An apicosome initiates self-organizing morphogenesis of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) self-organize into apicobasally polarized cysts, reminiscent of the lumenal epiblast stage, providing a model to explore key morphogenic processes in early human embryos. Here, we show that apical polarization begins on the interior of single hPSCs through the dynamic formation of a highly organized perinuclear apicosome structure. The membrane surrounding the apicosome is enriched in apical markers and displays microvilli and a primary cilium; its lumenal space is rich in Ca2+ Time-lapse imaging of isolated hPSCs reveals that the apicosome forms de novo in interphase, retains its structure during mitosis, is asymmetrically inherited after mitosis, and relocates to the recently formed cytokinetic plane, where it establishes a fully polarized lumen. In a multicellular aggregate of hPSCs, intracellular apicosomes from multiple cells are trafficked to generate a common lumenal cavity. Thus, the apicosome is a unique preassembled apical structure that can be rapidly used in single or clustered hPSCs to initiate self-organized apical polarization and lumenogenesis.

Stimulation of fibroblast proliferation by insoluble gadolinium salts.

The purpose of this study was to assess insoluble salts containing gadolinium (Gd(3+)) for effects on human dermal fibroblasts. Responses to insoluble Gd(3+) salts were compared to responses seen with Gd(3+) solubilized with organic chelators, as in the Gd(3+)-based contrast agents (GBCAs) used for magnetic resonance imaging. Insoluble particles of either Gd(3+) phosphate or Gd(3+) carbonate rapidly attached to the fibroblast cell surface and stimulated proliferation. Growth was observed at Gd(3+) concentrations between 12.5 and 125 µM, with toxicity at higher concentrations. Such a narrow window did not characterize GBCA stimulation. Proliferation induced by insoluble Gd(3+) salts was inhibited in the presence of antagonists of mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways (similar to chelated Gd(3+)) but was not blocked by an antibody to the platelet-derived growth factor receptor (different from chelated Gd(3+)). Finally, high concentrations of the insoluble Gd(3+) salts failed to prevent fibroblast lysis under low-Ca(2+) conditions, while similar concentrations of chelated Gd(3+) were effective. In conclusion, while insoluble Gd(3+) salts are capable of stimulating fibroblast proliferation, one should be cautious in assuming that GBCA dechelation must occur in vivo to produce the profibrotic changes seen in association with GBCA exposure in the subset of renal failure patients that develop nephrogenic systemic fibrosis.

Ordered assembly of the adhesive and electrochemical connections within newly formed intercalated disks in primary cultures of adult rat cardiomyocytes.

The intercalated disk (ID) is a complex structure that electromechanically couples adjoining cardiac myocytes into a functional syncitium. The integrity of the disk is essential for normal cardiac function, but how the diverse elements are assembled into a fully integrated structure is not well understood. In this study, we examined the assembly of new IDs in primary cultures of adult rat cardiac myocytes. From 2 to 5 days after dissociation, the cells flatten and spread, establishing new cell-cell contacts in a manner that recapitulates the in vivo processes that occur during heart development and myocardial remodeling. As cells make contact with their neighbors, transmembrane adhesion proteins localize along the line of apposition, concentrating at the sites of membrane attachment of the terminal sarcomeres. Cx43 gap junctions and ankyrin-G, an essential cytoskeletal component of voltage gated sodium channel complexes, were secondarily recruited to membrane domains involved in cell-cell contacts. The consistent order of the assembly process suggests that there are specific scaffolding requirements for integration of the mechanical and electrochemical elements of the disk. Defining the relationships that are the foundation of disk assembly has important implications for understanding the mechanical dysfunction and cardiac arrhythmias that accompany alterations of ID architecture.

Stoichiometry and Structure of Poly(amidoamine) Dendrimer-Lipid Complexes.

The energetics, stoichiometry, and structure of poly(amidoamine) (PAMAM) dendrimer-phospholipid interactions were measured with isothermal titration calorimetry (ITC), transmission electron microscopy (TEM), atomic force microscopy (AFM), dynamic light scattering (DLS), and molecular dynamics (MD) simulations. Dendrimers of sixth-generation and smaller interacted with the lipids at an average stoichiometry and enthalpy proportional to the number of primary amines per dendrimers (4.5 ± 0.1 lipids/primary amine and 6.3 ± 0.3 kJ/mol of primary amines, respectively). Larger dendrimers, however, demonstrated a decreased number of bound lipids and heat release per primary amine, presumably due to the steric restriction of dendrimer deformation on the lipid bilayer. For example, eighth-generation PAMAM dendrimers bound to 44% fewer lipids per primary amine and released 63% less heat per primary amine as compared to the smaller dendrimers. These differences in binding stoichiometry support generation-dependent models for dendrimer-lipid complexation, which are consistent with previously observed generation-dependent differences in dendrimer-induced membrane disruption. Dendrimers of seventh-generation and larger bound to lipids with an average stoichiometry consistent with each dendrimer having been wrapped by a bilayer of lipids, whereas smaller dendrimers did not.

ATF6alpha induces XBP1-independent expansion of the endoplasmic reticulum.

A link exists between endoplasmic reticulum (ER) biogenesis and the unfolded protein response (UPR), a complex set of signaling mechanisms triggered by increased demands on the protein folding capacity of the ER. The UPR transcriptional activator X-box binding protein 1 (XBP1) regulates the expression of proteins that function throughout the secretory pathway and is necessary for development of an expansive ER network. We previously demonstrated that overexpression of XBP1(S), the active form of XBP1 generated by UPR-mediated splicing of Xbp1 mRNA, augments the activity of the cytidine diphosphocholine (CDP-choline) pathway for biosynthesis of phosphatidylcholine (PtdCho) and induces ER biogenesis. Another UPR transcriptional activator, activating transcription factor 6alpha (ATF6alpha), primarily regulates expression of ER resident proteins involved in the maturation and degradation of ER client proteins. Here, we demonstrate that enforced expression of a constitutively active form of ATF6alpha drives ER expansion and can do so in the absence of XBP1(S). Overexpression of active ATF6alpha induces PtdCho biosynthesis and modulates the CDP-choline pathway differently than does enforced expression of XBP1(S). These data indicate that ATF6alpha and XBP1(S) have the ability to regulate lipid biosynthesis and ER expansion by mechanisms that are at least partially distinct. These studies reveal further complexity in the potential relationships between UPR pathways, lipid production and ER biogenesis.

Dendrimer-entrapped gold nanoparticles as a platform for cancer-cell targeting and imaging.

We present a general approach for the targeting and imaging of cancer cells using dendrimer-entrapped gold nanoparticles (Au DENPs). Au DENPs were found to be able to covalently link with targeting and imaging ligands for subsequent cancer-cell targeting and imaging. The Au DENPs linked with defined numbers of folic acid (FA) and fluorescein isothiocyanate (FI) molecules are water soluble, stable, and biocompatible. In vitro studies show that the FA- and FI-modified Au DENPs can specifically bind to KB cells (a human epithelial carcinoma cell line) that overexpress high-affinity folate receptors and they are internalized dominantly into lysosomes of target cells within 2 h. These findings demonstrate that Au DENPs may serve as a general platform for cancer imaging and therapeutics.

Islet microvasculature in islet hyperplasia and failure in a model of type 2 diabetes.

Gene expression profiling of islets from pre-diabetic male Zucker diabetic fatty (ZDF) rats showed increased expression of hypoxia-related genes, prompting investigation of the vascular integrity of the islets. The islet microvasculature was increased approximately twofold in young male ZDF rats by both morphometric analysis and quantifying mRNA levels of endothelial markers. ZDF rats at 12 weeks of age showed a significant reduction in the number of endothelial cells, which was prevented by pretreatment with pioglitazone. Light and electron microscopy of normoglycemic 7-week-old ZDF rats showed thickened endothelial cells with loss of endothelial fenestrations. By 12 weeks of age, there was disruption of the endothelium and intra-islet hemorrhage. Islets from 7- and 12-week-old ZDF rats showed an approximate three- and twofold increase in vascular endothelial growth factor (VEGF)-A mRNA and VEGF protein secretion, respectively, compared with lean controls. Thrombospondin-1 mRNA increased in 7- and 12-week-old rats by 2- and 10-fold, respectively, and was reduced by 50% in 12-week-old rats pretreated with pioglitazone. Islets from young male control rats induced migration of endothelial cells in a collagen matrix only after pretreatment with matrix metalloproteinase (MMP)-9. Islets from 7-week-old ZDF rats showed a fivefold increase in migration score compared with wild-type controls, even without MMP-9 treatment. Islets from 15-week-old ZDF rats did not induce migration; rather, they caused a significant rounding up of the duct-derived cells, suggesting a toxic effect. These data suggest that in the ZDF rat model of type 2 diabetes, an inability of the islet to maintain vascular integrity may contribute to beta-cell failure.

Regulation of Legionella phagosome maturation and infection through flagellin and host Ipaf.

Legionella pneumophila is an intracellular bacterium that causes an acute form of pneumonia called Legionnaires' disease. After infection of human macrophages, the Legionella-containing phagosome (LCP) avoids fusion with the lysosome allowing intracellular replication of the bacterium. In macrophages derived from most mouse strains, the LCP is delivered to the lysosome resulting in Legionella degradation and restricted bacterial growth. Mouse macrophages lacking the NLR protein Ipaf or its downstream effector caspase-1 are permissive to intracellular Legionella replication. However, the mechanism by which Ipaf restricts Legionella replication is not well understood. Here we demonstrate that the presence of flagellin and a competent type IV secretion system are critical for Legionella to activate caspase-1 in macrophages. Activation of caspase-1 in response to Legionella infection also required host Ipaf, but not TLR5. In the absence of Ipaf or caspase-1 activation, the LCP acquired endoplasmic reticulum-derived vesicles, avoided fusion with the lysosome, and allowed Legionella replication. Accordingly a Legionella mutant lacking flagellin did not activate caspase-1, avoided degradation, and replicated in wild-type macrophages. The regulation of phagosome maturation by Ipaf occurred within 2 h after infection and was independent of macrophage cell death. In vivo studies confirmed that flagellin and Ipaf play an important role in the control of Legionella clearance. These results reveal that Ipaf restricts Legionella replication through the regulation of phagosome maturation, providing a novel function for NLR proteins in host defense against an intracellular bacterium.

Encapsulation of submicrometer-sized 2-methoxyestradiol crystals into polymer multilayer capsules for biological applications.

A facile approach has been developed to encapsulate submicrometer-sized drug crystals into polymer multilayer capsules produced by sequential deposition of polymers onto the drug particle surfaces. 2-Methoxyestradiol (2-ME) is a hydrophobic metabolite of 17-beta estradiol, which has been demonstrated as a potential anticancer agent. It was selected as a model drug and was formulated into submicrometer-sized particles through fine milling followed by intense sonication in the presence of dipalmitoyl-dl-(R)-phosphatidylcholine (DPPC). The reserved positive charges on the 2-ME crystal surface by DPPC enhanced the water solubility of the particles and subsequent self-assembly of dextran sulfate (DS) and dextran (DN) multilayers through hydrogen bonding and physical adsorption. Upon the exposure of the drug capsules to ethanol, hollow DS/DN multilayer polymer shells can be formed. The encapsulation process and hollow polymer multilayer shell formation were confirmed by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM), while the surface morphology of the formed drug capsules was investigated using scanning electron microscopy (SEM). In vitro studies show that the inhibitory effect of the formed 2-ME capsules is the same as that of the conventional formulation of 2-ME in a concentrated ethanol solution, as demonstrated by dramatic changes in cell morphology and significantly decreased viability of target cells. We also demonstrate that the change of the outermost layer of the drug capsules does not significantly influence its bioactivity. The presented strategy to encapsulate submicrometer-sized hydrophobic drug particles is expected to provide a general pathway to fabricate drug capsules for various biological applications.