Direct Cellular Activity Research Articles

Control of cell membrane receptor condensation by adhesion to supported bilayers with nanoscale topography

Developing physical methods to modulate biomolecular clusters and condensates on cell membranes is of great importance for understanding physiological and pathological processes as well as for stimulating novel therapeutic strategies. Here, we propose an effective means to control receptor condensation on the cell membrane via specific adhesion to a supported lipid bilayer (SLB) with nanoscale topography. The specific adhesion is mediated by receptors in the cell membrane that bind their ligands anchored in the SLB. Using Monte Carlo simulations and mean-field theory, we demonstrate that the nanoscale topography of the SLB can enhance condensation of the receptors associated with lipid nanodomains. Our results indicate that SLBs with nanoscale topography proves an effective physical stimulus for tuning condensation of membrane adhesion proteins and lipids in cell membranes, and can serve as a feasible option to control and direct cellular activities, e.g., stem cell differentiation for biomedical and therapeutic applications.

Interactome of Long Non-Coding RNAs: Transcriptomic Expression Patterns and Shaping Cancer Cell Phenotypes.

RNA biology has gained extensive recognition in the last two decades due to the identification of novel transcriptomic elements and molecular functions. Cancer arises, in part, due to the accumulation of mutations that greatly contribute to genomic instability. However, the identification of differential gene expression patterns of wild-type loci has exceeded the boundaries of mutational study and has significantly contributed to the identification of molecular mechanisms that drive carcinogenic transformation. Non-coding RNA molecules have provided a novel avenue of exploration, providing additional routes for evaluating genomic and epigenomic regulation. Of particular focus, long non-coding RNA molecule expression has been demonstrated to govern and direct cellular activity, thus evidencing a correlation between aberrant long non-coding RNA expression and the pathological transformation of cells. lncRNA classification, structure, function, and therapeutic utilization have expanded cancer studies and molecular targeting, and understanding the lncRNA interactome aids in defining the unique transcriptomic signatures of cancer cell phenotypes.

The Role of Fc Gamma Receptors in Antibody-Mediated Rejection of Kidney Transplants.

For the past decades, complement activation and complement-mediated destruction of allograft cells were considered to play a central role in anti-HLA antibody-mediated rejection (AMR) of kidney transplants. However, also complement-independent mechanisms are relevant in the downstream immune activation induced by donor-specific antibodies, such as Fc-gamma receptor (FcγR)-mediated direct cellular activation. This article reviews the literature regarding FcγR involvement in AMR, and the potential contribution of FcγR gene polymorphisms to the risk for antibody mediated rejection of kidney transplants. There is large heterogeneity between the studies, both in the definition of the clinical phenotypes and in the technical aspects. The study populations were generally quite small, except for two larger study cohorts, which obviates drawing firm conclusions regarding the associations between AMR and specific FcγR polymorphisms. Although FcγR are central in the pathophysiology of AMR, it remains difficult to identify genetic risk factors for AMR in the recipient’s genome, independent of clinical risk factors, independent of the donor-recipient genetic mismatch, and in the presence of powerful immunosuppressive agents. There is a need for larger, multi-center studies with standardised methods and endpoints to identify potentially relevant FcγR gene polymorphisms that represent an increased risk for AMR after kidney transplantation.

The argonaut constructs its shell via physical self-organization and coordinated cell sensorial activity

SummaryThe shell of the cephalopod Argonauta consists of two layers of fibers that elongate perpendicular to the shell surfaces. Fibers have a high-Mg calcitic core sheathed by thin organic membranes (>100 nm) and configurate a polygonal network in cross section. Their evolution has been studied by serial sectioning with electron microscopy-associated techniques. During growth, fibers with small cross-sectional areas shrink, whereas those with large sections widen. It is proposed that fibers evolve as an emulsion between the fluid precursors of both the mineral and organic phases. When polygons reach big cross-sectional areas, they become subdivided by new membranes. To explain both the continuation of the pattern and the subdivision process, the living cells from the mineralizing tissue must perform contact recognition of the previously formed pattern and subsequent secretion at sub-micron scale. Accordingly, the fabrication of the argonaut shell proceeds by physical self-organization together with direct cellular activity.

Neurons and astrocytes of the chicken hypothalamus directly respond to lipopolysaccharide and chicken interleukin-6

In 4–5-month-old chicken, intravenous injections of bacterial lipopolysaccharide (LPS) induced a dose-dependent fever response and a pronounced increase of circulating interleukin-6 (IL-6). To assess a possible role for IL-6 in the brain of birds, a hypothalamic neuro-glial primary culture from 1-day-old chicken was established. Each well of cultured hypothalamic cells contained some 615 neurons, 1350 astrocytes, and 580 microglial cells on average. Incubation of chicken hypothalamic primary cultures with 10 or 100 µg/ml LPS induced a dose-dependent release of bioactive IL-6 into the supernatant. Populations of hypothalamic neurons (4%) and astrocytes (12%) directly responded to superfusion with buffer containing 10 µg/ml LPS with a transient increase of intracellular calcium, a sign of direct cellular activation. Stimulation of hypothalamic cultures with buffer containing 50 ng/ml chicken IL-6 induced calcium signaling in 11% of neurons and 22% of astrocytes investigated. These results demonstrate that IL-6 is produced in the periphery and in the hypothalamus in response to LPS in chicken. The observed cellular responses of hypothalamic cells to chicken IL-6 indicate that this cytokine may readily be involved in the manifestation of fever in the avian hypothalamus.

An Extreme Value Theory Model of Cross-Modal Sensory Information Integration in Modulation of Vertebrate Visual System Functions.

We propose a computational model of vision that describes the integration of cross-modal sensory information between the olfactory and visual systems in zebrafish based on the principles of the statistical extreme value theory. The integration of olfacto-retinal information is mediated by the centrifugal pathway that originates from the olfactory bulb and terminates in the neural retina. Motivation for using extreme value theory stems from physiological evidence suggesting that extremes and not the mean of the cell responses direct cellular activity in the vertebrate brain. We argue that the visual system, as measured by retinal ganglion cell responses in spikes/sec, follows an extreme value process for sensory integration and the increase in visual sensitivity from the olfactory input can be better modeled using extreme value distributions. As zebrafish maintains high evolutionary proximity to mammals, our model can be extended to other vertebrates as well.

Physical and Biological Determinants of the Fabrication of Molluscan Shell Microstructures

Molluscs are grand masters in the fabrication of shells, because these are composed of the largest variety of microstructures found among invertebrates. Molluscan microstructures are highly-ordered aggregates of either calcite or aragonite crystals with varied morphologies and three-dimensional arrangements. Classically, every aspect of the fabrication of microstructural aggregates is attributed to the action of proteins. There was, however, only direct evidence that the mineral phase, and indirect evidence that nucleation and the crystal shape, are determined by the types of soluble proteins. Some authors imply that crystal competition may also play a role. In addition, the fabrication of intergranular organic matrices typical of some microstructures (nacre, columnar prismatic) cannot have a protein-based explanation. Over the last decade I and collaborators have been applying a holistic view, based on analyzing and interpreting the features of both the organic (mantle, extrapallial space, periostracum, organic matrices) and inorganic (crystallite morphology, arrangement and crystallography) components of the biomineralization system. By interpreting them on biophysical principles, we have accumulated evidence that, in addition to the activity of proteins, other mechanisms contribute in an essential way to the organization of molluscan microstructures. In particular, we have identified processes such as: (1) crystal nucleation on preformed membranes, (2) nucleation and growth of crystals between and within self-organized membranes, (3) active subcellular processes of contact recognition and deposition. In summary, besides the activity of organic macromolecules, physical (crystal competition, self-organization) and /or biological (direct cellular activity) processes may operate in the fabrication of microstructures. The balance between the physical and biological varies among microstructures, with some being based exclusively on either physical or biological processes, and others having a mixed nature. Other calcifying invertebrates (e.g. corals, cirripeds, serpulids) secrete microstructures that are very similar to inorganic crystal aggregates, and only some brachiopods and, to a lesser extent, bryozoans may have secretory abilities comparable to those of molluscs. Here I provide a new perspective, which may allow microstructures to be understood in terms of evolutionary constraints, to compare the secretional abilities among taxa, and even to evaluate the probability of mimicking microstructures for the production of functional synthetic materials.

Mechanical strain induced phospho-proteomic signaling in uterine smooth muscle cells

Mechanical strain associated with the expanding uterus correlates with increased preterm birth rates. Mechanical signals result in a cascading network of protein phosphorylation events. These signals direct cellular activities and may lead to changes in contractile phenotype and calcium signaling. In this study, the complete phospho-proteome of uterine smooth muscle cells subjected to mechanical strain for 5 min was compared to un-strained controls. Statistically significant, differential phosphorylation events were annotated by Ingenuity Pathway Analysis to elucidate mechanically induced phosphorylation networks. Mechanical strain leads to the direct activation of ERK1/2, HSPB1, and MYL9, in addition to phosphorylation of PAK2, vimentin, DOCK1, PPP1R12A, and PTPN11 at previously unannotated sites. These results suggest a novel network reaction to mechanical strain and reveal proteins that participate in the activation of contractile mechanisms leading to preterm labor.

Calcium Phosphate Deposition With Normal Phosphate Concentration - Role of Pyrophosphate -

Calcium phosphate deposition (CPD) is the hallmark of vascular smooth muscle cell (VSMC) calcification. CPD is a thermodynamically-favored process under physiological conditions. Hydroxyapatite, the most common calcium phosphate in calcified arteries, is passively formed during VSMC calcification, independently on any direct cellular activity. Furthermore, in recent years it has been demonstrated there is an anti-calcifying effect by extracellular pyrophosphate, an endogenous inhibitor of CPD, both in vitro and in vivo, which directly blocks hydroxyapatite formation. We have used the in vitro calcification model without cellular activity, by treating confluent rat aortic VSMC with paraformaldehyde. Fixed cells were incubated with the indicated media to obtain or inhibit calcification. The calcium content was determined colorimetrically. Calcification was observed after 3 weeks (21 days) using a physiological concentration of calcium (1.8 mmol/L) and phosphate (1 mmol/L). Calcium deposition was directly proportional to the amount of phosphate in the media, with a calcification rate of 3.5, 7.5, and 14.3 µg·cm⁻²·day⁻¹, using 1, 2, and 4 mmol/L of phosphate, respectively. Under physiological conditions, pyrophosphate inhibits CPD with an IC₅₀ of ≍200 nmol/L. CPD occurs under a physiological concentration of calcium and phosphate, but this deposition is completely inhibited in the presence of a physiological concentration of pyrophosphate (3-5 µmol/L).

Protein S controls hypoxic/ischemic blood-brain barrier disruption through the TAM receptor Tyro3 and sphingosine 1-phosphate receptor

AbstractThe anticoagulant factor protein S (PS) has direct cellular activities. Lack of PS in mice causes lethal coagulopathy, ischemic/thrombotic injuries, vascular dysgenesis, and blood-brain barrier (BBB) disruption with intracerebral hemorrhages. Thus, we hypothesized that PS maintains and/or enhances the BBB integrity. Using a BBB model with human brain endothelial cells, we show PS inhibits time- and dose-dependently (half maximal effective concentration [EC50] = 27 ± 3 nM) oxygen/glucose deprivation-induced BBB breakdown, as demonstrated by measurements of the transmonolayer electrical resistance, permeability of endothelial monolayers to dextran (40 kDa), and rearrangement of F-actin toward the cortical cytoskeletal ring. Using Tyro-3, Axl, and Mer (TAM) receptor, tyrosine kinase silencing through RNA interference, specific N-terminus–blocking antibodies, Tyro3 phosphorylation, and Tyro3-, Axl- and Mer-deficient mouse brain endothelial cells, we show that Tyro3 mediates PS vasculoprotection. After Tyro3 ligation, PS activated sphingosine 1-phosphate receptor (S1P1), resulting in Rac1-dependent BBB protection. Using 2-photon in vivo imaging, we show that PS blocks postischemic BBB disruption in Tyro3+/+, Axl−/−, and Mer−/− mice, but not in Tyro3−/− mice or Tyro3+/+ mice receiving low-dose W146, a S1P1-specific antagonist. Our findings indicate that PS protects the BBB integrity via Tyro3 and S1P1, suggesting potentially novel treatments for neurovascular dysfunction resulting from hypoxic/ischemic BBB damage.

How to deal with large models?

Mol Syst Biol. 5: 240 The regulatory systems that allow cells to adapt to their environments are exceedingly complex, and although we know a great deal about the intricate mechanistic details of many of these systems, our ability to make accurate predictions about their system‐level behaviors is severely limited. We would like to make such predictions for a number of reasons. How can we reverse dysfunctional molecular changes of these systems that cause disease? More generally, how can we harness and direct cellular activities for beneficial purposes? Our ability to make accurate predictions about a system is also a measure of our fundamental understanding of that system. As evidenced by our mastery of technological systems, a useful understanding of a complex system can often be obtained through the development and analysis of a mathematical model, but predictive modeling of cellular regulatory systems, which necessarily relies on quantitative experimentation, is still in its infancy. There is much that we need to learn before modeling for practical applications becomes routine. In particular, we need to address a number of issues surrounding the large number of parameters that are typically found in a model for a cellular regulatory system. In a recent article published in Molecular Systems Biology , Peter Sorger et al report a significant contribution not only to our system‐level understanding of an important signal‐transduction system but also to our understanding of the methodology needed for developing and testing a large‐scale mathematical model for this type of system (Chen et al , 2009). The power of sensitivity analysis is demonstrated. In this study, to which William Chen, Birgit Schoeberl and Paul Jasper contributed equally, an ordinary differential equation (ODE) model for immediate‐early events in signaling by the epidermal growth factor (EGF) receptor (EGFR, which is also called ErbB1) (Schoeberl et …

Hybrid bone implants: Self-assembly of peptide amphiphile nanofibers within porous titanium

Over the past few decades there has been great interest in the use of orthopedic and dental implants that integrate into tissue by promoting bone ingrowth or bone adhesion, thereby eliminating the need for cement fixation. However, strategies to create bioactive implant surfaces to direct cellular activity and mineralization leading to osteointegration are lacking. We report here on a method to prepare a hybrid bone implant material consisting of a Ti–6Al–4V foam, whose 52% porosity is filled with a peptide amphiphile (PA) nanofiber matrix. These PA nanofibers can be highly bioactive by molecular design, and are used here as a strategy to transform an inert titanium foam into a potentially bioactive implant. Using scanning electron microscopy (SEM) and confocal microscopy, we show that PA molecules self-assemble into a nanofiber matrix within the pores of the metallic foam, fully occupying the foam's interconnected porosity. Furthermore, the method allows the encapsulation of cells within the bioactive matrix, and under appropriate conditions the nanofibers can nucleate mineralization of calcium phosphate phases with a Ca:P ratio that corresponds to that of hydroxyapatite. Cell encapsulation was quantified using a DNA measuring assay and qualitatively verified by SEM and confocal microscopy. An in vivo experiment was performed using a bone plug model in the diaphysis of the hind femurs of a Sprague Dawley rat and examined by histology to evaluate the performance of these hybrid systems after 4 weeks of implantation. Preliminary results demonstrate de novo bone formation around and inside the implant, vascularization around the implant, as well as the absence of a cytotoxic response. The PA–Ti hybrid strategy could be potentially tailored to initiate mineralization and direct a cellular response from the host tissue into porous implants to form new bone and thereby improve fixation, osteointegration, and long term stability of implants.

Importance of Snake Venom Metalloproteinases in Cell Biology: Effects on Platelets,Inflammatory and Endothelial Cells

Snake venom metalloproteinases (SVMPs) are widely distributed in snake venoms and play important roles in hemostatic disorders and local tissue damage that follows snakebite. The impact of SVMPs on hemostasis has been extensively studied showing diverse effects both on soluble factors and cellular components. The action of SVMPs involves catalytic and anti-adhesive properties, as well as direct cellular activation and/or the release of endogenous bioactive components. The purpose of this review is to overview the action of SVMPs on the inhibition of platelet functions; angiogenesis, particularly inducing apoptosis of endothelial cells; and regarding the pro-inflammatory reaction that follows snakebite. We discuss the structural features of the molecules that may be involved in such activities. The versatility and availability of SVMPs make them important tools for cell biology research into the mechanisms of action of endogenous metalloproteinases, for insights into cellular-matrix interactions and for clinical investigations into the treatment of snakebites.

BMK1 Mediates Growth Factor-induced Cell Proliferation through Direct Cellular Activation of Serum and Glucocorticoid-inducible Kinase

Activation of the mammalian mitogen-activated protein kinase known as BMK1 is required for growth factor-induced cell proliferation. To understand the mechanism by which BMK1 mediates this cellular response, this kinase was used as bait in a yeast two-hybrid-based library screening. Here, we report the identification of serum and glucocorticoid-inducible kinase (SGK) as a cellular protein that physically interacts with BMK1. During growth factor-induced cell stimulation, BMK1 activates SGK by phosphorylation at serine 78. This BMK1-mediated phosphorylation event is necessary for the activation of SGK and, more importantly, for cell proliferation induced by growth factors.

Functional tissue engineering: the role of biomechanics.

"Tissue engineering" uses implanted cells, scaffolds, DNA, protein, and/or protein fragments to replace or repair injured or diseased tissues and organs. Despite its early success, tissue engineers have faced challenges in repairing or replacing tissues that serve a predominantly biomechanical function. An evolving discipline called "functional tissue engineering" (FTE) seeks to address these challenges. In this paper, the authors present principles of functional tissue engineering that should be addressed when engineering repairs and replacements for load-bearing structures. First, in vivo stress/strain histories need to be measured for a variety of activities. These in vivo data provide mechanical thresholds that tissue repairs/replacements will likely encounter after surgery. Second, the mechanical properties of the native tissues must be established for subfailure and failure conditions. These "baseline data" provide parameters within the expected thresholds for different in vivo activities and beyond these levels if safety factors are to be incorporated. Third, a subset of these mechanical properties must be selected and prioritized. This subset is important, given that the mechanical properties of the designs are not expected to completely duplicate the properties of the native tissues. Fourth, standards must be set when evaluating the repairs/replacements after surgery so as to determine, "how good is good enough?" Some aspects of the repair outcome may be inferior, but other mechanical characteristics of the repairs and replacements might be suitable. New and improved methods must also be developed for assessing the function of engineered tissues. Fifth, the effects of physical factors on cellular activity must be determined in engineered tissues. Knowing these signals may shorten the iterations required to replace a tissue successfully and direct cellular activity and phenotype toward a desired end goal. Finally, to effect a better repair outcome, cell-matrix implants may benefit from being mechanically stimulated using in vitro "bioreactors" prior to implantation. Increasing evidence suggests that mechanical stress, as well as other physical factors, may significantly increase the biosynthetic activity of cells in bioartificial matrices. Incorporating each of these principles of functional tissue engineering should result in safer and more efficacious repairs and replacements for the surgeon and patient.