Dynamics of chromosome compaction during mitosis

We have quantitatively studied the space-time dynamics of mitotic chromosome compaction in cultured amphibian cells. After collecting digital phase-contrast images we have done digital image analysis to study spatial correlations in density. We find a characteristic distance at which the strongest correlations occur, which provides a quantitative measure of the size of patches of dense chromatin during interphase and early prophase. Later in mitosis, this length corresponds to the thickness of prophase and metaphase chromosomes. We find that during interphase strong correlations exist at a few-micrometer length; during prophase this correlation length progressively drops as the chromosomes are compacted. Our data are explained by a model based on assembly of chromatin loops onto already fiberlike interphase chromosomes. To test this model we have microinjected cobalt hexamine trichloride into interphase nuclei and have observed the rapid condensation of the interphase chromatin into thick fibers with a spacing similar to the native-state interphase correlation length determined from our image analysis.     

Blebs and apoptotic bodies are B cell autoantigens

Mounting evidence suggests that systemic lupus erythematosus autoantigens are derived from apoptotic cells. To characterize the potential interactions between apoptotic cells and B cells, the D56R/S76R variant of 3H9, a murine autoantibody that binds to DNA, chromatin, and anionic phospholipids, was compared with DNA4/1, a human anti-DNA autoantibody. Flow cytometry revealed that only D56R/S76R bound to Jurkat cells treated with either of three distinct proapoptotic stimuli, Ab binding was dependent on caspase activity, and immunoreactivity developed subsequent to annexin V binding. Confocal microscopy established a structural basis for the distinct kinetics of binding. D56R/S76R preferentially bound to membrane blebs of apoptotic cells, whereas annexin V binding did not require blebs. Inhibition of ROCK I kinase, an enzyme that stimulates nuclear fragmentation and fragment distribution into blebs, significantly reduced Ab binding. Because members of the collectin and pentraxin families of serum proteins bind to blebs on apoptotic cells and assist in the clearance of cellular remains, our results suggest that Abs to blebs could affect the recognition of apoptotic cells by cells of the innate immune system and thus modify tolerance to nuclear Ags.     

Protective immunity induced by irradiated third-stage larvae of the filaria Acanthocheilonema viteae is directed against challenge third-stage larvae before molting

Jirds (Meriones unguiculatus) were vaccinated with irradiated L3 third-stage larvae (L3) of Acanthocheilonema viteae, and the time required for killing of the challenge L3 was determined. The number of parasites recovered from vaccinated jirds was reduced to about 10% of the control values on the second day after challenge infection and later on. Histological studies revealed an eosinophil-rich infiltrate containing macrophages, neutrophils, and mast cells in the vicinity of the L3 on day 2 after challenge and destruction of the worms by day 4 after challenge. Ultrastructural studies confirmed these data and showed that eosinophils, macrophages, and mast cells were close to the L3 on day 2 after challenge. Flattening of the eosinophils onto the surface of the worms, degranulation of electron-dense material, and rupture of the L3 surface was observed on day 4 after challenge, followed by invasion of the inner of the worms by phagocytic cells. These data show that immune attack against the challenge L3 in vaccinated jirds is initiated between the first and the second day after challenge and that killing occurs around the fourth day after challenge, before the worms undergo their first molt.     

Use of frozen-hydrated axonemes to assess imaging parameters and resolution limits in cryoelectron tomography

Using a 400-kV cryoelectron microscope, we have obtained tomographic reconstructions of frozen-hydrated sea urchin axonemes with 8-10-nm resolution, as assessed by detection of characteristic components including doublet microtubules, radial spokes, central sheath projections, and outer dynein arms. We did not detect the inner dynein arms or the microtubule lattice. The 1/(8 nm) and 1/(16 nm) layer lines are consistently present in power spectra of both projection images and tomographic reconstructions. Strength and detection of the layer lines are dependent upon total electron dose and defocus. Both layer lines are surprisingly resistant to electron doses of up to 11000 electrons/nm(2). We present a summary of resolution considerations in cryoelectron tomography and conclude that the fundamental limitation is the total electron dose required for statistical significance. The electron dose can be fractionated among the numerous angular views in a tomographic data set, but there is an unavoidable fourth-power dependence of total dose on target resolution. Since higher-resolution features are more beam-sensitive, this dose requirement places an ultimate limit on the resolution of individual tomographic reconstructions. Instrumental and computational strategies to circumvent this limitation are discussed.     

Cardiac mechanoenergetics in silico

The aim of this thesis is to investigate the link between biochemical intracellular processes and mechanical contraction of the cardiac muscle. First, the regulation of intracellular energy fluxes between mitochondria and myofibrils is studied. It is shown, that the experimentally observed metabolic stability of the cardiac muscle is reproducible by a simple feedback regulation mechanism, i.e., ATP consumption in myofibrils and ATP production in mitochondria are balanced by the changes of the high energy phosphate concentrations. Second, an important property of energy transformation from biochemical form to mechanical work in the cardiac muscle, the linear relationship between the oxygen consumption and the stress-strain area, is replicated by a cross-bridge model. Third, by using the developed cross-bridge model, the correlation between ejection fraction of the left ventricle and heterogeneity of sarcomere strain, developed stress and ATP consumption in the left ventricular wall is established. Fourth, an experimentally observed linear relationship between oxygen consumption and the pressure-volume area can be predicted theoretically from a linear relationship between the oxygen consumption and the stress-strain area. Summing up, it is shown how the macrovariables of a cardiac muscle are interwoven with intracellular physiological processes into a whole.     

Focal adhesion protein FAP52 self-associates through a sequence conserved among the members of the PCH family proteins

FAP52 is a recently described focal adhesion-associated protein. It is a member of an emerging PCH (pombe Cdc15 homology) family of proteins characterized by a common domain organization and involvement in actin cytoskeleton organization, cytokinesis, and vesicular trafficking. Using gel filtration, surface plasmon resonance, and native polyacrylamide gel electrophoresis analysis, combined with chemical cross-linking of both native and recombinant protein, we show that FAP52 self-associates in vitro and suggest that it occurs predominantly as a trimer also in vivo. Analysis of the various domains of FAP52 by surface plasmon resonance showed that the highly alpha-helical region in the N-terminal half of the protein provides the self-association interface. Overexpression of the oligomerization domain in cultured cells was accompanied by major alterations in cellular morphology, actin organization, and the structure of focal adhesions, suggesting that an orderly coming together of FAP52 molecules is crucial for a proper actin filament organization and cytoskeletal structure. Comparison of the primary structures shows that all of the members of the PCH family have, in their N-terminal halves, a similar, highly alpha-helical region, suggesting that they all have a capacity to self-associate.     

Arginine residues in domain V have a central role for bacteria-binding activity of macrophage scavenger receptor MARCO

MARCO is a bacteria-binding macrophage-specific scavenger receptor that plays a role in innate immune response. MARCO has short intracellular and transmembrane domains, as well as a large extracellular domain composed of a spacer domain, a long collagenous domain, and a C-terminal scavenger receptor cysteine-rich domain (SRCR), domain V. As yet, no specific function has been assigned to the SRCR domain of scavenger receptors. In the present study, we generated several human and mouse MARCO variants with deletions or single amino acid substitutions and localized the primary bacteria-binding region to domain V. Furthermore, analysis of the MARCO variants containing only portions of domain V demonstrated a crucial role for an arginine-rich segment for this function. More precisely, the motif RXR was identified as an essential element for high-affinity bacterial binding. The results indicate that the binding properties of MARCO differ from those of the other class A scavenger receptors, SR-A and SRCL, whose ligand-binding function has been localized to the collagenous domain.     

Mammalian p55CDC Mediates Association of the Spindle Checkpoint Protein Mad2 with the Cyclosome/Anaphase-promoting Complex, and is Involved in Regulating Anaphase Onset and Late Mitotic Events

We have investigated the function of p55CDC, a mammalian protein related to Cdc20 and Hct1/Cdh1 in Saccharomyces cerevisiae, and Fizzy and Fizzy-related in Drosophila. Immunofluorescence studies and expression of a p55CDC-GFP chimera demonstrate that p55CDC is concentrated at the kinetochores in M phase cells from late prophase to telophase. Some p55CDC is also associated with the spindle microtubules and spindle poles, and some is diffuse in the cytoplasm. At anaphase, the concentration of p55CDC at the kinetochores gradually diminishes, and is gone by late telophase. In extracts prepared from M phase, but not from interphase HeLa cells, p55CDC coimmunoprecipitates with three important elements of the M phase checkpoint machinery: Cdc27, Cdc16, and Mad2. p55CDC is required for binding Mad2 with the Cdc27 and Cdc16. Thus, it is likely that p55CDC mediates the association of Mad2 with the cyclosome/anaphase-promoting complex. Microinjection of anti-p55CDC antibody into mitotic mammalian cells induces arrest or delay at metaphase, and impairs progression of late mitotic events. These studies suggest that mammalian p55CDC may be part of a regulatory and targeting complex for the anaphase-promoting complex.     

Construction of a xylanase A variant capable of polymerization

The aim of our work is to furnish enzymes with polymerization ability by creating fusion constructs with the polymerizable protein, flagellin, the main component of bacterial flagellar filaments. The D3 domain of flagellin, exposed on the surface of flagellar filaments, is formed by the hypervariable central portion of the polypeptide chain. D3 is not essential for filament formation. The concept in this project is to replace the D3 domain with suitable monomeric enzymes without adversely affecting polymerization ability, and to assemble these chimeric flagellins into tubular nanostructures. To test the feasibility of this approach, xylanase A (XynA) from B. subtilis was chosen as a model enzyme for insertion into the central part of flagellin. With the help of genetic engineering, a fusion construct was created in which the D3 domain was replaced by XynA. The flagellin-XynA chimera exhibited catalytic activity as well as polymerization ability. These results demonstrate that polymerization ability can be introduced into various proteins, and building blocks for rationally designed assembly of filamentous nanostructures can be created.     

The Psb27 assembly factor binds to the CP43 complex of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803

We have investigated the location of the Psb27 protein and its role in photosystem (PS) II biogenesis in the cyanobacterium Synechocystis sp. PCC 6803. Native gel electrophoresis revealed that Psb27 was present mainly in monomeric PSII core complexes but also in smaller amounts in dimeric PSII core complexes, in large PSII supercomplexes, and in the unassembled protein fraction. We conclude from analysis of assembly mutants and isolated histidine-tagged PSII subcomplexes that Psb27 associates with the "unassembled" CP43 complex, as well as with larger complexes containing CP43, possibly in the vicinity of the large lumenal loop connecting transmembrane helices 5 and 6 of CP43. A functional role for Psb27 in the biogenesis of CP43 is supported by the decreased accumulation and enhanced fragmentation of unassembled CP43 after inactivation of the psb27 gene in a mutant lacking CP47. Unexpectedly, in strains unable to assemble PSII, a small amount of Psb27 comigrated with monomeric and trimeric PSI complexes upon native gel electrophoresis, and Psb27 could be copurified with histidine-tagged PSI isolated from the wild type. Yeast two-hybrid assays suggested an interaction of Psb27 with the PsaB protein of PSI. Pull-down experiments also supported an interaction between CP43 and PSI. Deletion of psb27 did not have drastic effects on PSII assembly and repair but did compromise short-term acclimation to high light. The tentative interaction of Psb27 and CP43 with PSI raises the possibility that PSI might play a previously unrecognized role in the biogenesis/repair of PSII.