Multifunctional protein 4.1R regulates the asymmetric segregation of Numb during terminal erythroid maturation.

The asymmetric cell division of stem or progenitor cells generates daughter cells with distinct fates that balance proliferation and differentiation. Asymmetric segregation of Notch signaling regulatory protein Numb plays a crucial role in cell diversification. However, the molecular mechanism remains unclear. Here, we examined the unequal distribution of Numb in the daughter cells of murine erythroleukemia cells (MELCs) that undergo DMSO-induced erythroid differentiation. In contrast to the cytoplasmic localization of Numb during uninduced cell division, Numb is concentrated at the cell boundary in interphase, near the one-spindle pole in metaphase, and is unequally distributed to one daughter cell in anaphase in induced cells. The inheritance of Numb guides this daughter cell toward erythroid differentiation while the other cell remains a progenitor cell. Mitotic spindle orientation, critical for distribution of cell fate determinants, requires complex communication between the spindle microtubules and the cell cortex mediated by the NuMA-LGN-dynein/dynactin complex. Depletion of each individual member of the complex randomizes the position of Numb relative to the mitotic spindle. Gene replacement confirms that multifunctional erythrocyte protein 4.1R (4.1R) functions as a member of the NuMA-LGN-dynein/dynactin complex and is necessary for regulating spindle orientation, in which interaction between 4.1R and NuMA plays an important role. These results suggest that mispositioning of Numb is the result of spindle misorientation. Finally, disruption of the 4.1R-NuMA-LGN complex increases Notch signaling and decreases the erythroblast population. Together, our results identify a critical role for 4.1R in regulating the asymmetric segregation of Numb to mediate erythropoiesis.

Epithelial-specific isoforms of protein 4.1R promote adherens junction assembly in maturing epithelia.

Epithelial adherens junctions (AJs) and tight junctions (TJs) undergo disassembly and reassembly during morphogenesis and pathological states. The membrane-cytoskeleton interface plays a crucial role in junctional reorganization. Protein 4.1R (4.1R), expressed as a diverse array of spliceoforms, has been implicated in linking the AJ and TJ complex to the cytoskeleton. However, which specific 4.1 isoform(s) participate and the mechanisms involved in junctional stability or remodeling remain unclear. We now describe a role for epithelial-specific isoforms containing exon 17b and excluding exon 16 4.1R (4.1R+17b) in AJs. 4.1R+17b is exclusively co-localized with the AJs. 4.1R+17b binds to the armadillo repeats 1-2 of ß-catenin via its membrane-binding domain. This complex is linked to the actin cytoskeleton via a bispecific interaction with an exon 17b-encoded peptide. Exon 17b peptides also promote fodrin-actin complex formation. Expression of 4.1R+17b forms does not disrupt the junctional cytoskeleton and AJs during the steady-state or calcium-dependent AJ reassembly. Overexpression of 4.1R-17b forms, which displace the endogenous 4.1R+17b forms at the AJs, as well as depletion of the 4.1R+17b forms both decrease junctional actin and attenuate the recruitment of spectrin to the AJs and also reduce E-cadherin during the initial junctional formation of the AJ reassembly process. Expressing 4.1R+17b forms in depleted cells rescues junctional localization of actin, spectrin, and E-cadherin assembly at the AJs. Together, our results identify a critical role for 4.1R+17b forms in AJ assembly and offer additional insights into the spectrin-actin-4.1R-based membrane skeleton as an emerging regulator of epithelial integrity and remodeling.

Protein 4.1R Exon 16 3' Splice Site Activation Requires Coordination among TIA1, Pcbp1, and RBM39 during Terminal Erythropoiesis.

Exon 16 of protein 4.1R encodes a spectrin/actin-binding peptide critical for erythrocyte membrane stability. Its expression during erythroid differentiation is regulated by alternative pre-mRNA splicing. A UUUUCCCCCC motif situated between the branch point and the 3' splice site is crucial for inclusion. We show that the UUUU region and the last three C residues in this motif are necessary for the binding of splicing factors TIA1 and Pcbp1 and that these proteins appear to act in a collaborative manner to enhance exon 16 inclusion. This element also activates an internal exon when placed in a corresponding intronic position in a heterologous reporter. The impact of these two factors is further enhanced by high levels of RBM39, whose expression rises during erythroid differentiation as exon 16 inclusion increases. TIA1 and Pcbp1 associate in a complex containing RBM39, which interacts with U2AF65 and SF3b155 and promotes U2 snRNP recruitment to the branch point. Our results provide a mechanism for exon 16 3' splice site activation in which a coordinated effort among TIA1, Pcbp1, and RBM39 stabilizes or increases U2 snRNP recruitment, enhances spliceosome A complex formation, and facilitates exon definition through RBM39-mediated splicing regulation.

Rational design and characterization of the novel, broad and potent bispecific HIV-1 neutralizing antibody iMabm36.

BACKGROUND: Although broadly neutralizing monoclonal antibodies (bNAbs) have always been considered to be a potential therapeutic option for the prophylaxis and treatment of HIV infection, their lack of breadth against all HIV variants has been one of the limiting factors. To provide sufficient neutralization breadth and potency against diverse viruses, including neutralization escape mutants, strategies to combine different bNAbs have been explored recently. METHODS: We rationally designed and engineered a novel bispecific HIV-1-neutralizing antibody (bibNAb), iMabm36. The potency and breadth of iMabm36 against HIV were extensively characterized in vitro. RESULTS: iMabm36 comprises the anti-CD4 Ab ibalizumab (iMab) linked to 2 copies of the single-domain Ab m36, which targets a highly conserved CD4-induced epitope. iMabm36 neutralizes a majority of a large, multiclade panel of pseudoviruses (96%, n = 118) at an IC50 concentration of less than 10 µg/mL, with 83% neutralized at an IC50 concentration of less than 0.1 µg/mL. In addition, iMabm36 neutralizes a small panel of replication-competent transmitted-founder viruses to 100% inhibition at a concentration of less than 0.1 µg/mL in a peripheral blood mononuclear cell-based neutralizing assay. Mechanistically, the improved antiviral activity of iMabm36 is dependent on both the CD4-binding activity of the iMab component and the CD4i-binding activity of the m36 component. After characterizing that viral resistance to iMabm36 neutralization was due to mutations residing in the bridging sheet of gp120, an optimized m36 variant was engineered that, when fused to iMab, improved antiviral activity significantly. CONCLUSIONS: The interdependency of this dual mechanism of action enables iMabm36 to potently inhibit HIV-1 entry. These results demonstrate that mechanistic-based design of bibNAbs can generate potential preventive and therapeutic candidates for HIV/AIDS.

RBFOX2 promotes protein 4.1R exon 16 selection via U1 snRNP recruitment.

The erythroid differentiation-specific splicing switch of protein 4.1R exon 16, which encodes a spectrin/actin-binding peptide critical for erythrocyte membrane stability, is modulated by the differentiation-induced splicing factor RBFOX2. We have now characterized the mechanism by which RBFOX2 regulates exon 16 splicing through the downstream intronic element UGCAUG. Exon 16 possesses a weak 5' splice site (GAG/GTTTGT), which when strengthened to a consensus sequence (GAG/GTAAGT) leads to near-total exon 16 inclusion. Impaired RBFOX2 binding reduces exon 16 inclusion in the context of the native weak 5' splice site, but not the engineered strong 5' splice site, implying that RBFOX2 achieves its effect by promoting utilization of the weak 5' splice site. We further demonstrate that RBFOX2 increases U1 snRNP recruitment to the weak 5' splice site through direct interaction between its C-terminal domain (CTD) and the zinc finger region of U1C and that the CTD is required for the effect of RBFOX2 on exon 16 splicing. Our data suggest a novel mechanism for exon 16 5' splice site activation in which the binding of RBFOX2 to downstream intronic splicing enhancers stabilizes the pre-mRNA-U1 snRNP complex through interactions with U1C.

Volume polarization holographic recording in thick phenanthrenequinone-doped poly(methyl methacrylate) photopolymer.

Volume polarization holographic recording in phenanthrenequinone-doped poly (methyl methacrylate) photopolymer is obtained. Photoinduced birefringence in a 2 mm thick sample is measured by a phase-modulated ellipsometry. The birefringence induced in this material by linearly polarized beam at 514 nm reaches 1.2×10(-5). In addition, ability for recording volume polarization grating using two different polarization configurations is demonstrated and compared. The experimental results show that the diffraction efficiency of the hologram reaches to ∼40% by using two orthogonal circularly polarized beams.

Deassociate the initial temporal phase deviation provided by photoelastic modulator for stroboscopic illumination polarization modulated ellipsometry.

In addition to operating the imaging ellipsometric measurements by four-specific temporal phases in the photoelastic modulated ellipsometry, we added the fifth one to solve the initial phase of the photoelastic modulator. This methodology has been developed to conquer the slow imaging processing of charge-coupled device camera for the stroboscopic illumination in the polarization modulated imaging ellipsometry. Without any calibration in its initial phase, we can perform the ellipsometric measurement by the measurements of intensity at five-specific temporal phases. The intensities of a full cycle for a point on SiO(2)∕Si thin film were measured and analyzed for verifying this algorithm. The five stroboscopic illuminations were performed to measure the two-dimensional distribution of the same SiO(2)∕Si thin film.

Epitope mapping of ibalizumab, a humanized anti-CD4 monoclonal antibody with anti-HIV-1 activity in infected patients.

Ibalizumab is a humanized monoclonal antibody that binds human CD4, the primary receptor for human immunodeficiency virus type 1 (HIV-1). With its unique specificity for domain 2 of CD4, this antibody potently and broadly blocks HIV-1 infection in vitro by inhibiting a postbinding step required for viral entry but without interfering with major histocompatibility complex class II (MHC-II)-mediated immune function. In clinical trials, ibalizumab has demonstrated anti-HIV-1 activity in patients without causing immunosuppression. Thus, a characterization of the ibalizumab epitope was conducted in an attempt to gain insight into the underlying mechanism of its antiviral activity as well as its safety profile. By studying mouse/human chimeric CD4 molecules and site-directed point mutants of CD4, amino acids L96, P121, P122, and Q163 in domain 2 were found to be important for ibalizumab binding, with E77 and S79 in domain 1 also contributing. All these residues appear to cluster on the interface between domains 1 and 2 of human CD4 on a surface opposite the site where gp120 and the MHC-II molecule bind on domain 1. Separately, the epitope of M-T441, a weakly neutralizing mouse monoclonal antibody that competes with ibalizumab, was localized entirely within domain 2 on residues 123 to 125 and 138 to 140. The results reported herein not only provide an appreciation for why ibalizumab has not had significant adverse immunological consequences in infected patients to date but also raise possible steric hindrance mechanisms by which this antibody blocks HIV-1 entry into a CD4-positive cell.

Optimization of a four-temporal phase lock for photoelastic-modulated polarimetry.

A set of four-temporal phases in photoelastic-modulated polarimetry is proposed to measure the Stokes parameters. In comparison with the conventional polarimetry, which uses a set of four-spatial angles by rotating a quarter-wave plate to obtain the polarimetric parameters, this temporal type polarimetry not only can reduce the time consumption but also can avoid the measurement error from the beam deviation. In addition, based on singular value decomposition, the figure of merit of this temporal phase technique can improve its signal-to-noise ratio by a factor of 2 in comparison with the rotating quarter-wave plate.

Determining thickness of films on a curved substrate by use of ellipsometric measurements.

A three-intensity measurement technique has been employed in polarizer-sample-analyzer imaging ellipsometry to measure the two-dimensional ellipsometric parameters of a coated cylindrical lens. Since azimuth deviation alpha of a polarizer can also be measured with this three-intensity measurement technique, we tilted a well-calibrated thin-film wafer to identify the orientation of the measured alphawith respect to the incident plane. Using the analytic property of this measurement technique, we can correct the deviation and determine the thickness profile of the thin film coated on a cylindrical lens.

Analytical solutions of the azimuthal deviation of a polarizer and an analyzer by polarizer-sample-analyzer ellipsometry.

The analytical solutions of the azimuthal deviation of a polarizer and an analyzer were obtained by polarizer-sample-analyzer ellipsometry with a three-intensity measurement technique. By performing two sets of this three-intensity measurement with the polarizer's azimuth set at 45 degrees and at -45 degrees , we were able to obtain a set of ellipsometric parameters free from the azimuthal deviations of the polarizer and the analyzer.

Comparative evaluation of three different intramuscular delivery methods for DNA immunization in a nonhuman primate animal model.

Although plasmid DNA vaccines induce potent cell-mediated immune responses and prime for antibody responses in experimental laboratory animals, their immunogenicity in humans has been less remarkable. A number of strategies have been proposed to improve the immunogenicity of these vaccines, including using novel means of vaccine delivery. In the present study, the immunogenicity of three different methods of intramuscular plasmid DNA administration was compared in cynomolgus monkeys: needle and syringe, Biojector 2000, and Mini-Ject. The elicited cellular and humoral immune responses were comparable in monkeys immunized using these different delivery techniques, suggesting that the needle-free approaches to vaccine administration do not significantly improve the immunogenicity of the plasmid DNA vaccine used in the study.

Determination of optical parameters of a twisted-nematic liquid crystal by phase-sensitive optical heterodyne interferometric ellipsometry.

What is believed to be a novel phase-sensitive optical heterodyne interferometric ellipsometer is set up to characterize a twisted-nematic liquid crystal (TN-LC) by the elliptical parameters of the output polarization state. This ellipsometer presents the advantages of both polarized optical heterodyne interferometry and optical photometry, which introduce a polarization modulation that is capable of performing with high-sensitivity on phase detection in real time. The twist angle phi and the untwisted phase retardation gamma of TN-LC are measured precisely. The experimental results verify that a TN-LC can be treated as identical to an elliptical retarder.

Evaluation of CD62L expression as a marker for vaccine-elicited memory cytotoxic T lymphocytes.

The development of successful vaccination strategies for eliciting cytotoxic T lymphocytes (CTLs) will be facilitated by the definition of strategies for subdividing CTLs into functionally distinct subpopulations. We assessed whether surface expression of a number of cell-surface proteins could be used to define functionally distinct subpopulations of memory CTLs in mice immunized with a recombinant vaccinia virus expressing human immunodeficiency virus (HIV)-1 envelope (Env). We found changes in cell-surface expression of CD11a, CD44, CD45RB, CD49d, CD54 and CD62L on Env-specific CD8(+) T cells that appeared to differentiate them from other CD8(+) T cells within 1 week to 1 month following immunization. Further, we saw an up-regulation of CD62L surface expression on Env-specific CD8(+) memory T cells several months after immunization. However, CD62L expression did not correlate with differences in the abilities of CTLs to proliferate or produce interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) in vitro in response to Env peptide stimulation. Moreover, the expression of CD62L did not allow differentiation of CTLs into subpopulations with distinct expansion kinetics in vivo after adoptive transfer into naïve mice and subsequent boosting of these mice with a recombinant adenovirus expressing HIV-1 Env. Therefore, the definition of memory CD8(+) T-cell subpopulations on the basis of CD62L expression in mice does not allow the delineation of functionally distinct CTL subpopulations.

Recombinant poxvirus boosting of DNA-primed rhesus monkeys augments peak but not memory T lymphocyte responses.

Although a consensus has emerged that an HIV vaccine should elicit a cytotoxic T lymphocyte (CTL) response, the characteristics of an effective vaccine-induced T lymphocyte response remain unclear. We explored this issue in the simian human immunodeficiency virus/rhesus monkey model in the course of assessing the relative immunogenicity of vaccine regimens that included a cytokine-augmented plasmid DNA prime and a boost with DNA or recombinant pox vectors. Recombinant vaccinia virus, recombinant modified vaccinia Ankara (MVA), and recombinant fowlpox were comparable in their immunogenicity. Moreover, whereas the magnitude of the peak vaccine-elicited T lymphocyte responses in the recombinant pox virus-boosted monkeys was substantially greater than that seen in the monkeys immunized with plasmid DNA alone, the magnitudes of recombinant pox boosted CTL responses decayed rapidly and were comparable to those of the DNA-alone-vaccinated monkeys by the time of viral challenge. Consistent with these comparable memory T cell responses, the clinical protection seen in all groups of experimentally vaccinated monkeys was similar. This study, therefore, indicates that the steady-state memory, rather than the peak effector vaccine-elicited T lymphocyte responses, may be the critical immune correlate of protection for a CTL-based HIV vaccine.

Heterologous envelope immunogens contribute to AIDS vaccine protection in rhesus monkeys.

Because a strategy to elicit broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies has not yet been found, the role of an Env immunogen in HIV-1 vaccine candidates remains undefined. We sought to determine whether an HIV-1 Env immunogen genetically disparate from the Env of the challenge virus can contribute to protective immunity. We vaccinated Indian-origin rhesus monkeys with Gag-Pol-Nef immunogens, alone or in combination with Env immunogens that were either matched or mismatched with the challenge virus. These animals were then challenged with a pathogenic simian-human immunodeficiency virus. The vaccine regimen included a plasmid DNA prime and replication-defective adenoviral vector boost. Vaccine regimens that included the matched or mismatched Env immunogens conferred better protection against CD4(+) T-lymphocyte loss than that seen with comparable regimens that did not include Env immunogens. This increment in protective immunity was associated with anamnestic Env-specific cellular immunity that developed in the early days following viral challenge. These data suggest that T-lymphocyte immunity to Env can broaden the protective cellular immune response to HIV despite significant sequence diversity of the strains of the Env immunogens and can contribute to immune protection in this AIDS vaccine model.