Architecture and Characteristics of Bacterial Nanotubes.

Bacteria display an array of contact-dependent interaction systems that have evolved to facilitate direct cell-to-cell communication. We have previously identified a mode of bacterial communication mediated by nanotubes bridging neighboring cells. Here, we elucidate nanotube architecture, dynamics, and molecular components. Utilizing Bacillus subtilis as a model organism, we found that at low cell density, nanotubes exhibit remarkable complexity, existing as both intercellular tubes and extending tubes, with the latter frequently surrounding the cells in a "root-like" fashion. Observing nanotube formation in real time showed that these structures are formed in the course of minutes, displaying rapid movements. Utilizing a combination of super-resolution, light, and electron microscopy, we revealed that nanotubes are composed of chains of membranous segments harboring a continuous lumen. Furthermore, we discovered that a conserved calcineurin-like protein, YmdB, presents in nanotubes and is required for both nanotube production and intercellular molecular trade.

The macromolecular architecture of platelet-derived microparticles.

Platelets are essential for hemostasis and wound healing. They are involved in fundamental processes of vascular biology such as angiogenesis, tissue regeneration, and tumor metastasis. Upon activation, platelets shed small plasma membrane vesicles termed platelet-derived microparticles (PMPs). PMPs include functional cell adhesion machinery that comprises transmembrane receptors (most abundant are the αIIbß3 integrins), cytoskeletal systems and a large variety of adapter and signaling molecules. Glanzmann thrombasthenia (GT) is a condition characterized by platelets that are deficient of the integrin αIIbß3 heterodimer. Here, we use cryo-electron tomography (cryo-ET) to study the structural organization of PMPs (in both healthy and GT patients), especially the cytoskeleton organization and receptor architecture. PMPs purified from GT patients show a significantly altered cytoskeletal organization, characterized by a reduced number of filaments present, compared to the healthy control. Furthermore, our results show that incubating healthy PMPs with manganese ions (Mn(2+)), in the presence of fibrinogen, induces a major conformational change of integrin receptors, whereas thrombin activation yields a moderate response. These results provide the first insights into the native molecular organization of PMPs.

Structure and gating of the nuclear pore complex.

Nuclear pore complexes (NPCs) perforate the nuclear envelope and allow the exchange of macromolecules between the nucleus and the cytoplasm. To acquire a deeper understanding of this transport mechanism, we analyse the structure of the NPC scaffold and permeability barrier, by reconstructing the Xenopus laevis oocyte NPC from native nuclear envelopes up to 20 Å resolution by cryo-electron tomography in conjunction with subtomogram averaging. In addition to resolving individual protein domains of the NPC constituents, we propose a model for the architecture of the molecular gate at its central channel. Furthermore, we compare and contrast this native NPC structure to one that exhibits reduced transport activity and unveil the spatial properties of the NPC gate.

Developments in cryo-electron tomography for in situ structural analysis.

Structural analysis of macromolecular assemblies and their remodeling during physiological processes is instrumental to defining the fundament of cellular and molecular biology. Recent advances in computational and analytical tools for cryo-electron tomography have enabled the study of macromolecular structures in their native environment, providing unprecedented insights into cell function. Moreover, the recent implementation of direct electron detectors has progressed cryo-electron tomography to a stage where it can now be applied to the reconstruction of macromolecular structures at high resolutions. Here, we discuss some of the recent technical developments in cryo-electron tomography to reveal structures of macromolecular complexes in their physiological medium, focusing mainly on eukaryotic cells.

Developmental changes in opioid peptides and their receptors in Cpe(fat)/Cpe(fat) mice lacking peptide processing enzyme carboxypeptidase E.

Carboxypeptidase E (CPE) is involved in the biosynthesis of a number of neuropeptides including opioid peptides. A point mutation in this gene results in a loss of enzyme activity, decrease in mature neuroendocrine peptides, and development of late onset obesity as seen in Cpe(fat)/Cpe(fat) mice. In this study, we examined the processing of peptides derived from prodynorphin and proenkephalin in various brain regions of these mice during development. At 6 to 8 weeks, an age prior to the onset of obesity, levels of dynorphin peptides are decreased in all brain regions, whereas levels of ir-Met-enkephalin are differentially altered. There is an accumulation of C-terminally extended forms of all three opioid peptides in Cpe(fat)/Cpe(fat) mice, consistent with a lack of CPE activity. Thus, it appears that there is no direct correlation between the level of mature opioid peptides and the development of obesity in these mice. Since altered levels of peptides can influence the opioid receptor system, we examined the functional activity of mu and kappa opioid receptors using [35S]guanosine-5'-O-(gamma-thio)-triphosphate binding assays. We find no differences in kappa receptor activity in Cpe(fat)/Cpe(fat) compared with control littermate mice. In contrast, the mu receptor activity is differentially altered in select regions of Cpe(fat)/Cpe(fat) mice in response to a mu-specific ligand. Taken together, these results suggest that the lack of CPE activity leads to alterations in the level of opioid peptides during development and that changes in peptide levels differentially affect opioid receptor activity in vivo.