This study revealed that ubiquitously expressed transcript (UXT)-V1 is recruited to tumor necrosis factor (TNF) receptor complex I by interacting with TNF receptor-associated factor 2. UXT-V1 is a short-half-life protein, the degradation of which facilitates the formation of the apoptotic receptor complex II in response to TNF treatment. This study uncovers UXT-V1 as a novel regulator of TNF-induced apoptosis.
Proteins that directly regulate tumor necrosis factor (TNF) signaling have critical roles in determining cell death and survival. Previously we characterized ubiquitously expressed transcript (UXT)-V2 as a novel transcriptional cofactor to regulate nuclear factor-κB in the nucleus. Here we report that another splicing isoform of UXT, UXT-V1, localizes in cytoplasm and regulates TNF-induced apoptosis. UXT-V1 knockdown cells are hypersensitive to TNF-induced apoptosis. We demonstrated that UXT-V1 is a new component of TNF receptor signaling complex. We found that UXT-V1 binds to TNF receptor-associated factor 2 and prevents TNF receptor–associated death domain protein from recruiting Fas-associated protein with death domain. More importantly, UXT-V1 is a short-half-life protein, the degradation of which facilitates the formation of the apoptotic receptor complex II in response to TNF treatment. This study demonstrates that UXT-V1 is a novel regulator of TNF-induced apoptosis and sheds new light on the underlying molecular mechanism of this process.
Human Bre1, an E3 ligase for H2B monoubiquitination, binds p53 and enhances activator-dependent transcription. Ebp1, an ErbB3 receptor-binding protein, inhibits cell proliferation and acts as a tumor suppressor. Here, we show that hBre1 acts as an E3 ubiquitin ligase for Ebp1 tumor suppressor and promotes its polyubiquitination and degradation. Ebp1 is polyubiquitinated in cancer cells, which is regulated by its phosphorylation. We identified hBre1 acting as an E3 ligase for Ebp1 and increasing its polyubiquitination. Depletion of hBre1 blocks Ebp1's polyubiquitination and elevates its protein level, preventing cancer proliferation. hBre1 binds Ebp1 and suppresses its repressive effect on E2F-1. Moreover, Ebp1 protein level is substantially diminished in human cancers. It is robustly phosphorylated and localized in the nucleus of primary gliomas, correlating with hBre1 subcellular residency. Thus, hBre1 inhibits Ebp1's tumor suppressive activity through mediating its polyubiquitination and degradation.
Promyelocytic leukemia protein (PML) nuclear bodies (NBs) are dynamic subnuclear compartments that play roles in several cellular processes, including apoptosis, transcriptional regulation, and DNA repair. Histone deacetylase (HDAC) 7 is a potent corepressor that inhibits transcription by myocyte enhancer factor 2 (MEF2) transcription factors. We show here that endogenous HDAC7 and PML interact and partially colocalize in PML NBs. Tumor necrosis factor (TNF)-α treatment recruits HDAC7 to PML NBs and enhances association of HDAC7 with PML in human umbilical vein endothelial cells. Consequently, TNF-α promotes dissociation of HDAC7 from MEF2 transcription factors and the promoters of MEF2 target genes such as matrix metalloproteinase (MMP)-10, leading to accumulation of MMP-10 mRNA. Conversely, knockdown of PML enhances the association between HDAC7 and MEF2 and decreases MMP-10 mRNA accumulation. Accordingly, ectopic expression of PML recruits HDAC7 to PML NBs and leads to activation of MEF2 reporter activity. Notably, small interfering RNA knockdown of PML decreases basal and TNF-α-induced MMP-10 mRNA accumulation. Our results reveal a novel mechanism by which PML sequesters HDAC7 to relieve repression and up-regulate gene expression.
Nerve growth factor (NGF) induces neurite outgrowth and differentiation in a process that involves NGF binding to its receptor TrkA and endocytosis of the NGF–TrkA complex into signaling endosomes. Here, we find that biogenesis of signaling endosomes requires inactivation of Rab5 to block early endosome fusion. Expression of dominant-negative Rab5 mutants enhanced NGF-mediated neurite outgrowth, whereas a constitutively active Rab5 mutant or Rabex-5 inhibited this process. Consistently, inactivation of Rab5 sustained TrkA activation on the endosomes. Furthermore, NGF treatment rapidly decreased cellular level of active Rab5-GTP, as shown by pull-down assays. This Rab5 down-regulation was mediated by RabGAP5, which was shown to associate with TrkA by coimmunoprecipitation assays. Importantly, RNA interference of RabGAP5 as well as a RabGAP5 truncation mutant containing the TrkA-binding domain blocked NGF-mediated neurite outgrowth, indicating a requirement for RabGAP5 in this process. Thus, NGF signaling down-regulates Rab5 activity via RabGAP5 to facilitate neurite outgrowth and differentiation.
Ciliary neurotrophic factor (CNTF) is primarily known for its important cellular effects within the nervous system. However, recent studies indicate that its receptor can be highly expressed in denervated skeletal muscle. Here, we investigated the direct effect of CNTF on skeletal myoblasts of adult human. Surprisingly, we found that CNTF induced the myogenic lineage-committed myoblasts at a clonal level to dedifferentiate into multipotent progenitor cells—they not only could proliferate for over 20 passages with the expression absence of myogenic specific factors Myf5 and MyoD, but they were also capable of differentiating into new phenotypes, mainly neurons, glial cells, smooth muscle cells, and adipocytes. These “progenitor cells” retained their myogenic memory and were capable of redifferentiating into myotubes. Furthermore, CNTF could activate the p44/p42 MAPK and down-regulate the expression of myogenic regulatory factors (MRFs). Finally, PD98059, a specific inhibitor of p44/p42 MAPK pathway, was able to abolish the effects of CNTF on both myoblast fate and MRF expression. Our results demonstrate the myogenic lineage-committed human myoblasts can dedifferentiate at a clonal level and CNTF is a novel regulator of skeletal myoblast dedifferentiation via p44/p42 MAPK pathway.
Cell cycle progression is both regulated and accompanied by periodic changes in the expression levels of a large number of genes. To investigate cell cycle-regulated transcriptional programs in the fission yeast Schizosaccharomyces pombe, we developed a whole-genome oligonucleotide-based DNA microarray. Microarray analysis of both wild-type and cdc25 mutant cell cultures was performed to identify transcripts whose levels oscillated during the cell cycle. Using an unsupervised algorithm, we identified 747 genes that met the criteria for cell cycle-regulated expression. Peaks of gene expression were found to be distributed throughout the entire cell cycle. Furthermore, we found that four promoter motifs exhibited strong association with cell cycle phase-specific expression. Examination of the regulation of MCB motif-containing genes through the perturbation of DNA synthesis control/MCB-binding factor (DSC/MBF)-mediated transcription in arrested synchronous cdc10 mutant cell cultures revealed a subset of functional targets of the DSC/MBF transcription factor complex, as well as certain gene promoter requirements. Finally, we compared our data with those for the budding yeast Saccharomyces cerevisiae and found ∼140 genes that are cell cycle regulated in both yeasts, suggesting that these genes may play an evolutionarily conserved role in regulation of cell cycle-specific processes. Our complete data sets are available at http://giscompute.gis.a-star.edu.sg/~gisljh/CDC.
Reactive oxygen species can induce premature senescence. Caveolin-1 promotes oxidative stress–induced activation of the p53/p21Waf1/Cip1 pathway and development of premature senescence by acting as an endogenous inhibitor of the transcription factor Nrf2.
Reactive oxygen species (ROS) can induce premature cellular senescence, which is believed to contribute to aging and age-related diseases. The nuclear erythroid 2 p45–related factor-2 (Nrf2) is a transcription factor that mediates cytoprotective responses against stress. We demonstrate that caveolin-1 is a direct binding partner of Nrf2, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82–101) to the caveolin-binding domain of Nrf2 (amino acids 281–289). Biochemical studies show that Nrf2 is concentrated into caveolar membranes in human and mouse fibroblasts, where it colocalizes with caveolin-1, under resting conditions. After oxidative stress, caveolin-1 limits the movement of Nrf2 from caveolar membranes to the nucleus. In contrast, Nrf2 is constitutively localized to the nucleus before and after oxidative stress in caveolin-1–null mouse embryonic fibroblasts (MEFs), which do not express caveolin-1. Functional studies demonstrate that caveolin-1 acts as an endogenous inhibitor of Nrf2, as shown by the enhanced up-regulation of NQO1, an Nrf2 target gene, in caveolin-1–null MEFs and the activation or inhibition of a luciferase construct carrying an antioxidant responsive element (ARE) after down-regulation of caveolin-1 by small interfering RNA or overexpression of caveolin-1, respectively. Expression of a mutant form of Nrf2 that cannot bind to caveolin-1 (Φ→A-Nrf2) hyperactivates ARE and inhibits oxidative stress–induced activation of the p53/p21Waf1/Cip1 pathway and induction of premature senescence in fibroblasts. Finally, we show that overexpression of caveolin-1 in colon cancer cells inhibits oxidant-induced activation of Nrf2-dependent signaling, promotes premature senescence, and inhibits their transformed phenotype. Thus, by inhibiting Nrf2-mediated signaling, caveolin-1 links free radicals to the activation of the p53/senescence pathway.
Talin-mediated integrin activation drives integrin-based adhesions. A simple binary switch—vinculin competitively displacing RIAM from talin—is found to play a central role in the maturation and evolving functions of integrin-based adhesions.
Talin-mediated integrin activation drives integrin-based adhesions. Here we examine the roles of two proteins that induce talin–integrin interactions—vinculin and Rap1-GTP-interacting adaptor molecule (RIAM)—in the formation and maturation of integrin-based adhesions. RIAM-containing adhesions are primarily in the lamellipodium; RIAM is subsequently reduced in mature focal adhesions due to direct competition with vinculin for talin-binding sites. We show that vinculin binding to talin induces Rap1-independent association of talin with integrins and resulting integrin activation, in sharp contrast to Rap1-dependent RIAM-induced activation. Vinculin stabilizes adhesions, increasing their ability to transmit force, whereas RIAM played a critical role in lamellipodial protrusion. Thus displacement of RIAM by vinculin acts as a molecular switch that mediates the transition of integrin-based adhesions from drivers of lamellipodial protrusion to stable, force-bearing adhesions. Consequently changes in the abundance of two multiprotein modules within maturing adhesions, one regulated by Rap1 and one by tension, result in the temporal evolution of adhesion functions.
The interaction between astral microtubules and the cell cortex is accompanied by constant cortical release and transport of LGN/dynein complex, which is modulated by cortical actin filaments. Regulated cortical release and transport of LGN/dynein complex along astral microtubules may contribute to spindle positioning in mammalian cells.
Spindle positioning is believed to be governed by the interaction between astral microtubules and the cell cortex and involve cortically anchored motor protein dynein. How dynein is recruited to and regulated at the cell cortex to generate forces on astral microtubules is not clear. Here we show that mammalian homologue of Drosophila Pins (Partner of Inscuteable) (LGN), a Gαi-binding protein that is critical for spindle positioning in different systems, associates with cytoplasmic dynein heavy chain (DYNC1H1) in a Gαi-regulated manner. LGN is required for the mitotic cortical localization of DYNC1H1, which, in turn, also modulates the cortical accumulation of LGN. Using fluorescence recovery after photobleaching analysis, we show that cortical LGN is dynamic and the turnover of LGN relies, at least partially, on astral microtubules and DYNC1H1. We provide evidence for dynein- and astral microtubule–mediated transport of Gαi/LGN/nuclear mitotic apparatus (NuMA) complex from cell cortex to spindle poles and show that actin filaments counteract such transport by maintaining Gαi/LGN/NuMA and dynein at the cell cortex. Our results indicate that astral microtubules are required for establishing bipolar, symmetrical cortical LGN distribution during metaphase. We propose that regulated cortical release and transport of LGN complex along astral microtubules may contribute to spindle positioning in mammalian cells.
During vertebrate development, the PCP pathway controls multiple cellular processes. Loss of the gene for the PCP effector Fuzzy affects formation of primary cilia via mostly unknown mechanisms. We report that Fuzzy localizes to the primary cilia and orchestrates delivery of Rab8 and Dishevelled to the primary cilium; loss of Fuzzy affects cilia-dependent signaling.
The planar cell polarity (PCP) pathway controls multiple cellular processes during vertebrate development. Recently the PCP pathway was implicated in ciliogenesis and in ciliary function. The primary cilium is an apically projecting solitary organelle that is generated via polarized intracellular trafficking. Because it acts as a signaling nexus, defects in ciliogenesis or cilial function cause multiple congenital anomalies in vertebrates. Loss of the PCP effector Fuzzy affects PCP signaling and formation of primary cilia; however, the mechanisms underlying these processes are largely unknown. Here we report that Fuzzy localizes to the basal body and ciliary axoneme and is essential for ciliogenesis by delivering Rab8 to the basal body and primary cilium. Fuzzy appears to control subcellular localization of the core PCP protein Dishevelled, recruiting it to Rab8-positive vesicles and to the basal body and cilium. We show that loss of Fuzzy results in inhibition of PCP signaling and hyperactivation of the canonical WNT pathway. We propose a mechanism by which Fuzzy participates in ciliogenesis and affects both canonical WNT and PCP signaling.
Many signaling pathways important for hyphal development have been identified, but how Candida albicans coordinates information from these signaling pathways during hyphal development remains a major question. It is shown that reduced Tor1 signaling lowers the basal activity of the Hog1 MAP kinase to sustain hyphal elongation.
Candida albicans is able to undergo reversible morphological changes between yeast and hyphal forms in response to environmental cues. This morphological plasticity is essential for its pathogenesis. Hyphal development requires two temporally linked changes in promoter chromatin, which is sequentially regulated by temporarily clearing the transcription inhibitor Nrg1 upon activation of cAMP/protein kinase A and promoter recruitment of the histone deacetylase Hda1 under reduced target of rapamycin (Tor1) signaling. The GATA family transcription factor Brg1 recruits Hda1 to promoters for sustained hyphal development, and BRG1 expression is a readout of reduced Tor1 signaling. How Tor1 regulates BRG1 expression is not clear. Using a forward genetic screen for mutants that can sustain hyphal elongation in rich media, we found hog1, ssk2, and pbs2 mutants of the HOG mitogen-activated protein kinase pathway to express BRG1 irrespective of rapamycin. Furthermore, rapamycin lowers the basal activity of Hog1 through the functions of the two Hog1 tyrosine phosphatases Ptp2 and Ptp3. Active Hog1 represses the expression of BRG1 via the transcriptional repressor Sko1 as Sko1 disassociates from the promoter of BRG1 in the hog1 mutant or in rapamycin. Our data suggest that reduced Tor1 signaling lowers Hog1 basal activity via Hog1 phosphatases to activate BRG1 expression for hyphal elongation.
The present study shows that RNA-binding proteins CUGBP1 and HuR jointly regulate the translation of occludin and play a crucial role in the maintenance of tight junction integrity.
RNA-binding proteins CUG-binding protein 1 (CUGBP1) and HuR are highly expressed in epithelial tissues and modulate the stability and translation of target mRNAs. Here we present evidence that CUGBP1 and HuR jointly regulate the translation of occludin and play a crucial role in the maintenance of tight junction (TJ) integrity in the intestinal epithelial cell monolayer. CUGBP1 and HuR competed for association with the same occludin 3′-untranslated region element and regulated occludin translation competitively and in opposite directions. CUGBP1 overexpression decreased HuR binding to occludin mRNA, repressed occludin translation, and compromised the TJ barrier function, whereas HuR overexpression inhibited CUGBP1 association with occludin mRNA and promoted occludin translation, thereby enhancing the barrier integrity. Repression of occludin translation by CUGBP1 was due to the colocalization of CUGBP1 and tagged occludin RNA in processing bodies (P-bodies), and this colocalization was prevented by HuR overexpression. These findings indicate that CUGBP1 represses occludin translation by increasing occludin mRNA recruitment to P-bodies, whereas HuR promotes occludin translation by blocking occludin mRNA translocation to P-bodies via the displacement of CUGBP1.
This article reports that TLR9 signaling can reduce intrinsic microRNA-7 (miR-7) expression in human lung cancer cells and that overexpression of miR-7 can significantly inhibit TLR9 signaling–enhanced growth and metastatic potential of lung cancer cells in vitro and in vivo.
Recent evidence shows that microRNAs (miRNAs) contribute to the biological effects of Toll-like receptor (TLR) signaling on various cells. Our previous data showed that TLR9 signaling could enhance the growth and metastatic potential of human lung cancer cells. However, the potential role of miRNAs in the effects of TLR9 signaling on tumor biology remains unknown. In this paper, we first report that TLR9 signaling could reduce intrinsic miR-7 expression in human lung cancer cells. Furthermore, overexpression of miR-7 can significantly inhibit TLR9 signaling–enhanced growth and metastatic potential of lung cancer cells in vitro and in vivo. Notably, we identify phosphoinositide-3-kinase, regulatory subunit 3 (PIK3R3) as a novel target molecule of miR-7 in lung cancer cells by Western blotting and luciferase report assay. Further study shows that miR-7 inhibits the effects of TLR9 signaling on lung cancer cells through regulation of the PIK3R3/Akt pathway. These data suggest that miR-7 could act as a fine-tuner in regulating the biological effects of TLR9 signaling on human lung cancer cells, which might be helpful to the understanding of the potential role of miRNAs in TLR signaling effects on tumor biology.
Various agonists trigger β2-integrin activation in neutrophils, yet the mechanisms that regulate β2-integrin inside-out signaling remain obscure. Radil, a novel Rap downstream effector, is an important adapter in the pathway that links G protein–coupled chemoattractant receptors to adhesion complexes during neutrophil chemotaxis.
Integrin activation is required to facilitate multiple adhesion-dependent functions of neutrophils, such as chemotaxis, which is critical for inflammatory responses to injury and pathogens. However, little is known about the mechanisms that mediate integrin activation in neutrophils. We show that Radil, a novel Rap1 effector, regulates β1- and β2-integrin activation and controls neutrophil chemotaxis. On activation and chemotactic migration of neutrophils, Radil quickly translocates from the cytoplasm to the plasma membrane in a Rap1a-GTP–dependent manner. Cells overexpressing Radil show a substantial increase in cell adhesion, as well as in integrin/focal adhesion kinase (FAK) activation, and exhibit an elongated morphology, with severe tail retraction defects. This phenotype is effectively rescued by treatment with either β2-integrin inhibitory antibodies or FAK inhibitors. Conversely, knockdown of Radil causes severe inhibition of cell adhesion, β2-integrin activation, and chemotaxis. Furthermore, we found that inhibition of Rap activity by RapGAP coexpression inhibits Radil-mediated integrin and FAK activation, decreases cell adhesion, and abrogates the long-tail phenotype of Radil cells. Overall, these studies establish that Radil regulates neutrophil adhesion and motility by linking Rap1 to β2-integrin activation.
SCYL1BP1 is a new regulator of the p53 pathway, which is required for neurite outgrowth and regeneration. SCYL1BP1 suppresses neurite outgrowth by directly inducing Mdm2 transcription and consequently p53 inhibition, suggesting that it might be a novel transcriptional regulator for regulating neurite outgrowth and regeneration.
SCY1-like 1–binding protein 1 (SCYL1BP1) is a newly identified transcriptional activator domain containing a protein with many unknown biological functions. Recently emerging evidence has revealed that it is a novel regulator of the p53 pathway, which is required for neurite outgrowth and regeneration. Here we present evidence that SCYL1BP1 inhibits nerve growth factor–mediated neurite outgrowth in PC12 cells and affects morphogenesis of primary cortical neurons by strongly decreasing the p53 protein level in vitro, all of which depends on SCYL1BP1's transcriptional activator domain. Exogenous p53 rescues neurite outgrowth and neuronal morphogenesis defects caused by SCYL1BP1. Furthermore, SCYL1BP1 can directly induce Mdm2 transcription, whereas inhibiting the function of Mdm2 by specific small interfering RNAs results in partial rescue of neurite outgrowth and neuronal morphogenesis defects induced by SCYL1BP1. In vivo experiments show that SCYL1BP1 can also depress axonal regeneration, whereas inhibiting the function of SCYL1BP1 by specific short hairpin RNA enhances it. Taken together, these data strongly suggested that SCYL1BP1 is a novel transcriptional activator in neurite outgrowth by directly modulating the Mdm2/p53-dependent pathway, which might play an important role in CNS development and axonal regeneration after injury.
Oxysterol-binding protein (OSBP) is involved in endoplasmic reticulum (ER)-Golgi sterol transport, but how its activity is regulated is unknown. OSBP is phosphorylated at multiple sites, two of which regulate sterol binding and interaction with vesicle-associated, membrane protein–associated protein A in the ER. Phosphorylation does not affect phosphatidylinositol 4-phosphate binding, which could serve as a counter-ligand to facilitate sterol release in the Golgi.
The endoplasmic reticulum (ER)-Golgi sterol transfer activity of oxysterol-binding protein (OSBP) regulates sphingomyelin (SM) synthesis, as well as post-Golgi cholesterol efflux pathways. The phosphorylation and ER-Golgi localization of OSBP are correlated, suggesting this modification regulates the directionality and/or specificity of transfer activity. In this paper, we report that phosphorylation on two serine-rich motifs, S381-S391 (site 1) and S192, S195, S200 (site 2), specifically controls OSBP activity at the ER. A phosphomimetic of the SM/cholesterol-sensitive phosphorylation site 1 (OSBP-S5E) had increased in vitro cholesterol and 25-hydroxycholesterol–binding capacity, and cholesterol extraction from liposomes, but reduced transfer activity. Phosphatidylinositol 4-phosphate (PI(4)P) and cholesterol competed for a common binding site on OSBP; however, direct binding of PI(4)P was not affected by site 1 phosphorylation. Individual site 1 and site 2 phosphomutants supported oxysterol activation of SM synthesis in OSBP-deficient CHO cells. However, a double site1/2 mutant (OSBP-S381A/S3D) was deficient in this activity and was constitutively colocalized with vesicle-associated membrane protein–associated protein A (VAP-A) in a collapsed ER network. This study identifies phosphorylation regulation of sterol and VAP-A binding by OSBP in the ER, and PI(4)P as an alternate ligand that could be exchanged for sterol in the Golgi apparatus.
Genetic inactivation of the transcription factor GATA-6 in the embryoid body induces massive apoptosis at the early stage of ES cell differentiation. Evidence is provided that BMP-2 is a direct transcription target of GATA-6 and mediates GATA-6-dependent cell survival in concert with endoderm-derived basement membrane.
GATA-6 is a zinc-finger transcription factor essential for early embryogenesis. Ablation of GATA-6 in mice impairs endoderm differentiation and causes apoptosis of epiblast cells. The endoderm defects have been attributed to the loss of HNF4, disabled-2, and GATA-4. However, the mechanisms underlying epiblast apoptosis are unclear. In this study we used mouse embryonic stem cell–derived embryoid bodies (EBs) as a model for peri-implantation development and found that ablation of GATA-6 causes massive apoptosis during EB differentiation. Endoderm grafting experiments and ectopic basement membrane (BM) assembly suggest that both BM and non-BM factors contribute to cell survival. Furthermore, the increased cell death in mutant EBs is accompanied by reduced expression of bone morphogenetic protein 2 (BMP-2). Chromatin immunoprecipitation reveals direct binding of GATA-6 to the Bmp2 promoter. Treatment of the mutant EBs with BMP-2 markedly suppresses apoptosis, whereas stable overexpression of the BMP antagonist noggin or a dominant-negative BMP receptor in normal EBs leads to increased apoptosis. Last, activation of SMAD1/5 by phosphorylation is significantly inhibited in the absence of GATA-6, and this is reversed by exogenous BMP-2. Treatment of normal EBs with SMAD phosphorylation inhibitor increases apoptosis. Collectively these results suggest that GATA-6 promotes cell survival by regulating endoderm expression of BMP-2 and BM during embryonic epithelial morphogenesis.
Mutations in the connexin-43 gap junction protein cause the developmental disease known as oculodentodigital dysplasia. Structure and function approaches are used to demonstrate that the nature of the missense mutation in the amino-terminal domain of connexin-43 governs the mechanism that leads to loss of connexin-43 function.
Mutations in the gene encoding connexin-43 (Cx43) cause the human development disorder known as oculodentodigital dysplasia (ODDD). In this study, ODDD-linked Cx43 N-terminal mutants formed nonfunctional gap junction–like plaques and exhibited dominant-negative effects on the coupling conductance of coexpressed endogenous Cx43 in reference cell models. Nuclear magnetic resonance (NMR) protein structure determination of an N-terminal 23–amino acid polypeptide of wild-type Cx43 revealed that it folded in to a kinked α-helical structure. This finding predicted that W4 might be critically important in intramolecular and intermolecular interactions. Thus we engineered and characterized a W4A mutant and found that this mutant formed a regular, nonkinked α-helix but did not form functional gap junctions. Furthermore, a G2V variant peptide of Cx43 showed a kinked helix that now included V2 interactions with W4, resulting in the G2V mutant forming nonfunctional gap junctions. Also predicted from the NMR structures, a G2S mutant was found to relieve these interactions and allowed the protein to form functional gap junctions. Collectively, these studies suggest that the nature of the mutation conveys loss of Cx43 function by distinctly different mechanisms that are rooted in the structure of the N-terminal region.
The article describes characterization of the cilia protein Ttc26. The data show that Ttc26 is localized in the transition zone of primary cilia and photoreceptor cells. Knockdown of Ttc26 produced defective cilia in murine inner medullary collecting duct 3 cells and ciliogenesis defects in retinal photoreceptor and motile cilia in the pronephros in zebrafish.
In our effort to understand genetic disorders of the photoreceptor cells of the retina, we have focused on intraflagellar transport in photoreceptor sensory cilia. From previous mouse proteomic data we identified a cilia protein Ttc26, orthologue of dyf-13 in Caenorhabditis elegans, as a target. We localized Ttc26 to the transition zone of photoreceptor and to the transition zone of cilia in cultured murine inner medullary collecting duct 3 (mIMCD3) renal cells. Knockdown of Ttc26 in mIMCD3 cells produced shortened and defective primary cilia, as revealed by immunofluorescence and scanning electron microscopy. To study Ttc26 function in sensory cilia in vivo, we utilized a zebrafish vertebrate model system. Morpholino knockdown of ttc26 in zebrafish embryos caused ciliary defects in the pronephric kidney at 27 h postfertilization and distension/dilation of pronephros at 5 d postfertilization (dpf). In the eyes, the outer segments of photoreceptor cells appeared shortened or absent, whereas cellular lamination appeared normal in retinas at 5 dpf. This suggests that loss of ttc26 function prevents normal ciliogenesis and differentiation in the photoreceptor cells, and that ttc26 is required for normal development and differentiation in retina and pronephros. Our studies support the importance of Ttc26 function in ciliogenesis and suggest that screening for TTC26 mutations in human ciliopathies is justified.
Retromer complex mediates the sorting of cargo from the endosome to the Golgi apparatus. At the endosome, recognition of Ypt7 (Rab7) by the Vps35 retromer subunit is essential for the cargo export step of the retromer functional cycle. Retromer also controls Ypt7-regulated fusion dynamics of the late endovacuolar system.
The retromer complex, composed of sorting nexin subunits and a Vps26/Vps29/Vps35 trimer, mediates sorting of retrograde cargo from the endosome to the trans-Golgi network. The retromer trimer subcomplex is an effector of Rab7 (Ypt7 in yeast). Whereas endosome targeting of human retromer has been shown to require Rab7-GTP, targeting of yeast retromer to the endosome is independent of Ypt7-GTP and requires the Vps5 and Vps17 retromer sorting nexin subunits. An evolutionarily conserved amino acid segment within Vps35 is required for Ypt7/Rab7 recognition in vivo by both yeast and human retromer, establishing that Rab recognition is a conserved feature of this subunit. Recognition of Ypt7 by retromer is required for its function in retrograde sorting, and in yeast cells lacking the guanine nucleotide exchange factor for Ypt7, retrograde cargo accumulates in endosomes that are decorated with retromer, revealing an additional role for Rab recognition at the cargo export stage of the retromer functional cycle. In addition, yeast retromer trimer antagonizes Ypt7-regulated organelle tethering and fusion of endosomes/vacuoles via recognition of Ypt7. Thus retromer has dual roles in retrograde cargo export and in controlling the fusion dynamics of the late endovacuolar system.
Dying primary liver, NIH 3T3, and HeLa cells can reverse the advanced stage of apoptosis and survive even after incurring DNA damage. Some surviving cells harbor genetic alterations that result in phenotypic diversity, including oncogenic transformation.
Apoptosis serves as a protective mechanism by eliminating damaged cells through programmed cell death. After apoptotic cells pass critical checkpoints, including mitochondrial fragmentation, executioner caspase activation, and DNA damage, it is assumed that cell death inevitably follows. However, this assumption has not been tested directly. Here we report an unexpected reversal of late-stage apoptosis in primary liver and heart cells, macrophages, NIH 3T3 fibroblasts, cervical cancer HeLa cells, and brain cells. After exposure to an inducer of apoptosis, cells exhibited multiple morphological and biochemical hallmarks of late-stage apoptosis, including mitochondrial fragmentation, caspase-3 activation, and DNA damage. Surprisingly, the vast majority of dying cells arrested the apoptotic process and recovered when the inducer was washed away. Of importance, some cells acquired permanent genetic changes and underwent oncogenic transformation at a higher frequency than controls. Global gene expression analysis identified a molecular signature of the reversal process. We propose that reversal of apoptosis is an unanticipated mechanism to rescue cells from crisis and propose to name this mechanism “anastasis” (Greek for “rising to life”). Whereas carcinogenesis represents a harmful side effect, potential benefits of anastasis could include preservation of cells that are difficult to replace and stress-induced genetic diversity.
Two polarity proteins, partitioning defective 3 homologue (Par3) and mammalian homologues of Drosophila lethal(2)giant larvae (Lgl1/2), antagonize each other in modulating myosin II activation during cell–cell contact formation in Madin-Darby canine kidney cells. Altering the counteraction between Par3 and Lgl1/2 leads to entosis without matrix detachment.
Cell–cell contact formation following cadherin engagement requires actomyosin contraction along the periphery of cell–cell contact. The molecular mechanisms that regulate myosin activation during this process are not clear. In this paper, we show that two polarity proteins, partitioning defective 3 homologue (Par3) and mammalian homologues of Drosophila Lethal (2) Giant Larvae (Lgl1/2), antagonize each other in modulating myosin II activation during cell–cell contact formation in Madin-Darby canine kidney cells. While overexpression of Lgl1/2 or depletion of endogenous Par3 leads to enhanced myosin II activation, knockdown of Lgl1/2 does the opposite. Intriguingly, altering the counteraction between Par3 and Lgl1/2 induces cell–cell internalization during early cell–cell contact formation, which involves active invasion of the lateral cell–cell contact underneath the apical-junctional complexes and requires activation of the Rho–Rho-associated, coiled-coil containing protein kinase (ROCK)–myosin pathway. This is followed by predominantly nonapoptotic cell-in-cell death of the internalized cells and frequent aneuploidy of the host cells. Such effects are reminiscent of entosis, a recently described process observed when mammary gland epithelial cells were cultured in suspension. We propose that entosis could occur without matrix detachment and that overactivation of myosin or unbalanced myosin activation between contacting cells may be the driving force for entosis in epithelial cells.
Both gene knockout and chemical inhibition show that PKCδ is critical for efficient secretion of type I collagen by arterial smooth muscle cells. The data suggest that PKCδ regulates trafficking of collagen I by controlling its exit from the trans-Golgi network through a mechanism involving Cdc42.
Collagen type I is the most abundant component of extracellular matrix in the arterial wall. Mice knocked out for the protein kinase C δ gene (PKCδ KO) show a marked reduction of collagen I in the arterial wall. The lack of PKCδ diminished the ability of arterial smooth muscle cells (SMCs) to secrete collagen I without significantly altering the intracellular collagen content. Moreover, the unsecreted collagen I molecules accumulate in large perinuclear puncta. These perinuclear structures colocalize with the trans-Golgi network (TGN) marker TGN38 and to a lesser degree with cis-Golgi marker (GM130) but not with early endosomal marker (EEA1). Associated with diminished collagen I secretion, PKCδ KO SMCs exhibit a significant reduction in levels of cell division cycle 42 (Cdc42) protein and mRNA. Restoring PKCδ expression partially rescues Cdc42 expression and collagen I secretion in PKCδ KO SMCs. Inhibition of Cdc42 expression or activity with small interfering RNA or secramine A in PKCδ WT SMCs eliminates collagen I secretion. Conversely, restoring Cdc42 expression in PKCδ KO SMCs enables collagen I secretion. Taken together, our data demonstrate that PKCδ mediates collagen I secretion from SMCs, likely through a Cdc42-dependent mechanism.
During monocyte–macrophage differentiation, C/EBPα transcriptionally activates QKI, which in turn represses CSF1R and thus provides negative feedback to C/EBPα-induced macrophage differentiation. This feedback loop should be important in keeping the balance between cell proliferation and differentiation.
Differentiated macrophages are essential for the innate immune system; however, the molecular mechanisms underlying the generation of macrophages remain largely unknown. Here we show that the RNA-binding protein QKI, mainly QKI-5, is transcriptionally activated in the early differentiated monocytic progenitors when CCAAT/enhancer-binding protein (C/EBP) α is expressed. The forced expression of C/EBPα increases the endogenous expression of QKI. Chromatin immunoprecipitation analysis and reporter assays further confirm that C/EBPα activates the transcription of QKI, primarily by binding to the distal C/EBPα-binding site. Blocking the induction of QKI using RNA interference enhances the expression of endogenous CSF1R and facilitates macrophage differentiation. Further study of the mechanism reveals that QKI-5 facilitates the degradation of CSF1R mRNA by interacting with the distal QRE in the 3′ untranslated region. In summary, we show that in committed macrophage progenitors, C/EBPα-activated QKI-5 negatively regulates macrophage differentiation by down-regulating CSF1R expression, forming a negative feedback loop during macrophage differentiation.
Myosin-X, an unconventional myosin that has been studied primarily in fibroblast-like cells, has been shown to have important functions in polarized epithelial cell junction formation, regulation of paracellular permeability, and epithelial morphogenesis.
Myosin-X (Myo10) is an unconventional myosin that localizes to the tips of filopodia and has critical functions in filopodia. Although Myo10 has been studied primarily in nonpolarized, fibroblast-like cells, Myo10 is expressed in vivo in many epithelia-rich tissues, such as kidney. In this study, we investigate the localization and functions of Myo10 in polarized epithelial cells, using Madin-Darby canine kidney II cells as a model system. Calcium-switch experiments demonstrate that, during junction assembly, green fluorescent protein–Myo10 localizes to lateral membrane cell–cell contacts and to filopodia-like structures imaged by total internal reflection fluorescence on the basal surface. Knockdown of Myo10 leads to delayed recruitment of E-cadherin and ZO-1 to junctions, as well as a delay in tight junction barrier formation, as indicated by a delay in the development of peak transepithelial electrical resistance (TER). Although Myo10 knockdown cells eventually mature into monolayers with normal TER, these monolayers do exhibit increased paracellular permeability to fluorescent dextrans. Importantly, knockdown of Myo10 leads to mitotic spindle misorientation, and in three-dimensional culture, Myo10 knockdown cysts exhibit defects in lumen formation. Together these results reveal that Myo10 functions in polarized epithelial cells in junction formation, regulation of paracellular permeability, and epithelial morphogenesis.