Regulation of mitochondrial morphology by APC/CCdh1-mediated control of Drp1 stability

Homeostatic maintenance of cellular mitochondria requires a dynamic balance between fission and fusion, and controlled changes in morphology are important for processes such as apoptosis and cellular division. Interphase mitochondria have been described as an interconnected network that fragments as cells enter mitosis, and this mitotic mitochondrial fragmentation is known to be regulated by the dynamin-related GTPase Drp1 (dynamin-related protein 1), a key component of the mitochondrial division machinery. Loss of Drp1 function and the subsequent failure of mitochondrial division during mitosis lead to incomplete cytokinesis and the unequal distribution of mitochondria into daughter cells. During mitotic exit and interphase, the mitochondrial network reforms. Here we demonstrate that changes in mitochondrial dynamics as cells exit mitosis are driven in part through ubiquitylation of Drp1, catalyzed by the APC/C(Cdh1) (anaphase-promoting complex/cyclosome and its coactivator Cdh1) E3 ubiquitin ligase complex. Importantly, inhibition of Cdh1-mediated Drp1 ubiquitylation and proteasomal degradation during interphase prevents the normal G1 phase regrowth of mitochondrial networks following cell division.     

Arabidopsis has two redundant Cullin3 proteins that are essential for embryo development and that interact with RBX1 and BTB proteins to form multisubunit E3 ubiquitin ligase complexes in vivo

Cullin-based E3 ubiquitin ligases play important roles in the regulation of diverse developmental processes and environmental responses in eukaryotic organisms. Recently, it was shown in Schizosaccharomyces pombe, Caenorhabditis elegans, and mammals that Cullin3 (CUL3) directly associates with RBX1 and BTB domain proteins in vivo to form a new family of E3 ligases, with the BTB protein subunit functioning in substrate recognition. Here, we demonstrate that Arabidopsis thaliana has two redundant CUL3 (AtCUL3) genes that are essential for embryo development. Besides supporting anticipated specific AtCUL3 interactions with the RING protein AtRBX1 and representative Arabidopsis proteins containing a BTB domain in vitro, we show that AtCUL3 cofractionates and specifically associates with AtRBX1 and a representative BTB protein in vivo. Similar to the AtCUL1 subunit of the SKP1-CUL1-F-box protein-type E3 ligases, the AtCUL3 subunit of the BTB-containing E3 ligase complexes is subjected to modification and possible regulation by the ubiquitin-like protein Related to Ubiquitin in vivo. Together with the presence of large numbers of BTB proteins with diverse structural features and expression patterns, our data suggest that Arabidopsis has conserved AtCUL3-RBX1-BTB protein E3 ubiquitin ligases to target diverse protein substrates for degradation by the ubiquitin/proteasome pathway.     

The Directed Differentiation of Human iPS Cells into Kidney Podocytes

The loss of glomerular podocytes is a key event in the progression of chronic kidney disease resulting in proteinuria and declining function. Podocytes are slow cycling cells that are considered terminally differentiated. Here we provide the first report of the directed differentiation of induced pluripotent stem (iPS) cells to generate kidney cells with podocyte features. The iPS-derived podocytes share a morphological phenotype analogous with cultured human podocytes. Following 10 days of directed differentiation, iPS podocytes had an up-regulated expression of mRNA and protein localization for podocyte markers including synaptopodin, nephrin and Wilm’s tumour protein (WT1), combined with a down-regulation of the stem cell marker OCT3/4. In contrast to human podocytes that become quiescent in culture, iPS-derived cells maintain a proliferative capacity suggestive of a more immature phenotype. The transduction of iPS podocytes with fluorescent labeled-talin that were immunostained with podocin showed a cytoplasmic contractile response to angiotensin II (AII). A permeability assay provided functional evidence of albumin uptake in the cytoplasm of iPS podocytes comparable to human podocytes. Moreover, labeled iPS-derived podocytes were found to integrate into reaggregated metanephric kidney explants where they incorporated into developing glomeruli and co-expressed WT1. This study establishes the differentiation of iPS cells to kidney podocytes that will be useful for screening new treatments, understanding podocyte pathogenesis, and offering possibilities for regenerative medicine.     

Changing Museum Visitors’ Conceptions of Evolution

We examined whether a single visit to an evolution exhibition contributed to conceptual change in adult (n?=?30), youth, and child (n?=?34) museum visitors?? reasoning about evolution. The exhibition included seven current research projects in evolutionary science, each focused on a different organism. To frame this study, we integrated a developmental model of visitors?? understanding of evolution, which incorporates visitors?? intuitive beliefs, with a model of free-choice learning that includes personal, sociocultural, and contextual variables. Using pre- and post-measures, we assessed how visitors?? causal explanations about biological change, drawn from three reasoning patterns (evolutionary, intuitive, and creationist), were modified as a result of visiting the exhibition. Whatever their age, background beliefs, or prior intuitive reasoning patterns, visitors significantly increased their use of explanations from the evolutionary reasoning pattern across all measures and extended this reasoning across diverse organisms. Visitors also increased their use of one intuitive reasoning pattern, need-based (goal-directed) explanations, which, we argue, may be a step toward evolutionary reasoning. Nonetheless, visitors continued to use mixed reasoning (endorsing all three reasoning patterns) in explaining biological change. The personal, socio-cultural, and contextual variables were found to be related to these reasoning patterns in predictable ways. These findings are used to examine the structure of visitors?? reasoning patterns and those aspects of the exhibition that may have contributed to the gains in museum visitors?? understanding of evolution.     

A novel class of ammonium assimilation mutants of the photosynthetic bacterium Rhodobacter capsulatus

Nitrogen assimilation in Rhodobacter capsulatus has been shown to proceed via the coupled action of glutamine synthetase (GS) and glutamate synthase (GOGAT) with no measurable glutamate dehydrogenase (GDH) present. We have recently isolated a novel class of mutants of R. capsulatus strain B100 that lacks a detectable GOGAT activity but is able to grow at wild type rates under nitrogen-fixing conditions. While NH ₄ ⁺ -supported growth in the mutants was normal under anaerobic/photosynthetic conditions, the growth rate was decreased under aerobic conditions. Ammonium and methylammonium uptake experiments indicated that there was a clear difference in the ammonium assimilatory capabilities in these mutants under aerobic versus anaerobic growth. Regulation of expression of a nifH : : lacZ fusion in these mutants was not impaired. The possible existence of alternative ammonium assimilatory pathways is discussed.     

GENETIC AND ENVIRONMENTAL BASIS OF VARIABLE SEX RATIOS IN LABORATORY STRAINS OF POECILIOPSIS LUCIDA

Skewed sex ratios are common among several species of Poeciliopsis, a viviparous fish from northwestern Mexico. Since previous, unrelated studies from this laboratory (Angus and Schultz, 1983) suggested that deviation from a 1:1 sex ratio might be influenced by temperature, two inbred strains of P. lucida were tested for temperature-dependent sex determination by comparing sex ratios of offspring from pregnant females held at different water temperatures. Different sex ratios were produced by the two strains at the same temperature: one strain produced almost all-male offspring at 30°C and female-biased sex ratios at 24°C, while the other strain produced a 1:1 sex ratio at both temperatures. At intermediate temperatures, the labile strain produced sequentially fewer males with decreasing temperatures. The other strain produced a consistent sex ratio regardless of temperature. Poeciliopsis lucida apparently has a genetic polymorphism for temperature-influenced sex determination. An hypothesis is offered for the evolutionary origin of environmental sex determination.     

Downregulation of Rap1GAP in Human Tumor Cells Alters Cell/Matrix and Cell/Cell Adhesion

Rap1GAP expression is decreased in human tumors. The significance of its downregulation is unknown. We show that Rap1GAP expression is decreased in primary colorectal carcinomas. To elucidate the advantages conferred on tumor cells by loss of Rap1GAP, Rap1GAP expression was silenced in human colon carcinoma cells. Suppressing Rap1GAP induced profound alterations in cell adhesion. Rap1GAP-depleted cells exhibited defects in cell/cell adhesion that included an aberrant distribution of adherens junction proteins. Depletion of Rap1GAP enhanced adhesion and spreading on collagen. Silencing of Rap expression normalized spreading and restored E-cadherin, β-catenin, and p120-catenin to cell/cell contacts, indicating that unrestrained Rap activity underlies the alterations in cell adhesion. The defects in adherens junction protein distribution required integrin signaling as E-cadherin and p120-catenin were restored at cell/cell contacts when cells were plated on poly-l-lysine. Unexpectedly, Src activity was increased in Rap1GAP-depleted cells. Inhibition of Src impaired spreading and restored E-cadherin at cell/cell contacts. These findings provide the first evidence that Rap1GAP contributes to cell/cell adhesion and highlight a role for Rap1GAP in regulating cell/matrix and cell/cell adhesion. The frequent downregulation of Rap1GAP in epithelial tumors where alterations in cell/cell and cell/matrix adhesion are early steps in tumor dissemination supports a role for Rap1GAP depletion in tumor progression.Mammalian Rap proteins Rap1a/b and Rap2a/b/c are members of the Ras superfamily of small GTPases. Rap proteins are active when bound to GTP and inactive when bound to GDP. Cellular Rap activity is regulated by the concerted action of guanine nucleotide exchange factors that activate Rap (RapGEFs) and Rap-specific GTPase-activating proteins (RapGAPs) that inactivate Rap (reviewed in reference 10). The Rap1GAP family is composed of several members, including Rap1GAP, Rap1GAPII, Spa-1/SIPA1, and E6TP1/SIPA1L1. Several lines of evidence suggest that RapGAPs function as tumor and/or invasion suppressors. Downregulation of E6TP1 by human papillomavirus protein E6 contributes to cervical cancer (20, 21), and Spa-1 deficiency in mice induces a spectrum of myelodysplastic disorders similar to chronic myelogenous leukemia (26). The SPA1 gene was identified as a candidate for the metastasis efficiency modifier locus in mice (38). Although the relevance of this observation to humans is not yet clear, single-nucleotide polymorphisms in the SPA1 gene in human breast tumors have been associated with lymph node involvement and poor survival (15). Intriguingly, Spa-1 interacts with Brd4 (18) and Rrp-1b (13), the protein products of genes associated with patterns of extracellular matrix protein gene expression characteristic of metastatic tumors (14).The RAP1GAP gene maps to 1p35-36, a chromosomal region subject to copy number alterations in human tumors (36, 49). Rap1GAP protein levels are decreased in pancreatic adenocarcinomas (53), papillary thyroid carcinomas (37, 47, 57), and melanomas (56). Rap1GAP downregulation has been shown to arise as a consequence of proteasomal degradation (46), loss of heterozygosity (37, 53), and promoter methylation (56, 57). Mutations of unknown significance in RAP1GAP have been identified in breast cancer (42). Although downregulation of Rap1GAP is frequent in human tumors, the functional significance of decreased Rap1GAP expression is unknown. Up to now, studies assessing the role of Rap1GAP in tumor cells have relied exclusively on overexpression experiments. Overexpression of Rap1GAP in oropharyngeal squamous cell (54) and pancreatic (53) carcinoma lines impaired tumor formation in mouse xenograft models. In vitro, overexpression of Rap1GAP impaired tumor cell proliferation (34, 47, 53, 54, 56) and enhanced apoptosis (34, 53, 56). In some instances, overexpression of Rap1GAP inhibited tumor cell migration and invasion (3, 47, 53, 56), while in others, it enhanced invasion (34). While these studies provide insight into cellular processes that can be deregulated by overexpression, they do not assess the significance of depletion of endogenous Rap1GAP in human tumors.Colorectal cancer (CRC) is one of the leading causes of cancer deaths worldwide. The majority of CRC deaths arise as a consequence of distant metastases, most frequently to the liver. While the genetic basis of CRC is well understood (19, 48), less is known about the events that trigger the transition to metastatic disease. We report that Rap1GAP is highly expressed in normal colonic epithelium and that its expression is profoundly decreased in primary colorectal carcinomas. As one strategy to assess the significance of Rap1GAP depletion, the expression of Rap1GAP was silenced in human colon carcinoma cells. Silencing of Rap1GAP induced marked increases in Rap1 and Rap2 activity, the first evidence that Rap1GAP is an essential negative regulator of Rap GTPases in colon cancer. Rap1 regulates inside-out signaling through integrins (reviewed in references 8, 9, and 11) and is a target of outside-in signaling via cadherins (reviewed in reference 30). Downregulation of Rap1GAP induced profound alterations in cell/matrix and cell/cell adhesion. Suppressing Rap1GAP expression enhanced adhesion and spreading on collagen. Unexpectedly, based on the role of Rap1 in promoting cell/cell adhesion, silencing of Rap1GAP impaired cell/cell adhesion. These findings demonstrate a requirement for regulated Rap activity in the maintenance of epithelial cell structure and demonstrate a heretofore unappreciated role for Rap1GAP in the regulation of cell/cell adhesion. As the dissemination of tumor cells requires the weakening of cell/cell adhesion and an enhanced ability to adhere to collagen-rich interstitial matrices, our studies identify a potential mechanism through which loss of Rap1GAP contributes to tumor progression.     

Patency of the preterm fetal ductus arteriosus is regulated by endothelial nitric oxide synthase and is independent of vasa vasorum in the mouse

Patency of the fetal ductus arteriosus (DA) is maintained in an environment of low relative oxygen tension and a preponderance of vasodilating forces. In addition to prostaglandins, nitric oxide (NO), a potent vasodilator in the pulmonary and systemic vasculatures, has been implicated in regulation of the fetal DA. To further define the contribution of NO to DA patency, the expression and function of NO synthase (NOS) isoforms were examined in the mouse DA on days 17-19 of pregnancy and after birth. Our results show that endothelial NOS (eNOS) is the predominant isoform expressed in the mouse DA and is localized in the DA endothelium by in situ hybridization. Despite rapid constriction of the DA after birth, eNOS expression levels were unchanged throughout the fetal and postnatal period. Pharmacological inhibition of prostaglandin vs. NO synthesis in vivo showed that the preterm fetal DA on day 16 is more sensitive to NOS inhibition than the mature fetal DA on day 19, whereas prostaglandin inhibition results in marked DA constriction on day 19 but minimal effects on the day 16 DA. Combined prostaglandin and NO inhibition caused additional DA constriction on day 16. The contribution of vasa vasorum to DA regulation was also examined. Immunoreactive platelet endothelial cell adhesion molecule and lacZ tagged FLK1 localized to DA endothelial cells but revealed the absence of vasa vasorum within the DA wall. Similarly, there was no evidence of vasa vasorum by vascular casting. These studies indicate that eNOS is the primary source of NO in the mouse DA and that vasomotor tone of the preterm fetal mouse DA is regulated by eNOS-derived NO and is potentiated by prostaglandins. In contrast to other species, mechanisms for DA patency and closure appear to be independent of any contribution of the vasa vasorum.     

Small within-day increases in temperature affects boldness and alters personality in coral reef fish

Consistent individual differences in behaviour, termed personality, are common in animal populations and can constrain their responses to ecological and environmental variation, such as temperature. Here, we show for the first time that normal within-daytime fluctuations in temperature of less than 3°C have large effects on personality for two species of juvenile coral reef fish in both observational and manipulative experiments. On average, individual scores on three personality traits (PTs), activity, boldness and aggressiveness, increased from 2.5- to sixfold as a function of temperature. However, whereas most individuals became more active, aggressive and bold across temperature contexts (were plastic), others did not; this changed the individual rank order across temperatures and thus altered personality. In addition, correlations between PTs were consistent across temperature contexts, e.g. fish that were active at a given temperature also tended to be both bold and aggressive. These results (i) highlight the importance of very carefully controlling for temperature when studying behavioural variation among and within individuals and (ii) suggest that individual differences in energy metabolism may contribute to animal personality, given that temperature has large direct effects on metabolic rates in ectotherms.     

A Confocal microscopic study on colony morphology and sporulation of Bacillus sp.

Hexavalent chromium Cr(VI) is one of the dominant oxidation states of chromium that exist in the environment and is highly toxic to all forms of life. In the present study, we employ a confocal laser scanning microscope (CLSM) and investigate the effect of Cr(VI) on colony morphology of a Bacillus sp. isolated from soil exposed to tannery effluent. The colonies grown at chromium concentrations, control and 100 ppm are found to be opaque and beyond 200 ppm the colonies were translucent thus exhibiting phase variation. CLSM studies on colonies grown on control plates showed significant increase in height and in biovolume as a function of time whereas, the translucent colonies showed very little change in height and biovolume corresponding to the colony growth. Exopolymeric substance (EPS) content of translucent colonies was lesser than that of opaque colonies, indicating that EPS also plays a role in the observed phenomenon of phase variation. Studies on the effect of Cr(VI) on spore formation showed that Cr(VI) concentrations up to 100 ppm favored spore formation, while concentrations beyond 100 ppm showed a steady decline in spore formation.