Achilleas S. Frangakis

Proteome Organization in a Genome-Reduced Bacterium

Simply Mycoplasma The bacterium Mycoplasma pneumoniae, a human pathogen, has a genome of reduced size and is one of the simplest organisms that can reproduce outside of host cells. As such, it represents an excellent model organism in which to attempt a systems-level understanding of its biological organization. Now three papers provide a comprehensive and quantitative analysis of the proteome, the metabolic network, and the transcriptome of M. pneumoniae (see the Perspective by Ochman and Raghavan). Anticipating what might be possible in the future for more complex organisms, Kühner et al. (p. 1235) combine analysis of protein interactions by mass spectrometry with extensive structural information on M. pneumoniae proteins to reveal how proteins work together as molecular machines and map their organization within the cell by electron tomography. The manageable genome size of M. pneumoniae allowed Yus et al. (p. 1263) to map the metabolic network of the organism manually and validate it experimentally. Analysis of the network aided development of a minimal medium in which the bacterium could be cultured. Finally, G‡ell et al. (p. 1268) applied state-of-the-art sequencing techniques to reveal that this “simple” organism makes extensive use of noncoding RNAs and has exon- and intron-like structure within transcriptional operons that allows complex gene regulation resembling that of eukaryotes. The simplified proteome of a bacterium provides insight into the organization of proteins into molecular machines. The genome of Mycoplasma pneumoniae is among the smallest found in self-replicating organisms. To study the basic principles of bacterial proteome organization, we used tandem affinity purification–mass spectrometry (TAP-MS) in a proteome-wide screen. The analysis revealed 62 homomultimeric and 116 heteromultimeric soluble protein complexes, of which the majority are novel. About a third of the heteromultimeric complexes show higher levels of proteome organization, including assembly into larger, multiprotein complex entities, suggesting sequential steps in biological processes, and extensive sharing of components, implying protein multifunctionality. Incorporation of structural models for 484 proteins, single-particle electron microscopy, and cellular electron tomograms provided supporting structural details for this proteome organization. The data set provides a blueprint of the minimal cellular machinery required for life.

The molecular architecture of cadherins in native epidermal desmosomes

Desmosomes are cadherin-based adhesive intercellular junctions, which are present in tissues such as heart and skin. Despite considerable efforts, the molecular interfaces that mediate adhesion remain obscure. Here we apply cryo-electron tomography of vitreous sections from human epidermis to visualize the three-dimensional molecular architecture of desmosomal cadherins at close-to-native conditions. The three-dimensional reconstructions show a regular array of densities at ∼70 Å intervals along the midline, with a curved shape resembling the X-ray structure of C-cadherin, a representative ‘classical’ cadherin. Model-independent three-dimensional image processing of extracted sub-tomograms reveals the cadherin organization. After fitting the C-cadherin atomic structure into the averaged sub-tomograms, we see a periodic arrangement of a trans W-like and a cis V-like interaction corresponding to molecules from opposing membranes and the same cell membrane, respectively. The resulting model of cadherin organization explains existing two-dimensional data and yields insights into a possible mechanism of cadherin-based cell adhesion.

Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibers in mitotic chromosomes in situ

Although the formation of 30-nm chromatin fibers is thought to be the most basic event of chromatin compaction, it remains controversial because high-resolution imaging of chromatin in living eukaryotic cells had not been possible until now. Cryo-electron microscopy of vitreous sections is a relatively new technique, which enables direct high-resolution observation of the cell structures in a close-to-native state. We used cryo-electron microscopy and image processing to further investigate the presence of 30-nm chromatin fibers in human mitotic chromosomes. HeLa S3 cells were vitrified by high-pressure freezing, thin-sectioned, and then imaged under the cryo-electron microscope without any further chemical treatment or staining. For an unambiguous interpretation of the images, the effects of the contrast transfer function were computationally corrected. The mitotic chromosomes of the HeLa S3 cells appeared as compact structures with a homogeneous grainy texture, in which there were no visible 30-nm fibers. Power spectra of the chromosome images also gave no indication of 30-nm chromatin folding. These results, together with our observations of the effects of chromosome swelling, strongly suggest that, within the bulk of compact metaphase chromosomes, the nucleosomal fiber does not undergo 30-nm folding, but exists in a highly disordered and interdigitated state, which is, on the local scale, comparable with a polymer melt.

Identification of macromolecular complexes in cryoelectron tomograms of phantom cells

Electron tomograms of intact frozen-hydrated cells are essentially three-dimensional images of the entire proteome of the cell, and they depict the whole network of macromolecular interactions. However, this information is not easily accessible because of the poor signal-to-noise ratio of the tomograms and the crowded nature of the cytoplasm. Here, we describe a template matching algorithm that is capable of detecting and identifying macromolecules in tomographic volumes in a fully automated manner. The algorithm is based on nonlinear cross correlation and incorporates elements of multivariate statistical analysis. Phantom cells, i.e., lipid vesicles filled with macromolecules, provide a realistic experimental scenario for an assessment of the fidelity of this approach. At the current resolution of ≈4 nm, macromolecules in the size range of 0.5–1 MDa can be identified with good fidelity.

Quantitative and spatio-temporal features of protein aggregation in Escherichia coli and consequences on protein quality control and cellular ageing

The aggregation of proteins as a result of intrinsic or environmental stress may be cytoprotective, but is also linked to pathophysiological states and cellular ageing. We analysed the principles of aggregate formation and the cellular strategies to cope with aggregates in Escherichia coli using fluorescence microscopy of thermolabile reporters, EM tomography and mathematical modelling. Misfolded proteins deposited at the cell poles lead to selective re‐localization of the DnaK/DnaJ/ClpB disaggregating chaperones, but not of GroEL and Lon to these sites. Polar aggregation of cytosolic proteins is mainly driven by nucleoid occlusion and not by an active targeting mechanism. Accordingly, cytosolic aggregation can be efficiently re‐targeted to alternative sites such as the inner membrane in the presence of site‐specific aggregation seeds. Polar positioning of aggregates allows for asymmetric inheritance of damaged proteins, resulting in higher growth rates of damage‐free daughter cells. In contrast, symmetric damage inheritance of randomly distributed aggregates at the inner membrane abrogates this rejuvenation process, indicating that asymmetric deposition of protein aggregates is important for increasing the fitness of bacterial cell populations.

Human mitotic chromosomes consist predominantly of irregularly folded nucleosome fibres without a 30-nm chromatin structure

How a long strand of genomic DNA is compacted into a mitotic chromosome remains one of the basic questions in biology. The nucleosome fibre, in which DNA is wrapped around core histones, has long been assumed to be folded into a 30‐nm chromatin fibre and further hierarchical regular structures to form mitotic chromosomes, although the actual existence of these regular structures is controversial. Here, we show that human mitotic HeLa chromosomes are mainly composed of irregularly folded nucleosome fibres rather than 30‐nm chromatin fibres. Our comprehensive and quantitative study using cryo‐electron microscopy and synchrotron X‐ray scattering resolved the long‐standing contradictions regarding the existence of 30‐nm chromatin structures and detected no regular structure >11 nm. Our finding suggests that the mitotic chromosome consists of irregularly arranged nucleosome fibres, with a fractal nature, which permits a more dynamic and flexible genome organization than would be allowed by static regular structures.

Multiple large filament bundles observed in Caulobacter crescentus by electron cryotomography

While the absence of any cytoskeleton was once recognized as a distinguishing feature of prokaryotes, it is now clear that a number of different bacterial proteins do form filaments in vivo. Despite the critical roles these proteins play in cell shape, genome segregation and cell division, molecular mechanisms have remained obscure in part for lack of electron microscopy‐resolution images where these filaments can be seen acting within their cellular context. Here, electron cryotomography was used to image the widely studied model prokaryote Caulobacter crescentus in an intact, near‐native state, producing three‐dimensional reconstructions of these cells with unprecedented clarity and fidelity. We observed many instances of large filament bundles in various locations throughout the cell and at different stages of the cell cycle. The bundles appear to fall into four major classes based on shape and location, referred to here as ‘inner curvature’, ‘cytoplasmic’, ‘polar’ and ‘ring‐like’. In an attempt to identify at least some of the filaments, we imaged cells where crescentin and MreB filaments would not be present. The inner curvature and cytoplasmic bundles persisted, which together with their localization patterns, suggest that they are composed of as‐yet unidentified cytoskeletal proteins. Thus bacterial filaments are frequently found as bundles, and their variety and abundance is greater than previously suspected.

Visualization of macromolecular structures.

Structural biology is rapidly accumulating a wealth of detailed information about protein function, binding sites, RNA, large assemblies and molecular motions. These data are increasingly of interest to a broader community of life scientists, not just structural experts. Visualization is a primary means for accessing and using these data, yet visualization is also a stumbling block that prevents many life scientists from benefiting from three-dimensional structural data. In this review, we focus on key biological questions where visualizing three-dimensional structures can provide insight and describe available methods and tools.

Evidence for short-range helical order in the 30-nm chromatin fibers of erythrocyte nuclei

Chromatin folding in eukaryotes fits the genome into the limited volume of the cell nucleus. Formation of higher-order chromatin structures attenuates DNA accessibility, thus contributing to the control of essential genome functions such as transcription, DNA replication, and repair. The 30-nm fiber is thought to be the first hierarchical level of chromatin folding, but the nucleosome arrangement in the compact 30-nm fiber was previously unknown. We used cryoelectron tomography of vitreous sections to determine the structure of the compact, native 30-nm fiber of avian erythrocyte nuclei. The predominant geometry of the 30-nm fiber revealed by subtomogram averaging is a left-handed two-start helix with approximately 6.5 nucleosomes per 11 nm, in which the nucleosomes are juxtaposed face-to-face but are shifted off their superhelical axes with an axial translation of approximately 3.4 nm and an azimuthal rotation of approximately 54°. The nucleosomes produce a checkerboard pattern when observed in the direction perpendicular to the fiber axis but are not interdigitated. The nucleosome packing within the fibers shows larger center-to-center internucleosomal distances than previously anticipated, thus excluding the possibility of core-to-core interactions, explaining how transcription and regulation factors can access nucleosomes.

Visualization of cell microtubules in their native state

Background information. Over the past decades, cryo‐electron microscopy of vitrified specimens has yielded a detailed understanding of the tubulin and microtubule structures of samples reassembled in vitro from purified components. However, our knowledge of microtubule structure in vivo remains limited by the chemical treatments commonly used to observe cellular architecture using electron microscopy.