Synthesis and Characterization of Dual-Functionalized Core-Shell Fluorescent Microspheres for Bioconjugation and Cellular Delivery

The efficient transport of micron-sized beads into cells, via a non-endocytosis mediated mechanism, has only recently been described. As such there is considerable scope for optimization and exploitation of this procedure to enable imaging and sensing applications to be realized. Herein, we report the design, synthesis and characterization of fluorescent microsphere-based cellular delivery agents that can also carry biological cargoes. These core-shell polymer microspheres possess two distinct chemical environments; the core is hydrophobic and can be labeled with fluorescent dye, to permit visual tracking of the microsphere during and after cellular delivery, whilst the outer shell renders the external surfaces of the microspheres hydrophilic, thus facilitating both bioconjugation and cellular compatibility. Cross-linked core particles were prepared in a dispersion polymerization reaction employing styrene, divinylbenzene and a thiol-functionalized co-monomer. These core particles were then shelled in a seeded emulsion polymerization reaction, employing styrene, divinylbenzene and methacrylic acid, to generate orthogonally functionalized core-shell microspheres which were internally labeled via the core thiol moieties through reaction with a thiol reactive dye (DY630-maleimide). Following internal labeling, bioconjugation of green fluorescent protein (GFP) to their carboxyl-functionalized surfaces was successfully accomplished using standard coupling protocols. The resultant dual-labeled microspheres were visualized by both of the fully resolvable fluorescence emissions of their cores (DY630) and shells (GFP). In vitro cellular uptake of these microspheres by HeLa cells was demonstrated conventionally by fluorescence-based flow cytometry, whilst MTT assays demonstrated that 92% of HeLa cells remained viable after uptake. Due to their size and surface functionalities, these far-red-labeled microspheres are ideal candidates for in vitro, cellular delivery of proteins.     

The small GTPase Rap1 is activated by turbulence and is involved in integrin [alpha]IIb[beta]3-mediated cell adhesion in human megakaryocytes

The small GTPase Rap1, which is activated by a large variety of stimuli, functions in the control of integrin-mediated cell adhesion. Here we show that in human megakaryocytes and several other commonly used hematopoietic cell lines such as K562, Jurkat, and THP-1, stress induced by gentle tumbling of the samples resulted in rapid and strong activation of Rap1. This turbulence-induced activation could not be blocked by inhibitors previously shown to affect Rap1 activation in human platelets, such as the intracellular calcium chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and various protein kinase C inhibitors. Also inhibition of actin cytoskeleton dynamics did not influence this activation of Rap1, suggesting that this activation is mediated by cell surface receptors. Human platelets, however, were refractory to turbulence-induced activation of Rap1. To determine the consequences of Rap1 activation we measured adhesion of megakaryocytes to fibrinogen, which is mediated by the integrin alphaIIbbeta3, in the presence of inhibitors of Rap1 signaling. Introduction of both Rap1GAP and RalGDS-RBD in the megakaryoblastic cell line DAMI strongly reduced basal adhesion to immobilized fibrinogen. This inhibition was partially rescued by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate but not by alpha-thrombin. From these results we conclude that in megakaryocytes turbulence induces Rap1 activation that controls alphaIIbbeta3-mediated cell adhesion.     

Rap1 regulates E-cadherin-mediated cell-cell adhesion

The small GTPase Rap is best characterized as a critical regulator of integrin-mediated cell adhesion, although its mechanism of action is not understood. Rap also influences the properties of other cell-surface receptors and biological processes, although whether these are a consequence of effects on integrins is not clear. We show here that Rap also plays an important role in the regulation of cadherin-mediated cell-cell adhesion in epithelial cells. Expression of constitutively active Rap1A restored cadherin-mediated cell-cell contacts in mesenchymal Ras-transformed Madin-Darby canine kidney cells, resulting in reversion to an epithelial phenotype. Activation of endogenous Rap via the Rap exchange factor Epac1 also antagonized hepatocyte growth factor-induced disruption of adherens junctions. Inhibition of Rap signaling resulted in disruption of epithelial cell-cell contacts. Rap activity was required for adhesion of cells to recombinant E-cadherin extracellular domains, i.e. in the absence of integrin-mediated adhesion. These findings suggest that Rap signaling positively contributes to cadherin-mediated adhesion and that this occurs independently of effects on integrin-mediated adhesion. Our results imply that Rap may function in a broader manner to regulate the function of cell-surface adhesion receptors.     

Rap1A-deficient T and B cells show impaired integrin-mediated cell adhesion

Studies in tissue culture cells have demonstrated a role for the Ras-like GTPase Rap1 in the regulation of integrin-mediated cell-matrix and cadherin-mediated cell-cell contacts. To analyze the function of Rap1 in vivo, we have disrupted the Rap1A gene by homologous recombination. Mice homozygous for the deletion allele are viable and fertile. However, primary hematopoietic cells isolated from spleen or thymus have a diminished adhesive capacity on ICAM and fibronectin substrates. In addition, polarization of T cells from Rap1-/- cells after CD3 stimulation was impaired compared to that of wild-type cells. Despite this, these defects did not result in hematopoietic or cell homing abnormalities. Although it is possible that the relatively mild phenotype is a consequence of functional complementation by the Rap1B gene, our genetic studies confirm a role for Rap1A in the regulation of integrins.     

Makorin ring zinc finger protein 1 (MKRN1), a novel poly(A)-binding protein-interacting protein, stimulates translation in nerve cells

The poly(A)-binding protein (PABP), a key component of different ribonucleoprotein complexes, plays a crucial role in the control of mRNA translation rates, stability, and subcellular targeting. In this study we identify RING zinc finger protein Makorin 1 (MKRN1), a bona fide RNA-binding protein, as a binding partner of PABP that interacts with PABP in an RNA-independent manner. In rat brain, a so far uncharacterized short MKRN1 isoform, MKRN1-short, predominates and is detected in forebrain nerve cells. In neuronal dendrites, MKRN1-short co-localizes with PABP in granule-like structures, which are morphological correlates of sites of mRNA metabolism. Moreover, in primary rat neurons MKRN1-short associates with dendritically localized mRNAs. When tethered to a reporter mRNA, MKRN1-short significantly enhances reporter protein synthesis. Furthermore, after induction of synaptic plasticity via electrical stimulation of the perforant path in vivo, MKRN1-short specifically accumulates in the activated dendritic lamina, the middle molecular layer of the hippocampal dentate gyrus. Collectively, these data indicate that in mammalian neurons MKRN1-short interacts with PABP to locally control the translation of dendritic mRNAs at synapses.     

Rap2A links intestinal cell polarity to brush border formation

The microvillus brush border at the apex of the highly polarized enterocyte allows the regulated uptake of nutrients from the intestinal lumen. Here, we identify the small G protein Rap2A as a molecular link that couples the formation of microvilli directly to the preceding cell polarization. Establishment of apicobasal polarity, which can be triggered by the kinase LKB1 in single, isolated colon cells, results in enrichment of PtdIns(4,5)P(2) at the apical membrane. The subsequent recruitment of phospholipase D1 allows polarized accumulation of phosphatidic acid, which provides a local cue for successive signalling by the guanine nucleotide exchange factor PDZGEF, the small G protein Rap2A, its effector TNIK, the kinase MST4 and, ultimately, the actin-binding protein Ezrin. Thus, epithelial cell polarization is translated directly into the acquisition of brush borders through a small G protein signalling module whose action is positioned by a cortical lipid cue.     

Screening for dioxin-degrading basidiomycetes from temperate and tropical forests

A total of 500 strains of basidiomycetes isolated from temperate forests in Japan and 379 strains from tropical forests in Indonesia were subjected to a laboratory screening for dioxin-degrading ability. At first, about 200 fungal strains were selected by their ability to decolorize Remazol Brilliant Blue R dye as an indicator of ligninolytic activities. Next, for excluding the factor of dioxin sorption by mycelia, we prepared two series of living cultures exposed either long-term or short-term to 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), and compared the decreases in the remaining amounts of this model compound. We chose Bjerkandera adusta strain VH57 as a promising new candidate for dioxin degradation, because it gave 40% difference in 2,7-DCDD levels between the two treatments after 30 days of exposure.