Twisting in a crowd

Given our current understanding of nuclear structure, it is difficult to imagine how the nucleus performs its varied functions and controls the traffic of its many components. For example, how can densely packed chromatin be transcribed without the helical nature of the DNA resulting in entangled DNA and RNA and a stalled RNA polymerase? Here, Jacques Dubochet discusses a model of transcription in which DNA rotates around its axis, rather than RNA polymerase rotating around the DNA. Furthermore, he suggests that a view of chromatin as 'liquid' may help in understanding a wide range of nuclear functions

Bilayers of nucleosome core particles

Among the multiple effects involved in chromatin condensation and decondensation processes, interactions between nucleosome core particles are suspected to play a crucial role. We analyze them in the absence of linker DNA and added proteins, after the self-assembly of isolated nucleosome core particles under controlled ionic conditions. We describe an original lamellar mesophase forming tubules on the mesoscopic scale. High resolution imaging of cryosections of vitrified samples reveals how nucleosome core particles stack on top of one another into columns which themselves align to form bilayers that repel one another through a solvent layer. We deduce from this structural organization how the particles interact through attractive interactions between top and bottom faces and lateral polar interactions that originate in the heterogeneous charge distribution at the surface of the particle. These interactions, at work under conditions comparable with those found in the living cell, should be of importance in the mechanisms governing chromatin compaction in vivo

Electron microscopy of frozen biological suspensions

The methodology for preparing specimens in the frozen, hydrated state has been assessed using crystals and T4 bacteriophages. The methods have also been demonstrated with lambda bacteriophages, purple membrane of Halobacterium halobium and fibres of DNA. For particles dispersed in an aqueous environment, it is shown that optimum structural preservation is obtained from a thin, quench-frozen film with the bulk aqueous medium in the vitreous state. Crystallization of the bulk water may result in solute segregation and expulsion of the specimen from the film. Contrast measurements can be used to follow directly the state of hydration of a specimen during transition from the fully hydrated to the freeze-dried state and permit direct measurement of the water content of the specimen. By changing the concentration and composition of the aqueous medium the contrast of particles in a vitreous film can be controlled and any state of negative, positive or zero contrast may be obtained. At 100 K, frozen-hydrated, freeze-dried or sugar embedded crystals can withstand a three- to four-fold increase in electron exposure for the same damage when compared with similar sugar-embedded or freeze-dried samples at room temperature

Cryoelectron microscopy of vitrified sections: a new challenge for the analysis of functional nuclear architecture

Cryoelectron microscopy of vitrified sections has become a powerful tool for investigating the fine structural features of cellular compartments. In the present study, this approach has been applied in order to explore the ultrastructural morphology of the interphase nucleus in different mammalian cultured cells. Rat hepatoma, Chinese hamster ovary and Potorus kidney cells were cryofixed by high-pressure freezing and the cryosections were examined at low temperature by transmission electron microscopy. Our results show that while the contrast of nuclear structural domains is remarkably homogeneous in hydrated sections, some of them can be recognised due to their characteristic texture. Thus, condensed chromatin appears finely granular and the perichromatin region contains rather abundant fibro-granular elements suggesting the presence of dispersed chromatin fibres and of perichromatin fibrils and granules. The interchromatin space looks homogeneous and interchromatin granules have not been identified under these preparative conditions. In the nucleolus, the most striking feature is the granular component, while the other parts of the nucleolar body, which appear less contrasted, are difficult to resolve. The nuclear envelope is easily recognisable with its regular perinuclear space and nuclear pore complexes. Our observations are discussed in the context of results obtained by other, more conventional electron microscopic method

Cryo-electron microscopy of viruses

Thin vitrified layers of unfixed, unstained and unsupported virus suspensions can be prepared for observation by cryo-electron microscopy in easily controlled conditions. The viral particles appear free from the kind of damage caused by dehydration, freezing or adsorption to a support that is encountered in preparing biological samples for conventional electron microscopy. Cryo-electron microscopy of vitrified specimens offers possibilities for high resolution observations that compare favourably with any other electron microscopical method

Freezing: Facts and Hypothesis

Hexagonal ice crystals formed in frozen biological specimens are large and branched. They can produce severe structural damage by solute segregation but there are also cases where they seem to cause only minor damage. When cooling is more rapid, cubic ice crystals can be formed. These are small and in general, they cause little damage. These observations can be readily explained with the hypothesis that large hexagonal ice crystals can originate from the rewarming induced transformation of a large number of cubic ice crystals. This transformation would take place without significant solute displacement

Ups and downs in early electron cryo-microscopy.

This is a tale of two scientists who, in their younger days, had their scientific judgement clouded by the promise of a big discovery. Two years later, they found that their conclusions had been considerably exaggerated. They were lucky, though, as their later work would prove to be significant. Now, more than 30 years after those events, they met again and put in writing their understanding of what went wrong