Seeing gene expression in cells: the future of structural biology

Although much is known about the machinery that executes fundamental processes of gene expression in cells, much also remains to be learned about how that machinery works. A recent paper by O’Reilly et al. reports a major step forward in the direct visualization of central dogma processes at submolecular resolution inside bacterial cells frozen in a native state. The essential methodologies involved are cross-linking mass spectrometry (CLMS) and cryo-electron tomography (cryo-ET). In-cell CLMS provides in vivo protein interaction maps. Cryo-ET allows visualization of macromolecular complexes in their native environment. These methods have been integrated by O’Reilly et al. to describe a dynamic assembly in situ between a transcribing RNA polymerase (RNAP) and a translating ribosome – a complex known as the expressome – in the model bacterium Mycoplasma pneumoniae 1 . With the application of improved data processing and classification capabilities, this approach has allowed unprecedented insights into the architecture of this molecular assembly line, confirming the existence of a physical link between RNAP and the ribosome and identifying the transcription factor NusA as the linking molecule, as well as making it possible to see the structural effects of drugs that inhibit either transcription or translation. The work provides a glimpse into the future of integrative structural cell biology and can serve as a roadmap for the study of other molecular machineries in their native context.

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Cell biology of transcription and pre-mRNA splicing: nuclear architecture meets nuclear function

Journal of Cell Science ◽

10.1242/jcs.113.11.1841 ◽

2000 ◽

Vol 113 (11) ◽

pp. 1841-1849 ◽

Cited By ~ 13

Author(s):

T. Misteli

Keyword(s):

Gene Expression ◽

Cell Nucleus ◽

Cell Biology ◽

Nuclear Architecture ◽

Molecular Techniques ◽

Mrna Splicing ◽

Cellular Organization ◽

Control Of Gene Expression ◽

Expression Of Genes

Gene expression is a fundamental cellular process. The basic mechanisms involved in expression of genes have been characterized at the molecular level. A major challenge is now to uncover how transcription, RNA processing and RNA export are organized within the cell nucleus, how these processes are coordinated with each other and how nuclear architecture influences gene expression and regulation. A significant contribution has come from cell biological approaches, which combine molecular techniques with microscopy methods. These studies have revealed that the mammalian cell nucleus is a complex but highly organized organelle, which contains numerous subcompartments. I discuss here how two essential nuclear processes - transcription and pre-mRNA splicing - are spatially organized and coordinated in vivo, and how this organization might contribute to the control of gene expression. The dynamic nature of nuclear proteins and compartments indicates a high degree of plasticity in the cellular organization of nuclear functions. The cellular organization of transcription and splicing suggest that the morphology of nuclear compartments is largely determined by the activities of the nucleus.

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Environment and vascular bed origin influence differences in endothelial transcriptional profiles of coronary and iliac arteries

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00002.2010 ◽

2010 ◽

Vol 299 (3) ◽

pp. H837-H846 ◽

Cited By ~ 30

Author(s):

Kelley A. Burridge ◽

Morton H. Friedman

Keyword(s):

Gene Expression ◽

Cell Biology ◽

Dna Microarrays ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Local Environment ◽

Epigenetic Mechanism ◽

Transcriptional Profiles ◽

Vascular Bed

Atherosclerotic plaques tend to form in the major arteries at certain predictable locations. As these arteries vary in atherosusceptibility, interarterial differences in endothelial cell biology are of considerable interest. To explore the origin of differences observed between typical atheroprone and atheroresistant arteries, we used DNA microarrays to compare gene expression profiles of harvested porcine coronary (CECs) and iliac artery endothelial cells (IECs) grown in static culture out to passage 4. Fewer differences were observed between the transcriptional profiles of CECs and IECs in culture compared with in vivo, suggesting that most differences observed in vivo were due to distinct environmental cues in the two arteries. One-class significance of microarrays revealed that most in vivo interarterial differences disappeared in culture, as fold differences after passaging were not significant for 85% of genes identified as differentially expressed in vivo at 5% false discovery rate. However, the three homeobox genes, HOXA9, HOXA10, and HOXD3, remained underexpressed in coronary endothelium for all passages by at least nine-, eight-, and twofold, respectively. Continued differential expression, despite removal from the in vivo environment, suggests that primarily heritable or epigenetic mechanism(s) influences transcription of these three genes. Quantitative real-time polymerase chain reaction confirmed expression ratios for seven genes associated with atherogenesis and over- or underexpressed by threefold in CECs relative to IECs. The present study provides evidence that both local environment and vascular bed origin modulate gene expression in arterial endothelium. The transcriptional differences observed here may provide new insights into pathways responsible for coronary artery susceptibility.

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Unique Effects of p53−/− Leukemic Cells On Mesenchymal Stromal Cell Gene Expression Profile in Vitro

Blood ◽

10.1182/blood.v120.21.3468.3468 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3468-3468

Author(s):

Xiaoyang Ling ◽

Ye Chen ◽

Peter P. Ruvolo ◽

Vivian Ruvolo ◽

Zhiqiang Wang ◽

...

Keyword(s):

Gene Expression ◽

Cell Biology ◽

Stromal Cell ◽

Conflicts Of Interest ◽

Leukemic Cells ◽

Bone Marrow Niche ◽

In Vivo Experiments ◽

Cell Gene Expression

Abstract Abstract 3468 Mesenchymal stromal cells (MSC) participate in the generation of the microenvironmental bone marrow niche which protects normal and leukemic stem cells from injuries, including chemotherapy. MSC produce numerous factors that aid in this function; however, little is known about how leukemic cells affect MSC. In this study, paired murine AML cells, MLL/ENL/FIT3-ITD/p53−/− and MLL/ENL/FIT3-ITD/p53wt, originally derived from C57BL/6 mice (Zuber et al. Genes & Dev. 2009), were co-cultured with MSC from the same strain. After 48 hrs, MSC were isolated by FACS sorting using CD45−/PDGFr+ as markers. Total RNA was profiled on Illumina WG6 mouse whole-genome bead arrays by standard procedures. The significance analysis of microarrays (SAM) method identified 429 differentially-expressed genes (DEG) whose expression in MSC differed significantly (false discovery rate, 10%) in co-cultures with p53−/− (C78) vs. p53wt (C147) leukemic cells. Differences in these DEG were highly consistent in replicates (Figure 1). The results demonstrate that: 1) p53 status (p53−/− vs. p53wt) of AML cells affects GEP patterns in co-cultured MSC. Comparison of the GEP in MSC co-cultured with p53−/− (78) or p53wt (147) (Fig 1) identified the following 5 genes that showed the most significant differences (up- or down-regulated): up-regulated: WNT16, WNT5, IGFBp5, GCNT1, ATP1B1; down-regulated: NOS2, DCN, CCL7, CCL2, CAR9, CCL4. These were selected for qPCR validation, and the results confirmed the array data. In addition, immunohistochemical staining showed that WNT16 was up-regulated in MSC co-cultured with p53wt leukemic cells. In addition, CXCL5 was found up-regulated in MSC co-cultured with p53−/− leukemic cells. These results were consistent with the GEP data. 2) Leukemic cells alter MSC Signaling proteins in vitro: Western blotting showed that Stat3, Akt, PTEN, CXCL5 and HIF-1α were up- regulated in MSC co-cultured with p53−/− leukemic cells as compared to p53wt leukemic cells (48 hrs). Additional analyses showed that the downstream targets of HIF-1α, VEGFa and VEGFc, but not VEGFb, were up-regulated. Taken together, these results suggest that 1) leukemic cells with different p53 genetic background co-cultured with normal MSC have profoundly differential effects on GEP of normal MSC; 2) MSC co-cultured with p53−/− leukemic cells resulted in increased levels of onco-proteins such as Akt and HIF-1α when compared to MSC co-cultured with p53wt leukemic cells. Results suggest, for the first time, that the genetics of leukemic cells determines gene expression in co-cultured MSC. In vivo experiments are in progress to provide in vivo evidence for the existence of a novel model of leukemia-stroma interactions where the genetics of the tumor cell impacts stromal cell biology. Disclosures: No relevant conflicts of interest to declare.

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The future of mammary stem cell biology: the power of in vivo transplants

Breast Cancer Research ◽

10.1186/bcr1986 ◽

2008 ◽

Vol 10 (3) ◽

Cited By ~ 13

Author(s):

Geoffrey J Lindeman ◽

Jane E Visvader ◽

Matthew J Smalley ◽

Connie J Eaves

Keyword(s):

Stem Cell ◽

Cell Biology ◽

Mammary Stem Cell ◽

Stem Cell Biology ◽

The Future ◽

Mammary Stem

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New Twists and Turns in Bacterial Locomotion and Signal Transduction

Journal of Bacteriology ◽

10.1128/jb.00439-19 ◽

2019 ◽

Vol 201 (20) ◽

Cited By ~ 2

Author(s):

Kylie J. Watts ◽

Ady Vaknin ◽

Clay Fuqua ◽

Barbara I. Kazmierczak

Keyword(s):

Signal Transduction ◽

De Novo ◽

Electron Tomography ◽

Bacterial Cells ◽

Research Areas ◽

Flagellar Motors ◽

Two Component ◽

Two Component Signal Transduction

ABSTRACT Prokaryotic organisms occupy the most diverse set of environments and conditions on our planet. Their ability to sense and respond to a broad range of external cues remain key research areas in modern microbiology, central to behaviors that underlie beneficial and pathogenic interactions of bacteria with multicellular organisms and within complex ecosystems. Advances in our understanding of the one- and two-component signal transduction systems that underlie these sensing pathways have been driven by advances in imaging the behavior of many individual bacterial cells, as well as visualizing individual proteins and protein arrays within living cells. Cryo-electron tomography continues to provide new insights into the structure and function of chemosensory receptors and flagellar motors, while advances in protein labeling and tracking are applied to understand information flow between receptor and motor. Sophisticated microfluidics allow simultaneous analysis of the behavior of thousands of individual cells, increasing our understanding of how variance between individuals is generated, regulated, and employed to maximize fitness of a population. In vitro experiments have been complemented by the study of signal transduction and motility in complex in vivo models, allowing investigators to directly address the contribution of motility, chemotaxis, and aggregation/adhesion on virulence during infection. Finally, systems biology approaches have demonstrated previously uncharted areas of protein space in which novel two-component signal transduction pathways can be designed and constructed de novo. These exciting experimental advances were just some of the many novel findings presented at the 15th Bacterial Locomotion and Signal Transduction conference (BLAST XV) in January 2019.

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Electron tomography of cells

Quarterly Reviews of Biophysics ◽

10.1017/s0033583511000102 ◽

2011 ◽

Vol 45 (1) ◽

pp. 27-56 ◽

Cited By ~ 102

Author(s):

Lu Gan ◽

Grant J. Jensen

Keyword(s):

Electron Microscope ◽

Cell Biology ◽

3D Imaging ◽

Imaging Technique ◽

Electron Tomography ◽

Three Dimensional ◽

Molecular Organization ◽

Biological Structure ◽

Structural Cell ◽

Projection Images

AbstractThe electron microscope has contributed deep insights into biological structure since its invention nearly 80 years ago. Advances in instrumentation and methodology in recent decades have now enabled electron tomography to become the highest resolution three-dimensional (3D) imaging technique available for unique objects such as cells. Cells can be imaged either plastic-embedded or frozen-hydrated. Then the series of projection images are aligned and back-projected to generate a 3D reconstruction or ‘tomogram’. Here, we review how electron tomography has begun to reveal the molecular organization of cells and how the existing and upcoming technologies promise even greater insights into structural cell biology.

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Sphingosine kills bacteria by binding to cardiolipin

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.012325 ◽

2020 ◽

Vol 295 (22) ◽

pp. 7686-7696 ◽

Cited By ~ 3

Author(s):

Rabea Verhaegh ◽

Katrin Anne Becker ◽

Michael J. Edwards ◽

Erich Gulbins

Keyword(s):

Plasma Membrane ◽

Bactericidal Activity ◽

Cell Biology ◽

Plasma Membranes ◽

Central Element ◽

Lipid Binding ◽

Bacterial Cells ◽

Bacterial Killing

Sphingosine is a long-chain sphingoid base that has been shown to have bactericidal activity against many pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. We have previously demonstrated that sphingosine is present in nasal, tracheal, and bronchial epithelial cells and constitutes a central element of the defense of the airways against bacterial pathogens. Here, using assorted lipid-binding and cell biology assays, we demonstrate that exposing P. aeruginosa and S. aureus cells to sphingosine results in a very rapid, i.e. within minutes, permeabilization of the bacterial plasma membrane, resulting in leakiness of the bacterial cells, loss of ATP, and loss of bacterial metabolic activity. These alterations rapidly induced bacterial death. Mechanistically, we demonstrate that the presence of the protonated NH2 group in sphingosine, which is an amino-alcohol, is required for sphingosine's bactericidal activity. We also show that the protonated NH2 group of sphingosine binds to the highly negatively–charged lipid cardiolipin in bacterial plasma membranes. Of note, this binding was required for bacterial killing by sphingosine, as revealed by genetic experiments indicating that E. coli or P. aeruginosa strains that lack cardiolipin synthase are resistant to sphingosine, both in vitro and in vivo. We propose that binding of sphingosine to cardiolipin clusters cardiolipin molecules in the plasma membrane of bacteria. This clustering results in the formation of gel-like or even crystal-like structures in the bacterial plasma membrane and thereby promotes rapid permeabilization of the plasma membrane and bacterial cell death.

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Some Effects of Sound and Music on Organisms and Cells: A Review

Annual Research & Review in Biology ◽

10.9734/arrb/2019/v32i230080 ◽

2019 ◽

pp. 1-12

Author(s):

Jean-Marie Exbrayat ◽

Claire Brun

Keyword(s):

Immune System ◽

Cell Cultures ◽

Cell Biology ◽

Bacterial Cells ◽

Electrical Signals ◽

Therapeutic Applications ◽

Different Types ◽

The Future ◽

Unicellular Organisms ◽

The Brain

In animals, the sound vibrations are captured by the auditory cells, then transformed into electrical signals and conveyed to the nervous centers where they can be interpreted such as music. A lot of studies concern the effect of sound on the auditory cells and on the brain. Nevertheless, musical vibrations also affect other cells types in several organisms. These researches being not of the same nature, they need to be classified in order to provide elements of understanding the effects of music on cell biology. A lot of works were done on the effects of music on non-auditory cells. Effects on growth, apoptosis, immune system, protein activities in animal, plant and bacterial cells have been shown. These effects are of a physiological nature and require molecules and physicochemical mechanisms. Some works were performed on vegetal or animal total organisms, others directly on cells themselves, using cell cultures. Few works concern eukaryotic unicellular organisms. Results of these studies show music and sound exert effects on the physiology. But the experiments and results are still well disparate, with effects of different types of music on organisms via auditory on non-auditory cells, sometimes involving both auditory and non-auditory cells. Whatever the large variation of results, the study of the effects of sound and especially music on the cells is a subject on the future, considering the immense possibilities offered by music in modulating physiology, with potential therapeutic applications.

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Regulated Antisense RNA Eliminates Alpha-Toxin Virulence in Staphylococcus aureus Infection

Journal of Bacteriology ◽

10.1128/jb.181.21.6585-6590.1999 ◽

1999 ◽

Vol 181 (21) ◽

pp. 6585-6590 ◽

Cited By ~ 90

Author(s):

Yinduo Ji ◽

Andrea Marra ◽

Martin Rosenberg ◽

Gary Woodnutt

Keyword(s):

Gene Expression ◽

Staphylococcus Aureus ◽

Antisense Rna ◽

Expression System ◽

Toxin Gene ◽

Critical Element ◽

Bacterial Cells ◽

Alpha Toxin

ABSTRACT The ability to selectively disrupt gene function remains a critical element in elucidating information regarding gene essentiality for bacterial growth and/or pathogenesis. In this study, we adapted atet regulatory expression system for use inStaphylococcus aureus, with the goal of downregulating gene expression via induction of antisense RNA. We demonstrate that this system exhibits a 50- to 100-fold dose-dependent level of induction in bacterial cells grown in culture (i.e., in vitro) and also functions in mice (i.e., in vivo) following oral administration of inducer. To determine whether induced antisense RNA could interfere with chromosomally derived gene expression, we cloned a fragment of theS. aureus alpha-toxin gene (hla) in antisense orientation downstream of the tet promoter system and introduced the construct into S. aureus. Induced antisensehla RNA downregulated chromosomally derived hlagene expression in vitro approximately 14-fold. Similarly, induction ofhla antisense RNA in vivo dramatically reduced alpha-toxin expression in two different murine models of S. aureusinfection. Most importantly, this reduction completely eliminated the lethality of the infection. These results indicate that thetet regulatory system functions efficiently in S. aureus and induced antisense RNA can effectively downregulate chromosomal gene expression both in vitro and in vivo.

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