Faculty Bibliography

Electron microscopy (EM) and image analysis offer an effective approach for determining the three-dimensional structure of macromolecular complexes. The versatility of these methods means that molecular species not normally amenable to other structural methods, e.g., X-ray crystallography and NMR spectroscopy, can be analyzed. However, the resolution of EM structures is often too low to provide an atomic model directly by chain tracing. Instead, a combination of modeling and fitting can be an effective way to analyze the EM structure at an atomic level, thus allowing localization of subunits or evaluation of conformational changes. Here we describe the steps involved in this process: building a homology model, fitting this model to an EM map, and using computational methods for docking of additional domains to the model. As an example, we illustrate the methods using an integral membrane protein, CopA, which functions to pump copper across the membrane in an ATP-dependent manner. In this example, we build a homology model based on the published atomic coordinates for a related calcium pump from sarcoplasmic reticulum (SERCA). After fitting this homology model to a 17 A resolution EM map, computational software is used to dock a metal-binding domain (MBD) that is unique to the copper pump. Although this software identifies a number of plausible interfaces for docking, the constraints of the EM map steer us to select a unique solution. Thus, the synergy of these two methods allows us to describe both the location of the unknown MBD relative to the other cytoplasmic domains and the atomic details of the domain interface.

Immunoglobulin (Ig)G4-related ophthalmic disease belongs to a category of ocular adnexal lymphoproliferative disorders, the most frequent group of orbital tumors and simulating lesions. The aim of this study was to elucidate the number of IgG4-related diseases of orbital lymphoproliferative disorders and correlate ages and sex of such patients from 18 centers in Japan. One thousand and fourteen patients with orbital lymphoproliferative disorders were enrolled in this study. All had pathologically diagnosed lymphoproliferative disorders with surgical samples of ocular adnexal tissue. Patients with conjunctival lesions and intraocular lymphoma were excluded. Of the 1,014 cases of orbital lymphoproliferative disorders 404 (39.8 %) had extranodal mucosa-associated lymphoid tissue (MALT) lymphoma, 156 (15.4 %) had other malignant lymphomas, 191 (18.8 %) had non-IgG4 orbital inflammation, 219 (21.6 %) had IgG4-related orbital inflammation, and 44 (4.3 %) had IgG4-positive MALT lymphoma. Median age of the IgG4-related orbital inflammation group was 62 years, which is significantly lower than that of the MALT lymphoma group (median 66 years) and higher than the non-IgG4 orbital inflammation group (median 57 years). The male/female ratio was 105/114 in the IgG4-related orbital inflammation group. Nearly a quarter of orbital lymphoproliferative disorders in Japan are related to IgG4.

The Influenza A virus genome consists of eight negative sense, single-stranded RNA segments. Although it has been established that most virus particles contain a single copy of each of the eight viral RNAs, the packaging selection mechanism remains poorly understood. Influenza viral RNAs are synthesized in the nucleus, exported into the cytoplasm and travel to the plasma membrane where viral budding and genome packaging occurs. Due to the difficulties in analyzing associated vRNPs while preserving information about their positions within the cell, it has remained unclear how and where during cellular trafficking the viral RNAs of different segments encounter each other. Using a multicolor single-molecule sensitivity fluorescence in situ hybridization (smFISH) approach, we have quantitatively monitored the colocalization of pairs of influenza viral RNAs in infected cells. We found that upon infection, the viral RNAs from the incoming particles travel together until they reach the nucleus. The viral RNAs were then detected in distinct locations in the nucleus; they are then exported individually and initially remain separated in the cytoplasm. At later time points, the different viral RNA segments gather together in the cytoplasm in a microtubule independent manner. Viral RNAs of different identities colocalize at a high frequency when they are associated with Rab11 positive vesicles, suggesting that Rab11 positive organelles may facilitate the association of different viral RNAs. Using engineered influenza viruses lacking the expression of HA or M2 protein, we showed that these viral proteins are not essential for the colocalization of two different viral RNAs in the cytoplasm. In sum, our smFISH results reveal that the viral RNAs travel together in the cytoplasm before their arrival at the plasma membrane budding sites. This newly characterized step of the genome packaging process demonstrates the precise spatiotemporal regulation of the infection cycle.

During the first month of life, the murine posterior-frontal suture (PF) of the cranial vault closes through endochondral ossification, while other sutures remain patent. These processes are tightly regulated by canonical Wnt signaling. Low levels of active canonical Wnt signaling enable endochondral ossification and therefore PF-suture closure, whereas constitutive activation of canonical Wnt causes PF-suture patency. We therefore sought to test this concept with a knockout mouse model. PF-sutures of Axin2(-/-) mice, which resemble a state of constantly activated canonical Wnt signaling, were investigated during the physiological time course of PF-suture closure and compared in detail with wild type littermates. Histological analysis revealed that the architecture in Axin2(-/-) PF-sutures was significantly altered in comparison to wild type. The distance between the endocranial layers was dramatically increased and suture closure was significantly delayed. Moreover, physiological endochondral ossification did not occur, rather an ectopic cartilage appeared between the endocranial and ectocranial bone layers at P7 which eventually involutes at P13. Quantitative PCR analysis showed the lack of Col10alpha1 upregulation in Axin2(-/-) PF-suture. Immunohistochemistry and gene expression analysis also revealed high levels of type II collagen as compared to type I collagen and absence of Mmp-9 in the cartilage of Axin2(-/-) PF-suture. Moreover, TUNEL staining showed a high percentage of apoptotic chondrocytes in Axin2(-/-) PF-sutures at P9 and P11 as compared to wild type. These data indicated that Axin2(-/-) PF-sutures lack physiological endochondral ossification, contain ectopic cartilage and display delayed suture closure.

The Sonic Hedgehog (Shh) pathway is responsible for critical patterning events early in development and for regulating the delicate balance between proliferation and differentiation in the developing and adult vertebrate brain. Currently, our knowledge of the potential role of Shh in regulating neural stem cells (NSC) is largely derived from analyses of the mammalian forebrain, but for dorsal midbrain development it is mostly unknown. For a detailed understanding of the role of Shh pathway for midbrain development in vivo, we took advantage of mouse embryos with cell autonomously activated Hedgehog (Hh) signaling in a conditional Patched 1 (Ptc1) mutant mouse model. This animal model shows an extensive embryonic tectal hypertrophy as a result of Hh pathway activation. In order to reveal the cellular and molecular origin of this in vivo phenotype, we established a novel culture system to evaluate neurospheres (nsps) viability, proliferation and differentiation. By recreating the three-dimensional (3-D) microenvironment we highlight the pivotal role of endogenous Shh in maintaining the stem cell potential of tectal radial glial cells (RGC) and progenitors by modulating their Ptc1 expression. We demonstrate that during late embryogenesis Shh enhances proliferation of NSC, whereas blockage of endogenous Shh signaling using cyclopamine, a potent Hh pathway inhibitor, produces the opposite effect. We propose that canonical Shh signaling plays a central role in the control of NSC behavior in the developing dorsal midbrain by acting as a niche factor by partially mediating the response of NSC to epidermal growth factor (EGF) and fibroblast growth factor (FGF) signaling. We conclude that endogenous Shh signaling is a critical mechanism regulating the proliferation of stem cell lineages in the embryonic dorsal tissue.

Hepatic steatosis is a global epidemic that is thought to contribute to the pathogenesis of type 2 diabetes. MicroRNAs (miRs) are regulators that can functionally integrate a range of metabolic and inflammatory pathways in liver. We aimed to investigate the functional role of miR-155 in hepatic steatosis. Male C57BL/6 wild-type (WT) and miR-155(-/-) mice were fed either normal chow or high fat diet (HFD) for 6 months then lipid levels, metabolic and inflammatory parameters were assessed in livers and serum of the mice. Mice lacking endogenous miR-155 that were fed HFD for 6 months developed increased hepatic steatosis compared to WT controls. This was associated with increased liver weight and serum VLDL/LDL cholesterol and alanine transaminase (ALT) levels, as well as increased hepatic expression of genes involved in glucose regulation (Pck1, Cebpa), fatty acid uptake (Cd36) and lipid metabolism (Fasn, Fabp4, Lpl, Abcd2, Pla2g7). Using miRNA target prediction algorithms and the microarray transcriptomic profile of miR-155(-/-) livers, we identified and validated that Nr1h3 (LXRalpha) as a direct miR-155 target gene that is potentially responsible for the liver phenotype of miR-155(-/-) mice. Together these data indicate that miR-155 plays a pivotal role regulating lipid metabolism in liver and that its deregulation may lead to hepatic steatosis in patients with diabetes.

Chemotaxis assays are an invaluable tool for studying the biological activity of inflammatory mediators such as CC chemokines, which have been implicated in a wide range of chronic inflammatory diseases. Conventional chemotaxis systems such as the modified Boyden chamber are limited in terms of the data captured given that the assays are analysed at a single time-point. We report the optimisation and validation of a label-free, real-time cell migration assay based on electrical cell impedance to measure chemotaxis of different primary murine macrophage populations in response to a range of CC chemokines and other chemoattractant signalling molecules. We clearly demonstrate key differences in the migratory behavior of different murine macrophage populations and show that this dynamic system measures true macrophage chemotaxis rather than chemokinesis or fugetaxis. We highlight an absolute requirement for Galphai signaling and actin cytoskeletal rearrangement as demonstrated by Pertussis toxin and cytochalasin D inhibition. We also studied the chemotaxis of CD14(+) human monocytes and demonstrate distinct chemotactic profiles amongst different monocyte donors to CCL2. This real-time chemotaxis assay will allow a detailed analysis of factors that regulate macrophage responses to chemoattractant cytokines and inflammatory mediators.

Coronary artery disease, resulting from atherosclerosis, is the leading cause of death in the Western world. Most previous studies have subjected atherosclerotic arteries, a tissue of mixed cellular composition, to homogenization in order to identify the factors in plaque development, thereby obscuring information relevant to specific cell types. Because macrophage foam cells are critical mediators in atherosclerotic plaque advancement, we reasoned that performing gene analysis on those cells would provide specific insight in novel regulatory factors and potential therapeutic targets. We demonstrated for the first time in vascular biology that foam cell-specific RNA can be isolated by laser capture microdissection (LCM) of plaques. As expected, compared to whole tissue, a significant enrichment in foam cell-specific RNA transcripts was observed. Furthermore, because regression of atherosclerosis is a tantalizing clinical goal, we developed and reported a transplantation-based mouse model. This involved allowing plaques to form in apoE-/- mice and then changing the plaque's plasma environment from hyperlipidemia to normolipidemia. Under those conditions, rapid regression ensued in a process involving emigration of plaque foam cells to regional and systemic lymph nodes. Using LCM, we were able to show that under regression conditions, there was decreased expression in foam cells of inflammatory genes, but an up-regulation of cholesterol efflux genes. Interestingly, we also found that increased expression of chemokine receptor CCR7, a known factor in dendritic cell migration, was required for regression. In conclusion, the LCM methods described in this chapter, which have already lead to a number of striking findings, will likely further facilitate the study of cell type-specific gene expression in animal and human plaques during various stages of atherosclerosis, and after genetic, pharmacologic, and environmental perturbations.