor ◽

Caspase 8 ◽

Cleavage Sites ◽

Caspase Activation ◽

Functional Link ◽

Chromosomal Passenger Complex ◽

Caspase Cleavage ◽

Chromosomal Passenger ◽

Caspase 7 ◽

Induced Apoptosis

Despite the fact that the chromosomal passenger complex is well known to regulate kinetochore behavior in mitosis, no functional link has yet been established between the complex and kinetochore structure. In addition, remarkably little is known about how the complex targets to centromeres. Here, in a study of caspase-8 activation during death receptor-induced apoptosis in MCF-7 cells, we have found that cleaved caspase-8 rapidly translocates to the nucleus and that this translocation is correlated with loss of the centromere protein (CENP)-C, resulting in extensive disruption of centromeres. Caspase-8 activates cytoplasmic caspase-7, which is likely to be the primary caspase responsible for cleavage of CENP-C and INCENP, a key chromosomal passenger protein. Caspase-mediated cleavage of CENP-C and INCENP results in their mislocalization and the subsequent mislocalization of Aurora B kinase. Our results demonstrate that the chromosomal passenger complex is displaced from centromeres as a result of caspase activation. Furthermore, mutation of the primary caspase cleavage sites of INCENP and CENP-C and expression of noncleavable CENP-C or INCENP prevent the mislocalization of the passenger complex after caspase activation. Our studies provide the first evidence for a functional interplay between the passenger complex and CENP-C.

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Molecular characterization and expression of the stratification-related cytokeratins 4 and 15.

The Journal of Cell Biology ◽

10.1083/jcb.106.4.1249 ◽

1988 ◽

Vol 106 (4) ◽

pp. 1249-1261 ◽

Cited By ~ 98

Author(s):

R E Leube ◽

B L Bader ◽

F X Bosch ◽

R Zimbelmann ◽

T Achtstaetter ◽

...

Keyword(s):

Basal Cell ◽

Blot Hybridization ◽

Northern Blot Hybridization ◽

Type I ◽

Specific Expression ◽

Genes Encoding ◽

Cell Layers ◽

Hybridization In Situ ◽

Stratified Epithelia

A number of human cytokeratins are expressed during the development of stratified epithelia from one-layered polar epithelia and continue to be expressed in several adult epithelial tissues. For studies of the regulation of the synthesis of stratification-related cytokeratins in internal tissues, we have prepared cDNA and genomic clones encoding cytokeratin 4, as a representative of the basic (type II) cytokeratin subfamily and cytokeratin 15, as representative of the acidic (type I) subfamily, and determined their nucleotide sequences. The specific expression of mRNAs encoding these two polypeptides in certain stratified tissues and cultured cell lines is demonstrated by Northern blot hybridization. Hybridization in situ with antisense riboprobes and/or synthetic oligonucleotides shows the presence of cytokeratin 15 mRNA in all layers of esophagus, whereas cytokeratin 4 mRNA tends to be suprabasally enriched, although to degrees varying in different regions. We conclude that the expression of the genes encoding these stratification-related cytokeratins starts already in the basal cell layer and does not depend on vertical differentiation and detachment from the basal lamina. Our results also show that simple epithelial and stratification-related cytokeratins can be coexpressed in basal cell layers of certain stratified epithelia such as esophagus. Implications of these findings for epithelial differentiation and the formation of squamous cell carcinomas are discussed.

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Molecular properties of cardiac tail-anchored membrane protein SLMAP are consistent with structural role in arrangement of excitation-contraction coupling apparatus

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01015.2004 ◽

2005 ◽

Vol 288 (4) ◽

pp. H1810-H1819 ◽

Cited By ~ 18

Author(s):

Rosa M. Guzzo ◽

Maysoon Salih ◽

Edwin D. Moore ◽

Balwant S. Tuana

Keyword(s):

Potential Role ◽

Cardiac Myocyte ◽

Interaction Analysis ◽

Coiled Coil ◽

Spatial Arrangement ◽

Coupling Mechanism ◽

Developmentally Regulated ◽

Specific Expression ◽

Molecular Determinants ◽

Effective Excitation

The spatial arrangement of the cell-surface membranes (sarcolemma and transverse tubules) and internal membranes of the sarcoplasmic reticulum relative to the myofibril is critical for effective excitation-contraction (E-C) coupling in cardiac myocytes; however, the molecular determinants of this order remain to be defined. Here, we ascribe molecular and cellular properties to the coiled-coil, tail-anchored sarcolemmal membrane-associated protein (SLMAP) that are consistent with a potential role in organizing the E-C coupling apparatus of the cardiomyocyte. The expression of SLMAP was developmentally regulated and its localization was distinctly apparent at the level of the membranes involved in regulating the E-C coupling mechanism. Several SLMAP isoforms were expressed in the cardiac myocyte with unique COOH-terminal membrane anchors that could target this molecule to distinct subcellular membranes. Protein interaction analysis indicated that SLMAPs could self assemble and bind myosin in cardiac muscle. The cardiac-specific expression of SLMAP isoforms that can be targeted to distinct subcellular membranes, self assemble, and interact with the myofibril suggests a potential role for this molecule in the structural arrangement of the E-C coupling apparatus.

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A Novel Form of DAP5 Protein Accumulates in Apoptotic Cells as a Result of Caspase Cleavage and Internal Ribosome Entry Site-Mediated Translation

Molecular and Cellular Biology ◽

10.1128/mcb.20.2.496-506.2000 ◽

2000 ◽

Vol 20 (2) ◽

pp. 496-506 ◽

Cited By ~ 118

Author(s):

Sivan Henis-Korenblit ◽

Naomi Levy Strumpf ◽

Dan Goldstaub ◽

Adi Kimchi

Keyword(s):

Internal Ribosome Entry Site ◽

Dominant Negative ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Apoptotic Cells ◽

Entry Site ◽

Translation Rate ◽

Internal Ribosome Entry ◽

Caspase Cleavage ◽

Induced Apoptosis

ABSTRACT Death-associated protein 5 (DAP5) (also named p97 and NAT1) is a member of the translation initiation factor 4G (eIF4G) family that lacks the eIF4E binding site. It was previously implicated in apoptosis, based on the finding that a dominant negative fragment of the protein protected against cell death. Here we address its function and two distinct levels of regulation during apoptosis that affect the protein both at translational and posttranslational levels. DAP5 protein was found to be cleaved at a single caspase cleavage site at position 790, in response to activated Fas or p53, yielding a C-terminal truncated protein of 86 kDa that is capable of generating complexes with eIF4A and eIF3. Interestingly, while the overall translation rate in apoptotic cells was reduced by 60 to 70%, in accordance with the simultaneous degradation of the two major mediators of cap-dependent translation, eIF4GI and eIF4GII, the translation rate of DAP5 protein was selectively maintained. An internal ribosome entry site (IRES) element capable of directing the translation of a reporter gene when subcloned into a bicistronic vector was identified in the 5′ untranslated region of DAP5 mRNA. While cap-dependent translation from this transfected vector was reduced during Fas-induced apoptosis, the translation via the DAP5 IRES was selectively maintained. Addition of recombinant DAP5/p97 or DAP5/p86 to cell-free systems enhanced preferentially the translation through the DAP5 IRES, whereas neutralization of the endogenous DAP5 in reticulocyte lysates by adding a dominant negative DAP5 fragment interfered with this translation. The DAP5/p86 apoptotic form was more potent than DAP5/p97 in these functional assays. Altogether, the data suggest that DAP5 is a caspase-activated translation factor which mediates cap-independent translation at least from its own IRES, thus generating a positive feedback loop responsible for the continuous translation of DAP5 during apoptosis.

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Caspase-resistant Golgin-160 Disrupts Apoptosis Induced by Secretory Pathway Stress and Ligation of Death Receptors

Molecular Biology of the Cell ◽

10.1091/mbc.e04-11-0971 ◽

2005 ◽

Vol 16 (6) ◽

pp. 3019-3027 ◽

Cited By ~ 47

Author(s):

Rebecca S. Maag ◽

Marie Mancini ◽

Antony Rosen ◽

Carolyn E. Machamer

Keyword(s):

Er Stress ◽

Cell Lines ◽

Golgi Complex ◽

Secretory Pathway ◽

Coiled Coil ◽

Brefeldin A ◽

Resistant Mutant ◽

Death Receptors ◽

Drug Induced ◽

Caspase Cleavage

Golgin-160 is a coiled-coil protein on the cytoplasmic face of the Golgi complex that is cleaved by caspases during apoptosis. We assessed the sensitivity of cell lines stably expressing wild-type or caspase-resistant golgin-160 to several proapoptotic stimuli. Cells expressing a caspase-resistant mutant of golgin-160 were strikingly resistant to apoptosis induced by ligation of death receptors and by drugs that induce endoplasmic reticulum (ER) stress, including brefeldin-A, dithiothreitol, and thapsigargin. However, both cell lines responded similarly to other proapoptotic stimuli, including staurosporine, anisomycin, and etoposide. The caspase-resistant golgin-160 dominantly prevented cleavage of endogenous golgin-160 after ligation of death receptors or induction of ER stress, which could be explained by a failure of initiator caspase activation. The block in apoptosis in cells expressing caspase-resistant golgin-160 could not be bypassed by expression of potential caspase cleavage fragments of golgin-160, or by drug-induced disassembly of the Golgi complex. Our results suggest that some apoptotic signals (including those initiated by death receptors and ER stress) are sensed and integrated at Golgi membranes and that golgin-160 plays an important role in transduction of these signals.

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