Characterization of mitotically phosphorylated caldesmon.

Mitosis-specific phosphorylation by cdc2 kinase causes nonmuscle caldesmon to dissociate from microfilaments (Yamashiro, S., Yamakita, Y., Ishikawa, R., and Matsumura, F. (1990) Nature 344, 675-678; Yamashiro, S., Yamakita, Y., Hosoya, H., and Matsumura, F. (1991) Nature 349, 169-172). To explore the function of mitosis-specific phosphorylation of caldesmon, in vivo- and in vitro-phosphorylated caldesmons have been characterized. We have found that both in vivo and in vitro phosphorylation of caldesmon causes similar changes in the properties, including reduction in actin, calmodulin, and myosin binding of caldesmon, and a decrease in the inhibition of actomyosin ATPase by caldesmon. Rat non-muscle caldesmon is phosphorylated in vitro up to a ratio of 7 mol/mol of protein. Actin-binding constants of both a high affinity (K a = 1.2 x 10(7) M-1) and a low affinity (K a = 1 x 10(6) M-1) site of unphosphorylated caldesmon are reduced to less than 10(5) M-1 with 5 mol of phosphate incorporation per mol of protein. Actin-bound caldesmon can be phosphorylated by cdc2 kinase, which results in the dissociation of caldesmon from F-actin. Caldesmon has a second myosin-binding site in the C terminus, in addition to the N terminus myosin-binding domain previously reported, because the bacterially expressed C terminus of caldesmon shows binding to myosin. Phosphorylation of the C-terminal fragments decreases their myosin-binding affinity as observed with intact caldesmon. These results suggest that caldesmon loses most of its in vitro functions during mitosis as a result of phosphorylation, which may be required for the reorganization of microfilaments during mitosis.     

Multiple epitope tagging of expressed proteins for enhanced detection

Three FLAG epitopes have been incorporated into the mammalian expression vector pCMV-5 to create a transient expression vector, p3XFLAG-CMV-7. The vector was designed to express FLAG fusion proteins that can be detected at tenfold lower expression levels than the current FLAG fusion protein expression system. The usefulness of this expression and detection system was demonstrated by expression of bacterial alkaline phosphatase in COS-7 cells. In addition, 3XFLAG bacterial alkaline phosphatase was expressed in Escherichia coli, purified on anti-FLAG M2 affinity gel, and detection of 500 pg of purified protein by Western blot analysis is demonstrated.     

Transmission analysis of mitotically unstable B chromosomes in Locusta migratoria.

Seventeen controlled crosses in which the mitotically unstable B chromosome of Locusta migratoria was carried by one parent only have provided evidence that B chromosomes are significantly eliminated during sexual transmission in males, at a mean rate that almost counteracts the premeiotic accumulation derived from mitotic instability during germ line development. On the other hand, B chromosomes are significantly accumulated in females, presumably by their preferential migration to the secondary oocyte during the first meiotic division. These results substantially change the current knowledge about this B chromosome system, because the main B accumulation occurs in females and not in males, as was hitherto thought. Furthermore, this case shows that the maintenance of a single B system in natural populations may be the result of many different forces and mechanisms acting for and against B chromosomes.     

Nuclear localization of enhanced green fluorescent protein homomultimers

The green fluorescent protein (GFP) and its variants are used in many studies to determine the subcellular localization of other proteins by analyzing fusion proteins. The main problem for nuclear localization studies is the fact that, to some extent, GFP translocates to the nucleus on its own. Because the nuclear import could be due to unspecific diffusion of the relatively small GFP through the nuclear pores, we analyzed the localization of multimers of a GFP variant, the enhanced GFP (EGFP). By detecting the fluorescence of the expressed proteins in gels after nonreducing SDS-PAGE, we demonstrate the integrity of the expressed proteins. Nevertheless, even EGFP homotetramers and homohexamers are found in the nuclei of the five analyzed mammalian cell lines. The use of fusion constructs of small proteins with multimeric EGFP alone, therefore, is not adequate to prove nuclear import processes. Fusion to tetrameric EGFP in combination with a careful quantification of the fluorescence intensities in the nucleus and cytoplasm might be sufficient in many cases to identify a significant difference between the fusion protein and tetrameric EGFP alone to deduce a nuclear localization signal.     

Nuclear visions enhanced: chromatin structure, organization and dynamics

The EMBO Workshop on 'Chromatin Structure, Organization and Dynamics' took place in April 2011 in Prague, Czech Republic. Scientists from the life sciences, chemistry and biophysics presented their latest data on the generation of three-dimensional and, eventually, four-dimensional models of the genome, working to correlate changes in the organization of chromatin with the functional state of the genome.     

Element concentration changes in mitotically active and postmitotic enterocytes. An x-ray microanalysis study

Unfixed freeze-dried and uncoated tissue sections of the mouse duodenum were suspended across a hole in a carbon planchet and analyzed in a scanning electron microscope fitted with energy-dispersive x-ray analytical equipment. Computer analysis of the x-ray spectra allowed elemental microanalysis of the nucleus, cytoplasm, and late anaphase-early telophase chromatin regions in the cryptal and villus enterocytes. Elemental concentrations (mmol/kg dry wt) were measured for Na, Mg, P, S, Cl, K, and Ca. None of the elements were compartmentalized preferentially in either the nucleus or the cytoplasm of interphase enterocytes of crypts or in postmitotic enterocytes of villi. In contrast, Ca, S, and Cl are detectable in significantly higher concentrations in mitotic chromatin of dividing enterocytes of the crypt as compared to surrounding mitotic cytoplasm, but Na, Mg, and P are in lower concentrations in the mitotic chromatin as compared to mitotic cytoplasm. Interphase enterocytes of crypts have higher concentrations of Mg, P, and K, and lower concentrations of Na than do postmitotic enterocytes of villi.     

Multiple tandem epitope tagging for enhanced detection of protein expressed in mammalian cells

Epitope tagging is a valuable tool for quick detection, isolation, and analysis of protein-protein interaction, without prior knowledge of the target protein. The FLAG epitope tag, one of the most widely used tags, is an eight amino acid peptide that can be detected by anti-FLAG monoclonal antibody. In the present study, we have examined the detection sensitivity of a protein fused to three tandem FLAG epitopes by Western blot analysis, immunoprecipitation, and immunohistochemical analysis using anti-FLAG® M2 antibody. We find that the triple FLAG epitope significantly enhances the sensitivity of detection of fusion protein expressed in mammalian cells.     

Phosphorylation of vimentin in mitotically selected cells. In vitro cyclic AMP-independent kinase and calcium-stimulated phosphatase activities

The phosphorylation of the intermediate filament protein vimentin was examined under in vitro conditions. Cell cytosol and Triton-insoluble cytoskeleton preparations from nonmitotic and mitotically selected mouse L-929 cells exhibited vimentin kinase activity that is apparently cAMP and Ca2+ independent. The level of vimentin kinase activity was greater in preparations from mitotically selected cells than nonmitotic cells. Addition of Ca2+ to mitotic cytosol decreased net vimentin phosphorylation. Dephosphorylation experiments indicated that there is phosphatase activity in these preparations which is stimulated by addition of Ca2+. Fractionation of cytosol from nonmitotic cells on DEAE-Sephacel and phosphocellulose revealed a single major vimentin kinase activity (peak I). Fractionation of cytosol from mitotically selected cells yielded a similar activity (peak I) and an additional vimentin kinase activity (peak II) that was not found in nonmitotic preparations. Based on substrate specificity and lack of inhibition to characteristic inhibitors, the semipurified peak I and II vimentin kinase activities appear to be cAMP-independent enzymes that are distinct from casein kinases I and II. Phosphopeptide mapping studies indicated that both peak I and peak II vimentin kinases phosphorylate tryptic peptides in the NH2-terminal region of vimentin that are phosphorylated in intact cells. Electron microscopic examination of reconstituted vimentin filaments phosphorylated with both semipurified kinases indicated that phosphorylation induced filament disassembly. These experiments indicate that the increased phosphorylation of vimentin during mitosis may be catalyzed by a discrete cAMP-independent protein kinase. In addition, preparations from mitotic cells exhibited a Ca2+-stimulated phosphatase activity, suggesting that Ca2+ may play a regulatory role in vimentin dephosphorylation during mitosis.     

Severing of stable microtubules by a mitotically activated protein in Xenopus egg extracts.

Eukaryotic cells disassemble and reorganize their cytoskeleton during the cell cycle and in response to environmental cues. Disassembly of the actin cytoskeleton is aided by proteins that sever filamentous actin, but microtubule-severing proteins thus far have not been identified. Here, we describe an activity in extracts from Xenopus eggs that rapidly severs stable microtubules along their length. Severing is elicited by a protein(s) whose activity is greatly stimulated during mitosis through a posttranslational mechanism. The microtubule-severing factor may be involved in disassembling the interphase microtubule network prior to constructing the mitotic spindle.     

Nuclear magnetic resonance structure-based epitope mapping and modulation of dust mite group 13 allergen as a hypoallergen

IgE-mediated allergic response involves cross-linking of IgE bound on mast cells by specific surface epitopes of allergens. Structural studies on IgE epitopes of allergens are essential in understanding the characteristics of an allergen and for development of specific allergen immunotherapy. We have determined the structure of a group 13 dust mite allergen from Dermatophagoides farinae, Der f 13, using nuclear magnetic resonance. Sequence comparison of Der f 13 with homologous human fatty acid-binding proteins revealed unique surface charged residues on Der f 13 that may be involved in IgE binding and allergenicity. Site-directed mutagenesis and IgE binding assays have confirmed four surface charged residues on opposite sides of the protein that are involved in IgE binding. A triple mutant of Der f 13 (E41A_K63A_K91A) has been generated and found to have significantly reduced IgE binding and histamine release in skin prick tests on patients allergenic to group 13 dust mite allergens. The triple mutant is also able to induce PBMC proliferation in allergic patients with indices similar to those of wild-type Der f 13 and shift the secretion of cytokines from a Th2 to a Th1 pattern. Mouse IgG serum raised using the triple mutant is capable to block the binding of IgE from allergic patients to wild-type Der f 13, indicating potential for the triple mutant as a hypoallergen for specific immunotherapy. Findings in this study imply the importance of surface charged residues on IgE binding and allergenicity of an allergen, as was also demonstrated in other major allergens studied.     

Mutant conformation of p53. Precise epitope mapping using a filamentous phage epitope library.

Many naturally occurring point mutations in the p53 gene lead to a proportion of the encoded protein molecules adopting a distinct, "mutant" conformation characterized by exposure of a normally cryptic epitope recognized by the monoclonal antibody PAb240. Here the PAb240 epitope is defined using a filamentous phage epitope library. The hexapeptides displayed by the PAb240-binding phage isolated from the library were all highly related and allowed both direct localization of the epitope and prediction of a specific interaction between PAb240 and Xenopus TFIIIA. This study demonstrates for the first time the power of phage epitope libraries in the precise definition of previously unmapped epitopes. Identification of the PAb240 epitope precisely defines a region of the p53 molecule structurally altered by the mutation-induced conformational shift.     

The odd-even effect in mitotically unstable B chromosomes in grasshoppers

The odd-even effect, by which B chromosomes are more detrimental in odd numbers, has been reported in plants and animals. In grasshoppers, there are only a few reports of this effect and all were referred to as traits related to the formation of aberrant meiotic products (AMPs). Here we review the existing information about B chromosome effects on AMPs, chiasma frequency and the number of active nucleolus organizer regions (NORs) per cell. Polysomy for A chromosomes and B chromosomes are two kinds of chromosome polymorphism frequently found in grasshoppers. In some aspects, e.g. meiotic behaviour and mitotic instability leading to individual mosaicism (in the case of mitotically unstable Bs), polysomic As show similar characteristics to B chromosomes. In fact, polysomy is regarded as one of the main mechanisms for B chromosome origin. Here we review some features of meiotic behaviour in known cases of polysomy and mitotically unstable Bs in grasshoppers, in looking for possible causes for the odd-even effect. In all these traits, the odd-even effect was apparent, although its appearance was not universal in any case, with variation among species or populations within the same species. The equational division and lagging of the extra chromosomes, when univalents, could favour the appearance of abnormal meiotic products, and the formation of bivalents, when there are two or more extra chromosomes, inhibits this process. Therefore, the odd-even effect might be a consequence of the concomitant operation of both aspects of extra chromosome meiotic behaviour. The possibility that the odd-even effect might result from an increase in cell stress generated by odd numbers is suggested.     

Clonally diverse CTL response to a dominant viral epitope recognizes potential epitope variants.

RNA viruses undergo rapid sequence variation as the result of error-prone RNA replication mechanisms. When viable mutations arise in RNA regions encoding B or T cell epitopes, mutant viruses that can evade immune detection may be selected. In the carefully studied CTL response to the Gag p11C(C-M) epitope in SIVmac-infected Mamu-A*01(+) rhesus monkeys, it has been shown that CTL recognition of that epitope can occur even in the face of accruing mutations. To explore the underlying mechanism for this breadth of recognition, we have constructed Mamu-A*01 tetramers which discriminate T cells specific for epitope variants. Using these reagents we have defined discrete subsets of p11C(C-M)-specific T cells that cross-react with cells presenting variant peptides. We have found that individual Mamu-A*01(+) monkeys differ functionally in their ability to recognize epitope variants despite consistently strong recognition of the p11C(C-M) epitope. This functional difference is accounted for by the relative number of variant-specific T cells and by differences in the functionally relevant TCR repertoire of the infected monkeys. We have also found that monkeys immunized with DNA vaccine constructs encoding only the wild-type epitope sequence develop p11C(C-M)-specific CTL cross-reactive with variant peptides. Thus, cross-reactive CTL do not merely arise secondary to the emergence and immune presentation of viral CTL escape mutants but rather arise de novo following priming with a dominant epitope peptide sequence. Taken together, our results support the concept that the CTL response to a dominant viral epitope, although highly focused, can be clonally diverse and recognize potential epitope variants.     

Discovering peptide ligands using epitope libraries.

Epitope libraries are large collections of peptides. Each peptide is displayed on the surface of a bacteriophage particle and is encoded by a randomly mutated region of the phage genome, thus associating each unique peptide with the DNA molecule encoding it. Antibodies and other binding proteins are used to select specifically for rare, phage-bearing peptide ligands; sequencing of the corresponding viral DNA will reveal their amino acid sequences. Relatively high-affinity peptides for a variety of peptide- and non-peptide-binding ligates have been affinity-isolated from epitope libraries. This technology has been used to map epitopes on proteins and to find peptide mimics for non-peptide-binding ligates. The current challenge lies in developing epitope library technology so that tight-binding peptide ligands can be detected for a wider variety of ligates, including those that recognize folded proteins. Should this be accomplished, many powerful applications can be envisioned in the areas of drug design and the development of diagnostic markers and vaccines.     

Defined flanking spacers and enhanced proteolysis is essential for eradication of established tumors by an epitope string DNA vaccine

Loss of immunogenic epitopes by tumors has urged the development of vaccines against multiple epitopes. Recombinant DNA technologies have opened the possibility to develop multiepitope vaccines in a relatively rapid and efficient way. We have constructed four naked DNA-based multiepitope vaccines, containing CTL, Th cell, and B cell epitopes of the human papillomavirus type 16. Here we show that gene gun-mediated vaccination with an epitope-based DNA vaccine protects 100% of the vaccinated mice against a lethal tumor challenge. The addition of spacers between the epitopes was crucial for the epitope-induced tumor protection, as the same DNA construct without spacers was significantly less effective and only protected 50% of the mice. When tested for therapeutic potential, only the epitope construct with defined spacers significantly reduced the size of established tumors, but failed to induce tumor regression. Only after targeting the vaccine-encoded protein to the protein degradation pathway by linking it to ubiquitin, the vaccine-induced T cell-mediated eradication of 100% of 7-day established tumors in mice. The finding that defined flanking sequences around epitopes and protein targeting dramatically increased the efficacy of epitope string DNA vaccines against established tumors will be of importance for the further development of multiepitope DNA vaccines toward clinical application.     

Nuclear compartments and gene regulation.

Improvements in fluorescence microscopy have allowed us to explore the three-dimensional organization of the nucleus in ways that were impossible ten years ago, revealing subdomains or compartments within the nucleus defined by their enrichments of subsets of factors. Correlations have been drawn between the silencing of a gene and its proximity to a heterochromatic compartment or to the nuclear periphery. The application of genetics and high-resolution microscopy helps examine the creation, maintenance and impact of these compartments on gene expression.