Dynamic Localization of the Swe1 Regulator Hsl7 During the Saccharomyces cerevisiae Cell Cycle

In Saccharomyces cerevisiae, entry into mitosis requires activation of the cyclin-dependent kinase Cdc28 in its cyclin B (Clb)-associated form. Clb-bound Cdc28 is susceptible to inhibitory tyrosine phosphorylation by Swe1 protein kinase. Swe1 is itself negatively regulated by Hsl1, a Nim1-related protein kinase, and by Hsl7, a presumptive protein-arginine methyltransferase. In vivo all three proteins localize to the bud neck in a septin-dependent manner, consistent with our previous proposal that formation of Hsl1-Hsl7-Swe1 complexes constitutes a checkpoint that monitors septin assembly. We show here that Hsl7 is phosphorylated by Hsl1 in immune-complex kinase assays and can physically associate in vitro with either Hsl1 or Swe1 in the absence of any other yeast proteins. With the use of both the two-hybrid method and in vitro binding assays, we found that Hsl7 contains distinct binding sites for Hsl1 and Swe1. A differential interaction trap approach was used to isolate four single-site substitution mutations in Hsl7, which cluster within a discrete region of its N-terminal domain, that are specifically defective in binding Hsl1. When expressed in hsl7Delta cells, each of these Hsl7 point mutants is unable to localize at the bud neck and cannot mediate down-regulation of Swe1, but retains other functions of Hsl7, including oligomerization and association with Swe1. GFP-fusions of these Hsl1-binding defective Hsl7 proteins localize as a bright perinuclear dot, but never localize to the bud neck; likewise, in hsl1Delta cells, a GFP-fusion to wild-type Hsl7 or native Hsl7 localizes to this dot. Cell synchronization studies showed that, normally, Hsl7 localizes to the dot, but only in cells in the G1 phase of the cell cycle. Immunofluorescence analysis and immunoelectron microscopy established that the dot corresponds to the outer plaque of the spindle pole body (SPB). These data demonstrate that association between Hsl1 and Hsl7 at the bud neck is required to alleviate Swe1-imposed G2-M delay. Hsl7 localization at the SPB during G1 may play some additional role in fine-tuning the coordination between nuclear and cortical events before mitosis.     

Capillary Filling at the Microscale: Control of Fluid Front Using Geometry

We propose an experimental and theoretical framework for the study of capillary filling at the micro-scale. Our methodology enables us to control the fluid flow regime so that we can characterise properties of Newtonian fluids such as their viscosity. In particular, we study a viscous, non-inertial, non-Washburn regime in which the position of the fluid front increases linearly with time for the whole duration of the experiment. The operating shear-rate range of our apparatus extends over nearly two orders of magnitude. Further, we analyse the advancement of a fluid front within a microcapillary in a system of two immiscible Newtonian liquids. We observe a non-Washburn regime in which the front can accelerate or decelerate depending on the viscosity contrast between the two liquids. We then propose a theoretical model which enables us to study and explain both non-Washburn regimes. Furthermore, our theoretical model allows us to put forward ways to control the emergence of these regimes by means of geometrical parameters of the experimental set-up. Our methodology allows us to design and calibrate a micro-viscosimetre which works at constant pressure.     

First report of <Emphasis Type="Italic">Aspergillus sydowii</Emphasis> and <Emphasis Type="Italic">Aspergillus brasiliensis</Emphasis> as phosphorus solubilizers in maize

Maize is one of the most important crops worldwide. It provides food for humans and animals and is used in biotechnological and industrial processes to produce a wide variety of products. The phosphorus (P) requirement for its development and production is high, but the absorption efficiency of this nutrient is insufficient to meet its requirements. The use of P-solubilizing fungi can increase this efficiency, thus reducing the amount of fertilizers applied to the crops. Therefore, this study aimed to verify the potential use of A. sydowii and A. brasiliensis and their effect in the field as bioinoculants when associated with three P fertilization doses. The experiment was carried out in a maize field, and treatments were presence and absence of fungi associated with P mineral fertilization doses. The parameters evaluated were shoot dry matter, P content in the plant, and P content in the soil. A. sydowii caused the highest P content in the plant and soil at fertilization doses of 75% and 100%, contradicting the expected results from in vitro assays that indicated that A. brasiliensis would provide better rates of P uptake. There was no difference in the other fertilization doses or for dry matter when comparing all treatments. This result strongly suggests that the use of A. sydowii can improve the efficiency of P absorption with fertilization application. In addition, the molecular analysis of the two fungi performed in this study contributes novel information on the use of both species during the agricultural process.     

Prokaryotic and eukaryotic community structure affected by the presence of an acid mine drainage from an abandoned gold mine

The acid mine drainage that originates in the abandoned gold mine in San Luis, Argentina, is released into La Carolina stream. The aim of this study was to determine the influence of this mine drainage on the physicochemical parameters of the area studied and on both prokaryotic and eukaryotic community structure. In addition, specific relationships between microbial taxonomic groups and physicochemical parameters were established. The drainage that flows into La Carolina stream acidifies the stream and increases its sulfate, Zn, Cd and Te concentrations. Microbial analysis showed that prokaryotic community structure is mainly affected by pH values. Actinobacteria and Gammaproteobacteria were abundant in samples characterized by low pH values, while Nitrospirae, Chloroflexi, Deltaproteobacteria, Thaumarchaeota and Euryarchaeota were associated with high concentrations of heavy metals. Otherwise, Alphaproteobacteria was present in samples taken in sunlit areas. Regarding eukaryotic community structure, the sunlight had the greatest impact. Inside the mine, in the absence of light, fungi and protists members were the most abundant microorganisms, while those samples taken in the presence of light displayed algae (green algae and diatoms) as the most abundant ones. After receiving the mine drainage, the stream showed a decrease in the diatom abundance and green algae predominated.

     

Temporary immersion bioreactors (TIB) provide a versatile,cost-effective and reproducible in vitro analysis of the response of pineapple shoots to salinity and drought

Effects of salinity (NaCl) and the carbon source mannitol (0–200 mM) on micropropagation of pineapple cv. MD2 were analyzed in temporary immersion bioreactors (TIBs). Shoot multiplication rate, shoot cluster fresh weight and levels of aldehydes, chlorophylls, carotenoids and phenolics were determined in the plant material. The content of soluble phenolics in the culture medium was also evaluated. NaCl or mannitol above concentrations of 50 mM decreased pineapple shoot multiplication and fresh weight significantly. Two hundred mM NaCl decreased multiplication rate by 71.5% and cluster fresh weight by 40.0%. NaCl increased 2.4 times the levels of other aldehydes; 1.4 times the soluble phenolics in shoots; and 1.4 times the phenolics excreted to the culture medium. On the other hand, mannitol decreased the multiplication rate and cluster fresh weight by about 60%. Mannitol increased the contents of chlorophyll b 1.4 times and soluble phenolics 2.1 times. Results indicated that pineapple cv. MD2 is more sensitive to NaCl than to mannitol. Multiplication rates indicate that a 50% reduction was obtained with 37.4 mM NaCl and 66.5 mM mannitol. These concentrations can be used to stress shoots during micropropagation in TIBs and screen for/detect somaclonal variants with an increased salinity or drought tolerance.     

Rapid recycling of ClC‐2 chloride channels between plasma membrane and endosomes: Role of a tyrosine endocytosis motif in surface retrieval

ClC‐2 chloride channel is present in the brain and some transporting epithelia where its function is poorly understood. We have now demonstrated that the surface channels are rapidly internalised and approximately the 70% of the surface membrane protein recycles after 4‐ to 8‐min internalisation. Endocytosis of ClC‐2 was dependent upon tyrosine 179 located within an endocytic motif. Rapid recycling accompanied by an even faster internalisation could account for the abundant presence of ClC‐2 in intracellular membranous structures. At least a proportion of ClC‐2 resides in lipid rafts. Use of β‐cyclodextrin led to an increase in cell surface channel, but, surprisingly, a decrease in functionally active channels. We suggest that ClC‐2 requires residing in β‐cyclodextrin sensitive clusters with other molecules in order to remain active. Regulation of ClC‐2 trafficking to and within the membrane could be a means of modulating its activity. J. Cell. Physiol. 221: 650–657, 2009. © 2009 Wiley‐Liss, Inc.