Three-dimensional architecture of the rod sensory cilium and its disruption in retinal neurodegeneration.

Defects in primary cilia lead to devastating disease because of their roles in sensation and developmental signaling but much is unknown about ciliary structure and mechanisms of their formation and maintenance. We used cryo-electron tomography to obtain 3D maps of the connecting cilium and adjacent cellular structures of a modified primary cilium, the rod outer segment, from wild-type and genetically defective mice. The results reveal the molecular architecture of the cilium and provide insights into protein functions. They suggest that the ciliary rootlet is involved in cellular transport and stabilizes the axoneme. A defect in the BBSome membrane coat caused defects in vesicle targeting near the base of the cilium. Loss of the proteins encoded by the Cngb1 gene disrupted links between the disk and plasma membranes. The structures of the outer segment membranes support a model for disk morphogenesis in which basal disks are enveloped by the plasma membrane.

Enhancing and initiating phage-based therapies.

Drug development has typically been a primary foundation of strategy for systematic, long-range management of pathogenic cells. However, drug development is limited in speed and flexibility when response is needed to changes in pathogenic cells, especially changes that produce drug-resistance. The high replication speed and high diversity of phages are potentially useful for increasing both response speed and response flexibility when changes occur in either drug resistance or other aspects of pathogenic cells. We present strategy, with some empirical details, for (1) using modern molecular biology and biophysics to access these advantages during the phage therapy of bacterial infections, and (2) initiating use of phage capsid-based drug delivery vehicles (DDVs) with procedures that potentially overcome both drug resistance and other present limitations in the use of DDVs for the therapy of neoplasms. The discussion of phage therapy includes (a) historical considerations, (b) changes that appear to be needed in clinical tests if use of phage therapy is to be expanded, (c) recent work on novel phages and its potential use for expanding the capabilities of phage therapy and (d) an outline for a strategy that encompasses both theory and practice for expanding the applications of phage therapy. The discussion of DDVs starts by reviewing current work on DDVs, including work on both liposomal and viral DDVs. The discussion concludes with some details of the potential use of permeability constrained phage capsids as DDVs.

Visualizing the structural changes of bacteriophage Epsilon15 and its Salmonella host during infection.

The efficient mechanism by which double-stranded DNA bacteriophages deliver their chromosome across the outer membrane, cell wall, and inner membrane of Gram-negative bacteria remains obscure. Advances in single-particle electron cryomicroscopy have recently revealed details of the organization of the DNA injection apparatus within the mature virion for various bacteriophages, including epsilon15 (ɛ15) and P-SSP7. We have used electron cryotomography and three-dimensional subvolume averaging to capture snapshots of ɛ15 infecting its host Salmonella anatum. These structures suggest the following stages of infection. In the first stage, the tailspikes of ɛ15 attach to the surface of the host cell. Next, ɛ15's tail hub attaches to a putative cell receptor and establishes a tunnel through which the injection core proteins behind the portal exit the virion. A tube spanning the periplasmic space is formed for viral DNA passage, presumably from the rearrangement of core proteins or from cellular components. This tube would direct the DNA into the cytoplasm and protect it from periplasmic nucleases. Once the DNA has been injected into the cell, the tube and portal seals, and the empty bacteriophage remains at the cell surface.

Electron cryotomography reveals the portal in the herpesvirus capsid.

Herpes simplex virus type 1 is a human pathogen responsible for a range of illnesses from cold sores to encephalitis. The icosahedral capsid has a portal at one fivefold vertex which, by analogy to portal-containing phages, is believed to mediate genome entry and exit. We used electron cryotomography to determine the structure of capsids lacking pentons. The portal vertex appears different from pentons, being located partially inside the capsid shell, a position equivalent to that of bacteriophage portals. Such similarity in portal organization supports the idea of the evolutionary relatedness of these viruses.

Modular software platform for low-dose electron microscopy and tomography.

Transmission electron microscopy imaging protocols required by structural scientists vary widely and can be laborious without tailor-made applications. We present here the jeol automated microscopy expert system (james) api integrator, a programming library for computer control of transmission electron microscopy operations and equipment. james has been implemented on JEOL microscopes with Gatan CCDs but is designed to be modular so it can be adapted to run on different microscopes and detectors. We have used the james api integrator to develop two applications for low-dose digital imaging: james imaging application and the mr t tomographic imaging application. Both applications have been widely used within our NCRR-supported Center for routine data collection and are now made available for public download.