Functional Architecture of the Cell Nucleus
Dr. Karsten Richter
Using fluorescence live-cell imaging and electron microscopy, we are establishing how microcompartmentalization influences nuclear integrity. The cell nucleus is organized into many structural domains, which are large compared to the scale of single-molecule interactions: Chromatin is packed into chromosome band domains, chromosome arm domains and chromosome territories. Speckles, Cajal bodies and PML bodies are µm-sized structures, where specific sets of proteins and RNP's accumulate, and processes like DNA replication and RNA polymerization take place in factories with multiple sites of activity, using huge macromolecular machines. How are these domains established?
Our current research focuses on macromolecular crowding as a mechanism to establish microcompartments in cell nuclei. The concept takes into account the very special composition of nucleoplasms, which are packed with high-molecular chromatin, ribonucleo-particles and associated proteins. Macromolecular crowding drives bulky components into compact organizations without the explicit need of site-specific interactions. Experimentally, we challenge the crowding-status of the nucleoplasm by hypertonic extraction of water from living cells. These studies demonstrate a remarkable sensitivity of the nuclear ultrastructure upon hypertonic treatment: Within seconds, chromatin compaction becomes visible as part of a general partitioning of nuclear compounds into several, structurally distinct domains (Fig. 1). In particular, a complete reorganization of the nuclear periphery can be induced (Fig. 2). All these changes reversibly recover when cells are re-cultured in isotonic medium.
Realizing that macromolecular crowding provides a cooperative momentum for the assembly of macromolecular complexes, it may be functionally important as a parameter to stabilize nuclear processes like transcription and hnRNA maturation, which involve hundreds of factors. Contributing to robust expression patterns, the crowding state of the nucleoplasm may also have influence on the development of cancer.
Fig. 1
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Hypertonic incubation of living cells causes substantial structural reorganization of the nucleoplasm:
MCF7 cells were incubated for 20 minutes in hypertonic media, chemically fixed with aldehyde and osmium, embedded in epoxid-resin and processed for standard electron microscopy. Scale bar 1 µm.
Left: The nuclear content of untreated cells appears finely dispersed. Structural entities like chromatin (ch) and interchromatin granule clusters (ig) are difficult to individualize within the overall fibrogranular background of the nucleoplasm (cb: Cajal body).
Middle: Supplementation of 160 mM sucrose provoked compaction of chromatin (ch) and formation of small, dense bodies (db). The nucleoplasm became more differentially textured (ig: interchromatin granule cluster; no: nucleolus).
Right: Upon incubation with 320 mM sucrose, lacunae of fine-fibrillar material (fm) became distinct. Remarkably, the peripheral chromatin (ch) retracted from the nuclear envelope, which gave rise to a new peripheral domain (pl) filled with fine fibrillar material, while coarse granular material, including clusters of interchromatin granules (ig) accumulated at the interface with the peripheral chromatin.
Fig. 2
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The lamina-chromatin interaction defines the nuclear periphery as a peculiar nuclear compartment:
MCF7 cells were incubated with sucrose for 20 minutes (left: control, no sucrose; middle: 160 mM sucrose added to the growth medium, right: treatment with 320 mM sucrose), fixed with formaldehyde and fluorescence-labeled to reveal chromatin (red: ToPro3, Molecular Probes), speckles (green: anti-SC35, Sigma), the lamina (blue, upper row: anti lamin AC, kindly provided by H. Herrmann, DKFZ) and PML bodies (blue. lower row: anti-SP100, kindly provided by H. Will, HPI Hamburg). Scale bar 10 µm.
The chromatin separated by a gap from the lamin A-rim upon retraction from the nuclear periphery at 320 mM sucrose load (upper right image: double arrow). Neither speckles nor PML bodies ever appear at the nuclear periphery in normal control cells (left images). This still holds true with 160 mM sucrose treatment when chromatin compaction is induced (middle images), but peripheral chromatin remains associated with the lamina (middle images). However, as chromatin retracts form the lamina, speckles as well as PML bodies become enabled to settle the nuclear periphery (arrows).