Cell Environment Research Articles

The DnaA Protein Is Not the Limiting Factor for Initiation of Replication in Escherichia coli.

The bacterial replication cycle is driven by the DnaA protein which cycles between the active ATP-bound form and the inactive ADP-bound form. It has been suggested that DnaA also is the main controller of initiation frequency. Initiation is thought to occur when enough ATP-DnaA has accumulated. In this work we have performed cell cycle analysis of cells that contain a surplus of ATP-DnaA and asked whether initiation then occurs earlier. It does not. Cells with more than a 50% increase in the concentration of ATP-DnaA showed no changes in timing of replication. We suggest that although ATP-DnaA is the main actor in initiation of replication, its accumulation does not control the time of initiation. ATP-DnaA is the motor that drives the initiation process, but other factors will be required for the exact timing of initiation in response to the cell’s environment. We also investigated the in vivo roles of datA dependent DnaA inactivation (DDAH) and the DnaA-binding protein DiaA. Loss of DDAH affected the cell cycle machinery only during slow growth and made it sensitive to the concentration of DiaA protein. The result indicates that compromised cell cycle machines perform in a less robust manner.

Nucleotide composition bias and codon usage trends of gene populations in Mycoplasma capricolum subsp. capricolum and M. agalactiae

Because of the low GC content of the gene population, amino acids of the two mycoplasmas tend to be encoded by synonymous codons with an A or T end. Compared with the codon usage of ovine, Mycoplasma capricolum and M. agalactiae tend to select optimal codons, which are rare codons in ovine. Due to codon usage pattern caused by genes with key biological functions, the overall codon usage trends represent a certain evolutionary direction in the life cycle of the two mycoplasmas. The overall codon usage trends of a gene population of M. capricolum subsp. capricolum can be obviously separated from other mycoplasmas, and the overall codon usage trends of M. agalactiae are highly similar to those of M. bovis. These results partly indicate the independent evolution of the two mycoplasmas without the limits of the host cell's environment. The GC and AT skews estimate nucleotide composition bias at different positions of nucleotide triplets and the protein consideration caused by the nucleotide composition bias at codon positions 1 and 2 largely take part in synonymous codon usage patterns of the two mycoplasmas. The correlation between the codon adaptation index and codon usage variation indicates that the effect of codon usage on gene expression in M. capricolum subsp. capricolum is opposite to that of M. agalactiae, further suggesting independence of the evolutionary process influencing the overall codon usage trends of gene populations of mycoplasmas.

Induction of protein deletion through in utero electroporation to define deficits in neuronal migration in transgenic models.

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific Cre models, deletion of a protein throughout a tissue or cell population often leads to complex phenotypes resulting from multiple interacting mechanisms. Determining whether a phenotype results from disruption of a cell autonomous mechanism, which is intrinsic to the cell in question, or from a non-cell autonomous mechanism, which would result from impairment of that cell's environment, can be difficult to discern. To gain insight into protein function in an in vivo context, in utero electroporation (IUE) enables gene deletion in a small subset of cells within the developing cortex or some other selected brain region. IUE can be used to target specific brain areas, including the dorsal telencephalon, medial telencephalon, hippocampus, or ganglionic eminence. This facilitates observation of the consequences of cell autonomous gene deletion in the context of a healthy environment. The goal of this protocol is to show how IUE can be used to analyze a defect in radial migration in a floxed transgenic mouse line, with an emphasis on distinguishing between the cell autonomous and non-cell autonomous effects of protein deletion. By comparing the phenotype resulting from gene deletion within the entire cortex versus IUE-mediated gene deletion in a limited cell population, greater insight into protein function in brain development can be obtained than by using either technique in isolation.

3D Tracking of Single Quantum Dots through Off-Focus Imaging

Recently, tremendous improvements have been achieved in the precision of localization of single fluorescent molecules, allowing localization and tracking of biomolecules at the nm level. Since the behaviour of proteins and biological molecules is tightly influenced by the cell's environment, a growing number of microscopy techniques are moving from in vitro to live cell experiments. Looking at both diffusion and active transportation processes inside a cell requires three-dimensional localization over a few microns range, high SNR images and high temporal resolution (ms order of magnitude). To satisfy these requirements we developed an automated routine that allow 3D tracking of single fluorescent molecules in living cells with nanometer accuracy, by exploiting the properties of the point-spread-function of out-of-focus Quantum Dots bound to the protein of interest.

The ciliary baton: orchestrating neural crest cell development.

Primary cilia are cell surface, microtubule-based organelles that dynamically extend from cells to receive and process molecular and mechanical signaling cues. In the last decade, this organelle has gained increasing popularity due to its ability to act as a cellular antenna, receive molecular stimuli, and respond to the cell's environment. A growing field of data suggests that various tissues utilize and interpret the loss of cilia in different ways. Thus, careful examination of the role of cilia on individual cell types and tissues is necessary. Neural crest cells (NCCs) are an excellent example of cells that survey their environment for developmental cues. In this review, we discuss how NCCs utilize primary cilia during their ontogenic development, paying special attention to the role primary cilia play in processing developmental signals required for NCC specification, migration, proliferation, and differentiation. We also discuss how the loss of functional cilia on cranial and trunk NCCs affects the development of various organ systems to which they contribute. A deeper understanding of ciliary function could contribute greatly to understanding the molecular mechanisms guiding NCC development and differentiation. Furthermore, superimposing the ciliary contribution on our current understanding of NCC development identifies new avenues for therapeutic intervention in neurocristopathies.

Design of oligoaziridine-PEG coatings for efficient nanogold cellular biotagging

Biocompatible oligoaziridine-PEG coated gold nanoparticles overcome self-quenching while targeting the cell nucleus. The course of gold biotags within the cell's environment was tracked through confocal laser microscopy.

Integrin-mediated adhesion and mechano-sensing in cutaneous wound healing.

Integrin receptors mediate the interactions between cells and the extracellular matrix. They not only provide anchorage and a physical linkage to the matrix but also participate in cell signaling and the regulation of diverse cellular functions. In the epidermis of the skin, integrins are essential for tissue structure and integrity, and, under normal homeostatic conditions, the β1 subunit specifically controls the balance between proliferation and terminal differentiation. Integrin expression can also dynamically respond to changes in the cell's environment, and integrin-mediated adhesion is required for keratinocyte migration and re-epithelialization during wound repair. Importantly, integrins participate in keratinocyte mechanotransduction and could potentially regulate cell behavior within the altered mechanical microenvironment of a wound. While the complete functions of integrin receptors in cutaneous wound healing have yet to be determined, recent evidence suggests that cell-matrix interactions are perturbed in chronic and non-healing wounds. Integrins may therefore be a potential therapeutic target for improving wound repair and tissue regeneration.

Physical influences of the extracellular environment on cell migration.

The way in which a cell migrates is influenced by the physical properties of its surroundings, in particular the properties of the extracellular matrix. How the physical aspects of the cell's environment affect cell migration poses a considerable challenge when trying to understand migration in complex tissue environments and hinders the extrapolation of in vitro analyses to in vivo situations. A comprehensive understanding of these problems requires an integrated biochemical and biophysical approach. In this Review, we outline the findings that have emerged from approaches that span these disciplines, with a focus on actin-based cell migration in environments with different stiffness, dimensionality and geometry.

Classification of T cell movement tracks allows for prediction of cell function.

Using a unique combination of visual, statistical, and data mining methods, we tested the hypothesis that an immune cell's movement pattern can convey key information about the cell's function, antigen specificity, and environment. We applied clustering, statistical tests, and a support vector machine (SVM) to assess our ability to classify different datasets of imaged flouresently labelled T cells in mouse liver. We additionally saw clusters of different movement patterns of T cells of identical antigenic specificity. We found that the movement patterns of T cells specific and non-specific for malaria parasites are differentiable with 72% accuracy, and that specific cells have a higher tendency to move towards the parasite than non-specific cells. Movements of antigen-specific T cells in uninfected mice vs. infected mice were differentiable with 69.8% accuracy. We additionally saw clusters of different movement patterns of T cells of identical antigenic specificity. We concluded that our combination of methods has the potential to advance the understanding of cell movements in vivo.

Single-Cell Force Spectroscopy, an Emerging Tool to Quantify Cell Adhesion to Biomaterials

Cell adhesion receptors play a central role in sensing and integrating signals provided by the cellular environment. Thus, understanding adhesive interactions at the cell-biomaterial interface is essential to improve the design of implants that should emulate certain characteristics of the cell's natural environment. Numerous cell adhesion assays have been developed; among these, atomic force microscopy-based single-cell force spectroscopy (AFM-SCFS) provides a versatile tool to quantify cell adhesion at physiological conditions. Here we discuss how AFM-SCFS can be used to quantify the adhesion of living cells to biomaterials and give examples of using AFM-SCFS in tissue engineering and regenerative medicine. We anticipate that in the near future, AFM-SCFS will be established in the biomaterial field as an important technique to quantify cell-biomaterial interactions and thereby will contribute to the optimization of implants, scaffolds, and medical devices.

Single-Molecule Investigation of the Intraflagellar Transport of Membrane Signaling Proteins

In the past decade, cilia/flagella have come to be known as essential sensory organelles for cells. These cilia/flagella perform their sensory function via signaling pathways that include flagellar membrane signaling proteins and intraflagellar transport (IFT) machinery comprised of IFT particles and BBSome particles. While the membrane signaling proteins are responsible for collecting information regarding a cell's environment, the IFT machinery has been implicated in effecting the delivery of this information through the translocation and proper positioning of the signaling proteins along the flagellar body. Unfortunately, current understanding of this intraflagellar translocation and positioning is limited to rough measurements of average IFT speeds with little regard for speed fluctuations and the causes thereof. Further, the equally important diffusive motion of signaling proteins in the flagellar membrane has been largely ignored. As such, we use single-molecule fluorescence imaging methods to study the intraflagellar motion of BBS4-GFP, IFT20-GFP, and Pkd2-GFP, a transmembrane signaling protein, in Chlamydomonas reinhardtii. (i) In the flagellar entry region, we have found that Pkd2 largely performs Brownian diffusion, implicating lateral membrane diffusion as a primary flagellar entry mechanism. (ii) In the flagellar body, we have found that IFT trains exhibit speed changes of approximately ± 400 nm/s, which we interpret as events of dropping off or picking up membrane signaling proteins. Futhermore, we have observed the average speed of an IFT train with a BBSome attached to be 300 nm/s slower than the speed of a train without a BBSome, indicating that the BBSome carries at least one membrane signaling protein. (iii) Finally, at the flagellar tip, BBSomes remain bound to their associated membrane signaling proteins, diffusing in the membrane at the tip for an average of two seconds before undergoing retrograde IFT.

Primary cilia act as mechanosensors during bone healing around an implant

The primary cilium is an organelle that senses cues in a cell's local environment. Some of these cues constitute molecular signals; here, we investigate the extent to which primary cilia can also sense mechanical stimuli. We used a conditional approach to delete Kif3a in pre-osteoblasts and then employed a motion device that generated a spatial distribution of strain around an intra-osseous implant positioned in the mouse tibia. We correlated interfacial strain fields with cell behaviors ranging from proliferation through all stages of osteogenic differentiation. We found that peri-implant cells in the Col1Cre;Kif3afl/fl mice were unable to proliferate in response to a mechanical stimulus, failed to deposit and then orient collagen fibers to the strain fields caused by implant displacement, and failed to differentiate into bone-forming osteoblasts. Collectively, these data demonstrate that the lack of a functioning primary cilium blunts the normal response of a cell to a defined mechanical stimulus. The ability to manipulate the genetic background of peri-implant cells within the context of a whole, living tissue provides a rare opportunity to explore mechanotransduction from a multi-scale perspective.

Development of functional biomaterials with micro- and nanoscale technologies for tissue engineering and drug delivery applications.

Micro- and nanotechnologies have emerged as potentially effective fabrication tools for addressing the challenges faced in tissue engineering and drug delivery. The ability to control and manipulate polymeric biomaterials at the micron and nanometre scale with these fabrication techniques has allowed for the creation of controlled cellular environments, engineering of functional tissues and development of better drug delivery systems. In tissue engineering, micro- and nanotechnologies have enabled the recapitulation of the micro- and nanoscale detail of the cell's environment through controlling the surface chemistry and topography of materials, generating 3D cellular scaffolds and regulating cell-cell interactions. Furthermore, these technologies have led to advances in high-throughput screening (HTS), enabling rapid and efficient discovery of a library of materials and screening of drugs that induce cell-specific responses. In drug delivery, controlling the size and geometry of drug carriers with micro- and nanotechnologies have allowed for the modulation of parametres such as bioavailability, pharmacodynamics and cell-specific targeting. In this review, we introduce recent developments in micro- and nanoscale engineering of polymeric biomaterials, with an emphasis on lithographic techniques, and present an overview of their applications in tissue engineering, HTS and drug delivery.

Mob1: defining cell polarity for proper cell division

Mob1 is a component of both the mitotic exit network and Hippo pathway, being required for cytokinesis, control of cell proliferation and apoptosis. Cell division accuracy is crucial in maintaining cell ploidy and genomic stability and relies on the correct establishment of the cell division axis, which is under the control of the cell's environment and its intrinsic polarity. The ciliate Tetrahymena thermophila possesses a permanent anterior-posterior axis, left-right asymmetry and divides symmetrically. These unique features of Tetrahymena prompted us to investigate the role of Tetrahymena Mob1. Unexpectedly, we found that Mob1 accumulated in basal bodies at the posterior pole of the cell, and is the first molecular polarity marker so far described in Tetrahymena. In addition, Mob1 depletion caused the abnormal establishment of the cell division plane, providing clear evidence that Mob1 is important for its definition. Furthermore, cytokinesis was arrested and ciliogenesis delayed in Tetrahymena cells depleted of Mob1. This is the first evidence for an involvement of Mob1 in cilia biology. In conclusion, we show that Mob1 is an important cell polarity marker that is crucial for correct division plane placement, for cytokinesis completion and for normal cilia growth rates.

Epigenetics for ecotoxicologists

Do genes define your destiny? The field of epigeneticsexamines how genes and the environment interact to formthe basis of heredity and comes up with some surprisingfindings. We often think of deoxyribonucleic acid (DNA) asthe sole physical basis for heredity, the genetic code thatdetermines everything from eye and hair color to specificpersonality traits. The growing field of epigenetics suggeststhat this traditional paradigm is an oversimplification. Epi-genetics is the study of factors that are heritable, but whichoccur by mechanisms other than changes in the DNA codeitself. C.H. Waddington first used the term epigenetics in theearly 1940s to describe ‘‘the interactions of genes with theirenvironment, which bring the phenotype into being.’’ Epi-genetic marks are susceptible to environmental influences—both chemical and nonchemical—and can be inherited inways that may seem counterintuitive (see sidebar, ExpandingIdeas About Biological Inheritance). Effects of early lifeexperiences, such as parental care or nutrition, can showup later in life and even be passed on to future generations.This emerging area of research has interesting and importantimplications for the field of ecotoxicology—from basicscience to international policy.In classic genetics, genetic material is in the form of DNAand encodes all of the information necessary for life. Thisinformation is copied faithfully and passed down as cellsdivide. Every cell contains a complete copy of the DNAcode, but the pattern of gene expression—that is, whichgenes are turned ‘‘on’’ (expressed) or ‘‘off’’ (nonex-pressed)—determines the cell type and function. A skin cellon your arm contains the same DNA code as a nerve cell inyour brain, but their extreme differences in structure andfunction are due to the particular set of genes that each isexpressing. Epigenetics refers to an annotation in the form ofchemical marks on top of the DNA code; the prefix ‘‘epi’’comesfromaGreekwordmeaning‘‘over’’or‘‘above.’’Thesechemical marks, which are discussed in detail below, affectwhich genes are expressed and at what levels. Epigeneticmarks are highly influenced by the environment and can beinherited along with the genetic code as cells divide mitoti-cally, and in some cases meiotically, from one generation tothe next.Epigenetics is receiving significant attention in the field ofbiomedicine. A PubMed search for ‘‘epigenetic’’ revealedthat more than 1,300 review articles have been written on thetopic, of which more than 50% were published in the lastthree years alone. Epigenetics provides a mechanism for theBarker hypothesis, which postulates that nutrition and otherenvironmentalfactorsearlyindevelopmentcanaltersuscept-ibility to chronic diseases in adulthood. A classic exampleof this is that individuals who were conceived during theDutchWinterHunger(1944–1945)havepersistentepigeneticalterations on a characteristic gestational marker, the IGF2gene, as adults. Furthermore, individuals who experiencedthe Dutch Winter Hunger have higher rates of metabolicdisorders and cardiovascular disease, the epigenetic mecha-nisms of which are the focus of ongoing studies. Nutritionaldeprivation may also have an impact on human longevity;indeed, lifespanisnegativelycorrelated withfoodabundance

Identification and Quantification of S-Nitrosylation by Cysteine Reactive Tandem Mass Tag Switch Assay

Redox-switches are critical cysteine thiols that are modified in response to changes in the cell's environment conferring a functional effect. S-nitrosylation (SNO) is emerging as an important modulator of these regulatory switches; however, much remains unknown about the nature of these specific cysteine residues and how oxidative signals are interpreted. Because of their labile nature, SNO-modifications are routinely detected using the biotin switch assay. Here, a new isotope coded cysteine thiol-reactive multiplex reagent, cysTMT(6), is used in place of biotin, for the specific detection of SNO-modifications and determination of individual protein thiol-reactivity. S-nitrosylation was measured in human pulmonary arterial endothelia cells in vitro and in vivo using the cysTMT(6) quantitative switch assay coupled with mass spectrometry. Cell lysates were treated with S-nitrosoglutathione and used to identify 220 SNO-modified cysteines on 179 proteins. Using this approach it was possible to discriminate potential artifacts including instances of reduced protein disulfide bonds (6) and S-glutathionylation (5) as well as diminished ambiguity in site assignment. Quantitative analysis over a range of NO-donor concentrations (2, 10, 20 μm; GSNO) revealed a continuum of reactivity to SNO-modification. Cysteine response was validated in living cells, demonstrating a greater number of less sensitive cysteine residues are modified with increasing oxidative stimuli. Of note, the majority of available cysteines were found to be unmodified in the current treatment suggesting significant additional capacity for oxidative modifications. These results indicate a possible mechanism for the cell to gauge the magnitude of oxidative stimuli through the progressive and specific accumulation of modified redox-switches.

MDCK Cystogenesis Driven by Cell Stabilization within Computational Analogues

The study of epithelial morphogenesis is fundamental to increasing our understanding of organ function and disease. Great progress has been made through study of culture systems such as Madin-Darby canine kidney (MDCK) cells, but many aspects of even simple morphogenesis remain unclear. For example, are specific cell actions tightly coupled to the characteristics of the cell's environment or are they more often cell state dependent? How does the single lumen, single cell layer cyst consistently emerge from a variety of cell actions? To improve insight, we instantiated in silico analogues that used hypothesized cell behavior mechanisms to mimic MDCK cystogenesis. We tested them through in vitro experimentation and quantitative validation. We observed novel growth patterns, including a cell behavior shift that began around day five of growth. We created agent-oriented analogues that used the cellular Potts model along with an Iterative Refinement protocol. Following several refinements, we achieved a degree of validation for two separate mechanisms. Both survived falsification and achieved prespecified measures of similarity to cell culture properties. In silico components and mechanisms mapped to in vitro counterparts. In silico, the axis of cell division significantly affects lumen number without changing cell number or cyst size. Reducing the amount of in silico luminal cell death had limited effect on cystogenesis. Simulations provide an observable theory for cystogenesis based on hypothesized, cell-level operating principles.

Regenerative medicine spoilt for choice

Summary Embryonic stem cells, induced pluripotent cells, transdifferentiated cells – there is now a wide range of options for medical researchers trying to regenerate disease-affected tissues and organs. But which approach will win the day, and can science policy keep up with the bioethical implications of rapid progress? Michael Gross investigates.

New Multi Acid Side-Chain Ionomers for Proton Exchange Membrane Fuel Cells

This work describes the modification of the sulfonyl fluoride precursor of perfluorinated sulfonic acids (PFSAs) by the conversion of the sulfonyl fluoride group to a bis sulfonylimide group. The usefulness of these new polymers for fuel cell applications includes novel proton exchange membranes, catalyst additives or tie layers designed to be thermally and chemically robust while operating within a fuel cell's harsh environment at higher temperatures.

In Silico Translation of Cellular and Molecular Mechanisms to Clinical Phenomena in Atheroma Development with an Agent Based Model

Introduction: Atherosclerosis is the build-up of fatty deposits, cholesterol, cellular wastes, calcium and other substances as a “plaque” in the inner lining of arteries. These building plaques can rupture to form a blood clot, effectively blocking blood flow. Although the pathogenesis of atheromas as cell-mediated responses to inflammation have been postulated and shown in specific static models, the integration of this information into a model that captures the dynamics of the process is lacking. To develop effective treatment strategies and techniques, it is essential to characterize plaque formation through the integration of inflammatory responses, treatments, and predisposing factors. Agent-based modeling utilizes mechanistic basic science knowledge to develop a computational model that dynamically represents the inflammatory influences on atherosclerosis. Methods: A basic science literature review was conducted to identify the mechanisms of specific cell-cell interactions in inflammation and atheroma formation. This information was organized into influence diagrams of each cell type's behavior. Cells were treated as input-output devices, where the cell's local environment (molecules, mediators and neighboring cells) led to actions within the cells (expressed as mechanistic rules) to produce outputs back into the local environment. These cellular influence diagrams were converted into an abstract agent based model (ABM) using NetLogo. The resulting ABM included the multiple arterial layers: intima, media, and adventitia; and cellular agent classes: endothelial cells, smooth muscles cells, macrophages, foam cells, platelets, monocytes, t-lymphocytes and fibroblasts. Pattern oriented analysis was used to evaluate the model's behavior under differential shear forces associated with bifurcations of the vascular tree, where increased endothelial activation leading to atheroma formation occurs. Results: The arterial wall ABM successfully simulated general vascular wall cell interactions in macro-vessel endothelial activation, clot formation, intra-intimal inflammation and pre-atheroma formation. These behaviors matched those seen in histological examinations of atheroma formation. Furthermore, varying levels of predisposing factors, such as LDL and platelets, corresponded to respective changes in the likelihood and severity of atheroma development. When subjected to different abstract therapies for specified conditions, the ABM demonstrated behaviors similar to those seen in clinical situations. Conclusions: Combining atherosclerotic and generative inflammatory responses within an ABM provides a dynamic representation of the mechanistic hypothesis associated with atheroma formation. This model has multiple potential applications for modeling arterial wall responses to stent placements, angioplasty or surgical intervention. Agent based modeling, used as a means of dynamic knowledge representation, can be integrated into traditional biomedical research environments, where it can be the first step in the evaluation proposed mechanisms and interventions. Future work includes the incorporation of anti-platelet modalities, anti-nitric oxide therapies, anti-inflammatory therapies, and drug-eliciting stents.