Dynamics of Kv1 Channel Transport in Axons

Concerted actions of various ion channels that are precisely targeted along axons are crucial for action potential initiation and propagation, and neurotransmitter release. However, the dynamics of channel protein transport in axons remain unknown. Here, using time-lapse imaging, we found fluorescently tagged Kv1.2 voltage-gated K+ channels (YFP-Kv1.2) moved bi-directionally in discrete puncta along hippocampal axons. Expressing Kvβ2, a Kv1 accessory subunit, markedly increased the velocity, the travel distance, and the percentage of moving time of these puncta in both anterograde and retrograde directions. Suppressing the Kvβ2-associated protein, plus-end binding protein EB1 or kinesin II/KIF3A, by siRNA, significantly decreased the velocity of YFP-Kv1.2 moving puncta in both directions. Kvβ2 mutants with disrupted either Kv1.2-Kvβ2 binding or Kvβ2-EB1 binding failed to increase the velocity of YFP-Kv1.2 puncta, confirming a central role of Kvβ2. Furthermore, fluorescently tagged Kv1.2 and Kvβ2 co-moved along axons. Surprisingly, when co-moving with Kv1.2 and Kvβ2, EB1 appeared to travel markedly faster than its plus-end tracking. Finally, using fission yeast S. pombe expressing YFP-fusion proteins as reference standards to calibrate our microscope, we estimated the numbers of YFP-Kv1.2 tetramers in axonal puncta. Taken together, our results suggest that proper amounts of Kv1 channels and their associated proteins are required for efficient transport of Kv1 channel proteins along axons

BicaudalD Actively Regulates Microtubule Motor Activity in Lipid Droplet Transport

A great deal of sub-cellular organelle positioning, and essentially all minus-ended organelle transport, depends on cytoplasmic dynein, but how dynein's function is regulated is not well understood. BicD is established to play a critical role in mediating dynein function-loss of BicD results in improperly localized nuclei, mRNA particles, and a dispersed Golgi apparatus-however exactly what BicD's role is remains unknown. Nonetheless, it is widely believed that BicD may act to tether dynein to cargos. Here we use a combination of biophysical and biochemical studies to investigate BicD's role in lipid droplet transport during Drosophila embryogenesis.Functional loss of BicD impairs the embryo's ability to control the net direction of droplet transport; the developmentally controlled reversal in transport is eliminated. We find that minimal BicD expression (near-BicD(null)) decreases the average run length of both plus and minus end directed microtubule (MT) based transport. A point mutation affecting the BicD N-terminus has very similar effects on transport during cellularization (phase II), but in phase III (gastrulation) motion actually appears better than in the wild-type.In contrast to a simple static tethering model of BicD function, or a role only in initial dynein recruitment to the cargo, our data uncovers a new dynamic role for BicD in actively regulating transport. Lipid droplets move bi-directionally, and our investigations demonstrate that BicD plays a critical-and temporally changing-role in balancing the relative contributions of plus-end and minus-end motors to control the net direction of transport. Our results suggest that while BicD might contribute to recruitment of dynein to the cargo it is not absolutely required for such dynein localization, and it clearly contributes to regulation, helping activation/inactivation of the motors

Roles of Dynein and Dynactin in Early Endosome Dynamics Revealed Using Automated Tracking and Global Analysis

Microtubule-dependent movement is crucial for the spatial organization of endosomes in most eukaryotes, but as yet there has been no systematic analysis of how a particular microtubule motor contributes to early endosome dynamics. Here we tracked early endosomes labeled with GFP-Rab5 on the nanometer scale, and combined this with global, first passage probability (FPP) analysis to provide an unbiased description of how the minus-end microtubule motor, cytoplasmic dynein, supports endosome motility. Dynein contributes to short-range endosome movement, but in particular drives 85–98% of long, inward translocations. For these, it requires an intact dynactin complex to allow membrane-bound p150Glued to activate dynein, since p50 over-expression, which disrupts the dynactin complex, inhibits inward movement even though dynein and p150Glued remain membrane-bound. Long dynein-dependent movements occur via bursts at up to ∼8 µms−1 that are linked by changes in rate or pauses. These peak speeds during rapid inward endosome movement are still seen when cellular dynein levels are 50-fold reduced by RNAi knock-down of dynein heavy chain, while the number of movements is reduced 5-fold. Altogether, these findings identify how dynein helps define the dynamics of early endosomes

Plus- and Minus-End Directed Microtubule Motors Bind Simultaneously to Herpes Simplex Virus Capsids Using Different Inner Tegument Structures

Many viruses depend on host microtubule motors to reach their destined intracellular location. Viral particles of neurotropic alphaherpesviruses such as herpes simplex virus 1 (HSV1) show bidirectional transport towards the cell center as well as the periphery, indicating that they utilize microtubule motors of opposing directionality. To understand the mechanisms of specific motor recruitment, it is necessary to characterize the molecular composition of such motile viral structures. We have generated HSV1 capsids with different surface features without impairing their overall architecture, and show that in a mammalian cell-free system the microtubule motors dynein and kinesin-1 and the dynein cofactor dynactin could interact directly with capsids independent of other host factors. The capsid composition and surface was analyzed with respect to 23 structural proteins that are potentially exposed to the cytosol during virus assembly or cell entry. Many of these proteins belong to the tegument, the hallmark of all herpesviruses located between the capsid and the viral envelope. Using immunoblots, quantitative mass spectrometry and quantitative immunoelectron microscopy, we show that capsids exposing inner tegument proteins such as pUS3, pUL36, pUL37, ICP0, pUL14, pUL16, and pUL21 recruited dynein, dynactin, kinesin-1 and kinesin-2. In contrast, neither untegumented capsids exposing VP5, VP26, pUL17 and pUL25 nor capsids covered by outer tegument proteins such as vhs, pUL11, ICP4, ICP34.5, VP11/12, VP13/14, VP16, VP22 or pUS11 bound microtubule motors. Our data suggest that HSV1 uses different structural features of the inner tegument to recruit dynein or kinesin-1. Individual capsids simultaneously accommodated motors of opposing directionality as well as several copies of the same motor. Thus, these associated motors either engage in a tug-of-war or their activities are coordinately regulated to achieve net transport either to the nucleus during cell entry or to cytoplasmic membranes for envelopment during assembly

The Effect of Inoculants of Thiobacillus and Aspergillus on Corn Growth

Abstract\ud Phosphorus (P) is one of the essential macronutrients for growth and development of plant. Phosphorus is added to soil in the form of phosphatic fertilizers, part of which is utilized by plants and the remainder converted into insoluble fixed forms. Increasingly high cost of chemical fertilizers has been the major stimulus to search for an alternative, naturally-occurring, phosphate source. The researchers offered phosphorus rocks as a valuable alternative source for P. fertilizer. Unfortunately, rock phosphate is not plant available in soils with a pH greater than 6. One method to increase soluble form inorganic P is application of phosphate solublizing microorganisms and sulfur oxidizing bacteria (Thiobacillus) with rock phosphate. A greenhouse experiment was carried out with two bio fertilizers (bio fertilizers santes in incubation condition) in a soil with low available P on corn growth. The bio fertilizers were: rock phosphate with 20% sulfur, 15% vermicompost, Thiobacillus bacteria and Aspergillus fungi (BFS20V15) at three rates: 440 kg/ha (BF1) , 880 kg/ha (BF2), 1320kg/ha (BF3), rock phosphate with 20% sulfur, 15% vermicompost, Thiobacillus bacteria (BFS20V15) at three rates: 440 kg/ha (B1) , 880 kg/ha (B2), 1320kg/ha (B3), triple super phosphate (TSP), and control without phosphorus. In the greenhouse experiment, shoot dry matter, p uptake in plant and available p in soil were determined. The results showed that maximum yield obtained from BF3 with the shoot dry weight 7.2 g per plant and with no significant difference in relation to the triple super phosphate (7.5g) at 5% level. Also highest rate p-uptake resulted from BF3. There was significant difference between treatment BF3 and TSP on p-uptake. Results indicated that it could be possible to substitute rock phosphate inoculated with sulfur-oxidizing bacteria and phosphorous-solublizing fungus for super phosphate. \ud \ud Keywords: Uptake-p, pH Rock phosphate, Solfur, Vermicompos

The Effect of Nitrate Levels and Harvest Times on Fe, Zn, Cu, and K, Concentrations and Nitrate Reductase Activity in Lettuce and Spinach

Leafy vegetables are considered as the main sources of nitrate in the human diet. In order to investigate the effect of nitrate levels and harvest times on nitrate accumulation, nitrate reductase activity, concentrations of Fe, Zn, Cu and K in Lettuce and Spinach and their relation to nitrate accumulation in these leafy vegetables, two harvest times (29 and 46 days after transplanting), two vegetable species of lettuce and spinach and two concentrations of nitrate (10 and 20 mM) were used in a hydroponics greenhouse experiment with a completely randomized design and 3 replications. Modified Hoagland and Arnon nutrient solutions were used for the experiment. The results indicated that by increasing nitrate concentration of solution, nitrate accumulation in roots and shoots of lettuce and spinach increased significantly (P ≤ 0.05), and the same trend was observed for the nitrate reductase activity in the shoots of the two species. Increasing the nitrate concentrations of solution, reduced the shoot dry weight and the concentration of Fe and Cu in both species, where as it increased the K and Zn concentrations in the shoots of the two species in each both harvest times, the nitrate accumulation increased, but the nitrate reductase activity decreased in the shoots of the two species over the course of the growth. The Concentration of Fe, Cu and K decreased in the shoots of lettuce and the spinach with the time, despite the increase in Zn concentration in the shoots. The results also indicated that increasing nitrate concentrations of solution to the levels greater than the plant capacity for reduction and net uptake of nitrate, leads to the nitrate accumulation in the plants. Nitrate accumulation in plant tissue led to decreases in fresh shoot yield and Fe and Cu concentrations and nitrate reductase activities in both lettuce and spinach

Evaluation of Spatial-Temporal Variation of Soil Detachment Rate Potential in Rill Erosion, Case study: Doshmanziari Rainfed Lands, Fars province

Introduction: Soil erosion by water is one of the most widespread forms of land degradation and it has caused many undesirable consequences in last decades. On steep slopes, rill erosion is the most important type of erosion, which produces sediment and rill flow. It can be also considered as a vehicle for transporting soil particles detached from upland areas. Recent studies indicate that soil detachment rates are significantly influenced by land use. It is also known that there is a major difference between detachment rates of disturbed and natural soils (Zhang et al., 2003). Plowing rills especially in steep slopes increases sediment production. Sun et al. (2013) reported that the contribution of rill erosion in hill slope lands in china was more than 70%, which was approximately 50% of total soil erosion. In addition, measured soil loss is statistically related to hydraulic indicators such as slope, water depth, flow velocity, flow shear stress and stream power (Knapen et al., 2007). This study aims to evaluate the effects of hydraulic variables (shear stress and stream power) on spatial-temporal soil detachment rate. The focus is on the plowing rills in hillslope areas under wheat dry farming cultivation. Materials and Methods: The study area is located in hilly slopes with the slope of 22.56% under dry farming wheat cultivation at 60 km of west of Shiraz, Iran. Top-down conventional plowing was carried out in order to create 10 meters furrows. Slope and cross sections of rills were measured throughout the experiment at 1 m intervals by rill-meter. Water was added to the top of the rills for 10 minutes and inflow rates were 10, 15 and 20 L min-1. Hydraulic parameters such as shear stress and stream power were calculated measuring rill morphology and water depth. Flow velocity and hydraulic radius along the different rill experiments were also calculated. Sediment concentrations were measured in three equal regular time and distance intervals (measurement points (MPs)), they were considered to calculate sediment detachment rate in different times and sections of each rill experiment for spatial and temporal soil detachment rate evaluation. One-way analysis of variance (ANOVA) was employed to test the significance of differences of sediment detachment rate among different treatments. Results and Discussion: The results showed that the maximum values of shear stress and stream power were 14.07 Pa and 10.29 Wm-2 and the minimum values were 7.41 and 2.77 respectively. This research also indicated that changes in longitudinal profile of these hydraulic parameters along the rills affected the soil detachment rate values. Obtained average, minimum and maximum of the soil detachment rate were determined as 0.09, 0.02 and 0.22 kgm-2s-1, respectively. Due to Detachment-Transport Coupling mechanism, there was a significant difference between the initial and following MPs (