http://www.biomedcentral.com/bmccellbiol/ The latest research articles published by BMC Cell Biology 2012-05-30T00:00:00Z Background: Yeast has numerous mechanisms to survive stress. Deletion of myosin type II (myo1Delta) inSaccharomyces cerevisiae results in a cell that has defective cytokinesis. To survive thisgenetically induced stress, this budding yeast up regulates the PKC1 cell wall integritypathway (CWIP). More recently, our work indicated that TOR, another stress signalingpathway, was down regulated in myo1Delta strains. Since negative signaling by TOR is known toregulate PKC1, our objectives in this study were to understand the cross-talk between theTOR and PKC1 signaling pathways and to determine if they share upstream regulators formounting the stress response in myo1Delta strains Results: Here we proved that TORC1 signaling was down regulated in the myo1Delta strain. While ator1Delta mutant strain had increased viability relative to myo1Delta, a combined myo1Deltator1Deltamutant strain showed significantly reduced cell viability. Synthetic rescue of the tor2-21tslethal phenotype was observed in the myo1Delta strain in contrast to the chs2Delta strain, a chitinsynthase II null mutant that also activates the PKC1 CWIP and exhibits cytokinesis defectsvery similar to myo1Delta, where the rescue effect was not observed. We observed two pools ofSlt2p, the final Mitogen Activated Protein Kinase (MAPK) of the PKC1 CWIP; one pool thatis up regulated by heat shock and one that is up regulated by the myo1Delta stress. The cell wallstress sensor WSC1 that activates PKC1 CWIP under other stress conditions was shown to actas a negative regulator of TORC1 in the myo1Delta mutant. Finally, the repression of TORC1was inversely correlated with the activation of PKC1 in the myo1Delta strain. Conclusions: Regulated expression of TOR1 was important in the activation of the PKC1 CWIP in amyo1Delta strain and hence its survival. We found evidence that the PKC1 and TORC1 pathwaysshare a common upstream regulator associated with the cell wall stress sensor WSC1.Surprisingly, essential TORC2 functions were not required in the myo1Delta strain. Byunderstanding how yeast mounts a concerted stress response, one can further designpharmacological cocktails to undermine their ability to adapt and to survive. http://www.biomedcentral.com/1471-2121/13/13 Glorivee Pagán-Mercado Ednalise Santiago-Cartagena Pearl Akamine José Rodríguez-Medina BMC Cell Biology 2012, null:13 2012-05-30T00:00:00Z doi:10.1186/1471-2121-13-13 /content/figures/1471-2121-13-13-toc.gif BMC Cell Biology 1471-2121 ${item.volume} 13 2012-05-30T00:00:00Z PDF Background: The behaviour of tumour cells depends on factors such as genetics and the tumourmicroenvironment. The latter plays a crucial role in normal mammary gland development andalso in breast cancer initiation and progression. Breast cancer tissues tend to be highlydesmoplastic and dense matrix as a pre-existing condition poses one of the highest riskfactors for cancer development. However, matrix influence on tumour cell gene expressionand behaviour such as cell migration is not fully elucidated. Results: We generated high-density (HD) matrices that mimicked tumour collagen content of 20mg/cm3 that were ~14-fold stiffer than low-density (LD) matrix of 1 mg/cm3. Live-cellimaging showed breast cancer cells utilizing cytoplasmic streaming and cell bodycontractility for migration within HD matrix. Cell migration was blocked in the presence ofboth the ROCK inhibitor, Y-27632, and the MMP inhibitor, GM6001, but not by the drugsindividually. This suggests roles for ROCK1 and MMP in cell migration are complicated bycompensatory mechanisms. ROCK1 expression and protein activity, were significantlyupregulated in HD matrix but these were blocked by treatment with a histone deacetylase(HDAC) inhibitor, MS-275. In HD matrix, the inhibition of ROCK1 by MS-275 was indirectand relied upon protein synthesis and Notch1. Inhibition of Notch1 using pooled siRNA orDAPT abrogated the inhibition of ROCK1 by MS-275. Conclusion: Increased matrix density elevates ROCK1 activity, which aids in cell migration via cellcontractility. The upregulation of ROCK1 is epigenetically regulated in an indirect mannerinvolving the repression of Notch1. This is demonstrated from inhibition of HDACs by MS-275, which caused an upregulation of Notch1 levels leading to blockade of ROCK1expression. http://www.biomedcentral.com/1471-2121/13/12 Vanisri Raviraj Sandra Fok Jifei Zhao Hsin-Ya Chien J Guy Lyons Erik Thompson Lilian Soon BMC Cell Biology 2012, null:12 2012-05-14T00:00:00Z doi:10.1186/1471-2121-13-12 /content/figures/1471-2121-13-12-toc.gif BMC Cell Biology 1471-2121 ${item.volume} 12 2012-05-14T00:00:00Z PDF In the Methods section of our original manuscript [J.R.Beach et al. BMC.Cell Biol. 12, (2011)52] under the subheading "Creation of RLC mutants" the third sentence reads "MRLC2 cDNA (gene MYL12B; gene ID 103910) was purchased from ATCC". This sentence is incorrect. The manuscript should read "MRLC1 cDNA (gene MYL9; gene ID 10398) was purchased from ATCC". This correction does not in any way change the conclusions or any of the data in the manuscript. These two redundant genes produce essentially identical proteins, we simply listed the wrong one in the methods section. http://www.biomedcentral.com/1471-2121/13/11 Jordan Beach Lucila Licate James Crish Thomas Egelhoff BMC Cell Biology 2012, null:11 2012-04-18T00:00:00Z doi:10.1186/1471-2121-13-11 /content/figures/1471-2121-13-11-toc.gif BMC Cell Biology 1471-2121 ${item.volume} 11 2012-04-18T00:00:00Z PDF Background: Protein misfolding and subsequent aggregation are hallmarks of several human diseases. The cell has a variety of mechanisms for coping with misfolded protein stress, including ubiquitin-mediated protein degradation. In fact, the presence of ubiquitin at protein aggregates is a common feature of protein misfolding diseases. Ubiquitin conjugating enzymes (UBCs) are part of the cascade of enzymes responsible for the regulated attachment of ubiquitin to protein substrates. The specific UBC used during ubiquitination can determine the type of polyubiquitin chain linkage, which in turn plays an important role in determining the fate of the ubiquitinated protein. Thus, UBCs may serve an important role in the cellular response to misfolded proteins and the fate of protein aggregates. Results: The Q82 strain of C. elegans harbors a transgene encoding an aggregation prone tract of 82 glutamine residues fused to green fluorescent protein (Q82::GFP) that is expressed in the body wall muscle. When measured with time-lapse microscopy in young larvae, the initial formation of individual Q82::GFP aggregates occurs in approximately 58 minutes. This process is largely unaffected by a mutation in the C. elegans E1 ubiquitin activating enzyme. RNAi of ubc-22, a nematode homolog of E2-25 K, resulted in higher pre-aggregation levels of Q82::GFP and a faster initial aggregation rate relative to control. Knockdown of ubc-1 (RAD6 homolog), ubc-13, and uev-1 did not affect the kinetics of initial aggregation. However, RNAi of ubc-13 decreases the rate of secondary growth of the aggregate. This result is consistent with previous findings that aggregates in young adult worms are smaller after ubc-13 RNAi. mCherry::ubiquitin becomes localized to Q82::GFP aggregates during the fourth larval (L4) stage of life, a time point long after most aggregates have formed. FLIP and FRAP analysis indicate that mCherry::ubiquitin is considerably more mobile than Q82::GFP within aggregates. Conclusions: These data indicate that initial formation of Q82::GFP aggregates in C. elegans is not directly dependent on ubiquitination, but is more likely a spontaneous process driven by biophysical properties in the cytosol such as the concentration of the aggregating species. The effect of ubiquitination appears to be most significant in later, secondary aggregate growth. http://www.biomedcentral.com/1471-2121/13/10 Gregory Skibinski Lynn Boyd BMC Cell Biology 2012, null:10 2012-04-11T00:00:00Z doi:10.1186/1471-2121-13-10 /content/figures/1471-2121-13-10-toc.gif BMC Cell Biology 1471-2121 ${item.volume} 10 2012-04-11T00:00:00Z PDF Background: Recent studies have found that p53 and its' associated cell cycle pathways are major inhibitors of human induced pluripotent stem (iPS) cell generation. In the same family as p53 is p73, which shares sequence similarities with p53. However, p73 also has distinct properties of its own, such as two alternative promoters to express transactivation of p73 (TAp73) and N terminal deleted p73 (DNp73). Functionally, TAp73 acts similarly to p53 in tumor suppression. However, DNp73, on the other hand acts as an oncogene to suppress p53 and p73 induced apoptosis. Therefore, how can p73 have opposing roles in human iPS cell generation? Results: Transcription factors, Oct4, Sox2, Klf4 and cMyc (4TF, Yamanaka factors) are used as basal conditions to generate iPS cells. In addition, the factor of DNp73(actually alpha splicing DNp73, DNp73α) is used to generate iPS cells. The experiment found that the addition of DNp73 gene increases human iPS cell generation efficiency by 12.6 folds in comparison to human fibroblast cells transduced with only the basal conditions. Also, iPS cells generated with DNp73 expression are more resistant to in vitro and in vivo differentiation. Conclusions: This study found DNp73, a family member of p53, is also involved in the human iPS cell generation. Specifically, that the involvement of DNp73 generates iPS cells that are more resistant to in vitro and in vivo differentiation. Therefore, this data may prove to be useful in future developmental studies and cancer researches. http://www.biomedcentral.com/1471-2121/13/9 Yi Lin Zuxin Cheng Zhijian Yang Jingui Zheng Tongxiang Lin BMC Cell Biology 2012, null:9 2012-03-26T00:00:00Z doi:10.1186/1471-2121-13-9 /content/figures/1471-2121-13-9-toc.gif BMC Cell Biology 1471-2121 ${item.volume} 9 2012-03-26T00:00:00Z XML Background: Aurora kinases (Aurora-A, B and C) belong to a family of conserved serine/threonine kinases which are key regulators of cell cycle progression. Aurora-A and Aurora-B are expressed in somatic cells and involved in cell cycle regulation while aurora-C is meiotic chromosome passenger protein. As Aurora kinase C is rarely expressed in normal somatic cells and has been found over expressed in many cancer lines. It is suggested that Aurora-C-T191D is not hyperactive mutant.ResultAurora-C-T191D variant form was investigated and compared with wild type. The overexpression of Aurora-C-T191D was observed that it behaves like Aurora-C wild type (aurC-WT). Both Aurora-C-T191D and aurC-WT induce abnormal cell division resulting in centrosome amplification and multinucleation in transiently transfected cells as well as in stable cell lines. Similarly, Aurora-C-T191D and aurC-WT formed foci of colonies when grown on soft agar, indicating that a gain of Aurora-C activity is sufficient to transform cells. Furthermore, we reported that NIH-3 T3 stable cell lines overexpressing Aurora-C-T191D and its wild type partner induced tumour formation when injected into nude mice, demonstrating the oncogenic activity of enzymatically active Aurora kinase C. Interestingly enough tumour aggressiveness was positively correlated with the rate of kinase activity, making Aurora-C a potential anti-cancer therapeutic target. Conclusion: These findings proved that Aurora C-T191D is not hyperactive but is constitutively active mutant. http://www.biomedcentral.com/1471-2121/13/8 Jabbar Khan Sanaullah Khan Sobia Attaullah Ijaz Ali Shahid Khan BMC Cell Biology 2012, null:8 2012-03-26T00:00:00Z doi:10.1186/1471-2121-13-8 /content/figures/1471-2121-13-8-toc.gif BMC Cell Biology 1471-2121 ${item.volume} 8 2012-03-26T00:00:00Z XML Background: Interferon-α (IFN-α) exerts an anti-tumor effect at least through induction of apoptosis in a variety of types including B lymphoma cells. We recently found that IFN-α induced a sustained activation of c-Jun NH2-terminal kinase1 (JNK1), which is implicated in activation of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) promoter. In the present study, we explored upstream component(s) of the prolonged IFN-α-initiated activation of JNK1. Results: IFN-α caused activation of PKC-δ in Daudi B lymphoma cells and myeloma U266 cells, as detected by Western blotting using a monoclonal antibody specific for the phosphorylated form of PKC-δ. The dominant-negative form of mutant PKC-δ (dnPKC-δ) reduced the IFN-α-induced JNK1 activation, TRAIL promoter activity, loss of mitochondrial membrane potential (ΔΨm), and increase in propidium iodide (PI) positive cells. The IFN-α-induced activation of JNK1 and the TRAIL promoter was also attenuated by the PKC-δ inhibitor rottlerin. Moreover, a constitutively active form of mutant PKC-δ enhanced the IFN-α-induced TRAIL promoter activity and loss of ΔΨm in Daudi B lymphoma cells. In addition, IFN-α-induced Ser727 phosphorylation of Stat1 was also abrogated by dnPKC-δ. Conclusions: IFN-α induced JNK1 activation via PKC-δ, leading to upregulation of TRAIL. The interaction of the consequent enhanced TRAIL expression with TRAIL-receptor results in a loss of ΔΨm and increase in PI positive cells. The IFN-α-induced apoptotic events may also be affected by the Ser727-Stat1 induced by PKC-δ-mediated signaling component(s). http://www.biomedcentral.com/1471-2121/13/7 Noriko Yanase Miho Hayashida Yuki Kanetaka Akinori Hoshika Junichiro Mizuguchi BMC Cell Biology 2012, null:7 2012-03-21T00:00:00Z doi:10.1186/1471-2121-13-7 /content/figures/1471-2121-13-7-toc.gif BMC Cell Biology 1471-2121 ${item.volume} 7 2012-03-21T00:00:00Z XML Background: A cholesterol-palmitoyl interaction has been reported to occur in the dimeric interface of the β2-adrenergic receptor crystal structure. We sought to investigate whether a similar phenomenon could be observed with μ-opioid receptor (OPRM1), and if so, to assess the role of cholesterol in this class of G protein-coupled receptor (GPCR) signaling. Results: C3.55(170) was determined to be the palmitoylation site of OPRM1. Mutation of this Cys to Ala did not affect the binding of agonists, but attenuated receptor signaling and decreased cholesterol associated with the receptor signaling complex. In addition, both attenuation of receptor palmitoylation (by mutation of C3.55[170] to Ala) and inhibition of cholesterol synthesis (by treating the cells with simvastatin, a HMG-CoA reductase inhibitor) impaired receptor signaling, possibly by decreasing receptor homodimerization and Gαi2 coupling; this was demonstrated by co-immunoprecipitation, immunofluorescence colocalization and fluorescence resonance energy transfer (FRET) analyses. A computational model of the OPRM1 homodimer structure indicated that a specific cholesterol-palmitoyl interaction can facilitate OPRM1 homodimerization at the TMH4-TMH4 interface. Conclusions: We demonstrate that C3.55(170) is the palmitoylation site of OPRM1 and identify a cholesterol-palmitoyl interaction in the OPRM1 complex. Our findings suggest that this interaction contributes to OPRM1 signaling by facilitating receptor homodimerization and G protein coupling. This conclusion is supported by computational modeling of the OPRM1 homodimer. http://www.biomedcentral.com/1471-2121/13/6 Hui Zheng Elizabeth Pearsall Dow Hurst Yuhan Zhang Ji Chu Yali Zhou Patricia Reggio Horace Loh Ping-Yee Law BMC Cell Biology 2012, null:6 2012-03-19T00:00:00Z doi:10.1186/1471-2121-13-6 Role of palmitoylation in OPRM1 signaling The presence of a palmitoylation site on the μ-opioid receptor (OPRM1) and a cholesterol-palmitoyl interaction in the receptor complex may contribute to OPRM1 signaling by facilitating homodimerization and G protein coupling. /content/figures/1471-2121-13-6-toc.gif BMC Cell Biology 1471-2121 ${item.volume} 6 2012-03-19T00:00:00Z XML Background: The mammalian inner ear contains the organ of Corti which is responsible for the conversion of sound into neuronal signals. This specialised epithelial tissue is the product of a complex developmental process where a common precursor cell type differentiates into the sound transducing hair cells and the non-innervated supporting cells. We hypothesised that integrin proteins, which are involved in cell attachment to extracellular matrix proteins and cellular signalling, play a role in the differentiation process of the precursor inner ear epithelial cells. To test our hypothesis we have utilised a cell line (OC-2) derived from E13 embryonic immortomouse inner ears. In vitro, by switching the incubation temperature from 33°C to 39°C, the OC-2 cells can be induced to differentiate and express hair cells markers, such as Myosin VIIa. The OC-2 cells are thus a useful model system for testing mechanism of hair cells differentiation. Results: We have identified 4 integrin subunits which are expressed in OC-2 cells: α6, αv, β1 and β3. Among these, the relative level of expression of the αv, β1 and β3 subunits increased in a time dependent manner when the cells were exposed to the differentiating temperature of 39°C, most notably so for β3 which was not detectable at 33°C. Treatment of fully differentiated OC-2 cells with siRNA against the four integrin subunits reduced the expression of not only the respective integrin proteins but also of the hair cell marker Myosin VIIa. Conversely over-expression of β3 was sufficient to induce the expression of Myosin VIIa at 33°C. Conclusions: Our data demonstrate that modulation of integrin expression is associated with the differentiation process of the OC-2 cells. This suggests that the maturation of the organ of Corti, from where OC-2 cells are derived, may also depend on changes of gene expression associated with integrin expression. http://www.biomedcentral.com/1471-2121/13/5 Ivan Brunetta Stefano Casalotti Ian Hart Andrew Forge Louise Reynolds BMC Cell Biology 2012, null:5 2012-03-17T00:00:00Z doi:10.1186/1471-2121-13-5 /content/figures/1471-2121-13-5-toc.gif BMC Cell Biology 1471-2121 ${item.volume} 5 2012-03-17T00:00:00Z XML Background: Rab11 and its effector molecule, Rab11-FIP3 (FIP3), associate with recycling endosomes and traffic into the furrow and midbody of cells during cytokinesis. FIP3 also controls recycling endosome distribution during interphase. Here, we examine whether phosphorylation of FIP3 is involved in these activities. Results: We identify four sites of phosphorylation of FIP3 in vivo, S-102, S-280, S-347 and S-450 and identify S-102 as a target for Cdk1-cyclin B in vitro. Of these, we show that S-102 is phosphorylated in metaphase and is dephosphorylated as cells enter telophase. Over-expression of FIP3-S102D increased the frequency of binucleate cells consistent with a role for this phospho-acceptor site in cytokinesis. Mutation of S-280, S-347 or S-450 or other previously identified phospho-acceptor sites (S-488, S-538, S-647 and S-648) was without effect on binucleate cell formation and did not modulate the distribution of FIP3 during the cell cycle. In an attempt to identify a functional role for FIP3 phosphorylation, we report that the change in FIP3 distribution from cytosolic to membrane-associated observed during progression from anaphase to telophase is accompanied by a concomitant dephosphorylation of FIP3. However, the phospho-acceptor sites identified here did not control this change in distribution. Conclusions: Our data thus identify FIP3 as a cell cycle regulated phosphoprotein and suggest dephosphorylation of FIP3 accompanies its translocation from the cytosol to membranes during telophase. S102 is dephosphorylated during telophase; mutation of S102 exerts a modest effect on cytokinesis. Finally, we show that de/phosphorylation of the phospho-acceptor sites identified here (S-102, S-280, S-347 and S-450) is not required for the spatial control of recycling endosome distribution or function. http://www.biomedcentral.com/1471-2121/13/4 Louise Collins Glenn Simon Johanne Matheson Christine Wu M Clare Miller Tetsuhisa Otani Xinzi Yu Shigeo Hayashi Rytis Prekeris Gwyn Gould BMC Cell Biology 2012, null:4 2012-03-08T00:00:00Z doi:10.1186/1471-2121-13-4 /content/figures/1471-2121-13-4-toc.gif BMC Cell Biology 1471-2121 ${item.volume} 4 2012-03-08T00:00:00Z XML