Microtubule Binding and Trapping at the Tip of Neurites Regulate Tau Motion in Living Neurons

During the development of neurons, the microtubule-associated tau proteins show a graded proximo-distal distribution in axons. In tauopathies such as Alzheimer's disease, tau accumulates in the somatodendritic compartment. To scrutinize the determinants of tau's distribution and motion, we constructed photoactivatable green fluorescent protein (GFP)-tagged tau fusion proteins and recorded their distribution after focal activation in living cells. Simulation showed that the motion of tau was compatible with diffusion/reaction as opposed to active transport/reaction. Effective diffusion constants of 0.7–0.8 μm²/second were calculated in neurites of PC12 cells and primary cortical neurons. Furthermore, tau's amino terminal projection domain mediated binding and enrichment of tau at distal neurites indicating that the tip of a neurite acts as an adsorber trapping tau protein. Treatment with taxol, incorporation of disease-related tau modifications, experimentally induced hyperphosphorylation and addition of preaggregated amyloid β peptides (Aβ) increased the effective diffusion constant compatible with a decreased binding to microtubules. Distal enrichment was present after taxol treatment but was suppressed at disease-relevant conditions. The data suggest that (i) dynamic binding of tau to microtubules and diffusion along microtubules and (ii) trapping at the tip of a neurite both contribute to its distribution during development and disease.     

Evidence for a shift in life-history strategy during the secondary phase of a plant invasion

We investigated the correlated response of several key traits of Lythrum salicaria L. to water availability gradients in introduced (Iowa, USA) and native (Switzerland, Europe) populations. This was done to investigate whether plants exhibit a shift in life-history strategy during expansion into more stressful habitats during the secondary phase of invasion, as has recently been hypothesized by Dietz and Edwards (Ecology 87(6):1359, 2006). Plants in invaded habitats exhibited a correlated increase in longevity and decrease in overall size in the transition into more stressful mesic habitats. In contrast, plants in the native range only exhibited a decrease in height. Our findings are consistent with the hypothesis that secondary invasion is taking place in L. salicaria, allowing it to be more successful under the more stressful mesic conditions in the invaded range. If this trend continues, L. salicaria may become a more problematic species in the future.     

Evaluation of the wick method for in situ 13C and 15N labelling of annual plants using sugar-urea mixtures

To investigate the amount and fate of root-derived C and N, often tracer techniques are used, where plants are labelled with isotopes. In the present study, we evaluated the suitability of the cotton wick method for in situ labelling of peas (Pisum sativum L.) and oats (Avena sativa L.) with ¹³C and ¹⁵N simultaneously. With two greenhouse experiments we investigated how the wick method and aqueous urea and sugar solutions at a variety of concentrations affected plant development. In addition, we investigated the distribution of ¹³C and ¹⁵N in plants from column experiments under outdoor conditions. Solution was taken up by the plant from a small vial connected to the stem by a cotton wick which was passed through a hole in the stem of the plants. Generally, solution uptake varied between individual plants and decreased with increasing sugar concentrations. Below-ground, above-ground and total plant dry matter, were not significantly affected by the wick method and the applied solutions. Mixtures of aqueous glucose solutions at 2 to 4% and aqueous urea solutions at 1% are useful carriers of ¹³C and ¹⁵N. However, in the investigated plants isotopes were not homogeneously distributed among plant parts. Above-ground plant biomass was preferentially enriched with ¹³C and ¹⁵N, whereas below-ground plant biomass was generally lower enriched. Moreover, isotope distribution ratio of individual plants varied considerably, independent of plant part or timing of labelling. This must be taken into account when estimating root-derived C and N. Future studies comparing labelling methods need to present the isotope distribution ratios among plant parts to allow a true comparison of the methods and the evaluation of their suitability for estimating rhizodeposition.     

The classical pathway of melanogenesis is not essential for melanin synthesis in the adult retinal pigment epithelium

Premelanosomes are presumed to be essential for melanogenesis in melanocytes and pre-natal retinal pigment epithelium (RPE) cells. We analysed melanin synthesis in adenoviral-transduced tyrosinase-gene-expressing amelanotic RPE (ARPE) 19 cells to determine whether premelanosome formation is needed for post-natal melanogenesis. The synthesis of melanogenic proteins and melanin granules was investigated by immunocytochemistry and light and electron microscopy. The occurrence of tyrosinase was analysed ultrastructurally by dihydroxyphenylalanine histochemistry. The viability of transduced cell cultures was examined via MTT assay. We found active tyrosinase in small granule-like vesicles throughout the cytoplasm and in the endoplasmic reticulum and nuclear membrane. Tyrosinase was also associated with multi-vesicular and multi-lamellar organelles. Typical premelanosomes, structural protein PMEL17, tyrosinase-related protein 1 and classic melanosomal stages I–IV were not detected. Instead, melanogenesis took place inside multi-vesicular and multi-lamellar bodies of unknown origin. Viability was not affected up to 10 days after transduction. We thus demonstrate a pathway of melanin formation lacking typical hallmarks of melanogenesis.     

Drought responses of flood-tolerant trees in Amazonian floodplains

Background

Flood-tolerant tree species of the Amazonian floodplain forests are subjected to an annual dry period of variable severity imposed when low river-water levels coincide with minimal precipitation. Although the responses of these species to flooding have been examined extensively, their responses to drought, in terms of phenology, growth and physiology, have been neglected hitherto, although some information is found in publications that focus on flooding.

Scope

The present review examines the dry phase of the annual flooding cycle. It consolidates existing knowledge regarding responses to drought among adult trees and seedlings of many Amazonian floodplain species.

Main Findings

Flood-tolerant species display variable physiological responses to dry periods and drought that indicate desiccation avoidance, such as reduced photosynthetic activity and reduced root respiration. However, tolerance and avoidance strategies for drought vary markedly among species. Drought can substantially decrease growth, biomass and photosynthetic activity among seedlings in field and laboratory studies. When compared with the responses to flooding, drought can impose higher seedling mortality and slower growth rates, especially among evergreen species. Results indicate that tolerance and avoidance strategies for drought vary markedly between species. Both seedling recruitment and photosynthetic activity are affected by drought,

Conclusions

For many species, the effects of drought can be as important as flooding for survival and growth, particularly at the seedling phase of establishment, ultimately influencing species composition. In the context of climate change and predicted decreases in precipitation in the Amazon Basin, the effects of drought on plant physiology and species distribution in tropical floodplain forest ecosystems should not be overlooked.Key words: Drought responses, Amazonia, floodplain forests, tree ecology, várzea     

Ligand-induced formation of a transient tryptophan synthase complex with αββ subunit stoichiometry

The prototypical tryptophan synthases form a stable heterotetrameric αββα complex in which the constituting TrpA and TrpB1 subunits activate each other in a bidirectional manner. The hyperthermophilic archaeon Sulfolobus solfataricus does not contain a TrpB1 protein but instead two members of the phylogenetically distinct family of TrpB2 proteins, which are encoded within (sTrpB2i) and outside (sTrpB2a) the tryptophan operon. It has previously been shown that sTrpB2a does not functionally or structurally interact with sTrpA, whereas sTrpB2i substantially activates sTrpA in a unidirectional manner. However, in the absence of catalysis, no physical complex between sTrpB2i and sTrpA could be detected. In order to elucidate the structural requirements for complex formation, we have analyzed the interaction between sTrpA (α-monomer) and sTrpB2i (ββ-dimer) by means of spectroscopy, analytical gel filtration, and analytical ultracentrifugation, as well as isothermal titration calorimetry. In the presence of the TrpA ligand glycerol 3-phosphate (GP) and the TrpB substrate l-serine, sTrpA and sTrpB2i formed a physical complex with a thermodynamic dissociation constant of about 1 μM, indicating that the affinity between the α- and ββ-subunits is weaker by at least 1 order of magnitude than the affinity between the corresponding subunits of prototypical tryptophan synthases. The observed stoichiometry of the complex was 1 subunit of sTrpA per 2 subunits of sTrpB2i, which corresponds to a αββ quaternary structure and testifies to a strong negative cooperativity for the binding of the α-monomers to the ββ-dimer. The analysis of the interaction between sTrpB2i and sTrpA in the presence of several substrate, transition state, and product analogues suggests that the αββ complex remains stable during the whole catalytic cycle and disintegrates into α- and ββ-subunits upon the release of the reaction product tryptophan. The formation of a transient tryptophan synthase complex, together with the observed low affinity of sTrpB2i for l-serine, couples the rate of tryptophan biosynthesis in S. solfataricus to the cytosolic availability of l-serine.     

Routes of epithelial water flow: aquaporins versus cotransporters

The routes water takes through membrane barriers is still a matter of debate. Although aquaporins only allow transmembrane water movement along an osmotic gradient, cotransporters are believed to be capable of water transport against the osmotic gradient. Here we show that the renal potassium-chloride-cotransporter (KCC1) does not pump a fixed amount of water molecules per movement of one K⁺ and one Cl⁻, as was reported for the analogous transporter in the choroid plexus. We monitored water and potassium fluxes through monolayers of primary cultured renal epithelial cells by detecting tiny solute concentration changes in the immediate vicinity of the monolayer. KCC1 extruded K⁺ ions in the presence of a transepithelial K⁺ gradient, but did not transport water. KCC1 inhibition reduced epithelial osmotic water permeability P_f by roughly one-third, i.e., the effect of inhibitors was small in resting cells and substantial in hormonal stimulated cells that contained high concentrations of aquaporin-2 in their apical membranes. The furosemide or DIOA (dihydroindenyl-oxy-alkanoic acid)-sensitive water flux was much larger than expected when water passively followed the KCC1-mediated ion flow. The inhibitory effect of these drugs on water flux was reversed by the K⁺-H⁺ exchanger nigericin, indicating that KCC1 affects water transport solely by K⁺ extrusion. Intracellular K⁺ retention conceivably leads to cell swelling, followed by an increased rate of endocytic AQP2 retrieval from the apical membrane.     

Palmitoylation of pulmonary surfactant protein SP-C is critical for its functional cooperation with SP-B to sustain compression/expansion dynamics in cholesterol-containing surfactant films

Recent data suggest that a functional cooperation between surfactant proteins SP-B and SP-C may be required to sustain a proper compression-expansion dynamics in the presence of physiological proportions of cholesterol. SP-C is a dually palmitoylated polypeptide of 4.2 kDa, but the role of acylation in SP-C activity is not completely understood. In this work we have compared the behavior of native palmitoylated SP-C and recombinant nonpalmitoylated versions of SP-C produced in bacteria to get a detailed insight into the importance of the palmitic chains to optimize interfacial performance of cholesterol-containing surfactant films. We found that palmitoylation of SP-C is not essential for the protein to promote rapid interfacial adsorption of phospholipids to equilibrium surface tensions (∼22 mN/m), in the presence or absence of cholesterol. However, palmitoylation of SP-C is critical for cholesterol-containing films to reach surface tensions ≤1 mN/m at the highest compression rates assessed in a captive bubble surfactometer, in the presence of SP-B. Interestingly, the ability of SP-C to facilitate reinsertion of phospholipids during expansion was not impaired to the same extent in the absence of palmitoylation, suggesting the existence of palmitoylation-dependent and -independent functions of the protein. We conclude that palmitoylation is key for the functional cooperation of SP-C with SP-B that enables cholesterol-containing surfactant films to reach very low tensions under compression, which could be particularly important in the design of clinical surfactants destined to replacement therapies in pathologies such as acute respiratory distress syndrome.