Mammalian Cell Systems Research Articles

In vitro reporter assays for screening of chemicals that disrupt androgen signaling.

Endocrine disruptive chemicals (EDCs) modulate hormone signaling and cause developmental and reproductive anomalies. Today, there is a global concern regarding endocrine disruption effects, particularly those mediated by the androgen receptor (AR). Androgen or male hormones are critical for the development and maintenance of male characteristics and numerous EDCs exist in the environment with the potential to disrupt androgen action. The threat is more during critical developmental windows when there is increased sensitivity to these compounds. Timely screening and detection of the EDCs is essential to minimize deleterious effects produced by these toxic chemicals. As a first line of screening, in vitro transcription assays are very useful due to their speed, convenience, and cost effectiveness. In this paper, recent in vitro reporter assays for detecting androgenic or antiandrogenic activity of EDCs have been reviewed. Two important cell systems used for this purpose, namely, the mammalian or yeast cell systems, have been discussed. Use of reporter genes such as bacterial luciferase (lux) and green fluorescent protein (gfp) has significantly improved speed and sensitivity of detection. Also, many of the current reporter assay systems can be used in a high throughput format allowing speedy evaluation of multiple potential EDCs at a lower price.

Reduced folate carrier (RFC) as a predictive marker for response to pemetrexed in advanced non-small cell lung cancer (NSCLC)

RFC is the major transport system in mammalian cells for folate cofactors and antifolate therapeutics. The aim of this study was to assess the predictive value of RFC expression in patients receiving pemetrexed for advanced NSCLC. The study was carried out in a population of 48 patients with advanced NSCLC which have received pemetrexed monotherapy in second and third line. RFC expression was assessed using a two-step model of immunohistochemical staining in paraffin-embedded tissue samples. RFC expression was detected in 16 (33 %) patients. In the global population, the median progression free survival (PFS) and the median overall survival (OS) were 3.3 and 6.5 months respectively. The subgroup of patients with expression of RFC had a tendency to better median PFS (4.5 vs 2.8 months; p = 0.926) and median OS (11.7 vs 4.8; p = 0.150). In patients with adenocarcinoma histology and RFC expression median OS after treatment with pemetrexed was 14.4 months versus 5.0 in those with adenocarcinoma but without RFC expression (p = 0.039). These results suggest the possible relation between RFC expression and response to treatment with antifolates (pemetrexed) independently of the tumor histology. Further studies are required to confirm these results.

A Role for MeCP2 in Switching Gene Activity via Chromatin Unfolding and HP1γ Displacement

Methyl-CpG-binding protein 2 (MeCP2) is generally considered to act as a transcriptional repressor, whereas recent studies suggest that MeCP2 is also involved in transcription activation. To gain insight into this dual function of MeCP2, we assessed the impact of MeCP2 on higher-order chromatin structure in living cells using mammalian cell systems harbouring a lactose operator and reporter gene-containing chromosomal domain to assess the effect of lactose repressor-tagged MeCP2 (and separate MeCP2 domains) binding in living cells. Our data reveal that targeted binding of MeCP2 elicits extensive chromatin unfolding. MeCP2-induced chromatin unfolding is triggered independently of the methyl-cytosine-binding domain. Interestingly, MeCP2 binding triggers the loss of HP1γ at the chromosomal domain and an increased HP1γ mobility, which is not observed for HP1α and HP1β. Surprisingly, MeCP2-induced chromatin unfolding is not associated with transcriptional activation. Our study suggests a novel role for MeCP2 in reorganizing chromatin to facilitate a switch in gene activity.

A simple, robust and highly efficient transient expression system for producing antibodies

Transient expression systems in mammalian cells have become the method of choice for producing research quantities of antibodies. Both the speed and yield of the available transient systems and the natural posttranslational modifications favor these systems above expression in lower eukaryotes, prokaryotes or stable cell lines.We describe an optimized mammalian transient expression system, capable of producing up to 400mg/L of native secreted antibodies in less than a week. The system is composed of commercially available components and is based on expression in the fast growing suspension cell line, FreeStyle™ 293-F (HEK-293F).The method depends on an optimal combination of a gene transfer method, an expression vector and cotransfection with expression enhancing plasmids, encoding the large T antigen of the SV40 virus and the cell cycle inhibitors p21 and p27.Optimization of all components of the expression system, by experimental design techniques, yielded maximal expression levels (including antibody isotypes IgG1, 2, 3, 4 and Fab fragments of various species).Expression volumes were scalable from 0.1ml up to 1.2L in a simple shaker flask system in animal component free, low protein medium, enabling consistent production of relatively high amounts of a large number of native antibodies.

Lafora disease fibroblasts exemplify the molecular interdependence between thioredoxin 1 and the proteasome in mammalian cells

Thioredoxin 1 (Trx1) is a key regulator of cellular redox balance and participates in cellular signaling events. Recent evidence from yeast indicates that members of the Trx family interact with the 20S proteasome, indicating redox regulation of proteasome activity. However, there is little information about the interrelationship of Trx proteins with the proteasome system in mammalian cells, especially in the nucleus. Here, we have investigated this relationship under various cellular conditions in mammalian cells. We show that Trx1 levels and its subcellular localization (cytosol, endoplasmic reticulum, and nucleus) depend on proteasome activity during the cell cycle in NIH3T3 fibroblasts and under stress conditions, when proteasomes are inhibited. In addition, we also studied in these cells how the main cellular antioxidant systems are stimulated when proteasome activity is inhibited. Finally, we describe a reduction in Trx1 levels in Lafora disease fibroblasts and demonstrate that the nuclear colocalization of Trx1 with 20S proteasomes in laforin-deficient cells is altered compared with control cells. Our results indicate a close relationship between Trx1 and the 20S nuclear proteasome and give a new perspective to the study of diseases or physiopathological conditions in which defects in the proteasome system are associated with oxidative stress.

Standardized Transportation of Human Islets: An Islet Cell Resource Center Study of more than 2,000 Shipments

Preservation of cell quality during shipment of human pancreatic islets for use in laboratory research is a crucial, but neglected, topic. Mammalian cells, including islets, have been shown to be adversely affected by temperature changes in vitro and in vivo, yet protocols that control for thermal fluctuations during cell transport are lacking. To evaluate an optimal method of shipping human islets, an initial assessment of transportation conditions was conducted using standardized materials and operating procedures in 48 shipments sent to a central location by eight pancreas-processing laboratories using a single commercial airline transporter. Optimization of preliminary conditions was conducted, and human islet quality was then evaluated in 2,338 shipments pre- and postimplementation of a finalized transportation container and standard operating procedures. The initial assessment revealed that the outside temperature ranged from a mean of -4.6 ± 10.3°C to 20.9 ± 4.8°C. Within-container temperature drops to or below 15°C occurred in 16 shipments (36%), while the temperature was found to be stabilized between 15°C and 29°C in 29 shipments (64%). Implementation of an optimized transportation container and operating procedure reduced the number of within-container temperature drops (≤ 15°C) to 13% (n = 37 of 289 winter shipments), improved the number desirably maintained between 15°C and 29°C to 86% (n = 250), but also increased the number reaching or exceeding 29°C to 1% (n = 2; overall p < 0.0001). Additionally, postreceipt quality ratings of excellent to good improved pre- versus postimplantation of the standardized protocol, adjusting for preshipment purity/viability levels (p < 0.0001). Our results show that extreme temperature fluctuations during transport of human islets, occurring when using a commercial airline transporter for long distance shipping, can be controlled using standardized containers, materials, and operating procedures. This cost-effective and pragmatic standardized protocol for the transportation of human islets can potentially be adapted for use with other mammalian cell systems and is available online at http://iidp.coh.org/sops.aspx.

Anti-apoptotic mechanism of silkworm hemolymph in HeLa cell apoptosis

Silkworm hemolymph (SH) was found to exhibit anti-apoptotic activities in mammalian and insect cell systems. An anti-apoptotic mechanism of SH was investigated in a staurosporine-induced HeLa cell using flow cytometry, caspase assay, Immunoblot, and Immunochemistry. The addition of 5% SH to the medium resulted in lower intracellular activities of caspase-3 and caspase-9 after 0.6μM of staurosporine treatment; however, SH did not directly inhibit the activities of those enzymes. This suggests SH inhibits the event upstream of these caspase activation steps, such as mitochondrial level events. We found from Immunoblot and Immunochemistry that cytochrome c release from the mitochondria was blocked by SH. SH also inhibited Bax translocation to the mitochondria. On the contrary, SH did not block the apoptosis when Bax is not involved in promoting apoptosis. With these results, we propose that SH protects mitochondria from apoptosis signal via blocking Bax translocation, and the subsequent apoptotic events are then inhibited. The inhibition of apoptosis using SH and its components may lead to new approaches for the minimization of cell death during commercial animal cell cultures.

Reduced folate carrier (RFC) as a predictive marker for response to pemetrexed in advanced non-small cell lung cancer (NSCLC).

e19034 Background: RFC is the major transport system in mammalian cells for folate cofactors and antifolate therapeutics. The aim of this study was to assess the predictive value of RFC expression in patients receiving pemetrexed for advanced NSCLC. Methods: The study was carried out in a population of 48 patients with advanced NSCLC which have received pemetrexed monotherapy in second and third line. RFC expression was assessed using a two-step model of immunohistochemical staining in paraffin-embedded tissue samples. Results: RFC expression was detected in 16 (33%) patients. In the global population, the median progression free survival (PFS) and the median overall survival (OS) were 3.3 and 6.5 months respectively. The subgroup of patients with expression of RFC had a tendency to better median PFS (4.5 vs 2.8 months; p=0.926) and median OS (11.7 vs 4.8; p=0.150). In patients with adenocarcinoma histology and RFC expression median OS after treatment with pemetrexed was 14.4 months versus 5.0 in those with adenocarcinoma but without RFC expression (p=0.039). Conclusions: These results suggest the possible relation between RFC expression and response to treatment with antifolates (pemetrexed) independently of the tumor histology. Further studies are required to confirm these results.

The Asymmetrical Structure of Golgi Apparatus Membranes Revealed by In situ Atomic Force Microscope

The Golgi apparatus has attracted intense attentions due to its fascinating morphology and vital role as the pivot of cellular secretory pathway since its discovery. However, its complex structure at the molecular level remains elusive due to limited approaches. In this study, the structure of Golgi apparatus, including the Golgi stack, cisternal structure, relevant tubules and vesicles, were directly visualized by high-resolution atomic force microscope. We imaged both sides of Golgi apparatus membranes and revealed that the outer leaflet of Golgi membranes is relatively smooth while the inner membrane leaflet is rough and covered by dense proteins. With the treatment of methyl-β-cyclodextrin and Triton X-100, we confirmed the existence of lipid rafts in Golgi apparatus membrane, which are mostly in the size of 20 nm –200 nm and appear irregular in shape. Our results may be of significance to reveal the structure-function relationship of the Golgi complex and pave the way for visualizing the endomembrane system in mammalian cells at the molecular level.

Abstract 2917: The role of Methionine sulfoxide reductase A in metastasis of breast cancer.

Abstract The role of oxidative stress in several human diseases is very well established, but the effect of oxidative stress on cancer cells, especially resulting from mitochondrial dysfunction, appears to be quite complex. Warburg first showed that cancer cells have a defect in mitochondrial respiration which explains why these cells have a greater dependency on glycolysis for energy production than normal cells, even in the presence of adequate oxygen. Cancer cells have been reported to be more sensitive to reactive oxygen species (ROS) and are known to have lower levels of several antioxidant enzymes, which are thought to function as tumor suppressors. In support of this are recent findings that indicate increased levels of ROS make cancer cells more aggressive and promote metastasis. However, in contrast there are other studies that indicate elevated levels of antioxidant enzymes drive tumor cells to metastasize and also prolong tumor cell survival. Our goal is to clarify the role of oxidative stress and ROS on cancer cell viability and aggressiveness. The focus will be on an important protein repair and catalytic antioxidant enzyme system, methionine sulfoxide reductase (Msr). The Msr system in mammalian cells (comprised of MsrA, MsrB1, MsrB2, MsrB3) reduces the oxidized methionine residues (Met(O)) in proteins back to methionine. Furthermore, the Msr system plays an important role in scavenging ROS by permitting methionine residues in proteins to function as catalytic antioxidants. MsrA has been studied in most detail. This enzyme plays an important role in protection against increased oxidative stress in a variety of organisms and can affect the life span of yeast and animals. The objective of our study was to determine whether the expression of MsrA is altered during tumor progression and to identify the molecular targets that regulate MsrA. Our preliminary data show that MsrA is downregulated in several types of breast cancer. We have used two lines of human mammary epithelial 21T series that represent different stages of tumor progression from a single patient and compared with metastatic human breast cancer cell line MDA-MB-231. Our data indicates that non-metastatic 21PT has higher protein levels of MsrA compared to both metastatic 21MT-1 and MDA-MB-231 cell lines as evidenced by western blots. MsrA protein levels of nonmetastatic 21PT are much lower than normal epithelial cells. Levels of MsrA activity were higher in nonmetastatic 21PT than metastatic 21MT-1 as determined by enzymatic activity assays. Enzyme activity of MsrA is nearly two fold higher in nonmetastatic 21PT than the MsrA levels in metastatic MDA-MB-231 cells. Our preliminary results also indicate upregulation of oncogenic (c-Myc) protein levels in metastatic lines suggesting an inverse correlation with MsrA. It is therefore likely that MsrA may be an important antioxidant enzyme in breast cancer cells whose regulation drives tumor progression. Citation Format: Shailaja Kesaraju, Xiaoping Wu, Diane Baronas-Lowell, Herbert Weissbach. The role of Methionine sulfoxide reductase A in metastasis of breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2917. doi:10.1158/1538-7445.AM2013-2917

Rheb (Ras Homologue Enriched in Brain)-dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Becomes Indispensable for Cardiac Hypertrophic Growth after Early Postnatal Period

Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb(-/-)). Rheb(-/-) mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb(-/-) was lower than that in the control (Rheb(+/+)) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb(+/+) mice but not in Rheb(-/-) mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb(-/-) hearts during the neonatal period. Rheb(-/-) hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb(-/-) hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb(-/-) mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.

Trans-packaging of human immunodeficiency virus type 1 genome into Gag virus-like particles in Saccharomyces cerevisiae

BackgroundYeast is recognized as a generally safe microorganism and is utilized for the production of pharmaceutical products, including vaccines. We previously showed that expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts released Gag virus-like particles (VLPs) extracellularly, suggesting that the production system could be used in vaccine development. In this study, we further establish HIV-1 genome packaging into Gag VLPs in a yeast cell system.ResultsThe nearly full-length HIV-1 genome containing the entire 5′ long terminal repeat, U3-R-U5, did not transcribe gag mRNA in yeast. Co-expression of HIV-1 Tat, a transcription activator, did not support the transcription. When the HIV-1 promoter U3 was replaced with the promoter for the yeast glyceraldehyde-3-phosphate dehydrogenase gene, gag mRNA transcription was restored, but no Gag protein expression was observed. Co-expression of HIV-1 Rev, a factor that facilitates nuclear export of gag mRNA, did not support the protein synthesis. Progressive deletions of R-U5 and its downstream stem-loop-rich region (SL) to the gag start ATG codon restored Gag protein expression, suggesting that a highly structured noncoding RNA generated from the R-U5-SL region had an inhibitory effect on gag mRNA translation. When a plasmid containing the HIV-1 genome with the R-U5-SL region was coexpressed with an expression plasmid for Gag protein, the HIV-1 genomic RNA was transcribed and incorporated into Gag VLPs formed by Gag protein assembly, indicative of the trans-packaging of HIV-1 genomic RNA into Gag VLPs in a yeast cell system. The concentration of HIV-1 genomic RNA in Gag VLPs released from yeast was approximately 500-fold higher than that in yeast cytoplasm. The deletion of R-U5 to the gag gene resulted in the failure of HIV-1 RNA packaging into Gag VLPs, indicating that the packaging signal of HIV-1 genomic RNA present in the R-U5 to gag region functions similarly in yeast cells.ConclusionsOur data indicate that selective trans-packaging of HIV-1 genomic RNA into Gag VLPs occurs in a yeast cell system, analogous to a mammalian cell system, suggesting that yeast may provide an alternative packaging system for lentiviral RNA.

Molecular Chaperone Disorders: Defective Hsp60 in Neurodegeneration

Chaperonins, a subgroup of molecular chaperones, form ring-shaped structures and assist folding of proteins by enclosing them in their inner cavity. The mitochondrial Hsp60/Hsp10 chaperonin system is essential for cell viability and only a very small number of mutations causing human disease have so far been found that appear to selectively affect neuronal tissues. We here review the knowledge on the mammalian Hsp60/Hsp10 system and discuss evidence and observations, which may explain why this is the case. The Hsp60 mutations shown to be associated with neurodegenerative diseases mildly affect the protein and leave residual function. We present arguments for the notion that the neuron/glia specificity may be due to an effect of Hsp60 deficiency on myelination, a neuron-specific property. The substrates of the Hsp60/Hsp10 system are only poorly defined, but the combination of deficiency of a number of mitochondrial enzymes and proteins that are highly dependent on this system for folding is the likely trigger for deficient myelination. However, a number of experimental observations indicate that Hsp60 may also have roles outside mitochondria and deficiency of Hsp60 due to mutation may also affect myelination via these signaling pathways. Taken together, it appears that mild Hsp60 deficiency primarily affects neuronal and/or glia cells whereas more severe deficiency of Hsp60 would affect all tissues and not be compatible with life. We discuss in the end what approaches may lead to a further understanding of the functions of the Hsp60/Hsp10 system in mammalian cells and thus its role in disease conditions.

Appropriate &amp;lt;i&amp;gt;In Vitro&amp;lt;/i&amp;gt; Methods for Genotoxicity Testing of Silver Nanoparticles

ObjectivesWe investigated the genotoxic effects of 40-59 nm silver nanoparticles (Ag-NPs) by bacterial reverse mutation assay (Ames test), in vitro comet assay and micronucleus (MN) assay. In particular, we directly compared the effect of cytochalasin B (cytoB) and rat liver homogenate (S9 mix) in the formation of MN by Ag-NPs.MethodsBefore testing, we confirmed that Ag-NPs were completely dispersed in the experimental medium by sonication (three times in 1 minute) and filtration (0.2 µm pore size filter), and then we measured their size in a zeta potential analyzer. After that the genotoxicity were measured and especially, S9 mix and with and without cytoB were compared one another in MN assay.ResultsAmes test using Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains revealed that Ag-NPs with or without S9 mix did not display a mutagenic effect. The genotoxicity of Ag-NPs was also evaluated in a mammalian cell system using Chinese hamster ovary cells. The results revealed that Ag-NPs stimulated DNA breakage and MN formation with or without S9 mix in a dose-dependent manner (from 0.01 µg/mL to 10 µg/mL). In particular, MN induction was affected by cytoB.ConclusionsAll of our findings, with the exception of the Ames test results, indicate that Ag-NPs show genotoxic effects in mammalian cell system. In addition, present study suggests the potential error due to use of cytoB in genotoxic test of nanoparticles.

Critical roles of type III phosphatidylinositol phosphate kinase in murine embryonic visceral endoderm and adult intestine

The metabolism of membrane phosphoinositides is critical for a variety of cellular processes. Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P(2)] controls multiple steps of the intracellular membrane trafficking system in both yeast and mammalian cells. However, other than in neuronal tissues, little is known about the physiological functions of PtdIns(3,5)P(2) in mammals. Here, we provide genetic evidence that type III phosphatidylinositol phosphate kinase (PIPKIII), which produces PtdIns(3,5)P(2), is essential for the functions of polarized epithelial cells. PIPKIII-null mouse embryos die by embryonic day 8.5 because of a failure of the visceral endoderm to supply the epiblast with maternal nutrients. Similarly, although intestine-specific PIPKIII-deficient mice are born, they fail to thrive and eventually die of malnutrition. At the mechanistic level, we show that PIPKIII regulates the trafficking of proteins to a cell's apical membrane domain. Importantly, mice with intestine-specific deletion of PIPKIII exhibit diarrhea and bloody stool, and their gut epithelial layers show inflammation and fibrosis, making our mutants an improved model for inflammatory bowel diseases. In summary, our data demonstrate that PIPKIII is required for the structural and functional integrity of two different types of polarized epithelial cells and suggest that PtdIns(3,5)P(2) metabolism is an unexpected and critical link between membrane trafficking in intestinal epithelial cells and the pathogenesis of inflammatory bowel disease.

以杆状病毒为基础的表面展示与基因表达系统

杆状病毒表达载体系统已被广泛应用于真核和原核生物的各种蛋白生产。通过与杆状病毒囊膜蛋白gp64融合表达或与特异性的锚定部位结合，可以将外源蛋白质展示在杆状病毒表面。研究发现，携带哺乳动物启动子的重组杆状病毒能将外源基因传递入哺乳动物细胞，实现外源蛋白的有效表达，表达蛋白的结构更接近天然蛋白。杆状病毒作为一种新型的基因表面展示和基因传递系统，具有广阔的应用前景，本文对杆状病毒作为外源蛋白表面展示技术和作为基因传递载体在哺乳动物细胞中表达外源基因的研究进展进行了综述。 The baculovirus expression vector system has been used extensively to produce numerous proteins originating from both prokaryotic and eukaryotic sources. Through fusing to the entire membrane protein gp64 of baculovirus or combining with special membrane anchor domain, exotic proteins could be expressed on the surface of baculovirus. It was found that baculovirus harboring mammalian expression cassettes could efficiently deliver foreign genes into mammalian cells and successfully express foreign genes. The baculovirus as surface display and gene delivery vector in mammalian cells have extensive prospects. In this article, the progress of baculovirus surface display technology and gene delivery system in mammalian cells was reviewed.

Minimum set of mutations needed to optimize cyan fluorescent proteins for live cell imaging

Cyan fluorescent proteins (CFPs) are widely used as FRET donors in genetically encoded biosensors for live cell imaging. Recently, cyan variants with greatly improved fluorescence quantum yields have been developed by large scale random mutagenesis. We show that the introduction of only two mutations, T65S and H148G, is able to confer equivalent performances on the popular form ECFP, leading to Aquamarine (QY = 0.89, τ(f) = 4.12 ns). Besides an impressive pH stability (pK(1/2) = 3.3), Aquamarine shows a very low general sensitivity to its environment, and undetectable photoswitching reactions. Aquamarine gives efficient and bright expression in different mammalian cell systems, with a long and single exponential intracellular fluorescence lifetime mostly insensitive to the fusion or the subcellular location of the protein. Aquamarine was also able to advantageously replace the CFP donor in the FRET biosensor AKAR for ratiometric measurements of protein kinase A activity. The performances of Aquamarine show that only two rounds of straightforward single point mutagenesis can be a quick and efficient way to optimize the donor properties in FRET-based biosensors.

The GAP Portion of Pseudomonas Aeruginosa Type III Secreted Toxin ExoS Upregulates Total and Surface Levels of Wild Type CFTR

Background: Pseudomonas aeruginosa (PA) infections account for a large percentage of fatal hospital acquired pneumonias. One of the PA Type III secreted toxin (TTST) ExoS, a bifunctional protein with N-terminal GTPase activating protein (GAP) and C-terminal ADP rybosyl transferase (ADPRT) activities, significantly contributes to PA virulence by targeting small molecular weight G-proteins (SMWGP). In this study, we have looked at one of the mechanisms by which the GAP portion of ExoS (ExoS-GAP) mediates cellular toxicity. Methods: The effects of ExoS-GAP on CFTR trafficking were studied in CFBE41o- Kir 2.2 and MDCK cell lines stably expressing CFTR using a transient transfection system. Results: Transient transfection of ExoS-GAP increased the total and surface protein levels of mature wild type CFTR in epithelial cells stably expressing wild type (WT) CFTR. The effect of ExoS-GAP was specific to CFTR in bronchial epithelial cells since it did not affect the total protein levels of Na⁺/K⁺ATPase, another membrane protein. A point mutation in the ExoS GAP domain (R146K), known to disrupt its catalytic GAP activity, abolished the effect of ExoS-GAP on WT CFTR. Lysosomal inhibition studies with Bafilomycin A1 indicate that ExoS-GAP decreased lysosomal degradation of the mature WT CFTR with concomitant increase in the total levels of mature WT CFTR. However, ExoS-GAP did not increase the total protein levels of ∆F508CFTR. Conclusion: The GAP portion of the PA TTST ExoS increases the total and surface levels of wild type CFTR in vitro mammalian cell system. The effect of ExoS-GAP on WT CFTR total protein levels provides new insight into understanding the virulent pathophysiology of PA infections.

Hematopoiesis

Enormous numbers of adult blood cells are constantly regenerated throughout life from hematopoietic stem cells through a series of progenitor stages. Accessibility, robust functional assays, well-established prospective isolation, and successful clinical application made hematopoiesis the classical mammalian stem cell system. Most of the basic concepts of stem cell biology have been defined in this system. At the same time, many long-standing disputes in hematopoiesis research illustrate our still limited understanding. Here we discuss the embryonic development and lifelong maintenance of the hematopoietic system, its cellular components, and some of the hypotheses about the molecular mechanisms involved in controlling hematopoietic cell fates.

Predicting stem cell fate changes by differential cell cycle progression patterns

Stem cell self-renewal, commitment and reprogramming rely on a poorly understood coordination of cell cycle progression and execution of cell fate choices. Using existing experimental paradigms, it has not been possible to probe this relationship systematically in live stem cells in vitro or in vivo. Alterations in stem cell cycle kinetics probably occur long before changes in phenotypic markers are apparent and could be used as predictive parameters to reveal changes in stem cell fate. To explore this intriguing concept, we developed a single-cell tracking approach that enables automatic detection of cell cycle phases in live (stem) cells expressing fluorescent ubiquitylation-based cell-cycle indicator (FUCCI) probes. Using this tool, we have identified distinctive changes in lengths and fluorescence intensities of G1 (red fluorescence) and S/G2-M (green) that are associated with self-renewal and differentiation of single murine neural stem/progenitor cells (NSCs) and embryonic stem cells (ESCs). We further exploited these distinctive features using fluorescence-activated cell sorting to select for desired stem cell fates in two challenging cell culture settings. First, as G1 length was found to nearly double during NSC differentiation, resulting in progressively increasing red fluorescence intensity, we successfully purified stem cells from heterogeneous cell populations by their lower fluorescence. Second, as ESCs are almost exclusively marked by the green (S/G2-M) FUCCI probe due to their very short G1, we substantially augmented the proportion of reprogramming cells by sorting green cells early on during reprogramming from a NSC to an induced pluripotent stem cell state. Taken together, our studies begin to shed light on the crucial relationship between cell cycle progression and fate choice, and we are convinced that the presented approach can be exploited to predict and manipulate cell fate in a wealth of other mammalian cell systems.