E. coli transports aggregated proteins to the poles by a specific and energy-dependent process.

Aggregation of proteins due to failure of quality control mechanisms is deleterious to both eukaryotes and prokaryotes. We found that in Escherichia coli, protein aggregates are delivered to the pole and form a large polar aggregate (LPA). The formation of LPAs involves two steps: the formation of multiple small aggregates and the delivery of these aggregates to the pole to form an LPA. Formation of randomly distributed aggregates, their delivery to the poles, and LPA formation are all energy-dependent processes. The latter steps require the proton motive force, activities of the DnaK and DnaJ chaperones, and MreB. About 90 min after their formation, the LPAs are dissolved in a process that is dependent upon ClpB, DnaK, and energy. Our results confirm and substantiate the notion that the formation of LPAs allows asymmetric inheritance of the aggregated proteins to a small number of daughter cells, enabling their rapid elimination from most of the bacterial population. Moreover, the results show that the processing of aggregated proteins by the protein quality control system is a multi-step process with distinct spatial and temporal controls.

Bacteriophage infection is targeted to cellular poles.

The poles of bacteria exhibit several specialized functions related to the mobilization of DNA and certain proteins. To monitor the infection of Escherichia coli cells by light microscopy, we developed procedures for the tagging of mature bacteriophages with quantum dots. Surprisingly, most of the infecting phages were found attached to the bacterial poles. This was true for a number of temperate and virulent phages of E. coli that use widely different receptors and for phages infecting Yersinia pseudotuberculosis and Vibrio cholerae. The infecting phages colocalized with the polar protein marker IcsA-GFP. ManY, an E. coli protein that is required for phage lambda DNA injection, was found to localize to the bacterial poles as well. Furthermore, labelling of lambda DNA during infection revealed that it is injected and replicated at the polar region of infection. The evolutionary benefits that lead to this remarkable preference for polar infections may be related to lambda's developmental decision as well as to the function of poles in the ability of bacterial cells to communicate with their environment and in gene regulation.

Electron tomography of degenerating neurons in mice with abnormal regulation of iron metabolism.

Previous studies have shown that IRP1(+/-) IRP2(-/-) knockout mice develop progressive neurodegenerative symptoms similar to those observed in human movement disorders such as Parkinson's disease. Histological investigations using optical microscopy show that these IRP knockout mice display accumulation of ferritin in axonal tracts in the brain, suggesting a possible role for excess ferritin in mediating axonal degeneration. Direct observation of the 3D distribution of ferritin by electron tomography indicates that ferritin amounts are increased by 3- to 4-fold in selected regions of the brain, and structural damage is observed within the axon as evidenced by the loss of the internal network of filaments, and the invaginations of neighboring oligodendrocyte membranes into the axonal medium. While optical microscopic investigations suggest that there is a large increase in ferritin in the presumptive axonal regions of the IRP knockout mice, electron tomographic studies reveal that most of the excess ferritin is localized to double-walled vesicular compartments which are present in the interior of the axon and appear to represent invaginations of the oligodendrocyte cells into the axon. The amount of ferritin observed in the axonal space of the knockout mice is at least 10-fold less than the amount of ferritin observed in wild-type mouse axons. The surprising conclusion from our analysis, therefore, is that despite the overall increase in ferritin levels in the knockout mouse brain, ferritin is absent from axons of degenerating neurons, suggesting that trafficking is compromised in early stages of this type of neuronal degeneration.

Three-dimensional electron microscopic imaging of membrane invaginations in Escherichia coli overproducing the chemotaxis receptor Tsr.

Electron tomography is a powerful method for determining the three-dimensional structures of large macromolecular assemblies, such as cells, organelles, and multiprotein complexes, when crystallographic averaging methods are not applicable. Here we used electron tomographic imaging to determine the molecular architecture of Escherichia coli cells engineered to overproduce the bacterial chemotaxis receptor Tsr. Tomograms constructed from fixed, cryosectioned cells revealed that overproduction of Tsr led to formation of an extended internal membrane network composed of stacks and extended tubular structures. We present an interpretation of the tomogram in terms of the packing arrangement of Tsr using constraints derived from previous X-ray and electron-crystallographic studies of receptor clusters. Our results imply that the interaction between the cytoplasmic ends of Tsr is likely to stabilize the presence of the membrane networks in cells overproducing Tsr. We propose that membrane invaginations that are potentially capable of supporting axial interactions between receptor clusters in apposing membranes could also be present in wild-type E. coli and that such receptor aggregates could play an important role in signal transduction during bacterial chemotaxis.

Electron microscopic analysis of membrane assemblies formed by the bacterial chemotaxis receptor Tsr.

The serine receptor (Tsr) from Escherichia coli is representative of a large family of transmembrane receptor proteins that mediate bacterial chemotaxis by influencing cell motility through signal transduction pathways. Tsr and other chemotaxis receptors form patches in the inner membrane that are often localized at the poles of the bacteria. In an effort to understand the structural constraints that dictate the packing of receptors in the plane of the membrane, we have used electron microscopy to examine ordered assemblies of Tsr in membrane extracts isolated from cells engineered to overproduce the receptor. Three types of assemblies were observed: ring-like "micelles" with a radial arrangement of receptor subunits, two-dimensional crystalline arrays with approximate hexagonal symmetry, and "zippers," which are receptor bilayers that result from the antiparallel interdigitation of cytoplasmic domains. The registration among Tsr molecules in the micelle and zipper assemblies was sufficient for identification of the receptor domains and for determination of their contributions to the total receptor length. The overall result of this analysis is compatible with an atomic model of the receptor dimer that was constructed primarily from the X-ray crystal structures of the periplasmic and cytoplasmic domains. Significantly, the micelle and zipper structures were also observed in fixed, cryosectioned cells expressing the Tsr receptor at high abundance, suggesting that the modes of Tsr assembly found in vitro are relevant to the situation in the cell.