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Harald Herrmann

Publications and source records attributed to Harald Herrmann.

36 records · Page 2Linked to original sources
PubMed2004

Crystal structure of the human lamin A coil 2B dimer: implications for the head-to-tail association of nuclear lamins.

Nuclear intermediate filaments (IFs) are made from fibrous proteins termed lamins that assemble, in association with several transmembrane proteins of the inner nuclear membrane and an unknown number of chromatin proteins, into a filamentous scaffold called the nuclear lamina. In man, three types of lamins with significant sequence identity, i.e. lamin A/C, lamin B1 and B2, are expressed. The molecular characteristics of the filaments they form and the details of the assembly mechanism are still largely unknown. Here we report the crystal structure of the coiled-coil dimer from the second half of coil 2 from human lamin A at 2.2A resolution. Comparison to the recently solved structure of the homologous segment of human vimentin reveals a similar overall structure but a different distribution of charged residues and a different pattern of intra- and interhelical salt bridges. These features may explain, at least in part, the differences observed between the lamin and vimentin assembly pathways. Employing a modeled lamin A coil 1A dimer, we propose that the head-to-tail association of two lamin dimers involves strong electrostatic attractions of distinct clusters of negative charge located on the opposite ends of the rod domain with arginine clusters in the head domain and the first segment of the tail domain. Moreover, lamin A mutations, including several in coil 2B, have been associated with human laminopathies. Based on our data most of these mutations are unlikely to alter the structure of the dimer but may affect essential molecular interactions occurring in later stages of filament assembly and lamina formation.

Amino Acid Sequence↗
PubMed2004

Molecular and biophysical characterization of assembly-starter units of human vimentin.

We have developed an assembly protocol for the intermediate filament (IF) protein vimentin based on a phosphate buffer system, which enables the dynamic formation of authentic IFs. The advantage of this physiological buffer is that analysis of the subunit interactions by chemical cross-linking of internal lysine residues becomes feasible. By this system, we have analyzed the potential interactions of the coiled-coil rod domains with one another, which are assumed to make a crucial contribution to IF formation and stability. We show that headless vimentin, which dimerizes under low salt conditions, associates into tetramers of the A(22)-type configuration under assembly conditions, indicating that one of the effects of increasing the ionic strength is to favor coil 2-coil 2 interactions. Furthermore, in order to obtain insight into the molecular interactions that occur during the first phase of assembly of full-length vimentin, we employed a temperature-sensitive variant of human vimentin, which is arrested at the "unit-length filament" (ULF) state at room temperature, but starts to elongate upon raising the temperature to 37 degrees C. Most importantly, we demonstrate by cross-linking analysis that ULF formation predominantly involves A(11)-type dimer-dimer interactions. The presence of A(22) and A(12) cross-linking products in mature IFs, however, indicates that major rearrangements do occur during the longitudinal annealing and radial compaction steps of IF assembly.

Buffers↗
PubMed2004

The biology of desmin filaments: how do mutations affect their structure, assembly, and organisation?

Desmin, the major intermediate filament (IF) protein of muscle, is evolutionarily highly conserved from shark to man. Recently, an increasing number of mutations of the desmin gene has been described to be associated with human diseases such as certain skeletal and cardiac myopathies. These diseases are histologically characterised by intracellular aggregates containing desmin and various associated proteins. Although there is progress regarding our knowledge on the cellular function of desmin within the cytoskeleton, the impact of each distinct mutation is currently not understood at all. In order to get insight into how such mutations affect filament assembly and their integration into the cytoskeleton we need to establish IF structure at atomic detail. Recent progress in determining the dimer structure of the desmin-related IF-protein vimentin allows us to assess how such mutations may affect desmin filament architecture.

Amino Acid Sequence↗
PubMed2004

Film-loaded SAW waveguides for integrated acousto-optical polarization converters.

We report on a detailed theoretical and experimental investigation of film-loaded surface acoustic wave (SAW) waveguides in lithium niobate (LiNbO3) for integrated acousto-optical (AO) polarization converters. The numerical analysis is based on both a scalar and a full-vectorial model. Dispersion plots and figures of merit for several structures are given, which lead to design parameters for optimized polarization converters. It is pointed out that very attractive structures are metal/dielectric/LiNbO3 strip waveguides and dielectric/LiNbO3 slot waveguides, in which metal is either gold (Au) or aluminum (Al), and the dielectric film is an optical transparent material such as silicon oxide (SiO2), magnesium oxide (MgO), or aluminium oxide (Al2O3). Polarization converters with the designed acoustical waveguides have been realized and characterized by optical conversion and laser probing measurements.

Journal Article↗
PubMed2004

Intermediate filaments: molecular structure, assembly mechanism, and integration into functionally distinct intracellular Scaffolds.

The superfamily of intermediate filament (IF) proteins contains at least 65 distinct proteins in man, which all assemble into approximately 10 nm wide filaments and are principal structural elements both in the nucleus and the cytoplasm with essential scaffolding functions in metazoan cells. At present, we have only circumstantial evidence of how the highly divergent primary sequences of IF proteins lead to the formation of seemingly similar polymers and how this correlates with their function in individual cells and tissues. Point mutations in IF proteins, particularly in lamins, have been demonstrated to lead to severe, inheritable multi-systemic diseases, thus underlining their importance at several functional levels. Recent structural work has now begun to shed some light onto the complex fine tuning of structure and function in these fibrous, coiled coil forming multidomain proteins and their contribution to cellular physiology and gene regulation.

Amino Acid Sequence↗
PubMed2003

Diffusion-limited compartmentalization of mammalian cell nuclei assessed by microinjected macromolecules.

In order to investigate the accessibility of the nucleoplasm for macromolecules with different physical properties, we microinjected FITC-conjugated dextrans of different sizes as well as anionic FITC-dextrans and FITC-poly-L-lysine into mammalian cell nuclei. Small dextrans displayed a homogeneous nuclear distribution. With increasing molecular mass (42 to 2500 kDa), FITC-dextrans were progressively excluded from chromatin regions, accumulating in and thereby outlining an apparently extended interchromatin space. Anionic FITC-dextrans (500 kDa) showed complete exclusion from labeled chromatin regions, while the positively charged FITC-poly-L-lysine was to some extent present within the chromatin regions. Moreover, the FITC-poly-L-lysine preferentially localized at the nuclear periphery. We also found a size-dependent exclusion of FITC-dextrans from nucleoli regions, while the FITC-poly-L-lysine accumulated in the nucleoli. Thus, the distinct and restricted nuclear accessibility for macromolecules is dependent on molecule size and electrical charge.

Animals↗
PubMed2003

Mutations at the mouse ichthyosis locus are within the lamin B receptor gene: a single gene model for human Pelger-Huët anomaly.

The nature of the wild-type gene product at the mouse ichthyosis (ic) locus has been of great interest because mutations at this locus cause marked abnormalities in nuclear heterochromatin, similar to those observed in Pelger-Huët anomaly (PHA). We recently found that human PHA is caused by mutations in the gene (LBR) encoding lamin B receptor, an evolutionarily conserved inner nuclear membrane protein involved in nuclear assembly and chromatin binding. Mice homozygous for deleterious alleles at the ichthyosis (ic) locus present with a blood phenotype similar to PHA, and develop other phenotypic abnormalities, including alopecia, variable expression of syndactyly and hydrocephalus. The ic locus on mouse chromosome 1 shares conserved synteny with the chromosomal location of the human LBR locus on human chromosome 1. In this study, we identified one nonsense (815ins) and two frameshift mutations (1088insCC and 1884insGGAA) within the Lbr gene of mice homozygous for either of three independent mutations (ic, ic(J) and ic(4J), respectively) at the ichthyosis locus. These allelic mutations are predicted to result in truncated or severely impaired LBR protein. Our studies of mice homozygous for the ic(J) mutation revealed a complete loss of LBR protein as shown by immunofluorescence microscopy and immunoblotting. The findings provide the molecular basis for the heterochromatin clumping and other distinct phenotypes caused by ic mutations. These spontaneous Lbr mutations confirm the molecular basis of human PHA and provide a small animal model for determination of the precise function of LBR in normal and pathological states.

Animals↗
PubMed2003

Molecular architecture of intermediate filaments.

Together with microtubules and actin microfilaments, approximately 11 nm wide intermediate filaments (IFs) constitute the integrated, dynamic filament network present in the cytoplasm of metazoan cells. This network is critically involved in division, motility and other cellular processes. While the structures of microtubules and microfilaments are known in atomic detail, IF architecture is presently much less understood. The elementary 'building block' of IFs is a highly elongated, rod-like dimer based on an alpha-helical coiled-coil structure. Assembly of cytoplasmic IF proteins, such as vimentin, begins with a lateral association of dimers into tetramers and gradually into the so-called unit-length filaments (ULFs). Subsequently ULFs start to anneal longitudinally, ultimately yielding mature IFs after a compaction step. For nuclear lamins, however, assembly starts with a head-to-tail association of dimers. Recently, X-ray crystallographic data were obtained for several fragments of the vimentin dimer. Based on the dimer structure, molecular models of the tetramer and the entire filament are now a possibility.

Amino Acid Sequence↗
PubMed2003

Acute effects of desmin mutations on cytoskeletal and cellular integrity in cardiac myocytes.

Mutations in desmin have been associated with a subset of human myopathies. Symptoms typically appear in the second to third decades of life, but in the most severe cases can manifest themselves earlier. How desmin mutations lead to aberrant muscle function, however, remains poorly defined. We created a series of four mutations in rat desmin and tested their in vitro filament assembly properties. RDM-G, a chimera between desmin and green fluorescent protein, formed protofilament-like structures in vitro. RDM-1 and RDM-2 blocked in vitro assembly at the unit-length filament stage, while RDM-3 had more subtle effects on assembly. When expressed in cultured rat neonatal cardiac myocytes via adenovirus infection, these mutant proteins disrupted the endogenous desmin filament to an extent that correlated with their defects in in vitro assembly properties. Disruption of the desmin network by RDM-1 was also associated with disruption of plectin, myosin, and alpha-actinin organization in a significant percentage of infected cells. In contrast, expression of RDM-2, which is similar to previously characterized human mutant desmins, took longer to disrupt desmin and plectin organization and had no significant effect on myosin or alpha-actinin organization over the 5-day time course of our studies. RDM-3 had the mildest effect on in vitro assembly and no discernable effect on either desmin, plectin, myosin, or alpha-actinin organization in vivo. These results indicate that mutations in desmin have both direct and indirect effects on the cytoarchitecture of cardiac myocytes.

Adenoviridae↗
PubMed2003

Functional complexity of intermediate filament cytoskeletons: from structure to assembly to gene ablation.

The cell biology of intermediate filament (IF) proteins and their filaments is complicated by the fact that the members of the gene family, which in humans amount to at least 65, are differentially expressed in very complex patterns during embryonic development. Thus, different tissues and cells express entirely different sets and amounts of IF proteins, the only exception being the nuclear B-type lamins, which are found in every cell. Moreover, in the course of evolution the individual members of this family have, within one species, diverged so much from each other with regard to sequence and thus molecular properties that it is hard to envision a unifying kind of function for them. The known epidermolytic diseases, caused by single point mutations in keratins, have been used as an argument for a role of IFs in mechanical "stress resistance," something one would not have easily ascribed to the beaded chain filaments, a special type of IF in the eye lens, or to nuclear lamins. Therefore, the power of plastic dish cell biology may be limited in revealing functional clues for these structural elements, and it may therefore be of interest to go to the extreme ends of the life sciences, i.e., from the molecular properties of individual molecules including their structure at the atomic level to targeted inactivation of their genes in living animals, mouse, and worm to define their role more precisely in metazoan cell physiology.

Animals↗
PubMed2003

Nestin promotes the phosphorylation-dependent disassembly of vimentin intermediate filaments during mitosis.

The expression of the intermediate filament (IF) protein nestin is closely associated with rapidly proliferating progenitor cells during neurogenesis and myogenesis, but little is known about its function. In this study, we examine the effects of nestin expression on the assembly state of vimentin IFs in nestin-free cells. Nestin is introduced by transient transfection and is positively correlated with the disassembly of vimentin IFs into nonfilamentous aggregates or particles in mitotic but not interphase cells. This nestin-mediated disassembly of IFs is dependent on the phosphorylation of vimentin by the maturation/M-phase-promoting factor at ser-55 in the amino-terminal head domain. In addition, the disassembly of vimentin IFs during mitosis appears to be a unique feature of nestin-expressing cell types. Furthermore, when the expression of nestin is downregulated by the nestin-specific small interfering RNA in nestin-expressing cells, vimentin IFs remain assembled throughout all stages of mitosis. Previous studies suggest that nonfilamentous vimentin particles are IF precursors and can be transported rapidly between different cytoplasmic compartments along microtubule tracks. On the basis of these observations, we speculate that nestin may play a role in the trafficking and distribution of IF proteins and potentially other cellular factors to daughter cells during progenitor cell division.

Animals↗
PubMed2002

Mutations in the gene encoding the lamin B receptor produce an altered nuclear morphology in granulocytes (Pelger-Huët anomaly).

Pelger-Huët anomaly (PHA; OMIM *169400) is an autosomal dominant disorder characterized by abnormal nuclear shape and chromatin organization in blood granulocytes. Affected individuals show hypolobulated neutrophil nuclei with coarse chromatin. Presumed homozygous individuals have ovoid neutrophil nuclei, as well as varying degrees of developmental delay, epilepsy and skeletal abnormalities. Homozygous offspring in an extinct rabbit lineage showed severe chondrodystrophy, developmental anomalies and increased pre- and postnatal mortality. Here we show, by carrying out a genome-wide linkage scan, that PHA is linked to chromosome 1q41-43. We identified four splice-site, two frameshift and two nonsense mutations in LBR, encoding the lamin B receptor. The lamin B receptor (LBR), a member of the sterol reductase family, is evolutionarily conserved and integral to the inner nuclear membrane; it targets heterochromatin and lamins to the nuclear membrane. Lymphoblastoid cells from heterozygous individuals affected with PHA show reduced expression of the lamin B receptor, and cells homozygous with respect to PHA contain only trace amounts of it. We found that expression of the lamin B receptor affects neutrophil nuclear shape and chromatin distribution in a dose-dependent manner. Our findings have implications for understanding nuclear envelope-heterochromatin interactions, the pathogenesis of Pelger-like conditions in leukemia, infection and toxic drug reactions, and the evolution of neutrophil nuclear shape.

Cell Line↗
PubMed2002

Conserved segments 1A and 2B of the intermediate filament dimer: their atomic structures and role in filament assembly.

Intermediate filaments (IFs) are key components of the cytoskeleton in higher eukaryotic cells. The elementary IF 'building block' is an elongated coiled-coil dimer consisting of four consecutive alpha-helical segments. The segments 1A and 2B include highly conserved sequences and are critically involved in IF assembly. Based on the crystal structures of three human vimentin fragments at 1.4-2.3 A resolution (PDB entries 1gk4, 1gk6 and 1gk7), we have established the molecular organization of these two segments. The fragment corresponding to segment 1A forms a single, amphipatic alpha-helix, which is compatible with a coiled-coil geometry. While this segment might yield a coiled coil within an isolated dimer, monomeric 1A helices are likely to play a role in specific dimer-dimer interactions during IF assembly. The 2B segment reveals a double-stranded coiled coil, which unwinds near residue Phe351 to accommodate a 'stutter'. A fragment containing the last seven heptads of 2B interferes heavily with IF assembly and also transforms mature vimentin filaments into a new kind of structure. These results provide the first insight into the architecture and functioning of IFs at the atomic level.

Amino Acid Sequence↗
PubMed2001

Fully functional, naturally occurring and C-terminally truncated variant human immunodeficiency virus (HIV) Vif does not bind to HIV Gag but influences intermediate filament structure.

A variant human immunodeficiency virus type 1 (HIV-1) vif gene, vifA45-2, which encodes a protein lacking 19 amino acids at the C terminus but which is fully functional in supporting HIV replication in non-permissive cells has been described previously. By employing newly generated anti-VifA45 serum, further properties of VifA45 and its full-length counterpart, VifA45open, in comparison to Vif from HIV strain BH10 are reported in permissive HeLa and COS-7 cells. The results obtained using confocal microscopic localization studies and in vitro binding assays do not support a requirement for the direct interaction of HIV Gag with Vif. Furthermore and in contrast to previous conclusions, detergent solubility analyses do not demonstrate a role for the C terminus of Vif in mediating localization to the fraction containing cellular membrane proteins. Localization of Vif from HIV strain BH10 to perinuclear aggregates in a small fraction (about 10%) of transfected HeLa cells has been previously reported. The intermediate filament protein vimentin colocalizes to these structures. In contrast, VifA45 and VifA45open form perinuclear aggregates in nearly all transfected HeLa cells; vimentin as well as the cytoskeletal-bridging protein plectin, but not the microtubular protein tubulin, become relocalized to these structures. Interestingly, in COS-7 cells, all of the functional Vif proteins tested (Vif from strain BH10, VifA45 and VifA45open) predominantly localize in the cytoplasm but still induce dramatic aggregation of vimentin and plectin, i.e. in these cells the respective Vif proteins are influencing intermediate filament structure in the absence of colocalization.

Animals↗
PubMedDate not supplied

Characterization of early assembly intermediates of recombinant human keratins.

The intermediate filaments (IFs) form major structural elements of the cytoskeleton. In vitro analyses of these fibrous proteins reveal very different assembly properties for the nuclear and cytoplasmic IF proteins. However, keratins in particular, the largest and most heterogenous group of cytoplasmic IF proteins, have been difficult to analyze due to their rapid assembly dynamics under the near-physiological conditions used for other IF proteins. We show here that keratins, like other cytoplasmic IF proteins, go through a stage of assembling into full-width soluble complexes, i.e., "unit-length filaments" (ULFs). In contrast to other IF proteins, however, longitudinal annealing of keratin ULFs into long filaments quasi-coincides with their formation. In vitro assembly of IF proteins into filaments can be initiated by an increase of the ionic strength and/or lowering of the pH of the assembly buffer. We now document that 23-mer peptides from the head domains of various IF proteins can induce filament formation even under conditions of low salt and high pH. This suggests that the "heads" are involved in the formation and longitudinal association of the ULFs. Using a Tris-buffering protocol that causes formation of soluble oligomers at pH 9, the epidermal keratins K5/14 form less regular filaments and less efficiently than the simple epithelial keratins K8/18. In sodium phosphate buffers (pH 7.5), however, K5/14 were able to form long partially unraveled filaments which compacted into extended, regular filaments upon addition of 20 mM KCl. Applying the same assembly regimen to mutant K14 R125H demonstrated that mutations causing a severe disease phenotype and morphological filament abnormalities can form long, regular filaments with surprising efficiency in vitro.

Base Sequence↗
PubMedDate not supplied

Investigation of nuclear architecture with a domain-presenting expression system.

We have investigated the topogenic properties of the nucleus by ectopic expression of chimeric proteins consisting of a NLS-modified cytoplasmic filament-forming protein, Xenopus laevis vimentin, and domains of inner nuclear membrane proteins. Whereas the "carrier" without cargo, the NLS-vimentin alone, is deposited in a few nuclear body-type structures (J.M. Bridger, H. Herrmann, C. Münkel, P. Lichter, J. Cell Sci., 111, 1241-1253), the distribution is entirely changed upon coupling with the evolutionarily conserved domain of the lamin B tail, the entire lamin B tail, the amino-terminal nucleoplasmic segment of the lamin B receptor (LBR), and the LEM domain of emerin, respectively. Remarkably, every individual chimeric protein exhibits a completely different distribution. Therefore, we assume that the chimeric parts are specifically recognized by factors engaged in nucleus-specific topogenesis. Thus, the conserved domain of the lamin B tail results in the formation of many small accumulations spread all over the nucleus. The chimera with the complete lamin B tail is deposited in short fibrillar aggregates within the nucleus. It does not mediate the integration of the chimeric protein into the nuclear membrane in cultured cells, indicating that the lamin tail alone is not sufficient to direct the integration of a protein into the lamina in vivo. In contrast, in the nuclear assembly system of Xenopus laevis the recombinant NLS-vimentin-lamin tail protein is concentrated at the nuclear membrane. The LBR chimera is arranged in a "beaded-chain"-type fashion, quite different from the more random deposition of NLS-vimentin alone. To our surprise, the LEM domain of emerin induces the retention of most of the chimeric proteins within the cytoplasm. Hence, it appears to be engaged in a strong cytoplasmic interaction that overrides the nuclear localization signal. Finally, the lamin chimera with the conserved part of the lamin B tail is shown to recruit LBR to the nuclear vimentin bodies and, vice versa, the LBR chimera attracts lamin B in transfected cells, thereby demonstrating their bona fide interaction in vivo.

Animals↗