Over the last year, we made progress with using both tomographic and ion abrasion imaging for studying cellular architecture. Using cryo-electron microscopy and cryo-electron tomography to visualize attack phase Bdellovibrio bacteriovorus, we discovered that while B. bacteriovorus displays internal architectural elements that are very similar to other gram-negative bacteria, they can undergo rapid, dramatic changes in shape, as demonstrated by molding of bacterial shape to the topography of the carbon substrate in time periods less than 1-2 minutes. This is a new and unexpected discovery, and the mechanisms that lead to these shape changes remain to be determined. Coordinated changes must occur in the interior of cells in response to extracellular contact, although the nature of this molecular restructuring is unclear. The bacterial cytoskeleton is an attractive candidate that could be involved given its dynamic role in shape modulation of chemotactic eukaryotic cells. Careful examination of the cellular interior in Bdellovibrio tomograms revealed the presence of an extended network of internal filaments, with certain filaments parallel to the inner cell membrane, and others distributed in the transverse direction. We have also used 3D tomographic imaging to document the presence of other macromolecular assemblies constituent in each cell including the flagellar rotor, some with clearly discernible C-rings, and a single chemotaxis receptor array that localizes near the flagellum. The sheathed flagellum inserts through the inner and outer membranes at the posterior pole at a point that is off the longitudinal axis of the cell. This off-axis positioning may be essential for mono-flagellated cells such as Bdellovibrio to steer and change direction. Comparison of highly bent and moderately bent cells reveals no discernable differences in the cellular features noted above, including the nucleoid, which remains undistorted and closely follows the curvature of the bend. There was also no appreciable difference in the 25 nm spacing between inner and outer membranes at regions of the highest curvature relative to other areas of the cell, although the anterior entry pole typically had a wider (40 nm) spacing compared to the rest of the cell. In parallel studies, we have collaborated with Dr. Subramaniams group to use ion-abrasion scanning electron microscopy for determination of the 3D architectures of diatoms and cells in the immune system. From 3D reconstructions of developmentally synchronized diatoms captured at different stages, we showed that both micro- and nanoscale siliceous structures could be visualized at specific stages in their formation. Not only could the structures be visualized in a whole-cell context, but the studies demonstrated that fragile, early-stage structures are visible, and that this could be combined with elemental mapping of the exposed slice. Using a similar experimental strategy, we helped image entire HIV-infected human macrophages at resolutions high enough to see individual HIV virions and their location within the cell. This approach revealed that HIV is actually present in a system of nanoscale tubes, barely larger than a virus at some places, that connect internal virus factory chambers to the cell surface. These tubes could allow the macrophage to deliver HIV virions to bystander cells from its continually-replenished stores of ammunition, held deep within the cell.
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