The functions of the actin nucleator Cobl in cellular morphogenesis critically depend on syndapin I

Spatial control of cortical actin nucleation is indispensable for proper establishment and plasticity of cell morphology. Cobl is a novel WH2 domain-based actin nucleator. The cellular coordination of Cobl's nucleation activity, however, has remained elusive. Here, we reveal that Cobl's cellular functions are dependent on syndapin. Cobl/syndapin complexes form in vivo, as demonstrated by colocalization, coimmunoprecipitation and subcellular recruitment studies. In vitro reconstitutions and subcellular fractionations demonstrate that, via its lipid-binding Fer/CIP4 Homology (FCH)-Bin/Amphiphysin/Rvs (F-BAR) domain, syndapin recruits Cobl to membranes. Consistently, syndapin I RNAi impairs cortical localization of Cobl. Further functional studies in neurons show that Cobl and syndapin I work together in dendritic arbor development. Importantly, both proteins are crucial for dendritogenesis. Cobl-mediated functions in neuromorphogenesis critically rely on syndapin I and interestingly also on Arp3. Endogenous Cobl, syndapin I and the Arp2/3 complex activator and syndapin-binding partner N-WASP were present in one complex, as demonstrated by coimmunoprecipitations. Together, these data provide detailed insights into the molecular basis for Cobl-mediated functions and reveal that different actin nucleators are functionally intertwined by syndapin I during neuromorphogenesis.     

Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles

Background

Biolistic transfection is proving an increasingly popular method of incorporating DNA or RNA into cells that are difficult to transfect using traditional methods. The technique routinely uses 'microparticles', which are ~1 μm diameter projectiles, fired into tissues using pressurised gas. These microparticles are efficient at delivering DNA into cells, but cannot efficiently transfect small cells and may cause significant tissue damage, thus limiting their potential usefulness. Here we describe the use of 40 nm diameter projectiles - nanoparticles - in biolistic transfections to determine if they are a suitable alternative to microparticles.

Results

Examination of transfection efficiencies in HEK293 cells, using a range of conditions including different DNA concentrations and different preparation procedures, reveals similar behaviour of microparticles and nanoparticles. The use of nanoparticles, however, resulted in ~30% fewer damaged HEK293 cells following transfection. Biolistic transfection of mouse ear tissue revealed similar depth penetration for the two types of particles, and also showed that < 10% of nuclei were damaged in nanoparticle-transfected samples, compared to > 20% in microparticle-transfected samples. Visualising details of small cellular structures was also considerably enhanced when using nanoparticles.

Conclusions

We conclude that nanoparticles are as efficient for biolistic transfection as microparticles, and are more appropriate for use in small cells, when examining cellular structures and/or where tissue damage is a problem.     

Factors Affecting the Acetylene to 15N2 Conversion Ratio in Root Nodules of Myrica gale L

When nodules of actinorhizal plants are exposed to acetylene, there is often an initial peak rate of acetylene reduction followed by a decline and a partial recovery. Treatment of hydroponically grown Myrica gale L. with water deficiency or dark stress increased the magnitude of the acetylene-induced decline and decreased the extent of the recovery. When N2 fixation was measured with 15N2 in unstressed plants, the ratio of acetylene reduction (peak) to N2 fixation prior to acetylene exposure was 3.73 [plus or minus] 0.14 (mean [plus or minus] SE). This value does not differ significantly (P < 0.05) from the theoretical minimum value of 4.0. In water-stressed plants the conversion ratio for the peak rate was greater (4.32 [plus or minus] 0.10) and in dark-stressed plants it was lower (2.54 [plus or minus] 0.33) than 4.0. The conversion ratio for the recovered rate of acetylene reduction was much lower than 4.0 in all cases, with mean values ranging from 1.16 to 2.60. We conclude that the peak rate of acetylene reduction provides the most reliable estimate of N2 fixation. The recovered rate of acetylene reduction consistently underestimates N2 fixation, sometimes severely, and thus measurements of acetylene reduction made in closed systems also underestimate N2 fixation to varying degrees.     

Effect of spring flooding on endophyte differentiation,nitrogenase activity,root growth and shoot growth inMyrica gale

Summary Spring flooding was investigated as a possible limiting factor in the development of nitrogenase activity, root growth, and shoot growth inMyrica gale. Dormant, one year oldMyrica gale plants were placed in a greenhouse in early April and given three treatments: control (not flooded), flooded-water (flooded with water to 2.5 cm above the soil level) and flooded-peat (flooded with water-saturated peat to 4.0 cm above the soil level). Nitrogenase activity was absent at budbreak but appeared concurrently with the differentiation of vesicles by theFrankia sp. endophyte. Flooding delayed the onset of nitrogenase activity, substantially reduced the specific nitrogenase activity of the nodules, and also severely limited the production of the new nodule biomass. Consequently by 67 days past budbreak nitrogenase activity was much greater in the control plants (5.55±0.42 mol C₂H₄/plant.h; ± SE; N=9) than in the flooded-water (1.18±0.29) and flooded-peat (0.15±0.05) plants. Production of new secondary roots was substantially reduced in the flooded plants but adventitious roots were rapidly produced along the flooded portion of the stem in the better aerated zone near the surface. New nodules formed on several adventitious roots by 67 days indicating that the plants are able to replace their largely nonfunctional deeply flooded nodules with new nodules in the aerobic zone. Initially shoot growth was unaffected by flooding but by 67 days the flooded plants had substantially less leaf biomass, lower leaf and stem nitrogen concentrations, and less total shoot nitrogen content than the control plants.     

Assessing horizontal transfer of nifHDK genes in eubacteria: nucleotide sequence of nifK from Frankia strain HFPCcI3

The structural genes for nitrogenase, nifK, nifD, and nifH, are crucial for nitrogen fixation. Previous phylogenetic analysis of the amino acid sequence of nifH suggested that this gene had been horizontally transferred from a proteobacterium to the gram-positive/cyanobacterial clade, although the confounding effects of paralogous comparisons made interpretation of the data difficult. An additional test of nif gene horizontal transfer using nifD was made, but the NifD phylogeny lacked resolution. Here nif gene phylogeny is addressed with a phylogenetic analysis of a third and longer nif gene, nifK. As part of the study, the nifK gene of the key taxon Frankia was sequenced. Parsimony and some distance analyses of the nifK amino acid sequences provide support for vertical descent of nifK, but other distance trees provide support for the lateral transfer of the gene. Bootstrap support was found for both hypotheses in all trees; the nifK data do not definitively favor one or the other hypothesis. A parsimony analysis of NifH provides support for horizontal transfer in accord with previous reports, although bootstrap analysis also shows some support for vertical descent of the orthologous nifH genes. A wider sampling of taxa and more sophisticated methods of phylogenetic inference are needed to understand the evolution of nif genes. The nif genes may also be powerful phylogenetic tools. If nifK evolved by vertical descent, it provides strong evidence that the cyanobacteria and proteobacteria are sister groups to the exclusion of the firmicutes, whereas 16S rRNA sequences are unable to resolve the relationships of these three major eubacterial lineages.      

Nitrogen fixation by Myrica gale root nodules Massachusetts wetland

Summary Nitrogenase activity was measured by acetylene reduction in excised Myrica gale nodules collected throughout the growing season at two sites associated with a small lake in central Massachusetts. One site was in an open, weakly minerotrophic peatland dominated by M. gale and the other was on the lakeshore. Nitrogenase activity appeared in late May when the leaves unfolded, reached a peak in July when the maximum number of leaves was present, and declined until it was no longer measurable in late October several days after all leaves had fallen. Summer activities were substantially higher at the peatland site than the lakeshore. Maximum activities were 19.8±1.9 and 8.1±0.7 mol/h x g dry weight (x±SE; N=20) at the peatland and lakeshore sites respectively.Nitrogenase activities were very low at 5° C and increased linearly from 10 to 30° C, the highest temperature examined. The maximum soil temperature measured was 20° C, and no significant diurnal fluctuation in activity was detected.Annual nitrogen fixation calculated from the seasonal nitrogenase activity curve was 34 kg N/ha x yr at the peatland site with mean dry weight nodule biomass of 104 kg/ha, and 24 kg N/ha x yr at the lakeshore with 111 kg/ha nodule biomass. These rates of nitrogen fixation are equivalent to 4–5x the amount of nitrogen contained in bulk precipitation and are major components in the nitrogen budgets of the M. gale plants and wetlands in which they grow.     

Caerulein secretion by dermal glands in xenopus laevis

Since gastrin and its related peptides are secreted by a minority population of widely dispersed cells in mamamalian tissues it has, in the past, been difficult to study the subcellular aspects of their secretion. From published reports (1, 2) it seemed possible that a satisfactory system for such studies might be provided by the skin of certain amphibians such as Xenopus laevis since in these tissues high concentrations of peptides such as caerulein exist, and there is some indication (3) that this, or a similar gastrin-like peptide, may be a dermal gland secretory product. We have therefore explored this possibility by studying the structure, secretory process, and secretory product of the most prominent non mucous type of gland in the skin of X. laevis. These studies clearly demonstrate that most, if not all, of the caerulein in which the skin is contained in secretion granules within the dermal glands and that its release can be specifically evoked by adrenergic stimulation. The release process by a holocrine mechanism expels all of the stored secretion onto the skin surface and thus for biosynthetic studies it should now be possible to synchronize the processes which lead to the replenishment of the peptide.     

A comparison of vascular vegetation and protozoan communities in some freshwater wetlands of Northern Lower Michigan

Vascular vegetation and protozoan communities were sampled in seven wetland sites — two bogs, two fens, two marshes, and one swamp — in summer 1977. Two similarity indices were used to compare vascular vegetation and Protozoa from each site with all the other sites. Bog sites were the most distinct from other wetland types with respect to chemical and physical characteristics, dominant vascular vegetation, and protozoan species composition. The swamp site had the highest similarity to all other sites with respect to both dominant vascular vegetation and protozoan species. Protozoan communities from different wetland types were much more similar than dominant vascular species; however, the pattern of similarity between wetland sites was very similar for both groups (Pearson product-moment correlation coefficient = 0.76).Protozoan communities were also compared with those from several nearby lakes with respect to colonization rate onto polyurethane foam artificial substrates. The structure and dynamics of protozoan communities of wetlands were broadly different from those of other freshwaters, and somewhat unique to the other wetland types. Evidence for a high degree of eutrophy in certain bog lakes is presented.     

Recent evolutionary history of American Onchocerca volvulus, based on analysis of a tandemly repeated DNA sequence family

Polymerase chain reaction (PCR) products were characterized for a repeated sequence family (designated "O-150") of the human filarial parasite Onchocerca volvulus. In phylogenetic inferences, the O-150 sequences clustered into closely related groups, suggesting that concerted evolution maintains sequence homology in this family. Using a novel mathematical model based on a nested application of an analysis of variance, we demonstrated that African rainforest and savannah strain parasite populations are significantly different. In contrast, parasites collected in the New World are indistinguishable from African savannah strains of O. volvulus. This finding supports the hypothesis that onchocerciasis was recently introduced into the New World, possibly as a result of the slave trade.      

Hemoglobin in five genetically diverse Frankia strains

Five strains of Frankia were selected to represent a wide range of genetic diversity and examined for presence of hemoglobin. All five strains produced hemoglobin when grown on media without (-N) or with (+N) combined nitrogen. This indicates that hemoglobin is common in Frankia and is not directly associated with nitrogen fixation. Frankia strain EAN1(pec) was examined in more detail. It showed greater hemoglobin concentration when grown at 2% O2 than at 20% O2 in the -N treatment but no effect of oxygen on hemoglobin concentration in the +N treatment. At both oxygen levels, it produced substantially more biomass in +N than in -N culture. It also produced significantly more biomass when the medium contained 0.2% CO2 than in the absence of CO2. The molecular mass of the hemoglobin as determined by size exclusion chromatography was 13.4 +/- 0.2 kDa (mean +/- SE, n = 3) and is consistent with that of a truncated hemoglobin. The hemoglobin had absorption spectra that were typical of a hemoglobin. The oxygen dissociation rate constants for the hemoglobin were 131.2 +/- 5.8 s(-1) for -N culture and 166 +/- 8.2 s(-1) for +N culture. These rapid rates are consistent with a function in facilitated diffusion of oxygen.