Showing papers in "Molecular Biology of the Cell in 2007"
TL;DR: SCs are largely juxtavascular and reciprocally interact with ECs during differentiation to support angio-myogenesis, and differentiating myogenic cells were both proangiogenic in vitro and spatiotemporally associated with neoangiogenesis in muscular dystrophy.
Abstract: Monitoring Editor: Marianne Bronner-Fraser Genetically engineered mice (Myf5(nLacZ/+), Myf5(GFP-P/+)) allowing direct muscle satellite cell (SC) visualization indicate that, in addition to being located beneath myofiber basal laminae, SCs are strikingly close to capillaries. After GFP(+) bone marrow transplantation, blood-borne cells occupying SC niches previously depleted by irradiation, were similarly detected near vessels, thereby corroborating the anatomical stability of juxtavascular SC niches. BrdU pulse-chase experiments also localize quiescent and less quiescent SCs near vessels. SCs, and to a lesser extent myonuclei, were nonrandomly associated with capillaries in humans. Significantly, they were correlated with capillarisation of myofibers, regardless to their type, in normal muscle. They also varied in paradigmatic physiological and pathological situations associated with variations of capillary density, including amyopathic dermatomyositis, a unique condition in which muscle capillary loss occurs without myofiber damage, and in athletes, in whom capillaries increase in number. Endothelial cell (EC) cultures specifically enhanced SC growth, through IGF-1, HGF, bFGF, PDGF-BB and VEGF, and, accordingly, cycling SCs remained mainly juxtavascular. Conversely, differentiating myogenic cells were both proangiogenic in vitro and spatiotemporally associated with neoangiogenesis in muscular dystrophy. Thus, SCs are largely juxtavascular and reciprocally interact with ECs during differentiation to support angio-myogenesis.
581 citations
TL;DR: Stable reduction of ATG5 or ATG7 in MCF-10A acini enhances luminal apoptosis during morphogenesis and fails to elicit long-term luminal filling, even when combined with apoptotic inhibition mediated by Bcl-2 overexpression.
Abstract: Autophagy has been proposed to promote cell death during lumen formation in three-dimensional mammary epithelial acini because numerous autophagic vacuoles are observed in the dying central cells during morphogenesis Because these central cells die due to extracellular matrix (ECM) deprivation (anoikis), we have directly interrogated how matrix detachment regulates autophagy Detachment induces autophagy in both nontumorigenic epithelial lines and in primary epithelial cells RNA interference-mediated depletion of autophagy regulators (ATGs) inhibits detachment-induced autophagy, enhances apoptosis, and reduces clonogenic recovery after anoikis Remarkably, matrix-detached cells still exhibit autophagy when apoptosis is blocked by Bcl-2 overexpression, and ATG depletion reduces the clonogenic survival of Bcl-2-expressing cells after detachment Finally, stable reduction of ATG5 or ATG7 in MCF-10A acini enhances luminal apoptosis during morphogenesis and fails to elicit long-term luminal filling, even when combined with apoptotic inhibition mediated by Bcl-2 overexpression Thus, autophagy promotes epithelial cell survival during anoikis, including detached cells harboring antiapoptotic lesions
531 citations
TL;DR: It is demonstrated that intracellular trafficking of Glut1 is a regulated component of growth factor-stimulated glucose uptake and that Akt can promote Glut 1 activity and recycling as well as prevent GlUT1 internalization.
Abstract: Cells require growth factors to support glucose metabolism for survival and growth. It is unclear, however, how noninsulin growth factors may regulate glucose uptake and glucose transporters. We show that the hematopoietic growth factor interleukin (IL)3, maintained the glucose transporter Glut1 on the cell surface and promoted Rab11a-dependent recycling of intracellular Glut1. IL3 required phosphatidylinositol-3 kinase activity to regulate Glut1 trafficking, and activated Akt was sufficient to maintain glucose uptake and surface Glut1 in the absence of IL3. To determine how Akt may regulate Glut1, we analyzed the role of Akt activation of mammalian target of rapamycin (mTOR)/regulatory associated protein of mTOR (RAPTOR) and inhibition of glycogen synthase kinase (GSK)3. Although Akt did not require mTOR/RAPTOR to maintain surface Glut1 levels, inhibition of mTOR/RAPTOR by rapamycin greatly diminished glucose uptake, suggesting Akt-stimulated mTOR/RAPTOR may promote Glut1 transporter activity. In contrast, inhibition of GSK3 did not affect Glut1 internalization but nevertheless maintained surface Glut1 levels in IL3-deprived cells, possibly via enhanced recycling of internalized Glut1. In addition, Akt attenuated Glut1 internalization through a GSK3-independent mechanism. These data demonstrate that intracellular trafficking of Glut1 is a regulated component of growth factor-stimulated glucose uptake and that Akt can promote Glut1 activity and recycling as well as prevent Glut1 internalization.
517 citations
TL;DR: Ferroportin (Fpn) is the only known iron exporter in vertebrates and it is shown that after binding of hepcidin, Fpn is tyrosine phosphorylated at the plasma membrane, and adjacent tyrosines as the phosphorylation sites are identified.
Abstract: Ferroportin (Fpn) is the only known iron exporter in vertebrates. Hepcidin, a peptide secreted by the liver in response to iron or inflammation, binds to Fpn, inducing its internalization and degradation. We show that after binding of hepcidin, Fpn is tyrosine phosphorylated at the plasma membrane. Mutants of human Fpn that do not get internalized or that are internalized slowly show either absent or impaired phosphorylation. We identify adjacent tyrosines as the phosphorylation sites and show that mutation of both tyrosines prevents hepcidin-mediated Fpn internalization. Once internalized, Fpn is dephosphorylated and subsequently ubiquitinated. An inability to ubiquitinate Fpn does not prevent hepcidin-induced internalization, but it inhibits the degradation of Fpn. Ubiquitinated Fpn is trafficked through the multivesicular body pathway en route to degradation in the late endosome/lysosome. Depletion of proteins involved in multivesicular body trafficking (Endosome Sorting Complex Required for Transport proteins), by small-interfering RNA, reduces the trafficking of Fpn-green fluorescent to the lysosome.
439 citations
TL;DR: A novel mechanism in which hypoxia induces HIF-1alpha mRNA expression via the PI3K/AKT pathway and activation of NFkappaB is provided.
Abstract: The oxygen sensitive alpha-subunit of the hypoxia-inducible factor-1 (HIF-1) is a major trigger of the cellular response to hypoxia. Although the posttranslational regulation of HIF-1alpha by hypoxia is well known, its transcriptional regulation by hypoxia is still under debate. We, therefore, investigated the regulation of HIF-1alpha mRNA in response to hypoxia in pulmonary artery smooth muscle cells. Hypoxia rapidly enhanced HIF-1alpha mRNA levels and HIF-1alpha promoter activity. Furthermore, inhibition of the phosphatidylinositol 3-kinase (PI3K)/AKT but not extracellular signal-regulated kinase 1/2 pathway blocked the hypoxia-dependent induction of HIF-1alpha mRNA and HIF-1alpha promoter activity, suggesting involvement of a PI3K/AKT-regulated transcription factor. Interestingly, hypoxia also induced nuclear factor-kappaB (NFkappaB) nuclear translocation and activity. In line, expression of the NFkappaB subunits p50 and p65 enhanced HIF-1alpha mRNA levels, whereas blocking of NFkappaB by an inhibitor of nuclear factor-kappaB attenuated HIF-1alpha mRNA induction by hypoxia. Reporter gene assays revealed the presence of an NFkappaB site within the HIF-1alpha promoter, and mutation of this site abolished induction by hypoxia. In line, gel shift analysis and chromatin immunoprecipitation confirmed binding of p50 and p65 NFkappaB subunits to the HIF-1alpha promoter under hypoxia. Together, these findings provide a novel mechanism in which hypoxia induces HIF-1alpha mRNA expression via the PI3K/AKT pathway and activation of NFkappaB.
388 citations
TL;DR: The results raise questions as to the unknown force(s) that expand the nucleus as yeast cells grow, as nuclear expansion must now be factored into conceptual and mathematical models of budding yeast growth and division.
Abstract: It is not known how the volume of the cell nucleus is set, nor how the ratio of nuclear volume to cell volume (N/C) is determined. Here, we have measured the size of the nucleus in growing cells of...
383 citations
TL;DR: It is shown that the mitochondrial transcription factor TFAM, an abundant and highly conserved High Mobility Group box protein, binds DNA cooperatively with nanomolar affinity as a homodimer and that it is capable of coordinating and fully compacting several DNA molecules together to form spheroid structures.
Abstract: Packaging DNA into condensed structures is integral to the transmission of genomes. The mammalian mitochondrial genome (mtDNA) is a high copy, maternally inherited genome in which mutations cause a variety of multisystem disorders. In all eukaryotic cells, multiple mtDNAs are packaged with protein into spheroid bodies called nucleoids, which are the fundamental units of mtDNA segregation. The mechanism of nucleoid formation, however, remains unknown. Here, we show that the mitochondrial transcription factor TFAM, an abundant and highly conserved High Mobility Group box protein, binds DNA cooperatively with nanomolar affinity as a homodimer and that it is capable of coordinating and fully compacting several DNA molecules together to form spheroid structures. We use noncontact atomic force microscopy, which achieves near cryo-electron microscope resolution, to reveal the structural details of protein-DNA compaction intermediates. The formation of these complexes involves the bending of the DNA backbone, and DNA loop formation, followed by the filling in of proximal available DNA sites until the DNA is compacted. These results indicate that TFAM alone is sufficient to organize mitochondrial chromatin and provide a mechanism for nucleoid formation.
375 citations
TL;DR: The data imply that the cellular ataxin-2 concentration is important for the assembly of stress granules and P-bodies, which are main compartments for regulating and controlling mRNA degradation, stability, and translation.
Abstract: Tight control of translation is fundamental for eukaryotic cells, and deregulation of proteins implicated contributes to numerous human diseases. The neurodegenerative disorder spinocerebellar ataxia type 2 is caused by a trinucleotide expansion in the SCA2 gene encoding a lengthened polyglutamine stretch in the gene product ataxin-2, which seems to be implicated in cellular RNA-processing pathways and translational regulation. Here, we substantiate a function of ataxin-2 in such pathways by demonstrating that ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6, a component of P-bodies and stress granules, representing cellular structures of mRNA triage. We discovered that altered ataxin-2 levels interfere with the assembly of stress granules and cellular P-body structures. Moreover, ataxin-2 regulates the intracellular concentration of its interaction partner, the poly(A)-binding protein, another stress granule component and a key factor for translational control. Thus, our data imply that the cellular ataxin-2 concentration is important for the assembly of stress granules and P-bodies, which are main compartments for regulating and controlling mRNA degradation, stability, and translation.
318 citations
TL;DR: The results demonstrate that the HIF-alpha TADs, particularly the N-T ADs, confer HIF target gene specificity, by interacting with additional transcriptional cofactors.
Abstract: The basic helix-loop-helix-Per-ARNT-Sim-proteins hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha are the principal regulators of the hypoxic transcriptional response. Although highly related, they can activate distinct target genes. In this study, the protein domain and molecular mechanism important for HIF target gene specificity are determined. We demonstrate that although HIF-2alpha is unable to activate multiple endogenous HIF-1alpha-specific target genes (e.g., glycolytic enzymes), HIF-2alpha still binds to their promoters in vivo and activates reporter genes derived from such targets. In addition, comparative analysis of the N-terminal DNA binding and dimerization domains of HIF-1alpha and HIF-2alpha does not reveal any significant differences between the two proteins. Importantly, replacement of the N-terminal transactivation domain (N-TAD) (but not the DNA binding domain, dimerization domain, or C-terminal transactivation domain [C-TAD]) of HIF-2alpha with the analogous region of HIF-1alpha is sufficient to convert HIF-2alpha into a protein with HIF-1alpha functional specificity. Nevertheless, both the N-TAD and C-TAD are important for optimal HIF transcriptional activity. Additional experiments indicate that the ETS transcription factor ELK is required for HIF-2alpha to activate specific target genes such as Cited-2, EPO, and PAI-1. These results demonstrate that the HIF-alpha TADs, particularly the N-TADs, confer HIF target gene specificity, by interacting with additional transcriptional cofactors.
315 citations
TL;DR: It is suggested that the δEF1 family proteins, SIP1 and δ EF1, are necessary, but not sufficient, for TGF-β–induced EMT and that Ets1 induced by T GF-β may function as an upstream transcriptional regulator of SIP2 and εEF1.
Abstract: Epithelial–mesenchymal transition (EMT), a crucial event in cancer progression and embryonic development, is induced by transforming growth factor (TGF)-β in mouse mammary NMuMG epithelial cells. I...
300 citations
TL;DR: These results identify KLK5, a key actor of the desquamation process, as the major target of LEKTI, and disclose a new mechanism of skin homeostasis by which the epidermal pH gradient allows precisely regulated KLK 5 activity and corneodesmosomal cleavage in the most superficial layers of the stratum corneum.
Abstract: LEKTI is a 15-domain serine proteinase inhibitor whose defective expression underlies the severe autosomal recessive ichthyosiform skin disease, Netherton syndrome. Here, we show that LEKTI is produced as a precursor rapidly cleaved by furin, generating a variety of single or multidomain LEKTI fragments secreted in cultured keratinocytes and in the epidermis. The identity of these biological fragments (D1, D5, D6, D8-D11, and D9-D15) was inferred from biochemical analysis, using a panel of LEKTI antibodies. The functional inhibitory capacity of each fragment was tested on a panel of serine proteases. All LEKTI fragments, except D1, showed specific and differential inhibition of human kallikreins 5, 7, and 14. The strongest inhibition was observed with D8-D11, toward KLK5. Kinetics analysis revealed that this interaction is rapid and irreversible, reflecting an extremely tight binding complex. We demonstrated that pH variations govern this interaction, leading to the release of active KLK5 from the complex at acidic pH. These results identify KLK5, a key actor of the desquamation process, as the major target of LEKTI. They disclose a new mechanism of skin homeostasis by which the epidermal pH gradient allows precisely regulated KLK5 activity and corneodesmosomal cleavage in the most superficial layers of the stratum corneum.
TL;DR: PAS assembly during nonspecific autophagy is studied, using an atg11Delta mutant background to eliminate the PAS formation that occurs during vegetative growth and it is found that protein complexes containing the Atg1 kinase have two roles for PAS Formation during nonsPecific autophileagy.
Abstract: Autophagy is the major degradative process for recycling cytoplasmic constituents and eliminating unnecessary organelles in eukaryotic cells. Most autophagy-related (Atg) proteins are recruited to the phagophore assembly site (PAS), a proposed site for vesicle formation during either nonspecific or specific types of autophagy. Therefore, appropriate recruitment of Atg proteins to this site is critical for their function in autophagy. Atg11 facilitates PAS recruitment for the cytoplasm-to-vacuole targeting pathway, which is a specific, autophagy-like process that occurs under vegetative conditions. In contrast, it is not known how Atg proteins are recruited to the PAS, nor which components are involved in PAS formation under nonspecific autophagy-inducing, starvation conditions. Here, we studied PAS assembly during nonspecific autophagy, using an atg11Δ mutant background to eliminate the PAS formation that occurs during vegetative growth. We found that protein complexes containing the Atg1 kinase have two roles for PAS formation during nonspecific autophagy. The Atg1 C terminus mediates an interaction with Atg13 and Atg17, facilitating a structural role of Atg1 that is needed to efficiently organize an initial step of PAS assembly, whereas Atg1 kinase activity affects the dynamics of protein movement at the PAS involved in Atg protein cycling.
TL;DR: The present results suggest that autophagy is controlled by the signals from at least three partly separate nutrient-sensing pathways that include PKA, Sch9, and TORC1.
Abstract: Autophagy is a highly conserved, degradative process in eukaryotic cells. The rapamycin-sensitive Tor kinase complex 1 (TORC1) has a major role in regulating induction of autophagy; however, the regulatory mechanisms are not fully understood. Here, we find that the protein kinase A (PKA) and Sch9 signaling pathways regulate autophagy cooperatively in yeast. Autophagy is induced in cells when PKA and Sch9 are simultaneously inactivated. Mutant alleles of these kinases bearing a mutation that confers sensitivity to the ATP-analogue inhibitor C3-1'-naphthyl-methyl PP1 revealed that autophagy was induced independently of effects on Tor kinase. The PKA-Sch9-mediated autophagy depends on the autophagy-related 1 kinase complex, which is also essential for TORC1-regulated autophagy, the transcription factors Msn2/4, and the Rim15 kinase. The present results suggest that autophagy is controlled by the signals from at least three partly separate nutrient-sensing pathways that include PKA, Sch9, and TORC1.
TL;DR: The data show that BM-MSCs use a different pathway from HSCs for intramyocardial trafficking and engraftment, and are distinctively different from that reported for hematopoietic stem cells (HSCs).
Abstract: Recent evidence has demonstrated the importance of bone marrow-derived mesenchymal stem cells (BM-MSCs) in the repair of damaged myocardium. The molecular mechanisms of engraftment and migration of BM-MSCs in the ischemic myocardium are unknown. In this study, we developed a functional genomics approach toward the identification of mediators of engraftment and migration of BM-MSCs within the ischemic myocardium. Our strategy involves microarray profiling (>22,000 probes) of ischemic hearts, complemented by reverse transcription-polymerase chain reaction and fluorescence-activated cell sorting of corresponding adhesion molecule and cytokine receptors in BM-MSCs to focus on the coexpressed pairs only. Our data revealed nine complementary adhesion molecules and cytokine receptors, including integrin β1, integrin α4, and CXC chemokine receptor 4 (CXCR4). To examine their functional contributions, we first blocked selectively these receptors by preincubation of BM-MSCs with specific neutralizing antibodies, and then we administered these cells intramyocardially. A significant reduction in the total number of BM-MSC in the infarcted myocardium was observed after integrin β1 blockade but not integrin α4 or CXCR4 blockade. The latter observation is distinctively different from that reported for hematopoietic stem cells (HSCs). Thus, our data show that BM-MSCs use a different pathway from HSCs for intramyocardial trafficking and engraftment.
TL;DR: A link between the inhibition of UPS activity, the formation of cytoplasmic stress granules (SGs), and mRNA metabolism is established, suggesting that SGs could be considered as one of the players that mediate the early response of the cell to proteasome inhibitors by interfering temporarily with mRNA decay pathways.
Abstract: The inhibition of the ubiquitin-dependent proteasome system (UPS) via specific drugs is one type of approach used to combat cancer. Although it has been suggested that UPS inhibition prevents the rapid decay of AU-rich element (ARE)-containing messages, very little is known about the cellular mechanisms leading to this effect. Here we establish a link between the inhibition of UPS activity, the formation of cytoplasmic stress granules (SGs), and mRNA metabolism. The assembly of the SGs requires the phosphorylation of the translation initiation factor eIF2α by a mechanism involving the stress kinase GCN2. On prolonged UPS inhibition and despite the maintenance of eIF2α phosphorylation, SGs disassemble and translation recovers in an Hsp72 protein-dependent manner. The formation of these SGs coincides with the disassembly of processing bodies (PBs), known as mRNA decay entities. As soon as the SGs assemble, they recruit ARE-containing messages such as p21cip1 mRNA, which are stabilized under these conditions. Hence, our findings suggest that SGs could be considered as one of the players that mediate the early response of the cell to proteasome inhibitors by interfering temporarily with mRNA decay pathways.
TL;DR: It is indicated that regulated lysosomal acidification can potentiate fAbeta degradation by microglia, and this work found that microglian cells actually contain higher levels of many lysOSomal proteases than macrophages.
Abstract: Microglia are the main immune cells of the brain, and under some circumstances they can play an important role in removal of fibrillar Alzheimer amyloid β peptide (fAβ). Primary mouse microglia can...
TL;DR: It is reported that celastrol's biological effects, including inhibition of glucocorticoid receptor activity, can be blocked by the addition of excess free thiol, suggesting a chemical mechanism for biological activity based on modification of key reactive thiols by this natural product.
Abstract: Stress response pathways allow cells to sense and respond to environmental changes and adverse pathophysiological states. Pharmacological modulation of cellular stress pathways has implications in the treatment of human diseases, including neurodegenerative disorders, cardiovascular disease, and cancer. The quinone methide triterpene celastrol, derived from a traditional Chinese medicinal herb, has numerous pharmacological properties, and it is a potent activator of the mammalian heat shock transcription factor HSF1. However, its mode of action and spectrum of cellular targets are poorly understood. We show here that celastrol activates Hsf1 in Saccharomyces cerevisiae at a similar effective concentration seen in mammalian cells. Transcriptional profiling revealed that celastrol treatment induces a battery of oxidant defense genes in addition to heat shock genes. Celastrol activated the yeast Yap1 oxidant defense transcription factor via the carboxy-terminal redox center that responds to electrophilic compounds. Antioxidant response genes were likewise induced in mammalian cells, demonstrating that the activation of two major cell stress pathways by celastrol is conserved. We report that celastrol's biological effects, including inhibition of glucocorticoid receptor activity, can be blocked by the addition of excess free thiol, suggesting a chemical mechanism for biological activity based on modification of key reactive thiols by this natural product.
TL;DR: The results indicate that Pat1p can be a nuclear-cytoplasmic shuttling protein and acts early in P- body assembly, and the Lsm1-7p complex appears to primarily function in a rate limiting step after P-body assembly in triggering decapping.
Abstract: Recent experiments have defined cytoplasmic foci, referred to as processing bodies (P-bodies), that contain untranslating mRNAs in conjunction with proteins involved in translation repression and mRNA decapping and degradation. However, the order of protein assembly into P-bodies and the interactions that promote P-body assembly are unknown. To gain insight into how yeast P-bodies assemble, we examined the P-body accumulation of Dcp1p, Dcp2p, Edc3p, Dhh1p, Pat1p, Lsm1p, Xrn1p, Ccr4p, and Pop2p in deletion mutants lacking one or more P-body component. These experiments revealed that Dcp2p and Pat1p are required for recruitment of Dcp1p and of the Lsm1-7p complex to P-bodies, respectively. We also demonstrate that P-body assembly is redundant and no single known component of P-bodies is required for P-body assembly, although both Dcp2p and Pat1p contribute to P-body assembly. In addition, our results indicate that Pat1p can be a nuclear-cytoplasmic shuttling protein and acts early in P-body assembly. In contrast, the Lsm1-7p complex appears to primarily function in a rate limiting step after P-body assembly in triggering decapping. Taken together, these results provide insight both into the function of individual proteins involved in mRNA degradation and the mechanisms by which yeast P-bodies assemble.
TL;DR: E-cadherin homophilic binding independent of other cell contacts directly transduces growth inhibition by a beta-catenin-dependent mechanism that inhibits selective signaling functions of growth factor receptors.
Abstract: E-cadherin function leads to the density-dependent contact inhibition of cell growth. Because cadherins control the overall state of cell contact, cytoskeletal organization, and the establishment of many other kinds of cell interactions, it remains unknown whether E-cadherin directly transduces growth inhibitory signals. To address this question, we have selectively formed E-cadherin homophilic bonds at the cell surface of isolated epithelial cells by using functionally active recombinant E-cadherin protein attached to microspheres. We find that E-cadherin ligation alone reduces the frequency of cells entering the S phase, demonstrating that E-cadherin ligation directly transduces growth inhibitory signals. E-cadherin binding to β-catenin is required for cell growth inhibition, but β-catenin/T-cell factor transcriptional activity is not involved in growth inhibition resulting from homophilic binding. Neither E-cadherin binding to p120-catenin nor β-catenin binding to α-catenin, and thereby the actin cytoskeleton, is required for growth inhibition. E-cadherin ligation also inhibits epidermal growth factor (EGF) receptor-mediated growth signaling by a β-catenin–dependent mechanism. It does not affect EGF receptor autophosphorylation or activation of ERK, but it inhibits transphosphorylation of Tyr845 and activation of signal transducers and activators of transcription 5. Thus, E-cadherin homophilic binding independent of other cell contacts directly transduces growth inhibition by a β-catenin–dependent mechanism that inhibits selective signaling functions of growth factor receptors.
TL;DR: It is demonstrated that Kif2b function is required for spindle assembly and chromosome movement and that the microtubule depolymerase activities of K if2a, Kif 2b, and MCAK fulfill distinct functions during mitosis in human cells.
Abstract: The human genome has three unique genes coding for kinesin-13 proteins called Kif2a, Kif2b, and MCAK (Kif2c). Kif2a and MCAK have documented roles in mitosis, but the function of Kif2b has not been defined. Here, we show that Kif2b is expressed at very low levels in cultured cells and that GFP-Kif2b localizes predominately to centrosomes and midbodies, but also to spindle microtubules and transiently to kinetochores. Kif2b-deficient cells assemble monopolar or disorganized spindles. Chromosomes in Kif2b-deficient cells show typical kinetochore-microtubule attachments, but the velocity of movement is reduced ∼80% compared with control cells. Some Kif2b-deficient cells attempt anaphase, but the cleavage furrow regresses and cytokinesis fails. Like Kif2a-deficient cells, bipolar spindle assembly can be restored to Kif2b-deficient cells by simultaneous deficiency of MCAK or Nuf2 or treatment with low doses of nocodazole. However, Kif2b-deficient cells are unique in that they assemble bipolar spindles when the pole focusing activities of NuMA and HSET are perturbed. These data demonstrate that Kif2b function is required for spindle assembly and chromosome movement and that the microtubule depolymerase activities of Kif2a, Kif2b, and MCAK fulfill distinct functions during mitosis in human cells.
TL;DR: A model where the EVH2 domain of Ena/VASP induces and maintains clustering of the barbed ends of actin filaments, which putatively corresponds to a transition from lamellipodial to filopodial localization is proposed.
Abstract: Filopodia have been implicated in a number of diverse cellular processes including growth-cone path finding, wound healing, and metastasis. The Ena/VASP family of proteins has emerged as key to filopodia formation but the exact mechanism for how they function has yet to be fully elucidated. Using cell spreading as a model system in combination with small interfering RNA depletion of Capping Protein, we determined that Ena/VASP proteins have a role beyond anticapping activity in filopodia formation. Analysis of mutant Ena/VASP proteins demonstrated that the entire EVH2 domain was the minimal domain required for filopodia formation. Fluorescent recovery after photobleaching data indicate that Ena/VASP proteins rapidly exchange at the leading edge of lamellipodia, whereas virtually no exchange occurred at filopodial tips. Mutation of the G-actin-binding motif (GAB) partially compromised stabilization of Ena/VASP at filopodia tips. These observations led us to propose a model where the EVH2 domain of Ena/VASP induces and maintains clustering of the barbed ends of actin filaments, which putatively corresponds to a transition from lamellipodial to filopodial localization. Furthermore, the EVH1 domain, together with the GAB motif in the EVH2 domain, helps to maintain Ena/VASP at the growing barbed ends.
TL;DR: It is concluded that a central epigenetic reprogramming event is relaxation of chromatin at loci associated with pluripotency to create a conformation compatible with transcriptional activation.
Abstract: Analyses of molecular events associated with reprogramming somatic nuclei to pluripotency are scarce. We previously reported the reprogramming of epithelial cells by extract of undifferentiated embryonal carcinoma (EC) cells. We now demonstrate reprogramming of DNA methylation and histone modifications on regulatory regions of the developmentally regulated OCT4 and NANOG genes by exposure of 293T cells to EC cell extract. OCT4 and NANOG are transcriptionally up-regulated and undergo mosaic cytosine-phosphate-guanosine demethylation. OCT4 demethylation occurs as early as week 1, is enhanced by week 2, and is most prominent in the proximal promoter and distal enhancer. Targeted OCT4 and NANOG demethylation does not occur in 293T extract-treated cells. Retinoic acid-mediated differentiation of reprogrammed cells elicits OCT4 promoter remethylation and transcriptional repression. Chromatin immunoprecipitation analyses of lysines K4, K9, and K27 of histone H3 on OCT4 and NANOG indicate that primary chromatin remodeling determinants are acetylation of H3K9 and demethylation of dimethylated H3K9. H3K4 remains di- and trimethylated. Demethylation of trimethylated H3K9 and H3K27 also occurs; however, trimethylation seems more stable than dimethylation. We conclude that a central epigenetic reprogramming event is relaxation of chromatin at loci associated with pluripotency to create a conformation compatible with transcriptional activation.
TL;DR: Proper proteasomal processing of ubiquitylated substrates requires Rpn11 plus either Uch37 or Usp14, and this model for mammalian 26S proteasome function is suggested whereby their concerted action governs proteasomesome function by linking deubiquitylation to substrate hydrolysis.
Abstract: We determined composition and relative roles of deubiquitylating proteins associated with the 26S proteasome in mammalian cells. Three deubiquitylating activities were associated with the 26S proteasome: two from constituent subunits, Rpn11/S13 and Uch37, and one from a reversibly associated protein, Usp14. RNA interference (RNAi) of Rpn11/S13 inhibited cell growth, decreased cellular proteasome activity via disrupted 26S proteasome assembly, and inhibited cellular protein degradation. In contrast, RNAi of Uch37 or Usp14 had no detectable effect on cell growth, proteasome structure or proteolytic capacity, but accelerated cellular protein degradation. RNAi of both Uch37 and Usp14 also had no effect on proteasome structure or proteolytic capacity, but inhibited cellular protein degradation. Thus, proper proteasomal processing of ubiquitylated substrates requires Rpn11 plus either Uch37 or Usp14. Although the latter proteins feature redundant deubiquitylation functions, they also appear to exert noncatalyic effects on proteasome activity that are similar to but independent of one another. These results reveal unexpected functional relationships among multiple deubiquitylating proteins and suggest a model for mammalian 26S proteasome function whereby their concerted action governs proteasome function by linking deubiquitylation to substrate hydrolysis.
TL;DR: The present method enabled to capture the molecular distribution of lipids in the cell membrane, and demonstrated that GM1 and GM3 form clusters that are susceptible to cholesterol depletion and chilling.
Abstract: Presence of microdomains has been postulated in the cell membrane, but two-dimensional distribution of lipid molecules has been difficult to determine in the submicrometer scale. In the present paper, we examined the distribution of gangliosides GM1 and GM3, putative raft molecules in the cell membrane, by immunoelectron microscopy using quick-frozen and freeze-fractured specimens. This method physically immobilized molecules in situ and thus minimized the possibility of artifactual perturbation. By point pattern analysis of immunogold labeling, GM1 was shown to make clusters of <100 nm in diameter in normal mouse fibroblasts. GM1-null fibroblasts were not labeled, but developed a similar clustered pattern when GM1 was administered. On cholesterol depletion or chilling, the clustering of both endogenous and exogenously-loaded GM1 decreased significantly, but the distribution showed marked regional heterogeneity in the cells. GM3 also showed cholesterol-dependent clustering, and although clusters of GM1 and GM3 were found to occasionally coincide, these aggregates were separated in most cases, suggesting the presence of heterogeneous microdomains. The present method enabled to capture the molecular distribution of lipids in the cell membrane, and demonstrated that GM1 and GM3 form clusters that are susceptible to cholesterol depletion and chilling.
TL;DR: It is shown that BLOC-1 functions in selective cargo exit from early endosomes toward melanosomes, and indicates that melanosome maturation requires at least two cargo transport pathways directly from earlyendosomes to melanOSomes, one pathway mediated by AP-3 and one pathways mediated by BL OC-1 andBLOC-2, that are deficient in several forms of HPS.
Abstract: Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defects in the formation and function of lysosome-related organelles such as melanosomes. HPS in humans or mice is caused by mutations in any of 15 genes, five of which encode subunits of biogenesis of lysosome-related organelles complex (BLOC)-1, a protein complex with no known function. Here, we show that BLOC-1 functions in selective cargo exit from early endosomes toward melanosomes. BLOC-1-deficient melanocytes accumulate the melanosomal protein tyrosinase-related protein-1 (Tyrp1), but not other melanosomal proteins, in endosomal vacuoles and the cell surface due to failed biosynthetic transit from early endosomes to melanosomes and consequent increased endocytic flux. The defects are corrected by restoration of the missing BLOC-1 subunit. Melanocytes from HPS model mice lacking a different protein complex, BLOC-2, accumulate Tyrp1 in distinct downstream endosomal intermediates, suggesting that BLOC-1 and BLOC-2 act sequentially in the same pathway. By contrast, intracellular Tyrp1 is correctly targeted to melanosomes in melanocytes lacking another HPS-associated protein complex, adaptor protein (AP)-3. The results indicate that melanosome maturation requires at least two cargo transport pathways directly from early endosomes to melanosomes, one pathway mediated by AP-3 and one pathway mediated by BLOC-1 and BLOC-2, that are deficient in several forms of HPS.
TL;DR: Early steps of α2β1 integrin-mediated cell adhesion to a collagen type I matrix is characterized by using single-cell force spectroscopy, in agreement with the role of α 2β1 as a collagentype I matrix.
Abstract: We have characterized early steps of α2β1 integrin-mediated cell adhesion to a collagen type I matrix by using single-cell force spectroscopy. In agreement with the role of α2β1 as a collagen type ...
TL;DR: It is shown that both gh Relin and des-acyl ghrelin stimulate proliferating C2C12 skeletal myoblasts to differentiate and to fuse into multinucleated myotubes in vitro through activation of p38, and that C2 C12 cells do not express GHSR-1a, but they do contain a common high-affinity binding site recognized by both acylated and des
Abstract: Ghrelin is an acylated peptidyl gastric hormone acting on the pituitary and hypothalamus to stimulate appetite, adiposity, and growth hormone release, through activation of growth hormone secretagogue receptor (GHSR)-1a receptor. Moreover, ghrelin features several activities such as inhibition of apoptosis, regulation of differentiation, and stimulation or inhibition of proliferation of several cell types. Ghrelin acylation is absolutely required for both GHSR-1a binding and its central endocrine activities. However, the unacylated ghrelin form, des-acyl ghrelin, which does not bind GHSR-1a and is devoid of any endocrine activity, is far more abundant than ghrelin in plasma, and it shares with ghrelin some of its cellular activities. In here we show that both ghrelin and des-acyl ghrelin stimulate proliferating C2C12 skeletal myoblasts to differentiate and to fuse into multinucleated myotubes in vitro through activation of p38. Consistently, both ghrelin and des-acyl ghrelin inhibit C2C12 proliferation in growth medium. Moreover, the ectopic expression of ghrelin in C2C12 enhances differentiation and fusion of these myoblasts in differentiation medium. Finally, we show that C2C12 cells do not express GHSR-1a, but they do contain a common high-affinity binding site recognized by both acylated and des-acylated ghrelin, suggesting that the described activities on C2C12 are likely mediated by this novel, yet unidentified receptor for both ghrelin forms.
TL;DR: It is shown that Plk1, when inactivated by TAL, spreads over the arms of chromosomes, resembling the localization of its binding partner PICH, and that both proteins are mutually dependent on each other for correct localization.
Abstract: Polo-like kinase 1 (Plk1) is a key regulator of mitotic progression and cell division in eukaryotes. It is highly expressed in tumor cells and considered a potential target for cancer therapy. Here, we report the discovery and application of a novel potent small-molecule inhibitor of mammalian Plk1, ZK-Thiazolidinone (TAL). We have extensively characterized TAL in vitro and addressed TAL specificity within cells by studying Plk1 functions in sister chromatid separation, centrosome maturation, and spindle assembly. Moreover, we have used TAL for a detailed analysis of Plk1 in relation to PICH and PRC1, two prominent interaction partners implicated in spindle assembly checkpoint function and cytokinesis, respectively. Specifically, we show that Plk1, when inactivated by TAL, spreads over the arms of chromosomes, resembling the localization of its binding partner PICH, and that both proteins are mutually dependent on each other for correct localization. Finally, we show that Plk1 activity is essential for cleavage furrow formation and ingression, leading to successful cytokinesis.
TL;DR: Results show that replication forks moving from one origin, as well as from neighboring origins, tend to exhibit the same velocity, although the plasticity of the replication program allows for their adaptation to variable interorigin distances.
Abstract: The spatial organization of replicons into clusters is believed to be of critical importance for genome duplication in higher eukaryotes, but its functional organization still remains to be fully clarified. The coordinated activation of origins is insufficient on its own to account for a timely completion of genome duplication when interorigin distances vary significantly and fork velocities are constant. Mechanisms coordinating origin distribution with fork progression are still poorly elucidated, because of technical difficulties of visualizing the process. Taking advantage of a single molecule approach, we delineated and compared the DNA replication kinetics at the genome level in human normal primary and malignant cells. Our results show that replication forks moving from one origin, as well as from neighboring origins, tend to exhibit the same velocity, although the plasticity of the replication program allows for their adaptation to variable interorigin distances. We also found that forks that emanated from closely spaced origins tended to move slower than those associated with long replicons. Taken together, our results indicate a functional role for origin clustering in the dynamic regulation of genome duplication.
TL;DR: This work systematically analyzed SNARE sequences from 145 different species and established a highly accurate classification for all SNARE proteins, establishing 20 SNARE subclasses that probably represent the original repertoire of a eukaryotic cenancestor.
Abstract: Proteins of the SNARE (soluble N-ethylmalemide-sensitive factor attachment protein receptor) family are essential for the fusion of transport vesicles with an acceptor membrane. Despite considerable sequence divergence, their mechanism of action is conserved: heterologous sets assemble into membrane-bridging SNARE complexes, in effect driving membrane fusion. Within the cell, distinct functional SNARE units are involved in different trafficking steps. These functional units are conserved across species and probably reflect the conservation of the particular transport step. Here, we have systematically analyzed SNARE sequences from 145 different species and have established a highly accurate classification for all SNARE proteins. Principally, all SNAREs split into four basic types, reflecting their position in the four-helix bundle complex. Among these four basic types, we established 20 SNARE subclasses that probably represent the original repertoire of a eukaryotic cenancestor. This repertoire has been modulated independently in different lines of organisms. Our data are in line with the notion that the ur-eukaryotic cell was already equipped with the various compartments found in contemporary cells. Possibly, the development of these compartments is closely intertwined with episodes of duplication and divergence of a prototypic SNARE unit.