The glomerular capillary tuft is a highly specialized microcapillary that is dedicated to function as a sophisticated molecular sieve. The glomerulus filter has a unique molecular composition, and several essential glomerular proteins are expressed in the kidney exclusively by glomerular podocytes. A catalog of >300 glomerulus-upregulated transcripts that were identified using expressed sequence tag profiling and microarray analysis was published recently. This study characterized the expression profile of five glomerulus-upregulated transcripts/proteins (ehd3, dendrin, sh2d4a, plekhh2, and 2310066E14Rik) in detail. The expression pattern of these novel glomerular transcripts in various mouse tissues was studied using reverse transcriptase-PCR, Northern blotting, and in situ hybridization. For studying the distribution of corresponding proteins, polyclonal antibodies were raised against the gene products, and Western blotting, immunofluorescence, and immunoelectron microscopic analyses were performed. Remarkably, it was discovered that all five transcripts/proteins were expressed in the kidney exclusively by glomerular cells. Ehd3 was expressed only by glomerular endothelial cells. Importantly, ehd3 is the first gene ever shown to be expressed exclusively by glomerular endothelial cells and not by other endothelial cells in the kidney. Dendrin, sh2d4a, plekhh2, and 2310066E14Rik, however, were transcribed solely by podocytes. With the use of polyclonal antibodies, dendrin, sh2d4a, and plekhh2 proteins were localized to the slit diaphragm and the foot process, whereas 2310066E14Rik protein was localized to the podocyte major processes and cell body. This study provides fresh insights into glomerular biology and uncovers new possibilities to explore the role of these novel proteins in the glomerular physiology and pathology. T he glomerular capillary tuft of the kidney is a unique micro-organ that is specialized to operate as a sophisticated molecular sieve (1). The filtration barrier is composed of the glomerular endothelial cells (GEC), the glomerular basement membrane (GBM), and the podocyte cells. GEC differ from most other endothelial cells in that they are extraordinarily flattened and fenestrated (2). The fenestrated structure of GEC depends on podocyte-derived vascular endothelial growth factor A (3), but the mechanisms behind their unique structure largely are unknown. The GBM, like all basement membranes (BM), is composed of interconnected collagen type IV and laminin networks, where proteoglycans and other matrix components are attached. The GBM, however, is distinct from most other BM of the body, because it contains a unique composition of collagen type IV and laminin isoforms. The importance of these components is highlighted by the fact that defects in GBM-specific type IV collagen or laminin lead to Alport syndrome and Pierson syndrome, respectively (4 -6).The podocyte foot processes and the slit diaphragm serve as a final filtration barrier in the glomerulus. This filtration machinery contains a n...

Human cytomegalovirus (HCMV), a leading cause of congenital birth defects, forms an unusual cytoplasmic virion maturation site termed the "assembly compartment" (AC). Here, we show that the AC also acts as a microtubule-organizing center (MTOC) wherein centrosome activity is suppressed and Golgi-based microtubule (MT) nucleation is enhanced. This involved viral manipulation of discrete functions of MT plus-end-binding (EB) proteins. In particular, EB3, but not EB1 or EB2, was recruited to the AC and was required to nucleate MTs that were rapidly acetylated. EB3-regulated acetylated MTs were necessary for nuclear rotation prior to cell migration, maintenance of AC structure, and optimal virus replication. Independently, a myristoylated peptide that blocked EB3-mediated enrichment of MT regulatory proteins at Golgi regions of the AC also suppressed acetylated MT formation, nuclear rotation, and infection. Thus, HCMV offers new insights into the regulation and functions of Golgi-derived MTs and the therapeutic potential of targeting EB3.

The Crumbs family of transmembrane proteins has an important role in the differentiation of the apical membrane domain in various cell types, regulating such processes as epithelial cell polarization. The mammalian Crumbs protein family is composed of three members. Here, we inactivated the mouse Crb2 gene with gene-targeting techniques and found that the protein is crucial for early embryonic development with severe abnormalities appearing in Crb2-deficient embryos at late-gastrulation. Our findings indicate that the primary defect in the mutant embryos is disturbed polarity of the epiblast cells at the primitive streak, which affects epithelial to mesenchymal transition (EMT) during gastrulation, resulting in impaired mesoderm and endoderm formation, and embryonic lethality by embryonic day 12.5. These findings therefore indicate a novel role for the Crumbs family of proteins. Developmental Dynamics 240:2646-2656,

Human herpesvirus 6A (HHV-6A), a member of the betaherpesvirus family, is associated with several human diseases. Like all herpesviruses, HHV-6A establishes a lifelong, latent infection in its host. Reactivation of HHV-6A is frequent within the immunosuppressed and immunocompromised populations and results in lytic viral replication within multiple organs, often leading to severe disease. MicroRNAs (miRNAs) are key regulators of multiple cellular processes that regulate the translation of specific transcripts. miRNAs carried by herpesviruses play important roles in modulating the host cell, thereby facilitating a suitable environment for productive viral infection and/or latency. Currently, there are approximately 150 known human herpesvirusencoded miRNAs, although an miRNA(s) encoded by HHV-6A has yet to be reported. We hypothesized that HHV-6A, like other members of the human herpesvirus family, encodes miRNAs, which function to promote viral infection. We utilized deep sequencing of small RNA species isolated from cells harboring HHV-6A to identify five novel small noncoding RNA species that originate from the viral genome, one of which has the characteristics of a viral miRNA. These RNAs are expressed during productive infection by either bacterial artificial chromosome (BAC)-derived virus in Jjhan cells or wild-type HHV-6A strain U1102 virus in HSB2 cells and are associated with the RNA induced silencing complex (RISC) machinery. Growth analyses of mutant viruses that lack each individual miRNA revealed that a viral miRNA candidate (miR-U86) targets the HHV-6A IE gene U86, thereby regulating lytic replication. The identification and biological characterization of this HHV-6A-specific miRNA is the first step to defining how the virus regulates its life cycle. IMPORTANCEA majority of the human population is infected with human herpesvirus 6A (HHV-6A), a betaherpesvirus family member. Infections usually occur in young children, and upon resolution, the virus remains in a latent state within the host. Importantly, during times of weakened immune responses, the virus can reactivate and is correlated with significant disease states. Viruses encode many different types of factors that both undermine the host antiviral response and regulate viral replication, including small RNA species called microRNAs (miRNAs). Here we report that HHV-6A encodes at least one miRNA, which we named miR-U86. We have characterized the requirement of this viral miRNA and its impact on the viral life cycle and found that it functions to regulate a viral protein important for efficient viral replication. Our data suggest that viral miRNAs are important for HHV-6A and that they may serve as an important therapeutic target to inhibit the virus. Human herpesvirus 6 (HHV-6), a ubiquitous pathogen in the general population, is a member of the betaherpesvirus family. HHV-6 consists of two variants: HHV-6A and HHV-6B. Even though they are nearly 90% homologous at the genomic level and they have common cell tropism (e.g., T-lymphocytes)...

Unbiased transcriptome profiling and functional genomics approaches identified glucocorticoid-induced transcript 1 (GLCCI1) as being a transcript highly specific for the glomerulus, but its role in glomerular development and disease is unknown. Here, we report that mouse glomeruli express far greater amounts of Glcci1 protein compared with the rest of the kidney. RT-PCR and Western blotting demonstrated that mouse glomerular Glcci1 is approximately 60 kD and localizes to the cytoplasm of podocytes in mature glomeruli. In the fetal kidney, intense Glcci1 expression occurs at the capillary-loop stage of glomerular development. Using gene knockdown in zebrafish with morpholinos, morphants lacking Glcci1 function had collapsed glomeruli with foot-process effacement. Permeability studies of the glomerular filtration barrier in these zebrafish morphants demonstrated a disruption of the selective glomerular permeability filter. Taken together, these data suggest that Glcci1 promotes the normal development and maintenance of podocyte structure and function.