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Nuclear Architecture - 5th Junior Scientist Conference of the German Society for Cell Biology (DGZ) Held in Jena, September 25th to 27th, 2003

Posted on: Friday, 30 April 2004, 06:00 CDT

The "Junior Scientist Conference" of the German Society for Cell Biology (DGZ) has taken place for the fifth time. After having started biannually, it will from this year on take place every September. Last year's meeting was on "Embryonic and somatic stem cells in basic research and medicine" and was a success just like the third meeting on "Cell-cell and cell-matrix interactions during development and differentiation" in 2000. We are now slightly switching our conference language into English and we will expand the participation of foreign scientists. The subject of the next meeting, 26th - 28th September 2004, will be "Cytoskeletal Dynamics". These meetings have 14 invited speakers and up to 80 participants, mostly graduate and postdoctoral fellows. The meeting includes an extended session by Zeiss scientists giving an introduction into the latest developments of light microscopy. This year's meeting was opened by two talks from scientists working at Carl Zeiss. The first talk celebrated "Fifty Years of Phase Contrast: Nobel Prize for Frits Zernicke", given by Dr. Heinz Gundlach, and the second dealt with "Modern Methods in Laser Scanning Microscopy", given by Dr. Richard Ankerhold.

The biology of the cell nucleus is an active field of research, and exciting developments are under way in many areas including live cell imaging, small RNAs or nuclear scaffolding as well as chromosome segregation during mitosis.

Nuclear structural components

The presence of actin in the nucleus is known for more than twenty years, however its nuclear role is still being disputed. Susanne Illenberger (TU, Braunschweig) gave a brief review on nuclear aclin and its proposed roles. In particular, she summed up compelling evidence for the classification of actin as a clualcompartment protein. Together with Brigitte M. Jockusch she characterised a further cytoskeletal dual-compartment protein, Raver1, which was isolated based on its binding to mctavinculin. Raver1 is found both at the plasma membrane and within the nucleus where it is involved in the splicing process (Fig. 1). Further studies revealed that Raver1 not only interacts with metavinculin but also with actin directly. As this nuclear actin is detected by an anti-aclin antibody which recognises a specific actin conformation, an intriguing avenue on conformation studies of nuclear actin is opened. Not only actin is present in the nucleus but also a variety of actin-binding proteins such protein 4.1. Iakowos Karakesisoglou (Universitat zu Koln) reported two novel actin-binding proteins as components of the nucleus, NUANCE (Fig. 2) and Enaptin. Both are nuclear envelope proteins and are anchored in the nuclear membrane by a type II transmembrane domain. NUANCE and Enaptin are gigantic proteins with molecular masses of around 800 kDa and higher. They have a similar overall structure with an actinbinding domain of the alpha-actinin type at their N-terminus which is separated from the C-terminal transmembrane domain by a long coiled-coil region, and both interact with lamins.

Fig. 1. The dual-compartment protein raver1 translocates from the nucleus to the plasma membrane in differentiating cells. Raver1 is a partner of nuclear proteins involved in RNA splicing, but leaves the nucleus and migrates to the plasma membrane during cell differentiation (Huttelmaier et al., 2000, J. Cell Biol. 155, 775). The immunofluorescence image shows differentiating MDCK epithelial cells, demonstrating Raverl (red) in the nucleus as well as at the plasma membrane in distinct cytoplasmic "Raver-containing bodies" (RCBs). Cell-cell contacts are marked by the tight junction protein ZO-1 (green). Bar equals 10 m. (D. Fleckenstein, B. M. Jockusch and S. Illenberger, TU Braunschweig).

Fig. 2. NUANCE, a giant protein connecting (he nucleus and actin cyloskeleton. The protein consists of an N-terminal actin-binding domain followed by a large helical rod domain with several spectrin repeats and a C-terminal transmembrane domain. The immunofluorescence image shows the nuclear membrane localisation of NUANCE (green) in COS7 cells but also a diffuse cytoplasmic staining in a region nearby the nucleus. The F-actin cytoskeleton is labelled by TRITCphalloidin (red). Bar equals 10 m. (Figure provided by Thorsten Libotte)

The nuclear matrix appears to be another controversial topic in nuclear biology. The main proteins are hnRNPs, proteins involved in replication and transcription, DNA-binding factors and proteins of unknown functions. Frank Fackelmayer (University of Hamburg) discussed the problems of matrix isolation. He succeeded in the isolation of the matrix component SAF-A (scaffold attachment factor A) where he defined a SARbinding domain. This domain interacts with DNA elements, the SARs (scaffold attachment region), thought to partition eukaryotic genomes into independent chromatin loops by attaching the DNA to proteins of a nuclear scaffold or matrix. He presented data indicating that a disruption of the nuclear matrix interferes with nuclear processes such as DNA replication.

Cellular imaging

Jan Ellenberg (EMBL, Heidelberg) gave an impressive report on the dynamics of the nuclear pore complex. His group started out to solve the question whether the molecular architecture of the nuclear pore complex (NPC) is static or a result of a dynamic equilibrium of assembly and disassembly of the numerous NPC components. The exchange rates of 19 nucleoporins were analysed using FRAP (fluorescence recovery after photobleaching) at a confocal laser scanning microscope. These analyses revealed a differential pattern of nucleoporin dynamics: Putative components of the spoke ring complex turned out to be rather stable with average residency times of more than 20 h, whereas others were less stable and dissociated from the complex within 2 to 20 h. The NPC association of three components was extremely dynamic with residency times of shorter than 10 min, reflecting a possible role in regulation of nucleoplasmic transport.

Mitosis

Two talks dealt with cellular processes involved in proper segregation of nuclear material into daughter cells during mitosis. Ralph Graf (LMU, A.B.I. Zellbiologie, Munchen) reported about functional analyses of the centrosome, a nucleus-associated organelle that forms the spindle poles of the mitotic spindle. He uses Dictyostelium discoideum as a model system and characterised DdCP224 and DdLIS1, (wo interacting centrosomal proteins involved in cell dynamics. DdCP224 is required for centrosome duplication, cytokinesis and microtubule growth. DdLIS1 is a homologue of the human lissencephaly 1 protein. It is required for mitotic progression and cell migration which became evident in DdLIS1 mutants with spindle defects and altered actin dynamics. Moreover, both DdCP224 and DdLIS1 are involved in microtubule plus end interactions with the cell cortex which was demonstrated by live cell analysis of GFP-mutants employing 4D-confocal microscopy.

Sister chromatid cohesion, a further prerequisite for proper mitotic progression, was the subject of the presentation hy JanMichael Peters (IMP, Vienna, Austria). Cohesion prevents chromosome segregation prior to proper bipolar attachment of both centromeres to the mitotic spindle. Chromatid cohesion is mediated by cohesin complexes the dissociation of which is required for anaphase progression. This process is regulated by phosphorylation involving polo-like kinase (PIk1) activity. Aurora B is a further kinase involved in chromosome segregation. This was investigated using Hcsperadin/BIBI1489, a drug identified in collaboration with Boehringer Ingelheim. Hesperadin (Hespera was the opponent of Aurora in Greek mythology) interferes with chromosome alignment and segregation through inhibition of Aurora B. Jan-Michel Peters suggested that Aurora B is required to generate unattached kinetochores on mono-oriented chromosomes which in turn could promote bipolar attachment as well as maintain signalling of the spindle checkpoint.

Architectural principles of the nucleus

The molecular characterization of a novel nucleolar protein of Xenopus laevis, called protein NO145, was presented by Sandra Kneiel (DKFZ, Heidelberg). By immunohlotting, protein NO145 was identified in karyoskeletal fractions of Xenopus laevis oocytes and in all stages of oogencsis. Very remarkably, the protein is completely degraded during egg formation and is not expressed, in any significant amount, in early embryos and other tissues of adult frogs. Detailed immunolocalisation studies using light and electron microscopy have shown that protein NO 145 specifically localises within a thin cage-like cortical layer surrounding the nucleolus compatible with its existence as part of a filamentous meshwork (for an image see the front cover of Zellbiologie aktuell 3/2002). Protein NO 145 does, however, not co-localise with any of the well characterized nucleolar subcompartments and is therefore the first constituent described for a presumptive nucleolar skeleton in amphibian oocytes.

Michaela Reichenzeller (DKFZ, Heidelberg) reported on the structure and dynamics of the interchromatin compartment as investigated by live cell imaging utilizing GFP chimeras of nuclear as well as ectopically expresse\d cytoplasmic filamentforming polypeptides. These studies demonstrated that the space between the interphase chromosomal entities ("chromosome territories") is biochemically very active, since it not only passively harbours ectopically expressed factors - instead of degrading them - but also very distinctly processes these proteins enabling them to explore accessible nuclear space just like a probe. In addition, the accessibility of nuclear space was investigated by microinjection of fluorescently labelled dextran particles (see (Goerisch et al., Exp. Cell Res. 289 (2003) 282294)). These studies revealed that the concept of an interchromosomal domain compartment is only beginning to be understood in molecular terms, however, its activity and dynamics can be nicely followed in the living cell.

The nuclear envelope

Our understanding of the organization and function of the nuclear envelope is still far from being complete. We just have learnt that in man mutations of one of the structural components of the lamina, lamin A, at more than 130 different sites give rise to various multi- systemic inheritable diseases including premature aging. Katrin Hoffmann (MDC, Berlin) found that various mutations in another nuclear envelope protein, the inner nuclear membrane protein LBR (lamin B receptor) cause a complex spectrum of diseases in mammals previously described as Pelger-Huett anomaly. A pronounced feature of patients with reduced levels of LBR is a reduction in the nuclear lobulation and drastic alterations of chromatin organisation in granulocytcs. The molecular basis of the disease remains unclear, particularly if this involves the known sterol reductase activity of LBR. However, the clinical effects of LBR reduction in patients are severe including developmental delay, mental retardation and skeletal abnormalities.

In addition to their localisation at the nuclear envelope, lamins and lamin-binding proteins arc also found in the nuclear interior. Particularly, A-type lamins and their nucleoplasmic binding partner LAP2[alpha] are prominent structures in the G1 phase of the cell cycle. However, neither the structure nor the functions of intranuclear lamin-LAP2[alpha] complexes are known at present. Roland Foisner (Vienna Biocenter, Wien) discussed potential roles of lamins A and LAP2cx in the control of gene expression and cell cycle progression due to their association with the tumour suppressor retinoblastoma protein Rb. These are intriguing novel functions of lamina proteins that go beyond mere structural support of nuclear architecture and could also contribute to the observed lamin-linked disease phenotypes (Fig. 3).

One of the most powerful approaches to investigate the function of a protein in vivo is to "knock down" its expression. Reimer Stick (University of Bremen) has investigated to what extent one can interfere with the synthesis of B-type lamins, the ubiquitously expressed nuclear intermediate filament proteins, during the embryogenesis of amphibian species such as Xenopus laevis. Although a classical object of embryogenesis, this frog has a serious complication as the oocytcs have stockpiled lots of maternal proteins and the embryonic transcription is turned on rather late after fertilization. Nevertheless, using morpholino-antisense oligonucleotides Stick and colleagues have managed to knock down lamin B1 and lamin B2 one by one and both together. Although knockdown of either lamin B1 or B2 had little effect, the removal of most of both B-type lamins interfered with embryogenesis underlining their importance for development.

Nucleocytoplasmic transport (NCT) is a highly controlled, signal- dependent, receptor-mediated process. Ueli Aebi (Biozentrum, Basel) described the state-of-the-art structural organization of the nuclear pore complex (NPC), a 120-MDa supramolecular protein machine which is the sole gateway perforating the double-membrane nuclear envelope (NE) that separates the cytoplasm from the nucleus of all cukaryotic cells during interphase. More specifically, (i) atomic force microscopy (AFM) of fully native Xenopus oocyte NEs spread on a flat support has revealed the asymmetry of the cytoplasmic and nuclear periphery of the NPC in its physiological buffer environment, (ii) Cryo-electron tomography of the same specimen but this time embedded in a -250-nm thick amorphous ice film, has provided novel insights into the NPC architecture (Fig. 4). And (iii) the domain topography of individual nuclcoporins within the NPC has been dissected by immuno-gold EM using domain-specific anti- nucleoporin antibodies. Last but not least, time-lapse AFM of NPCs depicted in distinct transport states has allowed direct correlation of NPC structure with function. Taken together, this integrated analysis of NPC structure and function has led to a more mechanistic model of NCT, in particular, how "confined Brownian motion" might achieve vectorial translocation of cargoes through the NPC.

Modifications: sumoylation and acetylation

Another level of nuclear structure and regulation was covered in Frauke Melchior's presentation (MPI, Martinsried) discussing SUMO, a small ubiquitin-like molecule which predominantly modifies nuclear proteins. This modification can be compared to phosphorylation in its molecular consequences and in its dynamics. One important target protein is RanGAP which, in its sumoylated form, can hind RanBP2/ NUP358. RanBP2/NUP358 is an E3-ligase located at the nuclear pore. In complex with RanGAP it sumoylates proteins that are transported through the nuclear pore. In addition to its role in nucleocytoplasmic transport SUMO is also important for intranuclear targeting where it is required for formation of PML nuclear bodies.

In many sexually dimorphic species a mechanism is required to ensure equivalent levels of gene expression from the sex chromosomes. Quite different strategies can be followed. In mammals, dosage compensation is achieved by X-chromosome inactivation, whereas in Drosophila the X-chromosome is activated in male flies such that a twofold higher expression is achieved which makes X- linked gene expression equal in males and females. The epigenetic mechanism underlying this molecular strategy was discussed by Peter Becker (LMU, A.B.I., Molekularbiologie, Munchen). It involves a dosage compensation complex (DCC) consisting of several proteins including an acetyltransferase, MOF, which modifies histone H4 in the X-chromosome at position 16. This acetylation of the lysine residue 16 is sufficient to derepress transcription. Other subunits of the DCC are the MSL (male-specific lethal) proteins, which bind to non-coding roX RNAs. After binding of the DCC at specific sites along the X-chromosome which are being mapped by CHIP analysis, the complex spreads along the chromosome and changes the morphology of the chromosome by modification of the chromatin.

Fig. 3. Differential attachment sites of proteins at chromosomes during nuclear assembly. (A) Schematic representation of a halfspindle and separated sister chromatids at anaphase, showing preferred attachment of LAP2[alpha], BAF, and emerin at core regions, and LBR, lamin B and probably LAP2[beta] at peripheral regions of decondensing chromosomes. LEM domains and transmembrane regions of proteins arc indicated; LAP2[alpha] is labelled with a, LAP2[beta] with b, and emerin with e. (B) Confocal immunofluorescence image of a cell in anaphase expressing YFP- labelled LAP2[alpha] (green) and CFP-labelled LBR (red), and phase- contrast image of the same cell. Merge of red and green stain is shown to visualize the different localisation of proteins at chromosomes during nuclear assembly in anaphase. Image was kindly provided by Thomas Dechat and Roland Foisner. Bar represents 10 m. (Reprinted from: TheScientificWorldJOURNAL (2003) 3, 1-20, with permission).

Fig. 4. Tomographic 3D-reconstruction of thick-ice embedded nuclear pore complexes. CF, central framework; CR, cytoplasmic ring; CP, plug residing in the central pore; DR, distal ring-like structure; NR, nuclear ring. For details see: D. Stoffler et al. (2003) J. Mol. Biol. 328, 119-130 (Reprinted from: Journal of Molecular Biology (2003) 328, 119-130, 2003 Elsevier Science Ltd., with permission from Elsevier).

The scientific program of the meeting was complemented by a poster exhibition of the junior scientists, from which two sessions of 17 short talks were selected. We will not give further details here, since most of the work is still unpublished, but we hope to see these papers in the press soon.

Ralph Grafa, Harald Herrmann-Lerdonl)h, Angelika A. Noegele

a Ludwig-Maximilians-Universitat Munchen, Adolf-Butenandt- Institut, Zellbiologie, Munchen/Germany

b Deutsches Krebsforschungszentrum, Abteilung fur Zellbiologie, Heidelberg/Germany

c Universitat zu Koln, Medizinische Fakultat, Institut fur Biochemie I, Koln/Germany

1 Dr. Harald Herrmann-Lerdon, Deutsches Krebsforschungszentrum, Abteilung fur Zellbiologie, Technologiepark 3, Im Neuenheimer Feld 580, D-69120 Heidelberg/Germany, e-mail: h.herrmann@dkfz.de

Copyright Urban & Fischer Verlag Jan 2004

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