scholarly journals Brefeldin A-dependent Membrane Tubule Formation Reconstituted In Vitro Is Driven by a Cell Cycle–regulated Microtubule Motor

2000 ◽  
Vol 11 (3) ◽  
pp. 941-955 ◽  
Author(s):  
Alasdair M. Robertson ◽  
Victoria J. Allan
Keyword(s):  
Cell Cycle ◽  
Cultured Cells ◽  
Brefeldin A ◽  
Binding Activity ◽  
Membrane Dynamics ◽  
Dependent Manner ◽  
Tubule Formation ◽  
Early Endosomes

Treatment of cultured cells with brefeldin A (BFA) induces the formation of extensive membrane tubules from the Golgi apparatus,trans-Golgi network, and early endosomes in a microtubule-dependent manner. We have reconstituted this transport process in vitro using Xenopus egg cytosol and a rat liver Golgi-enriched membrane fraction. The presence of BFA results in the formation of an intricate, interconnected tubular membrane network, a process that, as in vivo, is inhibited by nocodazole, the H1 anti-kinesin monoclonal antibody, and by membrane pretreatment with guanosine 5′-O-(3-thiotriphosphate). Surprisingly, membrane tubule formation is not due to the action of conventional kinesin or any of the other motors implicated in Golgi membrane dynamics. Two candidate motors of ∼100 and ∼130 kDa have been identified using the H1 antibody, both of which exhibit motor properties in a biochemical assay. Finally, BFA-induced membrane tubule formation does not occur in metaphase cytosol, and because membrane binding of both candidate motors is not altered after incubation in metaphase compared with interphase cytosol, these results suggest that either the ATPase or microtubule-binding activity of the relevant motor is cell cycle regulated.

scholarly journals Targeting a Lipid Desaturation Enzyme, SCD1, Selectively Eliminates Colon Cancer Stem Cells through the Suppression of Wnt and NOTCH Signaling

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 106
Author(s):  
Yeongji Yu ◽  
Hyejin Kim ◽  
SeokGyeong Choi ◽  
JinSuh Yu ◽  
Joo Yeon Lee ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Notch Signaling ◽  
Cultured Cells ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Dependent Manner ◽  
Lipid Desaturation ◽  
Novel Target

The elimination of the cancer stem cell (CSC) population may be required to achieve better outcomes of cancer therapy. We evaluated stearoyl-CoA desaturase 1 (SCD1) as a novel target for CSC-selective elimination in colon cancer. CSCs expressed more SCD1 than bulk cultured cells (BCCs), and blocking SCD1 expression or function revealed an essential role for SCD1 in the survival of CSCs, but not BCCs. The CSC potential selectively decreased after treatment with the SCD1 inhibitor in vitro and in vivo. The CSC-selective suppression was mediated through the induction of apoptosis. The mechanism leading to selective CSC death was investigated by performing a quantitative RT-PCR analysis of 14 CSC-specific signaling and marker genes after 24 and 48 h of treatment with two concentrations of an inhibitor. The decrease in the expression of Notch1 and AXIN2 preceded changes in the expression of all other genes, at 24 h of treatment in a dose-dependent manner, followed by the downregulation of most Wnt- and NOTCH-signaling genes. Collectively, we showed that not only Wnt but also NOTCH signaling is a primary target of suppression by SCD1 inhibition in CSCs, suggesting the possibility of targeting SCD1 against colon cancer in clinical settings.

The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly

1998 ◽  
Vol 111 (5) ◽  
pp. 557-572 ◽  
Author(s):  
C. Roghi ◽  
R. Giet ◽  
R. Uzbekov ◽  
N. Morin ◽  
I. Chartrain ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Mitotic Spindle ◽  
Cultured Cells ◽  
Dependent Manner ◽  
Egg Extracts ◽  
Pericentriolar Material ◽  
Spindle Microtubules ◽  
Cycle Dependent

By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 ‘invades’ the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.

scholarly journals Mammalian Dynamin-like Protein DLP1 Tubulates Membranes

2001 ◽  
Vol 12 (9) ◽  
pp. 2894-2905 ◽  
Author(s):  
Yisang Yoon ◽  
Kelly R. Pitts ◽  
Mark A. McNiven
Keyword(s):  
Cultured Cells ◽  
Immunogold Labeling ◽  
Wild Type ◽  
Tubule Formation ◽  
Defective Mutant ◽  
Ring Structures ◽  
Membrane Tubules ◽  
Large Gtpases

Dynamins are large GTPases with mechanochemical properties that are known to constrict and tubulate membranes. A recently identified mammalian dynamin-like protein (DLP1) is essential for the proper cellular distribution of mitochondria and the endoplasmic reticulum in cultured cells. In this study, we investigated the ability of DLP1 to remodel membranes similar to conventional dynamin. We found that the expression of a GTPase-defective mutant, DLP1-K38A, in cultured cells led to the formation of large cytoplasmic aggregates. Electron microscopy (EM) of cells expressing DLP1-K38A revealed that these aggregates were comprised of membrane tubules of a consistent diameter. High-magnification EM revealed the presence of many regular striations along individual membrane tubules, and immunogold labeling confirmed the association of DLP1 with these structures. Biochemical experiments with the use of recombinant DLP1 and labeled GTP demonstrated that DLP1-K38A binds but does not hydrolyze or release GTP. Furthermore, the affinity of DLP1-K38A for membrane is increased compared with wild-type DLP1. To test whether DLP1 could tubulate membrane in vitro, recombinant DLP1 was combined with synthetic liposomes and nucleotides. We found that DLP1 protein alone assembled into sedimentable macromolecular structures in the presence of guanosine-5′-O-(3-thio)triphosphate (GTPγS) but not GTP. EM of the GTPγS-treated DLP1 revealed clusters of stacked helical ring structures. When liposomes were included with DLP1, formation of long membrane tubules similar in size to those formed in vivo was observed. Addition of GTPγS greatly enhanced membrane tubule formation, suggesting the GTP-bound form of DLP1 deforms liposomes into tubules as the DLP1-K38A does in vivo. These results provide the first evidence that the dynamin family member, DLP1, is able to tubulate membranes both in living cells and in vitro. Furthermore, these findings also indicate that despite the limited homology to conventional dynamins (35%) these proteins remodel membranes in a similar manner.

scholarly journals CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

2004 ◽  
Vol 279 (44) ◽  
pp. 45887-45896 ◽  
Author(s):  
Mark J. Demma ◽  
Serena Wong ◽  
Eugene Maxwell ◽  
Bimalendu Dasmahapatra
Keyword(s):  
Dna Binding ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Binding Activity ◽  
Mutant P53 ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

CNP gene expression is activated by Wnt signaling and correlates with Wnt4 expression during renal injury

2003 ◽  
Vol 284 (4) ◽  
pp. F653-F662 ◽  
Author(s):  
Kameswaran Surendran ◽  
Theodore C. Simon
Keyword(s):  
Wnt Signaling ◽  
Renal Injury ◽  
Cultured Cells ◽  
Proximal Promoter ◽  
Dependent Manner ◽  
Binding Factor ◽  
Identical Cell ◽  
Renal Tubular

C-type natriuretic peptide (CNP) regulates salt excretion, vascular tone, and fibroblast proliferation and activation. CNP inhibits fibroblast activation in vitro and fibrosis in vivo, but endogenous CNP gene ( Nppc) expression during tissue fibrosis has not been reported. We determined that Nppc is induced in renal tubular epithelia and then in interstitial myofibroblasts after unilateral ureteral obstruction (UUO). Induction of Nppcoccurred in identical cell populations to those in which Wnt4 is induced after renal injury. In addition, Nppc was activated in Wnt4-expressing cells during nephrogenesis. Wnt signaling components β-catenin and T cell factor/lymphoid enhancer binding factor (TCF/LEF) specifically bound to cognate elements in the Nppc proximal promoter. Wnt-4, β-catenin, and LEF-1 activated an Nppc transgene in cultured cells, and transgene activation by Wnt-4 and LEF-1 was dependent on the presence of intact cognate elements. These findings suggest that Wnt-4 stimulates Nppc in a TCF/LEF-dependent manner after renal injury and thus may contribute to limiting renal fibrosis.

scholarly journals Presence of a Poly(A) Binding Protein and Two Proteins with Cell Cycle-Dependent Phosphorylation in Crithidia fasciculata mRNA Cycling Sequence Binding Protein II

2004 ◽  
Vol 3 (5) ◽  
pp. 1185-1197 ◽  
Author(s):  
Bidyottam Mittra ◽  
Dan S. Ray
Keyword(s):  
Cell Cycle ◽  
Binding Protein ◽  
High Specificity ◽  
Binding Activity ◽  
Mrna Levels ◽  
Crithidia Fasciculata ◽  
Target Mrna ◽  
Cycle Dependent

ABSTRACT Crithidia fasciculata cycling sequence binding proteins (CSBP) have been shown to bind with high specificity to sequence elements present in several mRNAs that accumulate periodically during the cell cycle. The first described CSBP has subunits of 35.6 (CSBPA) and 42 kDa (CSBPB). A second distinct binding protein termed CSBP II has been purified from CSBPA null mutant cells, lacking both CSBPA and CSBPB proteins, and contains three major polypeptides with predicted molecular masses of 63, 44.5, and 33 kDa. Polypeptides of identical size were radiolabeled in UV cross-linking assays performed with purified CSBP II and 32P-labeled RNA probes containing six copies of the cycling sequence. The CSBP II binding activity was found to cycle in parallel with target mRNA levels during progression through the cell cycle. We have cloned genes encoding these three CSBP II proteins, termed RBP63, RBP45, and RBP33, and characterized their binding properties. The RBP63 protein is a member of the poly(A) binding protein family. Homologs of RBP45 and RBP33 proteins were found only among the kinetoplastids. Both RBP45 and RBP33 proteins and their homologs have a conserved carboxy-terminal half that contains a PSP1-like domain. All three CSBP II proteins show specificity for binding the wild-type cycling sequence in vitro. RBP45 and RBP33 are phosphoproteins, and RBP45 has been found to bind in vivo specifically to target mRNA containing cycling sequences. The levels of phosphorylation of both RBP45 and RBP33 were found to cycle during the cell cycle.

scholarly journals Di-Leucine Signals Mediate Targeting of Tyrosinase and Synaptotagmin to Synaptic-like Microvesicles within PC12 Cells

1999 ◽  
Vol 10 (11) ◽  
pp. 3979-3990 ◽  
Author(s):  
Anastasiya D. Blagoveshchenskaya ◽  
Eric W. Hewitt ◽  
Daniel F. Cutler
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Protein Traffic ◽  
C Terminus ◽  
Targeting Signal

One pathway in forming synaptic-like microvesicles (SLMV) involves direct budding from the plasma membrane, requires adaptor protein 2 (AP2) and is brefeldin A (BFA) resistant. A second route leads from the plasma membrane to an endosomal intermediate from which SLMV bud in a BFA-sensitive, AP3-dependent manner. Because AP3 has been shown to bind to a di-leucine targeting signal in vitro, we have investigated whether this major class of targeting signals is capable of directing protein traffic to SLMV in vivo. We have found that a di-leucine signal within the cytoplasmic tail of human tyrosinase is responsible for the majority of the targeting of HRP-tyrosinase chimeras to SLMV in PC12 cells. Furthermore, we have discovered that a Met-Leu di-hydrophobic motif within the extreme C terminus of synaptotagmin I supports 20% of the SLMV targeting of a CD4-synaptotagmin chimera. All of the traffic to the SLMV mediated by either di-Leu or Met-Leu is BFA sensitive, strongly suggesting a role for AP3 and possibly for an endosomal intermediate in this process. The differential reduction in SLMV targeting for HRP-tyrosinase and CD4-synaptotagmin chimeras by di-alanine substitutions or BFA treatment implies that different proteins use the two routes to the SLMV to differing extents.

scholarly journals The vacuolar kinase Yck3 maintains organelle fragmentation by regulating the HOPS tethering complex

2005 ◽  
Vol 168 (3) ◽  
pp. 401-414 ◽  
Author(s):  
Tracy J. LaGrassa ◽  
Christian Ungermann
Keyword(s):  
Cellular Membrane ◽  
Membrane Dynamics ◽  
Dependent Manner ◽  
Casein Kinase I ◽  
Functional Protein ◽  
Hypertonic Stress ◽  
Functional Studies ◽  
Vacuole Fusion

The regulation of cellular membrane flux is poorly understood. Yeast respond to hypertonic stress by fragmentation of the normally large, low copy vacuole. We used this phenomenon as the basis for an in vivo screen to identify regulators of vacuole membrane dynamics. We report here that maintenance of the fragmented phenotype requires the vacuolar casein kinase I Yck3: when Yck3 is absent, salt-stressed vacuoles undergo fission, but reassemble in a SNARE-dependent manner, suggesting that vacuole fusion is disregulated. Accordingly, when Yck3 is deleted, in vitro vacuole fusion is increased, and Yck3 overexpression blocks fusion. Morphological and functional studies show that Yck3 modulates the Rab/homotypic fusion and vacuole protein sorting complex (HOPS)-dependent tethering stage of vacuole fusion. Intriguingly, Yck3 mediates phosphorylation of the HOPS subunit Vps41, a bi-functional protein involved in both budding and fusion during vacuole biogenesis. Because Yck3 also promotes efficient vacuole inheritance, we propose that tethering complex phosphorylation is a part of a general, switch-like mechanism for driving changes in organelle architecture.

scholarly journals AMPK directly activates mTORC2 to promote cell survival during acute energetic stress

2019 ◽  
Vol 12 (585) ◽  
pp. eaav3249 ◽  
Author(s):  
Dubek Kazyken ◽  
Brian Magnuson ◽  
Cagri Bodur ◽  
Hugo A. Acosta-Jaquez ◽  
Deqiang Zhang ◽  
...  
Keyword(s):  
Cell Survival ◽  
Cultured Cells ◽  
Kinase Assay ◽  
Dependent Manner ◽  
Downstream Signaling ◽  
Energetic Stress ◽  
Diabetes Drug ◽  
Tumor Suppressive Function

AMP-activated protein kinase (AMPK) senses energetic stress and, in turn, promotes catabolic and suppresses anabolic metabolism coordinately to restore energy balance. We found that a diverse array of AMPK activators increased mTOR complex 2 (mTORC2) signaling in an AMPK-dependent manner in cultured cells. Activation of AMPK with the type 2 diabetes drug metformin (GlucoPhage) also increased mTORC2 signaling in liver in vivo and in primary hepatocytes in an AMPK-dependent manner. AMPK-mediated activation of mTORC2 did not result from AMPK-mediated suppression of mTORC1 and thus reduced negative feedback on PI3K flux. Rather, AMPK associated with and directly phosphorylated mTORC2 (mTOR in complex with rictor). As determined by two-stage in vitro kinase assay, phosphorylation of mTORC2 by recombinant AMPK was sufficient to increase mTORC2 catalytic activity toward Akt. Hence, AMPK phosphorylated mTORC2 components directly to increase mTORC2 activity and downstream signaling. Functionally, inactivation of AMPK, mTORC2, and Akt increased apoptosis during acute energetic stress. By showing that AMPK activates mTORC2 to increase cell survival, these data provide a potential mechanism for how AMPK paradoxically promotes tumorigenesis in certain contexts despite its tumor-suppressive function through inhibition of growth-promoting mTORC1. Collectively, these data unveil mTORC2 as a target of AMPK and the AMPK-mTORC2 axis as a promoter of cell survival during energetic stress.

Imatinib Induced Re-Activation of FoxO3 Transcription Factor in CML Is Responsible for the Induction of a Quiescent Status of CD34 Leukaemic Progenitor Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1090-1090
Author(s):  
Daniela Cilloni ◽  
Cristina Panuzzo ◽  
Francesca Messa ◽  
Francesca Arruga ◽  
Enrico Bracco ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Dna Binding ◽  
Progenitor Cells ◽  
Binding Activity ◽  
Mrna Levels ◽  
Dna Binding Activity ◽  
Cd34 Cells

Abstract The FoxO family of transcription factors is regulated by PI3K/Akt induced phosphorylation resulting in nuclear exclusion and degradation. Nuclear FoxO transcribes proapoptotic molecules and cell cycle inhibitors. In CML cells the TK activity of Bcr-Abl leads to the abnormal activation of downstream effectors including PI3K/Akt. The aim of this study was to investigate the role of FoxO3 in Bcr-Abl induced apoptotic arrest and cell growth and the effect of imatinib (IM) induced re-activation of FoxO3 activity in CML progenitor cells. BM cells were collected from 52 CML patients and 20 healthy donors. The expression level of FoxO3 was tested by RQ-PCR. The protein amount and localization was analyzed by Western blot and immunofluorescence, DNA binding activity was measured by EMSA. In addition, FoxO3 was analyzed in CML primary cells and CD34+ cells after IM incubation. Cell cycle and the expression levels of CD47, which has been demonstrated to increased during progression through the cell cycle and stem cell mobilization, was measured by FACS in CD34+ cell population. In addition K562 cells was transfected with pECE-FoxO3 to clarify FoxO3 effects on cell growth and apoptosis. Finally we used our already set up model of Drosophila melanogaster (Dm) transgenic for human Bcr-Abl to study the pathway leading to FoxO3 inactivation. We found that, despite either FoxO3 mRNA levels or protein amount are similar in CML cells compared to controls, FoxO3 protein is equally distributed in the nucleus and cytoplasm in controls but it is completely cytoplasmatic in CML cells and it enters the nucleus during in vivo IM treatment or in vitro IM incubation. Additionally, FoxO3 DNA binding activity in CML patients is completely absent at diagnosis and reappears after IM treatment. Moreover FoxO3 overexpression in transfected cells results into a 49±9 % reduction of proliferation which was further reduced of 75±5 % after IM incubation. Furthermore, we demonstrated that IM incubation results into the reactivation of FoxO3 in Ph+ CD34+ cells inducing quiescence into this population as demonstrated by the comparison of cell cycle kinetics and by a decreased expression of CD47. Finally, the progeny obtained from the crossbreeding of Bcr-Abl flies and flies transgenic for FoxO showed a rescue of FoxO phenotype demonstrating that FoxO inactivation is Bcr-Abl mediated. Overall, these in vitro and in vivo experiments suggest that FoxO3 is inactivated in CML cells and its delocalization is mainly dependant from Bcr-Abl activity. The antiproliferative activity of IM may be mediated by FoxO3 re-localization. On the other side, FoxO3 re-activation induced by IM results into a quiescence of Bcr-Abl CD34+ progenitor cells, which raises a hypothesis that FoxO3 could play a role in IM resistance. This investigation was conducted by CML Correlative Studies Network (CCSN), TOPS, which is sponsored by Novartis Oncology

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