http://www.biomedcentral.com/bmccellbiol/ The latest research articles published by BMC Cell Biology 2012-02-02T00:00:00Z Background: As many invertebrate species have multipotent cells that undergo cell growth and differentiation during regeneration and budding, many unique and interesting homeostatic factors are expected to exist in those animals. However, our understanding of such factors and global mechanisms remains very poor. Single zooids of the tunicate, Polyandrocarpa misakiensis, can give off as many as 40 buds during the life span. Bud development proceeds by means of transdifferentiation of very limited number of cells and tissues. TC14-3 is one of several different but closely related polypeptides isolated from P. misakiensis. It acts as a cytostatic factor that regulates proliferation, adhesion, and differentiation of multipotent cells, although the molecular mechanism remains uncertain. The Polycomb group (PcG) genes are involved in epigenetic control of genomic activity in mammals. In invertebrates except Drosophila, PcG and histone methylation have not been studied so extensively, and genome-wide gene regulation is poorly understood. Results: When Phe65 of TC14-3 was mutated to an acidic amino acid, the resultant mutant protein failed to dimerize. The replacement of Thr69 with Arg69 made dimers unstable. When Glu106 was changed to Gly106, the resultant mutant protein completely lost Ca2+ binding. All these mutant proteins lacked cytostatic activity, indicating the requirement of protein dimerization and calcium for the activity. Polyandrocarpa Eed, a component of PcG, is highly expressed during budding, like TC14-3. When wild-type and mutant TC14-3s were applied in vivo and in vitro to Polyandrocarpa cells, only wild-type TC14-3 could induce Eed without affecting histone methyltransferase gene expression. Eed-expressing cells underwent trimethylation of histone H3 lysine27. PmEed knockdown by RNA interference rescued cultured cells from the growth-inhibitory effects of TC14-3. Conclusion: These results show that in P. misakiensis, the cytostatic activity of TC14-3 is mediated by PmEed and resultant histone modification, and that the gene expression requires both the protein dimerization and Ca2+-binding of TC14-3. This system consisting of a humoral factor, PcG, and histone methylation would contribute to the homeostatic regulation of cell growth and terminal differentiation of invertebrate multipotent cells. http://www.biomedcentral.com/1471-2121/13/3 Kaz Kawamura Kohki Takakura Daigo Mori Kohki Ikeda Akio Nakamura Tomohiko Suzuki BMC Cell Biology 2012, null:3 2012-02-02T00:00:00Z doi:10.1186/1471-2121-13-3 /content/figures/1471-2121-13-3-toc.gif BMC Cell Biology 1471-2121 ${item.volume} 3 2012-02-02T00:00:00Z PDF Background: Erlins are highly conserved proteins associated with lipid rafts within the endoplasmic reticulum (ER). Biochemical studies in mammalian cell lines have shown that erlins are required for ER associated protein degradation (ERAD) of activated inositol-1,4,5-trisphosphate receptors (IP3Rs), implying that erlin proteins might negatively regulate IP3R signalling. In humans, loss of erlin function appears to cause progressive intellectual disability, motor dysfunction and joint contractures. However, it is unknown if defects in IP3R ERAD are the underlying cause of this disease phenotype, whether ERAD of activated IP3Rs is the only function of erlin proteins, and what role ERAD plays in regulating IP3R-dependent processes in the context of an intact animal or embryo. In this study, we characterize the erlin homologue of the nematode Caenorhabditis elegans and examine erlin function in vivo. We specifically set out to test whether C. elegans erlin modulates IP3R-dependent processes, such as egg laying, embryonic development and defecation rates. We also explore the possibility that erlin might play a more general role in the ERAD pathway of C. elegans. Results: We first show that the C. elegans erlin homologue, ERL-1, is highly similar to mammalian erlins with respect to amino acid sequence, domain structure, biochemical properties and subcellular location. ERL-1 is present throughout the C. elegans embryo; in adult worms, ERL-1 appears restricted to the germline. The expression pattern of ERL-1 thus only partially overlaps with that of ITR-1, eliminating the possibility of ERL-1 being a ubiquitous and necessary regulator of ITR-1. We show that loss of ERL-1 does not affect overall phenotype, or alter brood size, embryonic development or defecation cycle length in either wild type or sensitized itr-1 mutant animals. Moreover we show that ERL-1 deficient worms respond normally to ER stress conditions, suggesting that ERL-1 is not an essential component of the general ERAD pathway. Conclusions: Although loss of erlin function apparently causes a strong phenotype in humans, no such effect is seen in C. elegans. C. elegans erlin does not appear to be a ubiquitous major modulator of IP3 receptor activity nor does erlin appear to play a major role in ERAD. http://www.biomedcentral.com/1471-2121/13/2 Maja Hoegg Stephen Robbins James McGhee BMC Cell Biology 2012, null:2 2012-01-23T00:00:00Z doi:10.1186/1471-2121-13-2 /content/figures/1471-2121-13-2-toc.gif BMC Cell Biology 1471-2121 ${item.volume} 2 2012-01-23T00:00:00Z PDF Background: SM22 has long been studied as an actin-associated protein. Interestingly, levels of SM22 are often reduced in tumour cell lines, while they are increased during senescence possibly indicating a role for SM22 in cell fate decisions via its interaction with actin. In this study we aimed to determine whether reducing levels of SM22 could actively contribute to a tumourigenic phenotype. Results: We demonstrate that in REF52 fibroblasts, decreased levels of SM22 disrupt normal actin organization leading to changes in the motile behaviour of cells. Interestingly, SM22 depletion also led to an increase in the capacity of cells to spontaneously form podosomes with a concomitant increase in the ability to invade Matrigel. In PC3 prostate epithelial cancer cells by contrast, where SM22 is undetectable, re-expression of SM22 reduced the ability to invade Matrigel. Furthermore SM22 depleted cells also had reduced levels of reactive oxygen species when under serum starvation stress. Conclusions: These findings suggest that depletion of SM22 could contribute to tumourigenic properties of cells. Reduction in SM22 levels would tend to promote cell survival when cells are under stress, such as in a hypoxic tumour environment, and may also contribute to increases in actin dynamics that favour metastatic potential. http://www.biomedcentral.com/1471-2121/13/1 Oliver Thompson Jeelan Moghraby Kathryn Ayscough Steve Winder BMC Cell Biology 2012, null:1 2012-01-18T00:00:00Z doi:10.1186/1471-2121-13-1 /content/figures/1471-2121-13-1-toc.gif BMC Cell Biology 1471-2121 ${item.volume} 1 2012-01-18T00:00:00Z PDF Background: A characteristic feature of frog reproduction is external fertilization accomplished outside the female's body. Mature fertilization-competent frog eggs are arrested at the meiotic metaphase II with high activity of the key meiotic regulators, maturation promoting factor (MPF) and cytostatic factor (CSF), awaiting fertilization. If the eggs are not fertilized within several hours of ovulation, they deteriorate and ultimately die by as yet unknown mechanism. Results: Here, we report that the vast majority of naturally laid unfertilized eggs of the African clawed frog Xenopus laevis spontaneously exit metaphase arrest under various environmental conditions and degrade by a well-defined apoptotic process within 48 hours after ovulation. The main features of this process include cytochrome c release, caspase activation, ATP depletion, increase of ADP/ATP ratio, apoptotic nuclear morphology, progressive intracellular acidification, and egg swelling. Meiotic exit seems to be a prerequisite for execution of the apoptotic program, since (i) it precedes apoptosis, (ii) apoptotic events cannot be observed in the eggs maintaining high activity of MPF and CSF, and (iii) apoptosis in unfertilized frog eggs is accelerated upon early meiotic exit. The apoptotic features cannot be observed in the immature prophase-arrested oocytes, however, the maturation-inducing hormone progesterone renders oocytes susceptible to apoptosis. Conclusions: The study reveals that naturally laid intact frog eggs die by apoptosis if they are not fertilized. A maternal apoptotic program is evoked in frog oocytes upon maturation and executed after meiotic exit in unfertilized eggs. The meiotic exit is required for execution of the apoptotic program in eggs. The emerging anti-apoptotic role of meiotic metaphase arrest needs further investigation. http://www.biomedcentral.com/1471-2121/12/56 Alexander Tokmakov Sho Iguchi Tetsushi Iwasaki Yasuo Fukami BMC Cell Biology 2011, null:56 2011-12-23T00:00:00Z doi:10.1186/1471-2121-12-56 /content/figures/1471-2121-12-56-toc.gif BMC Cell Biology 1471-2121 ${item.volume} 56 2011-12-23T00:00:00Z XML Background: The human homologue of the Drosophila Discs-large tumor suppressor protein, hDlg, is a multi-domain cytoplasmic protein that localizes to the membrane at intercellular junction sites. At both synaptic junctions and epithelia cell-cell junctions, hDlg is known to recruit several signaling proteins into macromolecular complexes. hDlg is also found at the midbody, a small microtubule-rich structure bridging the two daughter cells during cytokinesis, but its function at this site is not clear. Results: Here we describe the interaction of hDlg with the activated form of MEK2 of the canonical RAF/MEK/ERK pathway, a protein that is found at the midbody during cytokinesis. We show that both proteins localize to a sub-structure of the midbody, the midbody ring, and that the interaction between the PDZ domains of hDlg and the C-terminal portion of MEK2 is dependent on the phosphorylation of MEK2. Finally, we found that E-cadherin also localizes to the midbody and that its expression is required for the isoform-specific recruitment of hDlg, but not activated MEK2, to that structure. Conclusion: Our results suggest that like at other cell-cell junction sites, hDlg is part of a macromolecular complex of structural and signaling proteins at the midbody. http://www.biomedcentral.com/1471-2121/12/55 Suzanne Gaudet Marie-Josee Langlois Robert Lue Nathalie Rivard Alain Viel BMC Cell Biology 2011, null:55 2011-12-20T00:00:00Z doi:10.1186/1471-2121-12-55 /content/figures/1471-2121-12-55-toc.gif BMC Cell Biology 1471-2121 ${item.volume} 55 2011-12-20T00:00:00Z XML Background: Transforming growth factor-beta 1 (TGF-β1) has been implicated in hyperoxia-induced cell death and impaired alveolarization in the developing lung. In addition, the c-JunNH2-terminal kinase (JNK) pathway has been shown to have a role for TGF-β1-mediated effects. We hypothesized that the JNK pathway is an important regulator of hyperoxia-induced pulmonary responses in the developing murine lung. Results: We used cultured human lung epithelial cells, fetal rat lung fibroblasts and a neonatal TGF-β1 transgenic mouse model. We demonstrate that hyperoxia inhibits cell proliferation, activates cell death mediators and causes cell death, and promotes myofibroblast transdifferentiation, in a dose-dependent manner. Except for fibroblast proliferation, the effects were mediated via the JNK pathway. In addition, since we observed increased expression of TGF-β1 by epithelial cells on exposure to hyperoxia, we used a TGF-β1 transgenic mouse model to determine the role of JNK activation in TGF-β1 induced effects on lung development and on exposure to hyperoxia. We noted that, in this model, inhibition of JNK signaling significantly improved the spontaneously impaired alveolarization in room air and decreased mortality on exposure to hyperoxia. Conclusions: When viewed in combination, these studies demonstrate that hyperoxia-induced cell death, myofibroblast transdifferentiation, TGF-β1- and hyperoxia-mediated pulmonary responses are mediated, at least in part, via signaling through the JNK pathway. http://www.biomedcentral.com/1471-2121/12/54 Zhang Li Rayman Choo-Wing Huanxing Sun Angara Sureshbabu Reiko Sakurai Virender Rehan Vineet Bhandari BMC Cell Biology 2011, null:54 2011-12-15T00:00:00Z doi:10.1186/1471-2121-12-54 /content/figures/1471-2121-12-54-toc.gif BMC Cell Biology 1471-2121 ${item.volume} 54 2011-12-15T00:00:00Z XML Background: Embryonic stem cells (ESCs) can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs). However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation. Results: Murine D3-MHC-neor ESCs formed embyroid bodies (EBs) and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin) was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. Conclusions: This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC differentiation protocols within suspension bioreactors demands a more complete understanding of the impacts of shear forces on the regulation of pluripotency and differentiation in pluripotent stem cells. http://www.biomedcentral.com/1471-2121/12/53 Mehdi Shafa Roman Krawetz Yuan Zhang Jerome Rattner Anna Godollei Henry Duff Derrick Rancourt BMC Cell Biology 2011, null:53 2011-12-14T00:00:00Z doi:10.1186/1471-2121-12-53 /content/figures/1471-2121-12-53-toc.gif BMC Cell Biology 1471-2121 ${item.volume} 53 2011-12-14T00:00:00Z XML Background: Phosphorylation of non-muscle myosin II regulatory light chain (RLC) at Thr18/Ser19 is well established as a key regulatory event that controls myosin II assembly and activation, both in vitro and in living cells. RLC can also be phosphorylated at Ser1/Ser2/Thr9 by protein kinase C (PKC). Biophysical studies show that phosphorylation at these sites leads to an increase in the Km of myosin light chain kinase (MLCK) for RLC, thereby indirectly inhibiting myosin II activity. Despite unequivocal evidence that PKC phosphorylation at Ser1/Ser2/Thr9 can regulate myosin II function in vitro, there is little evidence that this mechanism regulates myosin II function in live cells. Results: The purpose of these studies was to investigate the role of Ser1/Ser2/Thr9 phosphorylation in live cells. To do this we utilized phospho-specific antibodies and created GFP-tagged RLC reporters with phosphomimetic aspartic acid substitutions or unphosphorylatable alanine substitutions at the putative inhibitory sites or the previously characterized activation sites. Cell lines stably expressing the RLC-GFP constructs were assayed for myosin recruitment during cell division, the ability to complete cell division, and myosin assembly levels under resting or spreading conditions. Our data shows that manipulation of the activation sites (Thr18/Ser19) significantly alters myosin II function in a number of these assays while manipulation of the putative inhibitory sites (Ser1/Ser2/Thr9) does not. Conclusions: These studies suggest that inhibitory phosphorylation of RLC is not a substantial regulatory mechanism, although we cannot rule out its role in other cellular processes or perhaps other types of cells or tissues in vivo. http://www.biomedcentral.com/1471-2121/12/52 Jordan Beach Lucila Licate James Crish Thomas Egelhoff BMC Cell Biology 2011, null:52 2011-12-02T00:00:00Z doi:10.1186/1471-2121-12-52 /content/figures/1471-2121-12-52-toc.gif BMC Cell Biology 1471-2121 ${item.volume} 52 2011-12-02T00:00:00Z XML Background: Intermediate filaments (IFs) are major components of the mammalian cytoskeleton and expressed in cell-type-specific patterns. Morphological changes during cell differentiation are linked to IF network remodeling. However, little is known concerning the presence and the role of IFs in embryonic stem (ES) cells and during their differentiation. Results: We have examined the expression profile of synemin isoforms in mouse pluripotent ES cells and during their neural differentiation induced by retinoic acid. Using RT-PCR, Western blotting and immunostaining, we show that synemin M is present at both mRNA and protein levels in undifferentiated ES cells as early as pluripotency factor Oct-3/4 and IF keratin 8. Synemin H was produced only in neural precursors when neural differentiation started, concurrently with synemin M, nestin and glial fibrillary acidic protein. However, both synemin H and M were restricted to the progenitor line during the neural differentiation program. Our in vivo analysis also confirmed the expression of synemins H/M in multipotent neural stem cells in the subventricular zone of the adult brain, a neurogenic germinal niche of the mice. Knocking down synemin in ES cells by shRNA lentiviral particles transduction has no influence on expression of Oct4, Nanog and SOX2, but decreased keratin 8 expression. Conclusions: Our study shows a developmental stage specific regulation of synemin isoforms in ES cells and its neural derivatives. These findings represent the first evidence that synemins could potentially be useful markers for distinguishing multipotent ES cells from undifferentiated neural stem cells and more committed progenitor cells. http://www.biomedcentral.com/1471-2121/12/51 Sheila de Souza Martins Onnik Agbulut Nicolas Diguet Jean-Christophe Larcher Bruna Paulsen Stevens Rehen Vivaldo Moura-Neto Denise Paulin Zhenlin Li Zhigang Xue BMC Cell Biology 2011, null:51 2011-11-23T00:00:00Z doi:10.1186/1471-2121-12-51 /content/figures/1471-2121-12-51-toc.gif BMC Cell Biology 1471-2121 ${item.volume} 51 2011-11-23T00:00:00Z XML Background: SOX4 is a developmental transcription factor that is required for differentiation and proliferation in multiple tissues. SOX4 is overexpressed in many human malignancies, but the precise role of SOX4 in cancer progression is still not well understood. Thus, the identification of additional SOX4 binding partners is essential for elucidating the mechanism of SOX4-mediated effects in cancer progression. Results: Here, we have adapted a one-step affinity purification method that enables rapid purification of SOX4 complexes via intracellular biotinylation of the amino-terminus of SOX4 to perform large-scale proteomics analysis. We have discovered that junction plakoglobin (JUP) interacts with SOX4 in both the cytosol and the nucleus and the interaction between SOX4 and plakoglobin is significantly increased when prostate and breast cancer cells are stimulated with WNT3A. Interactions between SOX4 and plakoglobin were further enhanced by the nuclear export inhibitor leptomycin B (LMB), suggesting that plakoglobin promotes nuclear export of SOX4. The SOX4-plakoglobin complex affected the expression of Wnt pathway target genes and SOX4 downstream targets, such as AXIN2, DICER1, and DHX9. In addition, SOX4 DNA binding activity to the promoters of DICER1, AXIN2, DHX9 and SOX4 itself was reduced by conditions that promote SOX4-plakoglobin complex formation. Conditions that enhanced SOX4-plakoglobin interactions resulted in reduced transcriptional activity of β-catenin luciferase reporters. Conclusions: These data suggest that this newly identified interaction between SOX4 and plakoglobin is inhibitory and provides new insights into the role of SOX4 in key pathways in cell proliferation, development, and cancer progression. http://www.biomedcentral.com/1471-2121/12/50 Yu-Heng Lai Jessica Cheng Dongmei Cheng Mattie Feasel Kyle Beste Junmin Peng Asma Nusrat Carlos Moreno BMC Cell Biology 2011, null:50 2011-11-19T00:00:00Z doi:10.1186/1471-2121-12-50 /content/figures/1471-2121-12-50-toc.gif BMC Cell Biology 1471-2121 ${item.volume} 50 2011-11-19T00:00:00Z XML