111 research outputs found
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
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 methods
Luminal particles within cellular microtubules.
The regulation of microtubule dynamics is attributed to microtubule-associated proteins that bind to the microtubule outer surface, but little is known about cellular components that may associate with the internal side of microtubules. We used cryoelectron tomography to investigate in a quantitative manner the three dimensional structure of microtubules in intact mammalian cells. We show that the lumen of microtubules in this native state is filled with discrete, globular particles with a diameter of 7 nm and spacings between 8 and 20 nm in neuronal cells. Cross-sectional views of microtubules confirm the presence of luminal material in vitreous sections of brain tissue. Most of the luminal particles had connections to the microtubule wall, as revealed in tomograms. A higher accumulation of particles was seen near the retracting plus ends of microtubules. The luminal particles were abundant in neurons, but were also observed in other cells, such as astrocytes and stem cells
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COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with in vitro reconstitution systems using purified components. Previously we have determined a complete structural model of the in vitro reconstituted COPI coat (Dodonova et al., 2017). Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath beta'-COP. We find that all coat components disassemble simultaneously and relatively rapidly after budding. Structural analysis in situ, maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo content as they progress from cis to trans, but the structure of the coat machinery remains constant
Push-me-pull-you: how microtubules organize the cell interior
Dynamic organization of the cell interior, which is crucial for cell function, largely depends on the microtubule cytoskeleton. Microtubules move and position organelles by pushing, pulling, or sliding. Pushing forces can be generated by microtubule polymerization, whereas pulling typically involves microtubule depolymerization or molecular motors, or both. Sliding between a microtubule and another microtubule, an organelle, or the cell cortex is also powered by molecular motors. Although numerous examples of microtubule-based pushing and pulling in living cells have been observed, it is not clear why different cell types and processes employ different mechanisms. This review introduces a classification of microtubule-based positioning strategies and discusses the efficacy of pushing and pulling. The positioning mechanisms based on microtubule pushing are efficient for movements over small distances, and for centering of organelles in symmetric geometries. Mechanisms based on pulling, on the other hand, are typically more elaborate, but are necessary when the distances to be covered by the organelles are large, and when the geometry is asymmetric and complex. Thus, taking into account cell geometry and the length scale of the movements helps to identify general principles of the intracellular layout based on microtubule forces
In Vivo Chromatin Organization of Mouse Rod Photoreceptors Correlates with Histone Modifications
BACKGROUND: The folding of genetic information into chromatin plays important regulatory roles in many nuclear processes and particularly in gene transcription. Post translational histone modifications are associated with specific chromatin condensation states and with distinct transcriptional activities. The peculiar chromatin organization of rod photoreceptor nuclei, with a large central domain of condensed chromatin surrounded by a thin border of extended chromatin was used as a model to correlate in vivo chromatin structure, histone modifications and transcriptional activity. METHODOLOGY: We investigated the functional relationships between chromatin compaction, distribution of histone modifications and location of RNA polymerase II in intact murine rod photoreceptors using cryo-preparation methods, electron tomography and immunogold labeling. Our results show that the characteristic central heterochromatin of rod nuclei is organized into concentric domains characterized by a progressive loosening of the chromatin architecture from inside towards outside and by specific combinations of silencing histone marks. The peripheral heterochromatin is formed by closely packed 30 nm fibers as revealed by a characteristic optical diffraction signal. Unexpectedly, the still highly condensed most external heterochromatin domain contains acetylated histones, which are usually associated with active transcription and decondensed chromatin. Histone acetylation is thus not sufficient in vivo for complete chromatin decondensation. The euchromatin domain contains several degrees of chromatin compaction and the histone tails are hyperacetylated, enriched in H3K4 monomethylation and hypo trimethylated on H3K9, H3K27 and H4K20. The transcriptionally active RNA polymerases II molecules are confined in the euchromatin domain and are preferentially located at the vicinity of the interface with heterochromatin. CONCLUSIONS: Our results show that transcription is located in the most decondensed and highly acetylated chromatin regions, but since acetylation is found associated with compact chromatin it is not sufficient to decondense chromatin in vivo. We also show that a combination of histone marks defines distinct concentric heterochromatin domains
Condensed Mitotic Chromosome Structure at Nanometer Resolution Using PALM and EGFP- Histones
Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow <20 nm resolution. We further found that image pre-processing using a combination of denoising and deconvolution of the raw PALM images substantially improved the spatial resolution of the reconstruction from noisy images. Performing PALM on Drosophila mitotic chromosomes labeled with H2AvD-EGFP, a histone H2A variant, revealed filamentous components of ∼70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10–30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples
Regulation of Signaling at Regions of Cell-Cell Contact by Endoplasmic Reticulum-Bound Protein-Tyrosine Phosphatase 1B
Protein-tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed PTP that is anchored to the endoplasmic reticulum (ER). PTP1B dephosphorylates activated receptor tyrosine kinases after endocytosis, as they transit past the ER. However, PTP1B also can access some plasma membrane (PM)-bound substrates at points of cell-cell contact. To explore how PTP1B interacts with such substrates, we utilized quantitative cellular imaging approaches and mathematical modeling of protein mobility. We find that the ER network comes in close proximity to the PM at apparently specialized regions of cell-cell contact, enabling PTP1B to engage substrate(s) at these sites. Studies using PTP1B mutants show that the ER anchor plays an important role in restricting its interactions with PM substrates mainly to regions of cell-cell contact. In addition, treatment with PTP1B inhibitor leads to increased tyrosine phosphorylation of EphA2, a PTP1B substrate, specifically at regions of cell-cell contact. Collectively, our results identify PM-proximal sub-regions of the ER as important sites of cellular signaling regulation by PTP1B
Toward visualization of nanomachines in their native cellular environment
The cellular nanocosm is made up of numerous types of macromolecular complexes or biological nanomachines. These form functional modules that are organized into complex subcellular networks. Information on the ultra-structure of these nanomachines has mainly been obtained by analyzing isolated structures, using imaging techniques such as X-ray crystallography, NMR, or single particle electron microscopy (EM). Yet there is a strong need to image biological complexes in a native state and within a cellular environment, in order to gain a better understanding of their functions. Emerging methods in EM are now making this goal reachable. Cryo-electron tomography bypasses the need for conventional fixatives, dehydration and stains, so that a close-to-native environment is retained. As this technique is approaching macromolecular resolution, it is possible to create maps of individual macromolecular complexes. X-ray and NMR data can be ‘docked’ or fitted into the lower resolution particle density maps to create a macromolecular atlas of the cell under normal and pathological conditions. The majority of cells, however, are too thick to be imaged in an intact state and therefore methods such as ‘high pressure freezing’ with ‘freeze-substitution followed by room temperature plastic sectioning’ or ‘cryo-sectioning of unperturbed vitreous fully hydrated samples’ have been introduced for electron tomography. Here, we review methodological considerations for visualizing nanomachines in a close-to-physiological, cellular context. EM is in a renaissance, and further innovations and training in this field should be fully supported
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