Cryo-electron tomography reveals that dynactin recruits a team of dyneins for processive motility

AbstractA key player in the intracellular trafficking network is cytoplasmic dynein, a protein complex that transports molecular cargo along microtubule tracks. It has been shown that vertebrate dynein’s movement becomes strikingly enhanced upon interacting with a cofactor named dynactin and one of several cargo-adapters, such as BicaudalD2. However, the mechanisms responsible for this increase in transport efficiency are not well understood, largely due to a lack of structural information. We used cryo-electron tomography to visualize the first 3-dimensional structure of the intact dynein-dynactin complex bound to microtubules. Our structure reveals that the dynactin-cargo-adapter complex recruits and binds to two dimeric cytoplasmic dyneins. Interestingly, the dynein motor organization closely resembles that of axonemal dynein, suggesting that cytoplasmic dynein and axonemal dyneins may utilize similar mechanisms to coordinate multiple motors. We propose that grouping dyneins onto a single dynactin scaffold promotes collective force production as well as unidirectional processive motility. These findings provide a structural platform that facilitates a deeper biochemical and biophysical understanding of dynein regulation and cellular transport.</jats:p

In Situ Imaging and Structure Determination of Biomolecular Complexes Using Electron Cryo-Tomography

Electron cryo-tomography (cryo-ET) is a technique that allows the investigation of intact macromolecular complexes while they are in their cellular milieu. Over the years, cryo-ET has had a huge impact on our understanding of how large biomolecular complexes look like, how they assemble, disassemble, function, and evolve(d). Recent hardware and software developments and combining cryo-ET with other techniques, e.g., focused ion beam milling (FIB-milling) and cryo-light microscopy, has extended the realm of cryo-ET to include transient molecular complexes embedded deep in thick samples (like eukaryotic cells) and enhanced the resolution of structures obtained by cryo-ET. In this chapter, we will present an outline of how to perform cryo-ET studies on a wide variety of biological samples including prokaryotic and eukaryotic cells and biological plant tissues. This outline will include sample preparation, data collection, and data processing as well as hybrid approaches like FIB-milling, cryosectioning, and cryo-correlated light and electron microscopy (cryo-CLEM)

A live RSV vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation

Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization and there remains no pediatric vaccine. RSV live-attenuated vaccines (LAVs) have a history of safe testing in infants; however, achieving an effective balance of attenuation and immunogenicity has proven challenging. Here we seek to engineer an RSV LAV with enhanced immunogenicity. Genetic mapping identifies strain line 19 fusion (F) protein residues that correlate with pre-fusion antigen maintenance by ELISA and thermal stability of infectivity in live RSV. We generate a LAV candidate named OE4 which expresses line 19F and is attenuated by codon-deoptimization of non-structural (NS1 and NS2) genes, deletion of the small hydrophobic (SH) gene, codon-deoptimization of the attachment (G) gene and ablation of the secreted form of G. OE4 (RSV-A2-dNS1-dNS2-ΔSH-dG(m)-Gs(null)-line19F) exhibits elevated pre-fusion antigen levels, thermal stability, immunogenicity, and efficacy despite heavy attenuation in the upper and lower airways of cotton rats

Resolution in electron tomography

Electron microscopes yield point resolution on the order of one angstrom, however the density maps from electron tomography typically have resolutions in the nanometre range. In this chapter I qualitatively discuss the typical limitations that occur in electron tomography of biological samples depending on the imaging modalities, with the focus on cryo electron tomography and subtomogram averaging