Architectural insights into a ciliary partition.

Ciliary compartmentalization plays pivotal roles in ciliogenesis and in various signaling pathways. Here we describe a structure at the ciliary base that appears to have all the features required for compartmentalization and which we thus call the "ciliary partitioning system" (CPS). This complex consists of the terminal plate, which serves as a cytosolic "ciliary pore complex" (CPC), and a membrane region well suited to serve as a diffusion barrier. The CPC is a plate-shaped structure containing nine pores through which the microtubule doublets of the basal body pass. Each pore expands from the doublet B-tubule into an opening well suited for the passage of intraflagellar transport particles. The membrane diffusion barrier encompasses an extended region of detergent-resistant periciliary membrane (ciliary pocket) and a ring complex that connects the CPC to the membrane. Proteomics analysis shows involvement of the ciliary pocket in vesicle trafficking, suggesting that this region plays an active role in membrane transport. The CPC and the ring together form a complete partition defining the ciliary boundary.

Molecular architecture of the chick vestibular hair bundle.

Hair bundles of the inner ear have a specialized structure and protein composition that underlies their sensitivity to mechanical stimulation. Using mass spectrometry, we identified and quantified >1,100 proteins, present from a few to 400,000 copies per stereocilium, from purified chick bundles; 336 of these were significantly enriched in bundles. Bundle proteins that we detected have been shown to regulate cytoskeleton structure and dynamics, energy metabolism, phospholipid synthesis and cell signaling. Three-dimensional imaging using electron tomography allowed us to count the number of actin-actin cross-linkers and actin-membrane connectors; these values compared well to those obtained from mass spectrometry. Network analysis revealed several hub proteins, including RDX (radixin) and SLC9A3R2 (NHERF2), which interact with many bundle proteins and may perform functions essential for bundle structure and function. The quantitative mass spectrometry of bundle proteins reported here establishes a framework for future characterization of dynamic processes that shape bundle structure and function.

Mammary collective cell migration involves transient loss of epithelial features and individual cell migration within the epithelium.

Normal mammary morphogenesis involves transitions between simple and multilayered epithelial organizations. We used electron microscopy and molecular markers to determine whether intercellular junctions and apico-basal polarity were maintained in the multilayered epithelium. We found that multilayered elongating ducts had polarized apical and basal tissue surfaces both in three-dimensional culture and in vivo. However, individual cells were only polarized on surfaces in contact with the lumen or extracellular matrix. The basolateral marker scribble and the apical marker atypical protein kinase C zeta localized to all interior cell membranes, whereas PAR3 displayed a cytoplasmic localization, suggesting that the apico-basal polarity was incomplete. Despite membrane localization of E-cadherin and ß-catenin, we did not observe a defined zonula adherens connecting interior cells. Instead, interior cells were connected through desmosomes and exhibited complex interdigitating membrane protrusions. Single-cell labeling revealed that individual cells were both protrusive and migratory within the epithelial multilayer. Inhibition of Rho kinase (ROCK) further reduced intercellular adhesion on apical and lateral surfaces but did not disrupt basal tissue organization. Following morphogenesis, segregated membrane domains were re-established and junctional complexes re-formed. We observed similar epithelial organization during mammary morphogenesis in organotypic culture and in vivo. We conclude that mammary epithelial morphogenesis involves a reversible, spatially limited, reduction in polarity and intercellular junctions and active individualistic cell migration. Our data suggest that reductions in polarity and adhesion during breast cancer progression might reflect partial recapitulation of a normal developmental program.

Characterization of a UL49-null mutant: VP22 of herpes simplex virus type 1 facilitates viral spread in cultured cells and the mouse cornea.

Herpes simplex virus type 1 (HSV-1) virions, like those of all herpesviruses, contain a proteinaceous layer termed the tegument that lies between the nucleocapsid and viral envelope. The HSV-1 tegument is composed of at least 20 different viral proteins of various stoichiometries. VP22, the product of the U(L)49 gene, is one of the most abundant tegument proteins and is conserved among the alphaherpesviruses. Although a number of interesting biological properties have been attributed to VP22, its role in HSV-1 infection is not well understood. In the present study we have generated both a U(L)49-null virus and its genetic repair and characterized their growth in both cultured cells and the mouse cornea. While single-step growth analyses indicated that VP22 is dispensable for virus replication at high multiplicities of infection (MOIs), analyses of plaque morphology and intra- and extracellular multistep growth identified a role for VP22 in viral spread during HSV-1 infection at low MOIs. Specifically, VP22 was not required for either virion infectivity or cell-cell spread but was required for accumulation of extracellular virus to wild-type levels. We found that the absence of VP22 also affected virion composition. Intracellular virions generated by the U(L)49-null virus contained reduced amounts of ICP0 and glycoproteins E and D compared to those generated by the wild-type and U(L)49-repaired viruses. In addition, viral spread in the mouse cornea was significantly reduced upon infection with the U(L)49-null virus compared to infection with the wild-type and U(L)49-repaired viruses, identifying a role for VP22 in viral spread in vivo as well as in vitro.

Genetic and molecular in vivo analysis of herpes simplex virus assembly in murine visual system neurons.

Herpes simplex virus (HSV) infects both epithelial cells and neuronal cells of the human host. Although HSV assembly has been studied extensively for cultured epithelial and neuronal cells, cultured neurons are biochemically, physiologically, and anatomically significantly different than mature neurons in vivo. Therefore, it is imperative that viral maturation and assembly be studied in vivo. To study viral assembly in vivo, we inoculated wild-type and replication-defective viruses into the posterior chamber of mouse eyes and followed infection in retinal ganglion cell bodies and axons. We used PCR techniques to detect viral DNA and RNA and electron microscopy immunohistochemistry and Western blotting to detect viral proteins in specific portions of the optic tract. This approach has shown that viral DNA replication is necessary for viral DNA movement into axons. Movement of viral DNA along ganglion cell axons occurs within capsid-like structures at the speed of fast axonal transport. These studies show that the combined use of intravitreal injections of replication-defective viruses and molecular probes allows the genetic analysis of essential viral replication and maturation processes in neurons in vivo. The studies also provide novel direct evidence for the axonal transport of viral DNA and support for the subassembly hypothesis of viral maturation in situ.

Temporal expression of herpes simplex virus type 1 mRNA in murine retina.

PURPOSE: During maturation of herpes simplex virus type 1 (HSV) in infected murine retinal F strain ganglion cells, new viral components are axonally transported in two phases. The viral envelope protein (gD) appears 48 hr before the capsid protein (VP5). Our hypothesis was that delayed appearance of VP5 mRNA in the infected eye causes the delayed expression of the VP5 protein in the axon. METHODS: HSV was injected into the ocular posterior chamber. Three to 24 hr later, the mice were euthanized, and the posterior eye was isolated. RNA was extracted, DNAase-treated, and used for amplification by reverse transcription-polymerase chain reaction (RT-PCR) using primers specific to gD, VP5 and a tegument protein VP22. RESULTS: VP22 and gD mRNAs are expressed 6 hr and VP5 mRNA is first detected 9 hr after infection. CONCLUSIONS: The results establish that delayed transcription does not play a significant role in the 48-hr delay in VP5 appearance in the retinal axons.

The microvesicle as a vehicle for EMMPRIN in tumor-stromal interactions.

EMMPRIN is a transmembrane glycoprotein expressed at high levels by tumor cells. It has been identified as a tumor-derived factor that can stimulate matrix metalloproteinase expression in fibroblasts and hence facilitate tumor invasion and metastasis. Recent studies have shown that full-length EMMPRIN is released by tumor cells, but the mechanism of release remains unclear. Here, we show that EMMPRIN is released from the surface of NCI-H460 cells via microvesicle shedding. However, these vesicles are unstable and rapidly break down to release bioactive EMMPRIN. Although microvesicle shedding has been considered a constitutive process in tumor cells, our data show that it can be amplified upon cell exposure to PMA, elucidating at least one signalling cascade responsible for EMMPRIN release. This pathway is dependent on protein kinase C, calcium mobilization and mitogen-activated protein kinase kinase (MEK 1/2). Thus, the results outline a novel form of tumor-stromal interaction in which extracellular matrix degradation by fibroblasts is controlled through the microvesicular release of EMMPRIN from tumor cells.

A procedure to deliver herpes simplex virus to the murine trigeminal ganglion.

Although initial herpes simplex virus (HSV) infections of the cornea are relatively easily treated, recurrent infections following reactivation of latent virus in the sensory ganglion cells are more difficult to treat. Untreated infections may result in severe consequences, including corneal scarring, glaucoma, and encephalitis. To develop such treatments, an experimental in vivo model was needed in which HSV can be applied directly to trigeminal ganglion cells. We have previously developed such a model to examine the mechanisms of HSV spread from trigeminal neurons to corneal epithelial cells. The current paper describes in detail the technical steps required for implementation of that model. Immunocytochemistry and electron microscopy have been used to validate the efficacy of the described procedures. This technique will be useful for future in vivo studies of neurotrophic viral infections of trigeminal ganglion cells.

Axonal transport and sorting of herpes simplex virus components in a mature mouse visual system.

The time course for delivery and transport of two major proteins of herpes simplex virus (HSV) has been determined for mature mouse retinal ganglion cell axons in vivo. Twenty-four hours after intravitreal injection of HSV, valacyclovir was introduced into the drinking water of the mice to inhibit subsequent viral replication. Without treatment, viral spread and replication in periaxonal glial cells confound study of axonal transport. At 2 to 5 days after infection, the animals were sacrificed and contiguous segments of the optic pathway were removed. Immunofluorescence microscopy indicated that the number of infected astrocytes was reduced in the proximal optic nerve and eliminated in the optic tract. Western blots of the retina with antibodies for envelope and capsid components, glycoprotein D (gD) and VP5, respectively, revealed that both components were expressed in retinal homogenates by 2 days. Results of reverse transcription-PCR indicated that there was no gD mRNA present in the treated optic tract 5 days after infection. Therefore, we conclude that gD is transcribed from viral mRNA in the retinal ganglion cell bodies. The gD accumulated in the proximal ganglion cell axon by 2 days and reached the most distal segment after 3 days. The VP5 first appeared in the proximal axons at 4 days, about 48 h after the appearance of gD. Thus, gD entered the axon earlier and independent of VP5. These finding confirm the subassembly model of viral transport in neurons and suggest that there is a 4- to 5-day window for initiation of effective antiviral treatment with valacyclovir.