Changes in purine specificity in tandem GAF chimeras from cyanobacterial cyaB1 adenylate cyclase and rat phosphodiesterase 2

The C-terminal catalytic domains of the 11 mammalian phosphodiesterase families (PDEs) are important drug targets. Five of the 11 PDE families contain less well-characterized N-terminal GAF domains. cGMP is the ligand for the GAF domains in PDEs 2, 5, 6 and 11, and cAMP is the ligand for PDE10. Structurally related tandem GAF domains signalling via cAMP are present in the cyanobacterial adenylate cyclases cyaB1 and cyaB2. Because current high-resolution crystal structures of the tandem GAF domains of PDE2 and cyaB2 do not reveal how cNMP specificity is encoded, we generated chimeras between the tandem GAF domains of cyaB1 and PDE2. Both bind the ligand in the GAF B subdomains. Segmental replacements in the highly divergent beta1-beta3 region of the GAF B subdomain of cyaB1 by the corresponding PDE2 regions switched signalling from cAMP to cGMP. Using 10 chimeric constructs, we demonstrated that, for this switch in purine specificity, only 11% of the sequence of the cyanobacterial GAF B needs to be replaced by PDE2 sequences. We were unable, however, to switch the purine specificity of the PDE2 tandem GAF domain from cGMP to cAMP in reverse constructs, i.e. by replacement of PDE2 segments with those from the cyaB1 GAF tandem domain. The data provide a novel view on the structure-function relationships underlying the purine specificity of cNMP-binding GAF domains and indicate that, as potential drug targets, they must be characterized structurally and biochemically one by one.     

Macroscopic and Microscopic Morphological Features of Stromatolites Related To Activity of Eukaryote-Dominated Biofilms in an Acid Mine Drainage Environment: Biosignatures and Understanding Preservation of Stromatolites as Trace Fossils

Euglena-, diatom-, and algae-dominated biofilms are the principal producers of iron-rich biolaminates that result in biosedimentary structures, or stromatolites, in an acid mine drainage (AMD) environment in Indiana. These structures are considered trace fossils because they are produced by organism-sediment interactions and record physicochemical conditions of the environment. Our purpose was to link the biofilm types to specific micro- and micromorphological features and the physicochemical conditions under which they were formed. Analyses revealed that Euglena-dominated biofilm produced thin, porous microlaminae by trapping, binding, and relocating AMD precipitates as the biofilm kept pace with chemical sedimentation. More massive microlaminae were produced by high rates of chemical sedimentation brought on by increased discharge and dilution of acidity. Diatom- and algae-dominated biofilms produced thick, mm–cm-scale, porous, spongelike micro- to macrolaminae through oxygenic photosynthesis and/or metal uptake in extracellular polymeric substances, which promoted mineral precipitation on cell walls to create a rigid, porous structure. The variations in biolaminate textures within the stromatolites record seasonal changes in the microbial populations and physicochemical conditions of the AMD environment. These iron-rich stromatolites represent trace fossils that record morphological biosignatures of eukaryote-dominated microbial biofilms and may serve as appropriate proxies in the search for similar evidence of eukaryotic life in other iron-rich paleoenvironments, such as those on early Earth and Mars.     

Remodelling of the nuclear periphery during muscle cell differentiation in vitro

We have examined the composition and ultrastructure of the nuclear periphery during in vitro myogenesis of the rat myoblast cell line, L6E9. Immunofluorescence labelling and immunoblotting showed that lamins A/C and B were all present in undifferentiated cells, but that they increased significantly before extensive cell fusion had occurred, with lamins A/C increasing proportionately more. Electron microscopic observations were consistent with these results, showing an increase in the prominence of the lamina during differentiation. On the other hand, immunofluorescence labelling suggested that the P1 antigen began to disappear from the nuclear periphery as the cells were fusing, after the increase in lamin quantity, and was no longer detectable in multinucleated cells. Unexpectedly, however, P1 was readily detected in isolated nuclei, whether prepared from myoblast or differentiated cultures, as well as in both myoblast and myotube nuclear matrices. It appears probable, therefore, that the fading of P1 labelling is due to masking of the epitope by a soluble factor recruited to the nuclear periphery as cells differentiate. These data, together with evidence that the genome is substantially rearranged during L6E9 myogenesis [Chaly and Munro, 1996], suggest that L6E9 cells are a useful model system in which to study the interrelationship of nuclear envelope organization, chromatin spatial order, and nuclear function. © 1996 Wiley-Liss, Inc.     

Specific Single or Double Proline Substitutions in the “Spring-loaded” Coiled-Coil Region of the Influenza Hemagglutinin Impair or Abolish Membrane Fusion Activity

We tested the role of the “spring-loaded” conformational change in the fusion mechanism of the influenza hemagglutinin (HA) by assessing the effects of 10 point mutants in the region of high coiled-coil propensity, HA2 54–81. The mutants included proline substitutions at HA2 55, 71, and 80, as well as a double proline substitution at residues 55 and 71. Mutants were expressed in COS or 293T cells and assayed for cell surface expression and structural features as well as for their ability to change conformation and induce fusion at low pH. We found the following: Specific mutations affected the precise carbohydrate structure and folding of the HA trimer. All of the mutants, however, formed trimers that could be expressed at the cell surface in a form that could be proteolytically cleaved from the precursor, HA0, to the fusion-permissive form, HA1-S-S-HA2. All mutants reacted with an antibody against the major antigenic site and bound red blood cells. Seven out of ten mutants displayed a wild-type (wt) or moderately elevated pH dependence for the conformational change. V55P displayed a substantial reduction (~60– 80%) in the initial rate of lipid mixing. The other single mutants displayed efficient fusion with the same pH dependence as wt-HA. The double proline mutant V55P/ S71P displayed no fusion activity despite being well expressed at the cell surface as a proteolytically cleaved trimer that could bind red blood cells and change conformation at low pH. The impairment in fusion for both V55P and V55P/S71P was at the level of outer leaflet lipid mixing. We interpret our results in support of the hypothesis that the spring-loaded conformational change is required for fusion. An alternate model is discussed.     

Mechanisms underlying the impacts of exotic plant invasions

Although the impacts of exotic plant invasions on community structure and ecosystem processes are well appreciated, the pathways or mechanisms that underlie these impacts are poorly understood. Better exploration of these processes is essential to understanding why exotic plants impact only certain systems, and why only some invaders have large impacts. Here, we review over 150 studies to evaluate the mechanisms underlying the impacts of exotic plant invasions on plant and animal community structure, nutrient cycling, hydrology and fire regimes. We find that, while numerous studies have examined the impacts of invasions on plant diversity and composition, less than 5% test whether these effects arise through competition, allelopathy, alteration of ecosystem variables or other processes. Nonetheless, competition was often hypothesized, and nearly all studies competing native and alien plants against each other found strong competitive effects of exotic species. In contrast to studies of the impacts on plant community structure and higher trophic levels, research examining impacts on nitrogen cycling, hydrology and fire regimes is generally highly mechanistic, often motivated by specific invader traits. We encourage future studies that link impacts on community structure to ecosystem processes, and relate the controls over invasibility to the controls over impact.     

The Ran GTPase system in fission yeast affects microtubules and cytokinesis in cells that are competent for nucleocytoplasmic protein transport

Misregulation of the evolutionarily conserved GTPase Ran in fission yeast results in defects in several cellular processes in cells that are competent for nucleocytoplasmic protein transport. These results suggest that transport is neither the only nor the primary Ran-dependent process in living cells. The ability of Ran to independently regulate multiple cellular processes in vivo is demonstrated by showing that (i) eight different transport-competent RanGEF (guanine nucleotide exchange factor) mutants have defects in mitotic spindle formation; (ii) the RanGEF temperature-sensitive mutant pim1-d1 has abnormal actin ring structures at the septum. Overexpression of Imp2p, which specifically destabilizes these structures, restores viability. (iii) Ran-dependent processes differ in their requirements for active Ran in vivo. Microtubule function, cytokinesis, and nuclear envelope structure are the Ran-dependent processes most sensitive to the amount of Ran protein in the cell, whereas nucleocytoplasmic protein transport is the most robust. Therefore, the ability of Ran from Schizosaccharomyces pombe to independently regulate multiple cellular processes may reflect differences in its interactions with the binding proteins that mediate these functions and explain the complex phenotypic consequences of its misregulation in vivo.     

Export of Thermus thermophilus cytoplasmic beta-glycosidase via the E. coli Tat pathway

The Tat pathway is distinct from the Sec machinery given its unusual capacity to export folded proteins, which contain a twin-arginine (RR) signal peptide, across the plasma membrane. The functionality of the Tat pathway has been demonstrated for several Gram-negative and Gram-positive mesophilic bacteria. To assess the specificity of the Tat system, and to analyze the capacity of a mesophilic bacterial Tat system to translocate cytoplasmic proteins from hyperthermophilic bacteria, we fused the Thermus thermophilus beta-glycosidase (Glc) to the twin-arginine signal peptide of the E. coli TorA protein. When expressed in E. coli, the thermophilic RR-Glc chimera was successfully synthesized and efficiently translocated into the periplasm of the wild type strain. In contrast, the beta-glycosidase accumulated within the cytoplasm of all the tat mutants analyzed. The beta-glycosidase synthesized in these strains exhibited thermophilic properties. These results demonstrated, for the first time, the capacity of the E. coli Tat system to export cytoplasmic hyperthermophilic protein, implying an important potential of the Tat system for the production of thermostable enzymes used in bioprocessing applications.