Faculty Bibliography

The epithelial-specific adaptor AP1B sorts basolateral proteins, but the trafficking routes where it performs its sorting role remain controversial. Here, we used an RNAi approach to knock down the medium subunit of AP1B (mu1B) in the prototype epithelial cell line Madin-Darby canine kidney (MDCK). Mu1B-knocked down MDCK cells displayed loss of polarity of several endogenous and exogenous basolateral markers transduced via adenovirus vectors, but exhibited normal polarity of apical markers. We chose two well characterized basolateral protein markers, the transferrin receptor (TfR) and the vesicular stomatitis virus G protein, to study the sorting role of AP1B. A surface-capture assay introduced here showed that mu1B-knocked down MDCK cells plated on filters at confluency and cultured for 4.5 d, sorted TfR correctly in the biosynthetic route but incorrectly in the recycling route. In contrast, these same cells missorted vesicular stomatitis virus G apically in the biosynthetic route. Strikingly, recently confluent MDCK cells (1-3 d) displayed AP1B-dependence in the biosynthetic route of TfR, which decreased with additional days in culture. Sucrose density gradient analysis detected AP1B predominantly in TfR-rich endosomal fractions in MDCK cells confluent for 1 and 4 d. Our results are consistent with the following model: AP1B sorts basolateral proteins in both biosynthetic and recycling routes of MDCK cells, as a result of its predominant functional localization in recycling endosomes, which constitute a post-Golgi station in the biosynthetic route of some plasma membrane proteins. TfR utilizes a direct route from Golgi to basolateral membrane that is established as the epithelial monolayer matures.

A newly designed Gemini-shaped hexabenzocoronene amphiphile (1), carrying an isothiouronium ion-appended side chain, self-assembles in CH2Cl2 to form a nanotubular object, whose graphitic wall is densely covered by a positively charged molecular layer of isothiouronium ion pendants. The graphitic nanotube can be dispersed uniformly in aqueous media owing to effective hydration as well as electrostatic repulsion. Post-supramolecular functionalization of the nanotube surface is possible, without disruption of the tubular morphology, by taking advantage of a specific interaction of the isothiouronium ion pendants with oxoanion guests. Mixing with sodium poly(4-styrenesulfonate) results in wrapping of the nanotube, while complexation with an electron-accepting oxoanion such as anthraquinone carboxylate allows photoinduced electron transfer from the graphitic wall to the bound guest molecules.

Cancer stem cells are rare tumor cells characterized by their ability to self-renew and to induce tumorigenesis. They are present in gliomas and may be responsible for the lethality of these incurable brain tumors. In the most aggressive and invasive type, glioblastoma multiforme (GBM), an average of about one year spans the period between detection and death [1]. The resistence of gliomas to current therapies may be related to the existence of cancer stem cells [2-6]. We find that human gliomas display a stemness signature and demonstrate that HEDGEHOG (HH)-GLI signaling regulates the expression of stemness genes in and the self-renewal of CD133(+) glioma cancer stem cells. HH-GLI signaling is also required for sustained glioma growth and survival. It displays additive and synergistic effects with temozolomide (TMZ), the current chemotherapeutic agent of choice. TMZ, however, does not block glioma stem cell self-renewal. Finally, interference of HH-GLI signaling with cyclopamine or through lentiviral-mediated silencing demonstrates that the tumorigenicity of human gliomas in mice requires an active pathway. Our results reveal the essential role of HH-GLI signaling in controlling the behavior of human glioma cancer stem cells and offer new therapeutic possibilities.

Rhomboid peptidases are members of a family of regulated intramembrane peptidases that cleave the transmembrane segments of integral membrane proteins. Rhomboid peptidases have been shown to play a major role in developmental processes in Drosophila and in mitochondrial maintenance in yeast. Most recently, the function of rhomboid peptidases has been directly linked to apoptosis. We have solved the structure of the rhomboid peptidase from Haemophilus influenzae (hiGlpG) to 2.2-A resolution. The phasing for the crystals of hiGlpG was provided mainly by molecular replacement, by using the coordinates of the Escherichia coli rhomboid (ecGlpG). The structural results on these rhomboid peptidases have allowed us to speculate on the catalytic mechanism of substrate cleavage in a membranous environment. We have identified the relative disposition of the nucleophilic serine to the general base/acid function of the conserved histidine. Modeling a tetrapeptide substrate in the context of the rhomboid structure reveals an oxyanion hole comprising the side chain of a second conserved histidine and the main-chain NH of the nucleophilic serine residue. In both hiGlpG and ecGlpG structures, a water molecule occupies this oxyanion hole.

We investigated the ability of targeted immunomicelles to detect and assess macrophages in atherosclerotic plaque using MRI in vivo. There is a large clinical need for a noninvasive tool to assess atherosclerosis from a molecular and cellular standpoint. Macrophages play a central role in atherosclerosis and are associated with plaques vulnerable to rupture. Therefore, macrophage scavenger receptor (MSR) was chosen as a target for molecular MRI. MSR-targeted immunomicelles, micelles, and gadolinium-diethyltriaminepentaacetic acid (DTPA) were tested in ApoE-/- and WT mice by using in vivo MRI. Confocal laser-scanning microscopy colocalization, macrophage immunostaining and MRI correlation, competitive inhibition, and various other analyses were performed. In vivo MRI revealed that at 24 h postinjection, immunomicelles provided a 79% increase in signal intensity of atherosclerotic aortas in ApoE-/- mice compared with only 34% using untargeted micelles and no enhancement using gadolinium-DTPA. Confocal laser-scanning microscopy revealed colocalization between fluorescent immunomicelles and macrophages in plaques. There was a strong correlation between macrophage content in atherosclerotic plaques and the matched in vivo MRI results as measured by the percent normalized enhancement ratio. Monoclonal antibodies to MSR were able to significantly hinder immunomicelles from providing contrast enhancement of atherosclerotic vessels in vivo. Immunomicelles provided excellent validated in vivo enhancement of atherosclerotic plaques. The enhancement seen is related to the macrophage content of the atherosclerotic vessel areas imaged. Immunomicelles may aid in the detection of high macrophage content associated with plaques vulnerable to rupture.

All known eukaryotic and some viral mRNA capping enzymes (CEs) transfer a GMP moiety of GTP to the 5'-diphosphate end of the acceptor RNA via a covalent enzyme-GMP intermediate to generate the cap structure. In striking contrast, the putative CE of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand (NNS) RNA viruses including rabies, measles, and Ebola, incorporates the GDP moiety of GTP into the cap structure of transcribing mRNAs. Here, we report that the RNA-dependent RNA polymerase L protein of VSV catalyzes the capping reaction by an RNA:GDP polyribonucleotidyltransferase activity, in which a 5'-monophosphorylated viral mRNA-start sequence is transferred to GDP generated from GTP via a covalent enzyme-RNA intermediate. Thus, the L proteins of VSV and, by extension, other NNS RNA viruses represent a new class of viral CEs, which have evolved independently from known eukaryotic CEs.

Stress in the endoplasmic reticulum (ER stress) and its cellular response, the unfolded protein response (UPR), are implicated in a wide variety of diseases, but its significance in many disorders remains to be validated in vivo. Here, we analyzed a branch of the UPR mediated by xbp1 in Drosophila to establish its role in neurodegenerative diseases. The Drosophila xbp1 mRNA undergoes ire-1-mediated unconventional splicing in response to ER stress, and this property was used to develop a specific UPR marker, xbp1-EGFP, in which EGFP is expressed in frame only after ER stress. xbp1-EGFP responds specifically to ER stress, but not to proteins that form cytoplasmic aggregates. The ire-1/xbp1 pathway regulates heat shock cognate protein 3 (hsc3), an ER chaperone. xbp1 splicing and hsc3 induction occur in the retina of ninaE(G69D)-/+, a Drosophila model for autosomal dominant retinitis pigmentosa (ADRP), and reduction of xbp1 gene dosage accelerates retinal degeneration of these animals. These results demonstrate the role of the UPR in the Drosophila ADRP model and open new opportunities for examining the UPR in other Drosophila disease models

Nervous systems have evolved two basic mechanisms for increasing the conduction speed of the electrical impulse. The first is through axon gigantism: using axons several times larger in diameter than the norm for other large axons, as for example in the well-known case of the squid giant axon. The second is through encasing axons in helical or concentrically wrapped multilamellar sheets of insulating plasma membrane--the myelin sheath. Each mechanism, alone or in combination, is employed in nervous systems of many taxa, both vertebrate and invertebrate. Myelin is a unique way to increase conduction speeds along axons of relatively small caliber. It seems to have arisen independently in evolution several times in vertebrates, annelids and crustacea. Myelinated nerves, regardless of their source, have in common a multilamellar membrane wrapping, and long myelinated segments interspersed with 'nodal' loci where the myelin terminates and the nerve impulse propagates along the axon by 'saltatory' conduction. For all of the differences in detail among the morphologies and biochemistries of the sheath in the different myelinated animal classes, the function is remarkably universal.

We have developed recombinant vesicular stomatitis virus (VSV) vectors expressing the Yersinia pestis lcrV gene. These vectors, given intranasally to mice, induced high antibody titers to the LcrV protein and protected against intranasal (pulmonary) challenge with Y. pestis. High-level protection was dependent on using an optimized VSV vector that expressed high levels of the LcrV protein from an lcrV gene placed in the first position in the VSV genome, followed by a single boost. This VSV-based vaccine vector system has potential as a plague vaccine protecting against virulent strains lacking the F1 protein.

BACKGROUND:The choice of a stem cell to divide symmetrically or asymmetrically has profound consequences for development and disease. Unregulated symmetric division promotes tumor formation, whereas inappropriate asymmetric division affects organ morphogenesis. Despite its importance, little is known about how spindle positioning is regulated. In some tissues cell fate appears to dictate the type of cell division, whereas in other tissues it is thought that stochastic variation in spindle position dictates subsequent sibling cell fate. RESULTS:Here we investigate the relationship between neural progenitor identity and spindle positioning in the Drosophila optic lobe. We use molecular markers and live imaging to show that there are two populations of progenitors in the optic lobe: symmetrically dividing neuroepithelial cells and asymmetrically dividing neuroblasts. We use genetically marked single cell clones to show that neuroepithelial cells give rise to neuroblasts. To determine if a change in spindle orientation can trigger a neuroepithelial to neuroblast transition, we force neuroepithelial cells to divide along their apical/basal axis by misexpressing Inscuteable. We find that this does not induce neuroblasts, nor does it promote premature neuronal differentiation. CONCLUSION/CONCLUSIONS:We show that symmetrically dividing neuroepithelial cells give rise to asymmetrically dividing neuroblasts in the optic lobe, and that regulation of spindle orientation and division symmetry is a consequence of cell type specification, rather than a mechanism for generating cell type diversity.