The nematode Caenorhabditis elegans uses striated muscle in its body wall for locomotion. The myofilament lattice is organized such that all the thin filament attachment structures (dense bodies, analogous to Z-disks) and thick filament organizing centers (M-lines) are attached to the muscle cell membrane. Thus, the force of muscle contraction is transmitted through these structures and allows locomotion of the worm. Dense bodies and M-lines are compositionally similar to focal adhesions and costameres, and are based on integrin and associated proteins. Null mutants for many of the newly discovered dense body and M-line proteins do not have obvious locomotion defects when observed casually, or when assayed by counting the number of times a worm moves back and forth in liquid. We hypothesized that many of these proteins, located as they are in muscle focal adhesions, function in force transmission, but we had not used an appropriate or sufficiently sensitive assay to reveal this function. Recently, we have developed a new quantitative assay of C. elegans locomotion that measures the maximum bending amplitude of an adult worm as it moves backwards. The assay had been used to reveal locomotion defects for null mutants of genes encoding ATN-1 (α-actinin) and PKN-1 (protein kinase N). Here, we describe the details of this method, and apply it to 21 loss of function mutants in 17 additional genes, most of which encode components of muscle attachment structures. As compared to wild type, mutants in 11 genes were found to have less ability to bend, and mutants in one gene were found to have greater ability to bend. Loss of function mutants for eight proteins had been reported to have normal locomotion (ZYX-1 (zyxin), ALP-1 (Enigma), DIM-1, SCPL-1), or locomotion that was not previously investigated (FRG-1 (FRG1), KIN-32 (focal adhesion kinase), LIM-8), or had only slightly decreased locomotion (PFN-3 (profilin)).
Spleen tyrosine kinase (SYK) is crucial to cellular functions mediated by immunoreceptors and integrins. We have developed and characterized a new genetically-encoded Förster resonance energy transfer (FRET)-based biosensor for studying the dynamics of SYK activities in living cells at a subcellular level. It contains an N-terminal ECFP, SH2 domain, a peptide derived from a SYK substrate VAV2, and a C-terminal YPet. Upon the specific phosphorylation by SYK in vitro, the biosensor substrate peptide bound to the intramolecular SH2 domain to reduce the FRET efficiency. Transfection of the biosensor did not affect activation of the endogenous SYK in host cells. Phosphorylation of the biosensor followed the same kinetics as the endogenous VAV2. Using FRET imaging and ratiometric analysis with this SYK biosensor, we visualized and quantified the realtime activation of SYK in K562 cells upon IgG Fc engagement of Fcc receptor IIA and in mouse embryonic fibroblasts upon stimulation by the platelet derived growth factor. These results demonstrate our biosensor as a powerful tool for studying cellular signaling that involves SYK.
Dendritic cells (DCs) play a critical role in orchestrating the host responses to a wide variety of foreign antigens and are essential in maintaining immune tolerance. Distinct biomaterials have been shown to differentially affect the phenotype of DCs, which suggested that biomaterials may be used to modulate immune response toward the biologic component in combination products. The elucidation of biomaterial property-DC phenotype relationships is expected to inform rational design of immuno-modulatory biomaterials. In this study, DC response to a set of 12 polymethacrylates (pMAs) was assessed in terms of surface marker expression and cytokine profile. Principal component analysis (PCA) determined that surface carbon correlated with enhanced DC maturation, while surface oxygen was associated with an immature DC phenotype. Partial square linear regression, a multivariate modeling approach, was implemented and successfully predicted biomaterial-induced DC phenotype in terms of surface marker expression from biomaterial properties with R prediction2 = 0.76. Furthermore, prediction of DC phenotype was effective based on only theoretical chemical composition of the bulk polymers with Rprediction2 = 0.80. These results demonstrated that immune cell response can be predicted from biomaterial properties, and computational models will expedite future biomaterial design and selection.
The Wnt signaling pathway inhibitor Dickkopf-2 (Dkk2) regulates osteoblast differentiation on microstructured titanium (Ti) surfaces, suggesting involvement of Wnt signaling in this process. To test this, human osteoblast-like MG63 cells were cultured on tissue culture polystyrene or Ti (smooth PT (Ra = 0.2 μm), sand-blasted and acid-etched SLA (Ra = 3.22 μm), modSLA (hydrophilic SLA)). Expression of Wnt pathway receptors, activators and inhibitors was measured by qPCR. Non-canonical pathway ligands, receptors and intracellular signaling molecules, as well as bone morphogenetic proteins BMP2 and BMP4, were upregulated on SLA and modSLA, whereas canonical pathway members were downregulated. To confirm that non-canonical signaling was involved, cells were cultured daily with exogenous Wnt3a (canonical pathway) or Wnt5a (non-canonical pathway). Alternatively, cells were cultured with antibodies to Wnt3a or Wnt5a to validate that Wnt proteins secreted by the cells were mediating cell responses to the surface. Wnt5a, but not Wnt3a, increased MG63 cell differentiation and BMP2 and BMP4 proteins, suggesting Wnt5a promotes osteogenic differentiation through production of BMPs. Effects of exogenous and endogenous Wnt5a were synergistic with surface microstructure, suggesting the response also depends on cell maturation state. These results indicate a major role for the non-canonical, calcium-dependent Wnt pathway in differentiation of osteoblasts on microstructured titanium surfaces during implant osseointegration.
Enteric viruses encounter a multitude of environments as they traverse the gastrointestinal tract. The interaction of enteric eukaryotic viruses with members of the host microbiota impacts the outcome of infection. Infection with several enteric viruses is impaired in the absence of the gut microbiota, specifically bacteria. The effects of bacteria on virus biology are diverse. Poliovirus capsid stability and receptor engagement are positively impacted by bacteria and bacterial lipopolysaccharides. Norovirus utilizes histo-blood group antigens produced by enteric bacteria to attach and productively infect B cells. Lipopolysaccharides on the envelope of mouse mammary tumor virus promote a tolerogenic environment that allows for the establishment of viral persistence. Reovirus binds Gram negative and Gram-positive bacteria through bacterial envelope components to enhance virion thermostability. Through the direct engagement of bacteria and bacterial components, viruses evolved diverse ways to impact the outcome of infection.
Calretinin-expressing (CR+) interneurons are the most common type of striatal interneuron in primates. However, because CR+ interneurons are relatively scarce in rodent striatum, little is known about their molecular and other properties, and they are typically excluded from models of striatal circuitry. Moreover, CR+ interneurons are often treated in models as a single homogenous population, despite previous descriptions of their heterogeneous structures and spatial distributions in rodents and primates. Here, we demonstrate that, in rodents, the combinatorial expression of secretagogin (Scgn), specificity protein 8 (SP8) and/or LIM homeobox protein 7 (Lhx7) separates striatal CR+ interneurons into three structurally and topographically distinct cell populations. The CR+/Scgn+/SP8+/Lhx7− interneurons are small-sized (typically 7–11 µm in somatic diameter), possess tortuous, partially spiny dendrites, and are rostrally biased in their positioning within striatum. The CR+/Scgn−/SP8−/Lhx7− interneurons are medium-sized (typically 12–15 µm), have bipolar dendrites, and are homogenously distributed throughout striatum. The CR+/Scgn−/SP8−/Lhx7+ interneurons are relatively large-sized (typically 12–20 µm), and have thick, infrequently branching dendrites. Furthermore, we provide the first in vivo electrophysiological recordings of identified CR+ interneurons, all of which were the CR+/Scgn−/SP8−/Lhx7− cell type. In the primate striatum, Scgn co-expression also identified a topographically distinct CR+ interneuron population with a rostral bias similar to that seen in both rats and mice. Taken together, these results suggest that striatal CR+ interneurons comprise at least three molecularly, structurally, and topographically distinct cell populations in rodents. These properties are partially conserved in primates, in which the relative abundance of CR+ interneurons suggests that they play a critical role in striatal microcircuits.
miR-21, as an oncogene that overexpresses in most human tumors, is involved in radioresistance; however, the mechanism remains unclear. Here, we demonstrate that miR-21-mediated radioresistance occurs through promoting repair of DNA double strand breaks, which includes facilitating both non-homologous end-joining (NHEJ) and homologous recombination repair (HRR). The miR-21-promoted NHEJ occurs through targeting GSK3B (a novel target of miR-21), which affects the CRY2/PP5 pathway and in turn increases DNA-PKcs activity. The miR-21-promoted HRR occurs through targeting both GSK3B and CDC25A (a known target of miR-21), which neutralizes the effects of targeting GSK3B-induced CDC25A increase because GSK3B promotes degradation of both CDC25A and cyclin D1, but CDC25A and cyclin D1 have an opposite effect on HRR. A negative correlation of expression levels between miR-21 and GSK3β exists in a subset of human tumors. Our results not only elucidate miR-21-mediated radioresistance, but also provide potential new targets for improving radiotherapy.
Background: In resting platelets, endothelial cell specific adhesion molecule (ESAM) is located in alpha granules, increasing its cell surface expression following platelet activation. However, the function of ESAM on platelets is unknown. Objective: To determine whether ESAM has a role in thrombus formation. Methods and results: We found that following platelet activation ESAM localizes to the junctions between adjacent platelets, suggesting a role for this protein in contact-dependent events that regulate thrombus formation. To test this hypothesis we examined the effect of ESAM deletion on platelet function. In vivo, ESAM-/- mice achieved more stable hemostasis than wild-type mice following tail transection, and developed larger thrombi following laser injury of cremaster muscle arterioles. In vitro, ESAM-/- platelets aggregated at lower concentrations of G protein-dependent agonists than wild-type platelets, and were more resistant to disaggregation. In contrast, agonist-induced calcium mobilization, αIIbβ3 activation, alpha-granule secretion and platelet spreading, were normal in ESAM-deficient platelets. To understand the molecular mechanism by which ESAM regulates platelet activity, we utilized a PDZ domain array to identify the scaffold protein NHERF-1 as an ESAM binding protein, and further demonstrated that it associates with ESAM in both resting and activated platelets. Conclusions: These findings support a model in which ESAM localizes to platelet contacts following platelet activation in order to limit thrombus growth and stability so that the optimal hemostatic response occurs following vascular injury.
Magnetic resonance imaging (MRI) is routinely used to obtain anatomical images that have greatly advanced biomedical research and clinical health care today, but the full potential of MRI in providing functional, physiological, and molecular information is only beginning to emerge. In this work, we sought to provide a gene expression marker for MRI based on bacterial magnetosomes, tiny magnets produced by naturally occurring magnetotactic bacteria. Specifically, magA, a gene in magnetotactic bacteria known to be involved with iron transport, is expressed in a commonly used human cell line, 293FT, resulting in the production of magnetic, iron-oxide nanoparticles by these cells and leading to increased transverse relaxivity. MRI shows that these particles can be formed in vivo utilizing endogenous iron and can be used to visualize cells positive for magA. These results demonstrate that magA alone is sufficient to produce magnetic nanoparticles and that it is an appropriate candidate for an MRI reporter gene.
Biomaterials capable of providing localized and sustained presentation of bioactive proteins are critical for effective therapeutic growth factor delivery. However, current biomaterial delivery vehicles commonly suffer from limitations that can result in low retention of growth factors at the site of interest or adversely affect growth factor bioactivity. Heparin, a highly sulfated glycosaminoglycan, is an attractive growth factor delivery vehicle due to its ability to reversibly bind positively charged proteins, provide sustained delivery, and maintain protein bioactivity. This study describes the fabrication and characterization of heparin methacrylamide (HMAm) microparticles for recombinant growth factor delivery. HMAm microparticles were shown to efficiently bind several heparin-binding growth factors (e.g. bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (FGF-2)), including a wide range of BMP-2 concentrations that exceeds the maximum binding capacity of other common growth factor delivery vehicles, such as gelatin. BMP-2 bioactivity was assessed on the basis of alkaline phosphatase (ALP) activity induced in skeletal myoblasts (C2C12). Microparticles loaded with BMP-2 stimulated comparable C2C12 ALP activity to soluble BMP-2 treatment, indicating that BMP-2-loaded microparticles retain bioactivity and potently elicit a functional cell response. In summary, our results suggest that heparin microparticles stably retain large amounts of bioactive BMP-2 for prolonged periods of time, and that presentation of BMP-2 via heparin microparticles can elicit cell responses comparable to soluble BMP-2 treatment. Consequently, heparin microparticles present an effective method of delivering and spatially retaining growth factors that could be used in a variety of systems to enable directed induction of cell fates and tissue regeneration.