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1.
  • Abudu, YP, et al. (author)
  • SAMM50 acts with p62 in piecemeal basal- and OXPHOS-induced mitophagy of SAM and MICOS components
  • 2021
  • In: The Journal of cell biology. - : Rockefeller University Press. - 1540-8140 .- 0021-9525. ; 220:8
  • Journal article (peer-reviewed)abstract
    • Mitophagy is the degradation of surplus or damaged mitochondria by autophagy. In addition to programmed and stress-induced mitophagy, basal mitophagy processes exert organelle quality control. Here, we show that the sorting and assembly machinery (SAM) complex protein SAMM50 interacts directly with ATG8 family proteins and p62/SQSTM1 to act as a receptor for a basal mitophagy of components of the SAM and mitochondrial contact site and cristae organizing system (MICOS) complexes. SAMM50 regulates mitochondrial architecture by controlling formation and assembly of the MICOS complex decisive for normal cristae morphology and exerts quality control of MICOS components. To this end, SAMM50 recruits ATG8 family proteins through a canonical LIR motif and interacts with p62/SQSTM1 to mediate basal mitophagy of SAM and MICOS components. Upon metabolic switch to oxidative phosphorylation, SAMM50 and p62 cooperate to mediate efficient mitophagy.
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2.
  • Acuna, LIG, et al. (author)
  • Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator
  • 2020
  • In: The Journal of cell biology. - : Rockefeller University Press. - 1540-8140 .- 0021-9525. ; 219:9
  • Journal article (peer-reviewed)abstract
    • In mammals, argonaute (AGO) proteins have been characterized for their roles in small RNA–mediated posttranscriptional and also in transcriptional gene silencing. Here, we report a different role for AGO1 in estradiol-triggered transcriptional activation in human cells. We show that in MCF-7 mammary gland cells, AGO1 associates with transcriptional enhancers of estrogen receptor α (ERα) and that this association is up-regulated by treating the cells with estrogen (E2), displaying a positive correlation with the activation of these enhancers. Moreover, we show that AGO1 interacts with ERα and that this interaction is also increased by E2 treatment, but occurs in the absence of RNA. We show that AGO1 acts positively as a coactivator in estradiol-triggered transcription regulation by promoting ERα binding to its enhancers. Consistently, AGO1 depletion decreases long-range contacts between ERα enhancers and their target promoters. Our results point to a role of AGO1 in transcriptional regulation in human cells that is independent from small RNA binding.
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3.
  • Adelfalk, C, et al. (author)
  • Cohesin SMC1beta protects telomeres in meiocytes
  • 2009
  • In: The Journal of cell biology. - : Rockefeller University Press. - 1540-8140 .- 0021-9525. ; 187:2, s. 185-199
  • Journal article (peer-reviewed)abstract
    • Meiosis-specific mammalian cohesin SMC1β is required for complete sister chromatid cohesion and proper axes/loop structure of axial elements (AEs) and synaptonemal complexes (SCs). During prophase I, telomeres attach to the nuclear envelope (NE), but in Smc1β−/− meiocytes, one fifth of their telomeres fail to attach. This study reveals that SMC1β serves a specific role at telomeres, which is independent of its role in determining AE/SC length and loop extension. SMC1β is necessary to prevent telomere shortening, and SMC3, present in all known cohesin complexes, properly localizes to telomeres only if SMC1β is present. Very prominently, telomeres in Smc1β−/− spermatocytes and oocytes loose their structural integrity and suffer a range of abnormalities. These include disconnection from SCs and formation of large telomeric protein–DNA extensions, extended telomere bridges between SCs, ring-like chromosomes, intrachromosomal telomeric repeats, and a reduction of SUN1 foci in the NE. We suggest that a telomere structure protected from DNA rearrangements depends on SMC1β.
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4.
  • Adhikari, Deepak, et al. (author)
  • Mastl is required for timely activation of APC/C in meiosis I and Cdk1 reactivation in meiosis II.
  • 2014
  • In: The Journal of cell biology. - : Rockefeller University Press. - 1540-8140 .- 0021-9525. ; 206:7, s. 843-853
  • Journal article (peer-reviewed)abstract
    • In mitosis, the Greatwall kinase (called microtubule-associated serine/threonine kinase like [Mastl] in mammals) is essential for prometaphase entry or progression by suppressing protein phosphatase 2A (PP2A) activity. PP2A suppression in turn leads to high levels of Cdk1 substrate phosphorylation. We have used a mouse model with an oocyte-specific deletion of Mastl to show that Mastl-null oocytes resume meiosis I and reach metaphase I normally but that the onset and completion of anaphase I are delayed. Moreover, after the completion of meiosis I, Mastl-null oocytes failed to enter meiosis II (MII) because they reassembled a nuclear structure containing decondensed chromatin. Our results show that Mastl is required for the timely activation of anaphase-promoting complex/cyclosome to allow meiosis I exit and for the rapid rise of Cdk1 activity that is needed for the entry into MII in mouse oocytes.
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5.
  • Aguilera, Anabella, et al. (author)
  • C-ferroptosis is an iron-dependent form of regulated cell death in cyanobacteria
  • 2021
  • In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 221:2
  • Journal article (peer-reviewed)abstract
    • Ferroptosis is an oxidative and iron-dependent form of regulated cell death (RCD) recently described in eukaryotic organisms like animals, plants, and parasites. Here, we report that a similar process takes place in the photosynthetic prokaryote Synechocystis sp. PCC 6803 in response to heat stress. After a heat shock, Synechocystis sp. PCC 6803 cells undergo a cell death pathway that can be suppressed by the canonical ferroptosis inhibitors, CPX, vitamin E, Fer-1, liproxstatin-1, glutathione (GSH), or ascorbic acid (AsA). Moreover, as described for eukaryotic ferroptosis, this pathway is characterized by an early depletion of the antioxidants GSH and AsA, and by lipid peroxidation. These results indicate that all of the hallmarks described for eukaryotic ferroptosis are conserved in photosynthetic prokaryotes and suggest that ferroptosis might be an ancient cell death program.
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6.
  • Athie, Alejandro, et al. (author)
  • Analysis of copy number alterations reveals the lncRNA ALAL-1 as a regulator of lung cancer immune evasion
  • 2020
  • In: The Journal of cell biology. - : Rockefeller University Press. - 1540-8140 .- 0021-9525. ; 219:9
  • Journal article (peer-reviewed)abstract
    • Cancer is characterized by genomic instability leading to deletion or amplification of oncogenes or tumor suppressors. However, most of the altered regions are devoid of known cancer drivers. Here, we identify lncRNAs frequently lost or amplified in cancer. Among them, we found amplified lncRNA associated with lung cancer-1 (ALAL-1) as frequently amplified in lung adenocarcinomas. ALAL-1 is also overexpressed in additional tumor types, such as lung squamous carcinoma. The RNA product of ALAL-1 is able to promote the proliferation and tumorigenicity of lung cancer cells. ALAL-1 is a TNFα- and NF-κB-induced cytoplasmic lncRNA that specifically interacts with SART3, regulating the subcellular localization of the protein deubiquitinase USP4 and, in turn, its function in the cell. Interestingly, ALAL-1 expression inversely correlates with the immune infiltration of lung squamous tumors, while tumors with ALAL-1 amplification show lower infiltration of several types of immune cells. We have thus unveiled a pro-oncogenic lncRNA that mediates cancer immune evasion, pointing to a new target for immune potentiation.
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7.
  • Aucott, Rebecca, et al. (author)
  • HP1-beta is required for development of the cerebral neocortex and neuromuscular junctions
  • 2008
  • In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 183:4, s. 597-606
  • Journal article (peer-reviewed)abstract
    • HP1 proteins are thought to be modulators of chromatin organization in all mammals, yet their exact physiological function remains unknown. In a first attempt to elucidate the function of these proteins in vivo, we disrupted the murine Cbx1 gene, which encodes the HP1-beta isotype, and show that the Cbx1(-/-) null mutation leads to perinatal lethality. The newborn mice succumbed to acute respiratory failure, whose likely cause is the defective development of neuromuscular junctions within the endplate of the diaphragm. We also observe aberrant cerebral cortex development in Cbx1(-/-) mutant brains, which have reduced proliferation of neuronal precursors, widespread cell death, and edema. In vitro cultures of neurospheres from Cbx1(-/-) mutant brains reveal a dramatic genomic instability. Our results demonstrate that HP1 proteins are not functionally redundant and that they are likely to regulate lineage-specific changes in heterochromatin organization.
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8.
  • Baldanzi, Gianluca, et al. (author)
  • Ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI 3-kinase/AKT.
  • 2002
  • In: The Journal of cell biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 159:6, s. 1029-37
  • Journal article (peer-reviewed)abstract
    • Ghrelin is an acyl-peptide gastric hormone acting on the pituitary and hypothalamus to stimulate growth hormone (GH) release, adiposity, and appetite. Ghrelin endocrine activities are entirely dependent on its acylation and are mediated by GH secretagogue (GHS) receptor (GHSR)-1a, a G protein-coupled receptor mostly expressed in the pituitary and hypothalamus, previously identified as the receptor for a group of synthetic molecules featuring GH secretagogue (GHS) activity. Des-acyl ghrelin, which is far more abundant than ghrelin, does not bind GHSR-1a, is devoid of any endocrine activity, and its function is currently unknown. Ghrelin, which is expressed in heart, albeit at a much lower level than in the stomach, also exerts a cardio protective effect through an unknown mechanism, independent of GH release. Here we show that both ghrelin and des-acyl ghrelin inhibit apoptosis of primary adult and H9c2 cardiomyocytes and endothelial cells in vitro through activation of extracellular signal-regulated kinase-1/2 and Akt serine kinases. In addition, ghrelin and des-acyl ghrelin recognize common high affinity binding sites on H9c2 cardiomyocytes, which do not express GHSR-1a. Finally, both MK-0677 and hexarelin, a nonpeptidyl and a peptidyl synthetic GHS, respectively, recognize the common ghrelin and des-acyl ghrelin binding sites, inhibit cell death, and activate MAPK and Akt.These findings provide the first evidence that, independent of its acylation, ghrelin gene product may act as a survival factor directly on the cardiovascular system through binding to a novel, yet to be identified receptor, which is distinct from GHSR-1a.
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9.
  • Bao, WJ, et al. (author)
  • Integrin alphav-mediated inactivation of p53 controls a MEK1-dependent melanoma cell survival pathway in three-dimensional collagen
  • 2004
  • In: The Journal of cell biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 167:4, s. 745-756
  • Journal article (peer-reviewed)abstract
    • Integrin αv is required for melanoma cell survival and tumor growth in various models. To elucidate integrin αv-mediated melanoma cell survival mechanisms, we used a three-dimensional (3D) collagen gel model mimicking the pathophysiological microenvironment of malignant melanoma in the dermis. We found that integrin αv inactivated p53 and that suppression of p53 activity by dominant negative p53 or p53-small interfering RNA obviated the need for integrin αv for melanoma cell survival in 3D-collagen and for tumor growth in vivo. This indicates that integrin αv-mediated inactivation of p53 functionally controls melanoma cell survival. Furthermore, we found that melanoma cell integrin αv was required for MAPK kinase (MEK) 1 and extracellular signal-regulated kinase (ERK)1/2 activity in 3D-collagen, whereas inhibition of MEK1 activity induced apoptosis. Surprisingly, MEK1 and ERK1/2 activities were restored in integrin αv-negative melanoma cells by suppression of p53, whereas concomitant block of MEK1 induced apoptosis. This suggests that integrin αv controls melanoma cell survival in 3D-collagen through a pathway involving p53 regulation of MEK1 signaling.
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10.
  • BARTH, BU, et al. (author)
  • The oligomerization reaction of the Semliki Forest virus membrane protein subunits
  • 1995
  • In: The Journal of cell biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 128:3, s. 283-291
  • Journal article (peer-reviewed)abstract
    • The Semliki Forest virus (SFV) spike is composed of three copies of a membrane protein heterodimer. The two subunits of this heterodimer (p62 and E1) are synthesized sequentially from a common mRNA together with the capsid (C) in the order C-p62-E1. In this work heterodimerization of the spike proteins has been studied in BHK 21 cells. The results indicate that: (a) the polyprotein is cotranslationally cleaved into individual chains; (b) the two membrane protein subunits are initially not associated with each other in the endoplasmic reticulum (ER); (c) heterodimerization occurs predominantly between subunits that originate from the same translation product (heterodimerization in cis); (d) the kinetics of subunit association are very fast (t1/2 = 4 min); and (e) this heterodimerization is highly efficient. To explain the cis-directed heterodimerization reaction we suggest that the p62 protein, which is made before E1 during 26S mRNA translation, is retained at its translocation site until also the E1 chain has been synthesized and translocated at this same site. The mechanism for p62 retention could either be that the p62 anchor sequence cannot diffuse out from an "active" translocation site or that the p62 protein is complexed with a protein folding facilitating machinery that is physically linked to the translocation apparatus.
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11.
  • Bauren, G, et al. (author)
  • Demonstration of a dynamic, transcription-dependent organization of pre-mRNA splicing factors in polytene nuclei
  • 1996
  • In: The Journal of cell biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 133:5, s. 929-941
  • Journal article (peer-reviewed)abstract
    • We describe the dynamic organization of pre-mRNA splicing factors in the intact polytene nuclei of the dipteran Chironomus tentans. The snRNPs and an SR non-snRNP splicing factor are present in excess, mainly distributed throughout the interchromatin. Approximately 10% of the U2 snRNP and an SR non-snRNP splicing factor are associated with the chromosomes, highly enriched in active gene loci where they are bound to RNA. We demonstrate that the splicing factors are specifically recruited to a defined gene upon induction of transcription during physiological conditions. Concomitantly, the splicing factors leave gene loci in which transcription is turned off. We also demonstrated that upon general transcription inhibition, the splicing factors redistribute from active gene loci to the interchromatin. Our findings demonstrate the dynamic intranuclear organization of splicing factors and a tight linkage between transcription and the intranuclear organization of the splicing machinery.
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12.
  • Beck, Halfdan, et al. (author)
  • Regulators of cyclin-dependent kinases are crucial for maintaining genome integrity in S phase
  • 2010
  • In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 188:5, s. 629-638
  • Journal article (peer-reviewed)abstract
    • Maintenance of genome integrity is of critical importance to cells. To identify key regulators of genomic integrity, we screened a human cell line with a kinome small interfering RNA library. WEE1, a major regulator of mitotic entry, and CHK1 were among the genes identified. Both kinases are important negative regulators of CDK1 and -2. Strikingly, WEE1 depletion rapidly induced DNA damage in S phase in newly replicated DNA, which was accompanied by a marked increase in single-stranded DNA. This DNA damage is dependent on CDK1 and -2 as well as the replication proteins MCM2 and CDT1 but not CDC25A. Conversely, DNA damage after CHK1 inhibition is highly dependent on CDC25A. Furthermore, the inferior proliferation of CHK1-depleted cells is improved substantially by codepletion of CDC25A. We conclude that the mitotic kinase WEE1 and CHK1 jointly maintain balanced cellular control of Cdk activity during normal DNA replication, which is crucial to prevent the generation of harmful DNA lesions during replication.
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13.
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14.
  • Benz, PM, et al. (author)
  • Cytoskeleton assembly at endothelial cell-cell contacts is regulated by alphaII-spectrin-VASP complexes
  • 2008
  • In: The Journal of cell biology. - : Rockefeller University Press. - 1540-8140 .- 0021-9525. ; 180:1, s. 205-219
  • Journal article (peer-reviewed)abstract
    • Directed cortical actin assembly is the driving force for intercellular adhesion. Regulated by phosphorylation, vasodilator-stimulated phosphoprotein (VASP) participates in actin fiber formation. We screened for endothelial proteins, which bind to VASP, dependent on its phosphorylation status. Differential proteomics identified αII-spectrin as such a VASP-interacting protein. αII-Spectrin binds to the VASP triple GP5-motif via its SH3 domain. cAMP-dependent protein kinase–mediated VASP phosphorylation at Ser157 inhibits αII-spectrin–VASP binding. VASP is dephosphorylated upon formation of cell–cell contacts and in confluent, but not in sparse cells, αII-spectrin colocalizes with nonphosphorylated VASP at cell–cell junctions. Ectopic expression of the αII-spectrin SH3 domain at cell–cell contacts translocates VASP, initiates cortical actin cytoskeleton formation, stabilizes cell–cell contacts, and decreases endothelial permeability. Conversely, the permeability of VASP-deficient endothelial cells (ECs) and microvessels of VASP-null mice increases. Reconstitution of VASP-deficient ECs rescues barrier function, whereas αII-spectrin binding-deficient VASP mutants fail to restore elevated permeability. We propose that αII-spectrin–VASP complexes regulate cortical actin cytoskeleton assembly with implications for vascular permeability.
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15.
  • Bergink, S, et al. (author)
  • Recognition of DNA damage by XPC coincides with disruption of the XPC-RAD23 complex
  • 2012
  • In: The Journal of cell biology. - : Rockefeller University Press. - 1540-8140 .- 0021-9525. ; 196:6, s. 681-688
  • Journal article (peer-reviewed)abstract
    • The recognition of helix-distorting deoxyribonucleic acid (DNA) lesions by the global genome nucleotide excision repair subpathway is performed by the XPC–RAD23–CEN2 complex. Although it has been established that Rad23 homologs are essential to protect XPC from proteasomal degradation, it is unclear whether RAD23 proteins have a direct role in the recognition of DNA damage. In this paper, we show that the association of XPC with ultraviolet-induced lesions was impaired in the absence of RAD23 proteins. Furthermore, we show that RAD23 proteins rapidly dissociated from XPC upon binding to damaged DNA. Our data suggest that RAD23 proteins facilitate lesion recognition by XPC but do not participate in the downstream DNA repair process.
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16.
  • Björk, Petra, et al. (author)
  • Intranuclear binding in space and time of exon junction complex and NXF1 to premRNPs/mRNPs in vivo
  • 2015
  • In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 211:1, s. 63-75
  • Journal article (peer-reviewed)abstract
    • Eukaryotic gene expression requires the ordered association of numerous factors with precursor messenger RNAs (premRNAs)/messenger RNAs (mRNAs) to achieve efficiency and regulation. Here, we use the Balbiani ring (BR) genes to demonstrate the temporal and spatial association of the exon junction complex (EJC) core with gene-specific endogenous premRNAs and mRNAs. The EJC core components bind cotranscriptionally to BR premRNAs during or very rapidly after splicing. The EJC core does not recruit the nonsense-mediated decay mediaters UPF2 and UPF3 until the BR messenger RNA protein complexes (mRNPs) enter the interchromatin. Even though several known adapters for the export factor NXF1 become part of BR mRNPs already at the gene, NXF1 binds to BR mRNPs only in the interchromatin. In steady state, a subset of the BR mRNPs in the interchromatin binds NXF1, UPF2, and UPF3. This binding appears to occur stochastically, and the efficiency approximately equals synthesis and export of the BR mRNPs. Our data provide unique in vivo information on how export competent eukaryotic mRNPs are formed.
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17.
  • Björk, Petra, et al. (author)
  • Specific combinations of SR proteins associate with single pre-messenger RNAs in vivo and contribute different functions
  • 2009
  • In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 184:4, s. 555-568
  • Journal article (peer-reviewed)abstract
    • Serine/arginine-rich (SR) proteins are required for messenger RNA (mRNA) processing, export, surveillance, and translation. We show that in Chironomus tentans, nascent transcripts associate with multiple types of SR proteins in specific combinations. Alternative splicing factor (ASF)/SF2, SC35, 9G8, and hrp45/SRp55 are all present in Balbiani ring (BR) pre-messenger ribonucleoproteins (mRNPs) preferentially when introns appear in the pre-mRNA and when cotranscriptional splicing takes place. However, hrp45/SRp55 is distributed differently in the pre-mRNPs along the gene compared with ASF/SF2, SC35, and 9G8, suggesting functional differences. All four SR proteins are associated with the BR mRNPs during export to the cytoplasm. Interference with SC35 indicates that SC35 is important for the coordination of splicing, transcription, and 3' end processing and also for nucleocytoplasmic export. ASF/SF2 is associated with polyribosomes, whereas SC35, 9G8, and hrp45/SRp55 cosediment with mono-ribosomes. Thus, individual endogenous pre-mRNPs/mRNPs bind multiple types of SR proteins during transcription, and these SR proteins accompany the mRNA and play different roles during the gene expression pathway in vivo.
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18.
  • Blokzijl, A, et al. (author)
  • Cross-talk between the Notch and TGF-beta signaling pathways mediated by interaction of the Notch intracellular domain with Smad3
  • 2003
  • In: The Journal of cell biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 163:4, s. 723-728
  • Journal article (peer-reviewed)abstract
    • The Notch and transforming growth factor-β (TGF-β) signaling pathways play critical roles in the control of cell fate during metazoan development. However, mechanisms of cross-talk and signal integration between the two systems are unknown. Here, we demonstrate a functional synergism between Notch and TGF-β signaling in the regulation of Hes-1, a direct target of the Notch pathway. Activation of TGF-β signaling up-regulated Hes-1 expression in vitro and in vivo. This effect was abrogated in myogenic cells by a dominant-negative form of CSL, an essential DNA-binding component of the Notch pathway. TGF-β regulated transcription from the Hes-1 promoter in a Notch-dependent manner, and the intracellular domain of Notch1 (NICD) cooperated synergistically with Smad3, an intracellular transducer of TGF-β signals, to induce the activation of synthetic promoters containing multimerized CSL- or Smad3-binding sites. NICD and Smad3 were shown to interact directly, both in vitro and in cells, in a ligand-dependent manner, and Smad3 could be recruited to CSL-binding sites on DNA in the presence of CSL and NICD. These findings indicate that Notch and TGF-β signals are integrated by direct protein–protein interactions between the signal-transducing intracellular elements from both pathways.
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19.
  • Bloom, O, et al. (author)
  • Colocalization of synapsin and actin during synaptic vesicle recycling
  • 2003
  • In: The Journal of cell biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 161:4, s. 737-747
  • Journal article (peer-reviewed)abstract
    • It has been hypothesized that in the mature nerve terminal, interactions between synapsin and actin regulate the clustering of synaptic vesicles and the availability of vesicles for release during synaptic activity. Here, we have used immunogold electron microscopy to examine the subcellular localization of actin and synapsin in the giant synapse in lamprey at different states of synaptic activity. In agreement with earlier observations, in synapses at rest, synapsin immunoreactivity was preferentially localized to a portion of the vesicle cluster distal to the active zone. During synaptic activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known function in clustering of vesicles in the reserve pool, synapsin migrates from the synaptic vesicle cluster and participates in the organization of the actin-rich cytomatrix in the endocytic zone during synaptic activity.
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20.
  • Boban, M, et al. (author)
  • Asi1 is an inner nuclear membrane protein that restricts promoter access of two latent transcription factors
  • 2006
  • In: The Journal of cell biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 173:5, s. 695-707
  • Journal article (peer-reviewed)abstract
    • Stp1 and Stp2 are homologous transcription factors in yeast that are synthesized as latent cytoplasmic precursors with NH2-terminal regulatory domains. In response to extracellular amino acids, the plasma membrane–localized Ssy1–Ptr3–Ssy5 (SPS) sensor endoproteolytically processes Stp1 and Stp2, an event that releases the regulatory domains. The processed forms of Stp1 and Stp2 efficiently target to the nucleus and bind promoters of amino acid permease genes. In this study, we report that Asi1 is an integral component of the inner nuclear membrane that maintains the latent characteristics of unprocessed Stp1 and Stp2. In cells lacking Asi1, full-length forms of Stp1 and Stp2 constitutively induce SPS sensor–regulated genes. The regulatory domains of Stp1 and Stp2 contain a conserved motif that confers Asi1-mediated control when fused to an unrelated DNA-binding protein. Our results indicate that latent precursor forms of Stp1 and Stp2 inefficiently enter the nucleus; however, once there, Asi1 restricts them from binding SPS sensor–regulated promoters. These findings reveal an unanticipated role of inner nuclear membrane proteins in controlling gene expression.
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21.
  • Bridge, Eileen, et al. (author)
  • Spliced exons of adenovirus late RNAs colocalize with snRNP in a specific nuclear domain
  • 1996
  • In: Journal of Cell Biology. - 0021-9525 .- 1540-8140. ; 135:2, s. 303-314
  • Journal article (peer-reviewed)abstract
    • Posttranscriptional steps in the production of mRNA include well characterized polyadenylation and splicing reactions, but it is also necessary to understand how RNA is transported within the nucleus from the site of its transcription to the nuclear pore, where it is translocated to the cytoplasmic compartment. Determining the localization of RNA within the nucleus is an important aspect of understanding RNA production and may provide clues for investigating the trafficking of RNA within the nucleus and the mechanism for its export to the cytoplasm. We have previously shown that late phase adenovirus-infected cells contain large clusters of snRNP and non-snRNP splicing factors; the presence of these structures is correlated with high levels of viral late gene transcription. The snRNP clusters correspond to enlarged interchromatin granules present in late phase infected cells. Here we show that polyadenylated RNA and spliced tripartite leader exons from the viral major late transcription unit are present in these same late phase snRNP-containing structures. We find that the majority of the steady state viral RNA present in the nucleus is spliced at the tripartite leader exons. Tripartite leader exons are efficiently exported from the nucleus at a time when we detect their accumulation in interchromatin granule clusters. Since the enlarged interchromatin granules contain spliced and polyadenylated RNA, we suggest that viral RNA may accumulate in this late phase structure during an intranuclear step in RNA transport.
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22.
  • Carvalho, T, et al. (author)
  • Targeting of adenovirus E1A and E4-ORF3 proteins to nuclear matrix-associated PML bodies
  • 1995
  • In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 131:1, s. 45-56
  • Journal article (peer-reviewed)abstract
    • The PML protein was first identified as part of a fusion product with the retinoic acid receptor alpha (RAR alpha), resulting from the t(15;17) chromosomal translocation associated with acute promyelocytic leukemia (APL). It has been previously demonstrated that PML, which is tightly bound to the nuclear matrix, concentrates in discrete subnuclear compartments that are disorganized in APL cells due to the expression of the PML-RAR alpha hybrid. Here we report that adenovirus infection causes a drastic redistribution of PML from spherical nuclear bodies into fibrous structures. The product encoded by adenovirus E4-ORF3 is shown to be responsible for this reorganization and to colocalize with PML into these fibers. In addition, we demonstrate that E1A oncoproteins concentrate in the PML domains, both in infected and transiently transfected cells, and that this association requires the conserved amino acid motif (D)LXCXE, common to all viral oncoproteins that bind pRB or the related p107 and p130 proteins. The SV-40 large T antigen, another member of this oncoprotein family is also found in close association with the PML nuclear bodies. Taken together, the present data indicate that the subnuclear domains containing PML represent a preferential target for DNA tumor viruses, and therefore suggest a more general involvement of the PML nuclear bodies in oncogenic processes.
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23.
  • Chapman, G, et al. (author)
  • High levels of Notch signaling down-regulate Numb and Numblike
  • 2006
  • In: The Journal of cell biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 175:4, s. 535-540
  • Journal article (peer-reviewed)abstract
    • Inhibition of Notch signaling by Numb is critical for many cell fate decisions. In this study, we demonstrate a more complex relationship between Notch and the two vertebrate Numb homologues Numb and Numblike. Although Numb and Numblike at low levels of Notch signaling negatively regulated Notch, high levels of Notch signaling conversely led to a reduction of Numb and Numblike protein levels in cultured cells and in the developing chick central nervous system. The Notch intracellular domain but not the canonical Notch downstream proteins Hes 1 and Hey 1 caused a reduction of Numb and Numblike. The Notch-mediated reduction of Numblike required the PEST domain in the Numblike protein and was blocked by the proteasome inhibitor MG132. Collectively, these observations reveal a reciprocal negative regulation between Notch and Numb/Numblike, which may be of relevance for stabilizing asymmetric cell fate switches and for tumor development.
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24.
  • Charpentier, E, et al. (author)
  • Plakoglobin suppresses epithelial proliferation and hair growth in vivo.
  • 2000
  • In: Journal of Cell Biology. - 0021-9525 .- 1540-8140. ; 149:2, s. 503-520
  • Journal article (peer-reviewed)abstract
    • Plakoglobin regulates cell adhesion by providing a modulatable connection between both classical and desmosomal cadherins and their respective cytoskeletal linker proteins. Both plakoglobin and the related protein beta-catenin are posttranscriptionally upregulated in response to Wnt-1 in cultured cells. Upregulation of beta-catenin has been implicated in potentiating hyperproliferation and tumor formation. To investigate the role of plakoglobin in these functions we expressed a full-length (PG) and an NH(2)-terminally truncated form of plakoglobin (DeltaN80PG) in mouse epidermis and hair follicles, tissues which undergo continuous and easily observed postnatal renewal and remodeling. Expression of these constructs results in stunted hair growth, a phenotype that has also been observed in transgenic mice expressing Wnt3 and Dvl2 (Millar et al. 1999). Hair follicles from PG and DeltaN80PG mice show premature termination of the growth phase (anagen) of the hair cycle, an event that is regulated in part by FGF5 (Hebert et al. 1994). The proliferative rate of the epidermal cells was reduced and apoptotic changes, which are associated with entry into the regressive phase of the hair follicle cycle (catagen), occurred earlier than usual.
  •  
25.
  • Chernyavsky, AI, et al. (author)
  • Novel signaling pathways mediating reciprocal control of keratinocyte migration and wound epithelialization through M3 and M4 muscarinic receptors
  • 2004
  • In: The Journal of cell biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 166:2, s. 261-272
  • Journal article (peer-reviewed)abstract
    • To test the hypothesis that keratinocyte (KC) migration is modulated by distinct muscarinic acetylcholine (ACh) receptor subtypes, we inactivated signaling through specific receptors in in vitro and in vivo models of reepithelialization by subtype-selective antagonists, small interfering RNA, and gene knockout in mice. KC migration and wound reepithelialization were facilitated by M4 and inhibited by M3. Additional studies showed that M4 increases expression of “migratory” integrins α5β1, αVβ5, and αVβ6, whereas M3 up-regulates “sedentary” integrins α2β1 and α3β1. Inhibition of migration by M3 was mediated through Ca2+-dependent guanylyl cyclase–cyclic GMP–protein kinase G signaling pathway. The M4 effects resulted from inhibition of the inhibitory pathway involving the adenylyl cyclase–cyclic AMP–protein kinase A pathway. Both signaling pathways intersected at Rho, indicating that Rho kinase provides a common effector for M3 and M4 regulation of cell migration. These findings offer novel insights into the mechanisms of ACh-mediated modulation of KC migration and wound reepithelialization, and may aid the development of novel methods to promote wound healing.
  •  
26.
  • Dacquin, R, et al. (author)
  • Amylin inhibits bone resorption while the calcitonin receptor controls bone formation in vivo
  • 2004
  • In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 164:4, s. 509-514
  • Journal article (peer-reviewed)abstract
    • Amylin is a member of the calcitonin family of hormones cosecreted with insulin by pancreatic beta cells. Cell culture assays suggest that amylin could affect bone formation and bone resorption, this latter function after its binding to the calcitonin receptor (CALCR). Here we show that Amylin inactivation leads to a low bone mass due to an increase in bone resorption, whereas bone formation is unaffected. In vitro, amylin inhibits fusion of mononucleated osteoclast precursors into multinucleated osteoclasts in an ERK1/2-dependent manner. Although Amylin +/- mice like Amylin-deficient mice display a low bone mass phenotype and increased bone resorption, Calcr +/- mice display a high bone mass due to an increase in bone formation. Moreover, compound heterozygote mice for Calcr and Amylin inactivation displayed bone abnormalities observed in both Calcr +/- and Amylin +/- mice, thereby ruling out that amylin uses CALCR to inhibit osteoclastogenesis in vivo. Thus, amylin is a physiological regulator of bone resorption that acts through an unidentified receptor.
  •  
27.
  • Daigle, N, et al. (author)
  • Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells
  • 2001
  • In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 154:1, s. 71-84
  • Journal article (peer-reviewed)abstract
    • The nuclear pore complex (NPC) and its relationship to the nuclear envelope (NE) was characterized in living cells using POM121-green fluorescent protein (GFP) and GFP-Nup153, and GFP-lamin B1. No independent movement of single pore complexes was found within the plane of the NE in interphase. Only large arrays of NPCs moved slowly and synchronously during global changes in nuclear shape, strongly suggesting mechanical connections which form an NPC network. The nuclear lamina exhibited identical movements. NPC turnover measured by fluorescence recovery after photobleaching of POM121 was less than once per cell cycle. Nup153 association with NPCs was dynamic and turnover of this nucleoporin was three orders of magnitude faster. Overexpression of both nucleoporins induced the formation of annulate lamellae (AL) in the endoplasmic reticulum (ER). Turnover of AL pore complexes was much higher than in the NE (once every 2.5 min). During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153) in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase. Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis.
  •  
28.
  • Danen, EHJ, et al. (author)
  • The fibronectin-binding integrins alpha 5 beta 1 and alpha v beta 3 differentially modulate RhoA-GTP loading, organization of cell matrix adhesions, and fibronectin fibrillogenesis
  • 2002
  • In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 159:6, s. 1071-1086
  • Journal article (peer-reviewed)abstract
    • We have studied the formation of different types of cell matrix adhesions in cells that bind to fibronectin via either alpha5beta1 or alphavbeta3. In both cases, cell adhesion to fibronectin leads to a rapid decrease in RhoA activity. However, alpha5beta1 but not alphavbeta3 supports high levels of RhoA activity at later stages of cell spreading, which are associated with a translocation of focal contacts to peripheral cell protrusions, recruitment of tensin into fibrillar adhesions, and fibronectin fibrillogenesis. Expression of an activated mutant of RhoA stimulates alphavbeta3-mediated fibrillogenesis. Despite the fact that alpha5beta1-mediated adhesion to the central cell-binding domain of fibronectin supports activation of RhoA, other regions of fibronectin are required for the development of alpha5beta1-mediated but not alphavbeta3-mediatecl focal contacts. Using chimeras of beta1 and beta3 subunits, we find that the extracellular domain of beta1 controls RhoA activity. By expressing both beta1 and beta3 at high levels, we show that beta1-mediated control of the levels of beta3 is important for the distribution of focal contacts. Our findings demonstrate that the pattern of fibronectin receptors expressed on a cell dictates the ability of fibronectin to stimulate RhoA-mediated organization of cell matrix adhesions.
  •  
29.
  • Dantuma, NP, et al. (author)
  • A dynamic ubiquitin equilibrium couples proteasomal activity to chromatin remodeling
  • 2006
  • In: The Journal of cell biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 173:1, s. 19-26
  • Journal article (peer-reviewed)abstract
    • Protein degradation, chromatin remodeling, and membrane trafficking are critically regulated by ubiquitylation. The presence of several coexisting ubiquitin-dependent processes, each of crucial importance to the cell, is remarkable. This brings up questions on how the usage of this versatile regulator is negotiated between the different cellular processes. During proteotoxic stress, the accumulation of ubiquitylated substrates coincides with the depletion of ubiquitylated histone H2A and chromatin remodeling. We show that this redistribution of ubiquitin during proteotoxic stress is a direct consequence of competition for the limited pool of free ubiquitin. Thus, the ubiquitin cycle couples various ubiquitin-dependent processes because of a rate-limiting pool of free ubiquitin. We propose that this ubiquitin equilibrium may allow cells to sense proteotoxic stress in a genome-wide fashion.
  •  
30.
  • Dantuma, NP, et al. (author)
  • Ubiquitin versus misfolding: The minimal requirements for inclusion body formation
  • 2016
  • In: The Journal of cell biology. - : Rockefeller University Press. - 1540-8140 .- 0021-9525. ; 213:2, s. 147-149
  • Journal article (peer-reviewed)abstract
    • Ubiquitin-containing inclusion bodies are characteristic features of numerous neurodegenerative diseases, but whether ubiquitin plays a functional role in the formation of these protein deposits is unclear. In this issue, Bersuker et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201511024) report that protein misfolding without ubiquitylation is sufficient for translocation into inclusion bodies.
  •  
31.
  • Daste, Frederic, et al. (author)
  • Control of actin polymerization via the coincidence of phosphoinositides and high membrane curvature
  • 2017
  • In: Journal of Cell Biology. - : ROCKEFELLER UNIV PRESS. - 0021-9525 .- 1540-8140. ; 216:11, s. 3745-3765
  • Journal article (peer-reviewed)abstract
    • The conditional use of actin during clathrin-mediated endocytosis in mammalian cells suggests that the cell controls whether and how actin is used. Using a combination of biochemical reconstitution and mammalian cell culture, we elucidate a mechanism by which the coincidence of PI(4,5)P-2 and PI(3)P in a curved vesicle triggers actin polymerization. At clathrin-coated pits, PI(3) P is produced by the INPP4A hydrolysis of PI(3,4)P-2, and this is necessary for actin-driven endocytosis. Both Cdc42.guanosine triphosphate and SNX9 activate N-WASP-WIP-and Arp2/3-mediated actin nucleation. Membrane curvature, PI(4,5)P-2, and PI(3) P signals are needed for SNX9 assembly via its PX-BAR domain, whereas signaling through Cdc42 is activated by PI(4,5)P-2 alone. INPP4A activity is stimulated by high membrane curvature and synergizes with SNX9 BAR domain binding in a process we call curvature cascade amplification. We show that the SNX9-driven actin comets that arise on human disease-associated oculocerebrorenal syndrome of Lowe (OCRL) deficiencies are reduced by inhibiting PI(3) P production, suggesting PI(3) P kinase inhibitors as a therapeutic strategy in Lowe syndrome.
  •  
32.
  • Ekholm-Reed, S, et al. (author)
  • Deregulation of cyclin E in human cells interferes with prereplication complex assembly
  • 2004
  • In: The Journal of cell biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 165:6, s. 789-800
  • Journal article (peer-reviewed)abstract
    • Deregulation of cyclin E expression has been associated with a broad spectrum of human malignancies. Analysis of DNA replication in cells constitutively expressing cyclin E at levels similar to those observed in a subset of tumor-derived cell lines indicates that initiation of replication and possibly fork movement are severely impaired. Such cells show a specific defect in loading of initiator proteins Mcm4, Mcm7, and to a lesser degree, Mcm2 onto chromatin during telophase and early G1 when Mcm2–7 are normally recruited to license origins of replication. Because minichromosome maintenance complex proteins are thought to function as a heterohexamer, loading of Mcm2-, Mcm4-, and Mcm7-depleted complexes is likely to underlie the S phase defects observed in cyclin E–deregulated cells, consistent with a role for minichromosome maintenance complex proteins in initiation of replication and fork movement. Cyclin E–mediated impairment of DNA replication provides a potential mechanism for chromosome instability observed as a consequence of cyclin E deregulation.
  •  
33.
  • Eliceiri, BP, et al. (author)
  • Integrin alphavbeta3 requirement for sustained mitogen-activated protein kinase activity during angiogenesis
  • 1998
  • In: The Journal of cell biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 140:5, s. 1255-1263
  • Journal article (peer-reviewed)abstract
    • Angiogenesis depends on growth factors and vascular cell adhesion events. Integrins and growth factors are capable of activating the ras/MAP kinase pathway in vitro, yet how these signals influence endothelial cells during angiogenesis is unknown. Upon initiation of angiogenesis with basic fibroblast growth factor (bFGF) on the chick chorioallantoic membrane (CAM), endothelial cell mitogen-activated protein (MAP) kinase (ERK) activity was detected as early as 5 min yet was sustained for at least 20 h. The initial wave of ERK activity (5–120 min) was refractory to integrin antagonists, whereas the sustained activity (4–20 h) depended on integrin αvβ3, but not β1 integrins. Inhibition of MAP kinase kinase (MEK) during this sustained αvβ3-dependent ERK signal blocked the formation of new blood vessels while not influencing preexisting blood vessels on the CAM. Inhibition of MEK also blocked growth factor induced migration but not adhesion of endothelial cells in vitro. Therefore, angiogenesis depends on sustained ERK activity regulated by the ligation state of both a growth factor receptor and integrin αvβ3.
  •  
34.
  •  
35.
  • Espada, J, et al. (author)
  • Nuclear envelope defects cause stem cell dysfunction in premature-aging mice
  • 2008
  • In: The Journal of cell biology. - : Rockefeller University Press. - 1540-8140 .- 0021-9525. ; 181:1, s. 27-35
  • Journal article (peer-reviewed)abstract
    • Nuclear lamina alterations occur in physiological aging and in premature aging syndromes. Because aging is also associated with abnormal stem cell homeostasis, we hypothesize that nuclear envelope alterations could have an important impact on stem cell compartments. To evaluate this hypothesis, we examined the number and functional competence of stem cells in Zmpste24-null progeroid mice, which exhibit nuclear lamina defects. We show that Zmpste24 deficiency causes an alteration in the number and proliferative capacity of epidermal stem cells. These changes are associated with an aberrant nuclear architecture of bulge cells and an increase in apoptosis of their supporting cells in the hair bulb region. These alterations are rescued in Zmpste24−/−Lmna+/− mutant mice, which do not manifest progeroid symptoms. We also report that molecular signaling pathways implicated in the regulation of stem cell behavior, such as Wnt and microphthalmia transcription factor, are altered in Zmpste24−/− mice. These findings establish a link between age-related nuclear envelope defects and stem cell dysfunction.
  •  
36.
  • Facanha, A L O, et al. (author)
  • The endoplasmic reticulum cation P-type ATPase Cta4p is required for control of cell shape and microtubule dynamics
  • 2002
  • In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 157:6, s. 1029-1039
  • Journal article (peer-reviewed)abstract
    • Here we describe the phenotypic characterization of the cta(4+) gene, encoding a novel member of the P4 family of P-type ATPases of fission yeast. The cta4Delta mutant is temperature sensitive and cold sensitive lethal and displays several morphological defects in cell polarity and cytokinesis. Microtubules are generally destabilized in cells lacking Cta4p. The microtubule length is decreased, and the number of microtubules per cell is increased. This is concomitant with an increase in the number of microtubule catastrophe events in the midzone of the cell. These defects are likely due to a general imbalance in cation homeostasis. Immunofluorescence microscopy and membrane fractionation experiments revealed that green fluorescent protein-tagged Cta4 localizes to the ER. Fluorescence resonance energy transfer experiments in living cells using the yellow cameleon indicator for Ca2+ indicated that Cta4p regulates the cellular Ca2+ concentration. Thus, our results reveal a link between cation homeostasis and the control of cell shape, microtubule dynamics, and cytokinesis, and appoint Ca2+ as a key ion in controlling these processes.
  •  
37.
  • FRISEN, J, et al. (author)
  • Rapid, widespread, and longlasting induction of nestin contributes to the generation of glial scar tissue after CNS injury
  • 1995
  • In: The Journal of cell biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 131:2, s. 453-464
  • Journal article (peer-reviewed)abstract
    • Neuronal regeneration does generally not occur in the central nervous system (CNS) after injury, which has been attributed to the generation of glial scar tissue. In this report we show that the composition of the glial scar after traumatic CNS injury in rat and mouse is more complex than previously assumed: expression of the intermediate filament nestin is induced in reactive astrocytes. Nestin induction occurs within 48 hours in the spinal cord both at the site of lesion and in degenerating tracts and lasts for at least 13 months. Nestin expression is induced with similar kinetics in the crushed optic nerve. In addition to the expression in reactive astrocytes, we also observed nestin induction within 48 hours after injury in cells close to the central canal in the spinal cord, while nestin expressing cells at later timepoints were found progressively further out from the central canal. This dynamic pattern of nestin induction after injury was mimicked by lacZ expressing cells in nestin promoter/lacZ transgenic mice, suggesting that defined nestin regulatory regions mediate the injury response. We discuss the possibility that the spatiotemporal pattern of nestin expression reflects a population of nestin positive cells, which proliferates and migrates from a region close to the central canal to the site of lesion in response to injury.
  •  
38.
  • Fusco, Ludovico, et al. (author)
  • Computer vision profiling of neurite outgrowth dynamics reveals spatio-temporal modularity of Rho GTPase signaling
  • 2016
  • In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 212:1, s. 91-111
  • Journal article (peer-reviewed)abstract
    • Rho guanosine triphosphatases (GTPases) control the cytoskeletal dynamics that power neurite outgrowth. This process consists of dynamic neuriteinitiation, elongation, retraction, and branching cycles that are likely to be regulated by specific spatiotemporal signaling networks, which cannot be resolved with static, steady-state assays. We present Neurite-Tracker, a computer-vision approach to automatically segment and track neuronal morphodynamics in time-lapse datasets. Feature extraction then quantifies dynamic neurite outgrowth phenotypes. We identify a set of stereotypic neurite outgrowth morphodynamic behaviors in a cultured neuronal cell system. Systematic RNA interference perturbation of a Rho GTPase interactome consisting of 219 proteins reveals a limited set of morphodynamic phenotypes. As proof of concept, we show that loss of function of two distinct RhoA-specific GTPase-activating proteins (GAPs) leads to opposite neurite outgrowth phenotypes. Imaging of RhoA activation dynamics indicates that both GAPs regulate different spatiotemporal Rho GTPase pools, with distinct functions. Our results provide a starting point to dissect spatiotemporal Rho GTPase signaling networks that regulate neurite outgrowth.
  •  
39.
  • Fässler, R, et al. (author)
  • Lack of ß1 integrin gene in embryonic stem cells affects morphology, adhesion, and migration but not integration into the inner cell mass of blastocysts
  • 1995
  • In: Journal of Cell Biology. - 0021-9525 .- 1540-8140. ; 128:5, s. 979-988
  • Journal article (peer-reviewed)abstract
    • A gene trap-type targeting vector was designed to inactivate the beta 1 integrin gene in embryonic stem (ES) cells. Using this vector more than 50% of the ES cell clones acquired a disruption in the beta 1 integrin gene and a single clone was mutated in both alleles. The homozygous mutant did not produce beta 1 integrin mRNA or protein, while alpha 3, alpha 5, and alpha 6 integrin subunits were transcribed but not detectable on the cell surface. Heterozygous mutants showed reduced beta 1 expression and surface localization of alpha/beta 1 heterodimers. The alpha V subunit expression was not impaired on any of the mutants. Homozygous ES cell mutants lacked adhesiveness for laminin and fibronectin but not for vitronectin and showed a reduced association with a fibroblast feeder layer. Furthermore, they did not migrate towards chemoattractants in fibroblast medium. None of these functions were impaired in heterozygous mutants. Scanning electron microscopy revealed that homozygous cells showed fewer cell-cell junctions and had many microvilli not usually found on wild type and heterozygous cells. This profound change in cell shape is not associated with gross alterations in the expression and distribution of cytoskeletal components. Unexpectedly, microinjection into blastocysts demonstrated full integration of homozygous and heterozygous mutants into the inner cell mass. This will allow studies of the consequences of beta 1 integrin deficiency in several in vivo situations.
  •  
40.
  • Gekara, Nelson O. (author)
  • DNA damage-induced immune response : Micronuclei provide key platform
  • 2017
  • In: Journal of Cell Biology. - : ROCKEFELLER UNIV PRESS. - 0021-9525 .- 1540-8140. ; 216:10, s. 2999-3001
  • Journal article (other academic/artistic)abstract
    • DNA damage-induced activation of the cytoplasmic DNA sensor cGAS influences the outcome of infections, autoinflammation, and cancer. Recent studies by Harding et al. (2017. Nature. http://dx.doi.org/10.1038/nature23470), Mackenzie et al. (2017. Nature. http://dx.doi.org/10.1038/nature23449), and Bartsch et al. (2017. Human Molecular Genetics. https://doi.org/10.1093/hmg/ddx283) demonstrate a role for micronuclei formation in DNA damage-induced immune activation.
  •  
41.
  • Giampietro, Costanza, et al. (author)
  • The actin-binding protein EPS8 binds VE-cadherin and modulates YAP localization and signaling
  • 2015
  • In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 211:6, s. 1177-1192
  • Journal article (peer-reviewed)abstract
    • Vascular endothelial (VE)-cadherin transfers intracellular signals contributing to vascular hemostasis. Signaling through VE-cadherin requires association and activity of different intracellular partners. Yes-associated protein (YAP)/TAZ transcriptional cofactors are important regulators of cell growth and organ size. We show that EPS8, a signaling adapter regulating actin dynamics, is a novel partner of VE-cadherin and is able to modulate YAP activity. By biochemical and imaging approaches, we demonstrate that EPS8 associates with the VE-cadherin complex of remodeling junctions promoting YAP translocation to the nucleus and transcriptional activation. Conversely, in stabilized junctions, 14-3-3-YAP associates with the VE-cadherin complex, whereas Eps8 is excluded. Junctional association of YAP inhibits nuclear translocation and inactivates its transcriptional activity both in vitro and in vivo in Eps8-null mice. The absence of Eps8 also increases vascular permeability in vivo, but did not induce other major vascular defects. Collectively, we identified novel components of the adherens junction complex, and we introduce a novel molecular mechanism through which the VE-cadherin complex controls YAP transcriptional activity.
  •  
42.
  • Gongrich, C, et al. (author)
  • ALK4 coordinates extracellular and intrinsic signals to regulate development of cortical somatostatin interneurons
  • 2020
  • In: The Journal of cell biology. - : Rockefeller University Press. - 1540-8140 .- 0021-9525. ; 219:1
  • Journal article (peer-reviewed)abstract
    • Although the role of transcription factors in fate specification of cortical interneurons is well established, how these interact with extracellular signals to regulate interneuron development is poorly understood. Here we show that the activin receptor ALK4 is a key regulator of the specification of somatostatin interneurons. Mice lacking ALK4 in GABAergic neurons of the medial ganglionic eminence (MGE) showed marked deficits in distinct subpopulations of somatostatin interneurons from early postnatal stages of cortical development. Specific losses were observed among distinct subtypes of somatostatin+/Reelin+ double-positive cells, including Hpse+ layer IV cells targeting parvalbumin+ interneurons, leading to quantitative alterations in the inhibitory circuitry of this layer. Activin-mediated ALK4 signaling in MGE cells induced interaction of Smad2 with SATB1, a transcription factor critical for somatostatin interneuron development, and promoted SATB1 nuclear translocation and repositioning within the somatostatin gene promoter. These results indicate that intrinsic transcriptional programs interact with extracellular signals present in the environment of MGE cells to regulate cortical interneuron specification.
  •  
43.
  • Grim, Jonathan E., et al. (author)
  • Isoform- and cell cycle-dependent substrate degradation by the Fbw7 ubiquitin ligase
  • 2008
  • In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 181:6, s. 913-920
  • Journal article (peer-reviewed)abstract
    • The SCF(FBW7) ubiquitin ligase degrades proteins involved in cell division, growth, and differentiation and is commonly mutated in cancers. The Fbw7 locus encodes three protein isoforms that occupy distinct subcellular localizations, suggesting that each has unique functions. We used gene targeting to create isoform-specific Fbw7-null mutations in human cells and found that the nucleoplasmic Fbw7alpha isoform accounts for almost all Fbw7 activity toward cyclin E, c-Myc, and sterol regulatory element binding protein 1. Cyclin E sensitivity to Fbw7 varies during the cell cycle, and this correlates with changes in cyclin E-cyclin-dependent kinase 2 (CDK2)-specific activity, cyclin E autophosphorylation, and CDK2 inhibitory phosphorylation. These data suggest that oscillations in cyclin E-CDK2-specific activity during the cell cycle regulate the timing of cyclin E degradation. Moreover, they highlight the utility of adeno-associated virus-mediated gene targeting in functional analyses of complex loci.
  •  
44.
  • Gruschke, Steffi, et al. (author)
  • Cbp3-Cbp6 interacts with the yeast mitochondrial ribosomal tunnel exit and promotes cytochrome b synthesis and assembly
  • 2011
  • In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 193:6, s. 1101-1114
  • Journal article (peer-reviewed)abstract
    • Mitochondria contain their own genetic system to express a small number of hydrophobic polypeptides, including cytochrome b, an essential subunit of the bc(1) complex of the respiratory chain. In this paper, we show in yeast that Cbp3, a bc(1) complex assembly factor, and Cbp6, a regulator of cytochrome b translation, form a complex that associates with the polypeptide tunnel exit of mitochondrial ribosomes and that exhibits two important functions in the biogenesis of cytochrome b. On the one hand, the interaction of Cbp3 and Cbp6 with mitochondrial ribosomes is necessary for efficient translation of cytochrome b messenger ribonucleic acid or transcript. On the other hand, the Cbp3-Cbp6 complex interacts directly with newly synthesized cytochrome b in an assembly intermediate that is not ribosome bound and that contains the assembly factor Cbp4. Our results suggest that synthesis of cytochrome b occurs preferentially on those ribosomes that have the Cbp3-Cbp6 complex bound to their tunnel exit, an arrangement that may ensure tight coordination of cytochrome b synthesis and assembly.
  •  
45.
  • Gruschke, Steffi, et al. (author)
  • The Cbp3-Cbp6 complex coordinates cytochrome b synthesis with bc(1) complex assembly in yeast mitochondria
  • 2012
  • In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 199:1, s. 137-150
  • Journal article (peer-reviewed)abstract
    • Respiratory chain complexes in mitochondria are assembled from subunits derived from two genetic systems. For example, the bc1 complex consists of nine nuclear encoded subunits and the mitochondrially encoded subunit cytochrome b. We recently showed that the Cbp3-Cbp6 complex has a dual function for biogenesis of cytochrome b: it is both required for efficient synthesis of cytochrome b and for protection of the newly synthesized protein from proteolysis. Here, we report that Cbp3-Cbp6 also coordinates cytochrome b synthesis with bc1 complex assembly. We show that newly synthesized cytochrome b assembled through a series of four assembly intermediates. Blocking assembly at early and intermediate steps resulted in sequestration of Cbp3-Cbp6 in a cytochrome b-containing complex, thereby making Cbp3-Cbp6 unavailable for cytochrome b synthesis and thus reducing overall cytochrome b levels. This feedback loop regulates protein synthesis at the inner mitochondrial membrane by directly monitoring the efficiency of bc1 complex assembly.
  •  
46.
  • Gucciardo, E, et al. (author)
  • Proactive for invasion: Reuse of matrix metalloproteinase for structural memory
  • 2016
  • In: The Journal of cell biology. - : Rockefeller University Press. - 1540-8140 .- 0021-9525. ; 213:1, s. 11-13
  • Journal article (peer-reviewed)abstract
    • Migratory cells translocate membrane type-1 matrix metalloproteinase (MT1-MMP) to podosomes or invadosomes to break extracellular matrix barriers. In this issue, El Azzouzi et al. (2016. J. Cell. Biol. http://dx.doi.org/10.1083/jcb.201510043) describe an unexpected function for the MT1-MMP cytoplasmic domain in imprinting spatial memory for podosome reformation via assembly in membrane islets.
  •  
47.
  • Hellstrom, M, et al. (author)
  • Lack of pericytes leads to endothelial hyperplasia and abnormal vascular morphogenesis
  • 2001
  • In: The Journal of cell biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 153:3, s. 543-553
  • Journal article (peer-reviewed)abstract
    • The association of pericytes (PCs) to newly formed blood vessels has been suggested to regulate endothelial cell (EC) proliferation, survival, migration, differentiation, and vascular branching. Here, we addressed these issues using PDGF-B– and PDGF receptor-β (PDGFR-β)–deficient mice as in vivo models of brain angiogenesis in the absence of PCs. Quantitative morphological analysis showed that these mutants have normal microvessel density, length, and number of branch points. However, absence of PCs correlates with endothelial hyperplasia, increased capillary diameter, abnormal EC shape and ultrastructure, changed cellular distribution of certain junctional proteins, and morphological signs of increased transendothelial permeability. Brain endothelial hyperplasia was observed already at embryonic day (E) 11.5 and persisted throughout development. From E 13.5, vascular endothelial growth factor-A (VEGF-A) and other genes responsive to metabolic stress became upregulated, suggesting that the abnormal microvessel architecture has systemic metabolic consequences. VEGF-A upregulation correlated temporally with the occurrence of vascular abnormalities in the placenta and dilation of the heart. Thus, although PC deficiency appears to have direct effects on EC number before E 13.5, the subsequent increased VEGF-A levels may further abrogate microvessel architecture, promote vascular permeability, and contribute to formation of the edematous phenotype observed in late gestation PDGF-B and PDGFR-β knock out embryos.
  •  
48.
  • Heusermann, W, et al. (author)
  • Exosomes surf on filopodia to enter cells at endocytic hot spots, traffic within endosomes, and are targeted to the ER
  • 2016
  • In: The Journal of cell biology. - : Rockefeller University Press. - 1540-8140 .- 0021-9525. ; 213:2, s. 173-184
  • Journal article (peer-reviewed)abstract
    • Exosomes are nanovesicles released by virtually all cells, which act as intercellular messengers by transfer of protein, lipid, and RNA cargo. Their quantitative efficiency, routes of cell uptake, and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake, which saturates with dose and time and reaches near 100% transduction efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. After internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction, and suggest unanticipated routes of subcellular cargo delivery.
  •  
49.
  • Hildenbeutel, Markus, et al. (author)
  • Assembly factors monitor sequential hemylation of cytochrome b to regulate mitochondria! translation
  • 2014
  • In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 205:4, s. 511-524
  • Journal article (peer-reviewed)abstract
    • Mitochondrial respiratory chain complexes convert chemical energy into a membrane potential by connecting electron transport with charge separation. Electron transport relies on redox cofactors that occupy strategic positions in the complexes. How these redox cofactors are assembled into the complexes is not known. Cytochrome b, a central catalytic subunit of complex III, contains two henne bs. Here, we unravel the sequence of events in the mitochondrial inner membrane by which cytochrome b is hemylated. Heme incorporation occurs in a strict sequential process that involves interactions of the newly synthesized cytochrome b with assembly factors and structural complex III subunits. These interactions are functionally connected to cofactor acquisition that triggers the progression of cytochrome b through successive assembly intermediates. Failure to hemylate cytochrome b sequesters the Cbp3-Cbp6 complex in early assembly intermediates, thereby causing a reduction in cytochrome b synthesis via a feedback loop that senses hemylation of cytochrome b.
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50.
  • Hirsch, E, et al. (author)
  • Defective Rac-mediated proliferation and survival after targeted mutation of the beta(1) integrin cytodomain
  • 2002
  • In: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 157:3, s. 481-492
  • Journal article (peer-reviewed)abstract
    • Cell matrix adhesion is required for cell proliferation and survival. Here we report that mutation by gene targeting of the cytoplasmic tail of beta(1) integrin leads to defective proliferation and survival both in vivo and in vitro. Primary murine embryonic fibroblasts (MEFs) derived from mutant homozygotes display defective cell cycle coupled to impaired activation of the FAK-P13K-Akt and Rac-JNK signaling pathways. Expression in homozygous MEFs of a constitutively active form of Rac is able to rescue proliferation, survival, and JNK activation. Moreover, although showing normal Erk phosphorylation, mutant cells fail to display Erk nuclear translocation upon fibronectin adhesion. However, expression of the constitutively activated form of Rac restores Erk nuclear localization, suggesting that adhesion-dependent Rac activation is necessary to integrate signals directed to promote MAPK activity. Altogether, our data provide the evidence for an epistatic interaction between the beta(1) integrin cytoplasmic domain and Rac, and indicate that this anchorage-dependent signaling pathway is crucial for cell growth control.
  •  
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