Cytosolic protein delivery using ultrasound-guided vaporization of perfluorocarbon nano-droplets.

Ultrasound-guided protein delivery is promising for site-specific control of cellular functions in the deep interior of the body in a noninvasive manner. Herein, we propose a method for cytosolic protein delivery based on ultrasound-guided intracellular vaporization of perfluorocarbon nano-droplets. The nano-droplets were conjugated with cargo proteins through a bio-reductively cleavable linker and introduced into living cells via antibody-mediated binding to a cell-surface receptor, which gets internalized through endocytosis. After the cells were exposed to ultrasound for endosomal escape of proteins, the ultrasound-responsive cytosolic release of a cargo enzyme was confirmed by visualizing the hydrolysis of the fluorogenic substrate using confocal microscopy. Moreover, a significant decrease in cell viability was achieved via the release of a cytotoxic protein in response to ultrasound treatment. The results of this study provide the proof of a principle that protein-conjugated nano-droplets can be used as carriers in ultrasound-guided cytosolic delivery of proteins.

Bioorthogonal Photoreactive Surfaces for Single-Cell Analysis of Intercellular Communications.

Methods to construct single-cell pairs of heterogeneous cells attract attention because of their potential in cell biological and medical applications for analyzing individual intercellular communications such as immune and nerve synaptic interactions. Photoactivatable substrate surfaces for cell anchoring are promising tools to achieve single-cell pairing. However, conventional surfaces that photoactivate a single type of cell anchoring moiety restrict the combination of cell pair types and their applications. We developed a photoresponsive material comprising a bioorthogonal photoreactive moiety and non-cell adhesive hydrophilic polymer. The material-coated surface allows conjugation with various cell anchoring molecules in response to light at specific timings and consequently achieves light-induced anchoring of a variety of cells at defined regions. Using the platform surface, an array of cancer cell and natural-killer (NK) cell pairs was constructed on a flat substrate surface and the dynamic morphological changes of the cancer cells were monitored by cytotoxic interaction with NK cells at a single-cell level. The photoreactive surface is a useful tool for image-based investigation of the communications between a variety of cell types.

Selective intracellular delivery of perfluorocarbon nanodroplets for cytotoxicity threshold reduction on ultrasound-induced vaporization.

BACKGROUND: Phase-change nanodroplets (PCNDs), which are liquid perfluorocarbon nanoparticles, have garnered much attention as ultrasound-responsive nanomedicines. The vaporization phenomenon has been employed to treat tumors mechanically. However, the ultrasound pressure applied to induce vaporization must be low to avoid damage to nontarget tissues. AIMS: Here, we report that the pressure threshold for vaporization to induce cytotoxicity can be significantly reduced by selective intracellular delivery of PCNDs into targeted tumors. METHODS AND RESULTS: In vitro experiments revealed that selective intracellular delivery of PCNDs induced PCND aggregation specifically inside the targeted cells. This close-packed configuration decreased the pressure threshold for vaporization to induce cytotoxicity. Moreover, following ultrasound exposure, significant decrease was observed in the viability of cells that incorporated PCNDs (35%) but not in the viability of cells that did not incorporate PCNDs (88%). CONCLUSIONS: Intracellular delivery of PCNDs reduced ultrasound pressure applied for vaporization to induce cytotoxicity. Confocal laser scanning microscopy and flow cytometry revealed that prolonged PCND-cell incubation increased PCND uptake and aggregation. This aggregation effect might have contributed to the cytotoxicity threshold reduction effect.

Epiregulin-blocking antibody inhibits epiregulin-dependent EGFR signaling.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase involved in many cellular functions including cell growth and migration. EGFR may be activated by EGF family ligands such as EGF and epiregulin (EREG). EREG is overexpressed in human colon and breast cancers, implying that EREG plays roles in tumorigenesis. Although EGF family members share a receptor, it is not well known whether their signaling pathways differ. In order to investigate EREG signaling, we established the anti-EREG antibody that inhibits EGFR downstream signaling stimulated by EREG but not by EGF. While the anti-EREG antibody has little effect on cell growth, it inhibits cell adhesion of EREG-expressing autocrine cancer cell lines. Our results suggest that anti-EREG antibodies represent valuable tools for elucidating EREG-specific signaling pathways, and may serve as therapeutic candidates for the treatment of cancers.

Epiregulin Recognition Mechanisms by Anti-epiregulin Antibody 9E5: STRUCTURAL, FUNCTIONAL, AND MOLECULAR DYNAMICS SIMULATION ANALYSES.

Epiregulin (EPR) is a ligand of the epidermal growth factor (EGF) family that upon binding to its epidermal growth factor receptor (EGFR) stimulates proliferative signaling, especially in colon cancer cells. Here, we describe the three-dimensional structure of the EPR antibody (the 9E5(Fab) fragment) in the presence and absence of EPR. Among the six complementarity-determining regions (CDRs), CDR1-3 in the light chain and CDR2 in the heavy chain predominantly recognize EPR. In particular, CDR3 in the heavy chain dramatically moves with cis-trans isomerization of Pro(103). A molecular dynamics simulation and mutational analyses revealed that Arg(40) in EPR is a key residue for the specific binding of 9E5 IgG. From isothermal titration calorimetry analysis, the dissociation constant was determined to be 6.5 nm. Surface plasmon resonance analysis revealed that the dissociation rate of 9E5 IgG is extremely slow. The superimposed structure of 9E5(Fab)·EPR on the known complex structure of EGF·EGFR showed that the 9E5(Fab) paratope overlaps with Domains I and III on the EGFR, which reveals that the 9E5(Fab)·EPR complex could not bind to the EGFR. The 9E5 antibody will also be useful in medicine as a neutralizing antibody specific for colon cancer.

Construction and characterization of functional anti-epiregulin humanized monoclonal antibodies.

Growth factors are implicated in several processes essential for cancer progression. Specifically, epidermal growth factor (EGF) family members, including epiregulin (EREG), are important prognostic factors in many epithelial cancers, and treatments targeting these molecules have recently become available. Here, we constructed and expressed humanized anti-EREG antibodies by variable domain resurfacing based on the three-dimensional (3D) structure of the Fv fragment. However, the initial humanized antibody (HM0) had significantly decreased antigen-binding affinity. Molecular modeling results suggested that framework region (FR) residues latently important to antigen binding included residue 49 of the light chain variable region (VL). Back mutation of the VL49 residue (tyrosine to histidine) generated the humanized version HM1, which completely restored the binding affinity of its murine counterpart. Importantly, only one mutation in the framework may be necessary to recover the binding capability of a humanized antibody. Our data support that HM1 exerts potent antibody-dependent cellular cytotoxicity (ADCC). Hence, this antibody may have potential for further development as a candidate therapeutic agent and research tool.

A single amino acid of toll-like receptor 4 that is pivotal for its signal transduction and subcellular localization.

Toll-like receptor 4 (TLR4) is essential for recognizing a Gram-negative bacterial component, lipopolysaccharide (LPS). A single amino acid mutation at position 712 of murine TLR4 leads to hyporesponsiveness to LPS. In this study we determined that an amino acid, a leucine at position 815 of human TLR4, is also pivotal for LPS responsiveness and subcellular distribution. By replacing the leucine with alanine, the mutant TLR4 lost responsiveness to LPS and did not localize on the plasma membrane. In addition, it does not coprecipitate with myeloid differentiation-2, an accessory protein that is necessary for TLR4 to recognize LPS. These results suggest that the leucine at position 815 is required for the normal maturation of TLR4 and for formation of the TLR4.MD-2 complex.

Chlamydophilal antigens induce foam cell formation via c-Jun NH2-terminal kinase.

Chlamydophila pneumoniae is known to be associated with atherosclerosis. Recent studies have reported that components of Chlamydophila pneumoniae (chlamydophilal antigens) induce foam cell formation in macrophages. However, the mechanism of foam cell formation induced by chlamydophilal antigens has yet to be elucidated. In this paper, we first found that mitogen-activated protein kinases including extracellular signal-regulated kinase, p38 and c-Jun NH2 terminal kinase are phosphorylated after stimulation by chlamydophilal antigens. We then showed that chlamydophilal antigens induce foam cell formation mainly via c-Jun NH2 terminal kinase. Finally, we demonstrated that foam cell formation and phosphorylation of mitogen-activated protein kinases induced by chlamydophilal antigens are mainly recognized through Toll-like receptor 2. These results collectively indicated that chlamydophilal antigens induce foam cell formation mainly via Toll-like receptor 2 and c-Jun NH2 terminal kinase.

Biphasic regulation of levofloxacin on lipopolysaccharide-induced IL-1beta production.

Fluoroquinolones have been known to exert modulatory activity on immune responses to microbial infection. However, the mechanism of this immunomodulation has not been well elucidated. In this study, we investigated the effect of levofloxacin on lipopolysaccharide (LPS)-induced production of interleukin-1beta (IL-1beta) in RAW264.7 cells. We showed that LPS-stimulated release of pre-synthesized IL-1beta was promoted by levofloxacin, in part via the p38 mitogen-activated protein kinase (MAPK) pathway. On the other hand, newly synthesized IL-1beta production was inhibited by levofloxacin. This immunoregulatory function of levofloxacin in the later phase as well as promotion of pre-synthesized IL-1beta release by levofloxacin in the early phase might be advantageous in the host defense to microbial pathogens.

Cooperation of phosphoinositides and BAR domain proteins in endosomal tubulation.

Phosphorylated derivatives of phosphatidylinositol (PtdIns) regulate many intracellular events, including vesicular trafficking and actin remodeling, by recruiting proteins to their sites of function. PtdIns(4,5)-bisphosphate [PI(4,5)P2] and related phosphoinositides are mainly synthesized by type I PtdIns-4-phosphate 5-kinases (PIP5Ks). We found that PIP5K induces endosomal tubules in COS-7 cells. ADP-ribosylation factor (ARF) 6 has been shown to act upstream of PIP5K and regulate endocytic transport and tubulation. ARF GAP with coiled-coil, ankyrin repeat, and pleckstrin homology domains 1 (ACAP1) has guanosine triphosphatase-activating protein (GAP) activity for ARF6. While there were few tubules induced by the expression of ACAP1 alone, numerous endosomal tubules were induced by coexpression of PIP5K and ACAP1. ACAP1 has a pleckstrin homology (PH) domain known to bind phosphoinositide and a Bin/amphiphysin/Rvs (BAR) domain that has been reported to detect membrane curvature. Truncated and point mutations in the ACAP1 BAR and PH domains revealed that both BAR and PH domains are required for tubulation. These results suggest that two ARF6 downstream molecules, PIP5K and ACAP1, function together in endosomal tubulation and that phosphoinositide levels may regulate endosomal dynamics.

Phosphatidylinositol-4-phosphate 5-kinase gamma is associated with cell-cell junction in A431 epithelial cells.

Cell to cell contact in epithelial cells is crucial for tissue integrity and is maintained by junctional complexes, such as the adherens junction (AJ). Actin polymerization has been shown to be important for AJ formation; however, the molecular mechanisms have yet to be clarified. It has been shown that increased phosphatidylinositol-4,5-bisphosphate (PIP2) induces actin polymerization. It is thus of interest to know more about the production of PIP2 during cell-cell adhesion formation in epithelial cells. The distribution of phosphatidylinositol-4-phosphate 5-kinase gamma635 (PIP5Kgamma635), an isoform of the PIP2 synthesizing enzymes, was examined in epithelial cell line A431. It was found that, in non-contact cells, PIP5Kgamma635 was not concentrated at the plasma membrane. However, in cells that were in contact, PIP5Kgamma635 localized to the intercellular contact sites and colocalized with E-cadherin and beta-catenin, two components of AJ, and with polymerized actin, but did not colocalize with focal adhesion, integrin-mediated cell-substratum complex. Decreasing calcium ion concentration induced both disruption of intercellular adhesion and the dissociation of both PIP5Kgamma635 and actin from the contact site. These results suggest that PIP5K has an important role in actin polymerization in epithelial cell-cell adhesion.

Association of active gamma-secretase complex with lipid rafts.

Cholesterol has been implicated in the pathogenesis of Alzheimer's disease (AD). Although the underlying mechanisms are not yet clear, several studies have provided evidence for the involvement of cholesterol-rich lipid rafts in the production of amyloid beta peptide (Abeta), the major component of amyloid deposits in AD. In this regard, the gamma-secretase complex is responsible for the final cleavage event in the processing of beta-amyloid precursor protein (betaAPP), resulting in Abeta generation. The gamma-secretase complex is a multiprotein complex composed of presenilin, nicastrin (NCT), APH-1, and PEN-2. Recent reports have suggested that gamma-secretase activity is predominantly localized in lipid rafts, and presenilin and NCT have been reported to be localized in lipid rafts. In this study, various biochemical methods, including coimmunoprecipitation, in vitro gamma-secretase assay, and methyl-beta-cyclodextrin (MbetaCD) treatment, are employed to demonstrate that all four components of the active endogenous gamma-secretase complex, including APH-1 and PEN-2, are associated with lipid rafts in human neuroblastoma cells (SH-SY5Y). Treatment with statins, 3-hydroxy-3-methylglutaryl-CoA-reductase inhibitors, significantly decreased the association of the gamma-secretase complex with lipid rafts without affecting the distribution of flotillin-1. This effect was partially abrogated by the addition of geranylgeraniol. These results suggest that both cholesterol and protein isoprenylation influence the active gamma-secretase complex association with lipid rafts.

Dibasic amino acid residues at the carboxy-terminal end of kinase homology domain participate in the plasma membrane localization and function of phosphatidylinositol 5-kinase gamma.

Type I phosphatidylinositol 4-phosphate (PI(4)P) 5-kinases (PIP5Ks) catalyze the synthesis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), an essential lipid molecule involved in various cellular processes such as regulation of actin cytoskeleton and membrane traffic. The protein localizes to the plasma membrane where its activity has been shown to be regulated by small GTPase ARFs and/or phosphatidic acid. Deletion analysis of amino- or carboxy-terminal sequences of PIP5Kgamma fused with EGFP demonstrated that the presence of central kinase homology domain (KHD), a 380 amino acid-long region highly conserved among PIP5K family, was necessary and sufficient for the plasma membrane localization of PIP5Kgamma. Particularly, the dibasic Arg-Lys sequence located at the carboxy-terminal end of KHD was shown to be crucial for the plasma membrane targeting of PIP5Kgamma, since the deletion or charge-reversal mutation of this dibasic sequence resulted in the mislocalization of the protein to the cytoplasm. Mislocalized mutants also failed to complement the temperature-sensitive growth of Saccharomyces cerevisiae mss4-1 mutant defective in PIP5K function. The presence of dibasic residues at the C-terminal end of KHD was conserved among mammalian as well as invertebrate PIP5K family members, but not in the type II PIPKs that are not targeted to the plasma membrane, suggesting that the conserved dibasic motif provides a mechanism essential for the proper localization and cellular function of PIP5Ks.