Patronin-mediated minus end growth is required for dendritic microtubule polarity.

Microtubule minus ends are thought to be stable in cells. Surprisingly, in Drosophila and zebrafish neurons, we observed persistent minus end growth, with runs lasting over 10 min. In Drosophila, extended minus end growth depended on Patronin, and Patronin reduction disrupted dendritic minus-end-out polarity. In fly dendrites, microtubule nucleation sites localize at dendrite branch points. Therefore, we hypothesized minus end growth might be particularly important beyond branch points. Distal dendrites have mixed polarity, and reduction of Patronin lowered the number of minus-end-out microtubules. More strikingly, extra Patronin made terminal dendrites almost completely minus-end-out, indicating low Patronin normally limits minus-end-out microtubules. To determine whether minus end growth populated new dendrites with microtubules, we analyzed dendrite development and regeneration. Minus ends extended into growing dendrites in the presence of Patronin. In sum, our data suggest that Patronin facilitates sustained microtubule minus end growth, which is critical for populating dendrites with minus-end-out microtubules

Improving Effect of the Policosanol from Ericerus pela Wax on Learning and Memory Impairment Caused by Scopolamine in Mice

Policosanol (PC) is a mixture of long-chain fatty alcohols that exhibits multiple biological activities, such as reducing blood lipid and cholesterol levels, lowering blood pressure, and extenuating liver inflammation. To assess PC’s impact on cognitive behavior and function, PC was prepared from Ericerus pela wax using a reduction method and analyzed using gas chromatography (GC). A total of 60 mice were randomly divided into six groups of 10 animals each: control (0.5% CMC-Na solution, i.g.), model (0.5% CMC-Na solution, i.g.), donepezil (3 mg/kg, i.g.), PC low- (2 g/kg, i.g.), medium (4 g/kg, i.g.), and high- (6 g/kg, i.g.) dose groups. All the groups were administered daily for 28 consecutive days. There were four parameters—escape latency, crossings of platform, swimming distance, and time spent in the target quadrant—that were recorded to evaluate the cognitive performance of mice in the Morris Water Maze (MWM). After MWM testing, the levels of acetylcholine (ACh), acetylcholinesterase (AChE), superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) that were present in brain tissue were determined using assay kits. The GC data showed that PC consisted of four major components: tetracosanol (14.40%), hexacosanol (48.97%), octacosanol (25.40%), and triacontanol (4.80%). In the MWM test, PC significantly decreased the escape latency (p < 0.05) and increased the crossings of the platform (p < 0.05) and swimming distance (p < 0.05) and time in the target quadrant (p < 0.05) in rodents compared to that in the model group. Moreover, PC increased the levels of ACh, SOD, and GSH; inhibited AChE; and reduced MDA in the brain tissue of the tested animals. This is the first report to evaluate the efficacy of PC for cognitive behavior and function in animals. Our findings demonstrate that PC from E. pela wax is likely to exert an enhancing effect on learning and memory by promoting the cholinergic system and attenuating oxidative stress, which will provide a new insight into the efficacy of PC and expand its application in the food, nutraceutical, and beverage industries

Systematic Analysis of the Functions of Lysine Acetylation in the Regulation of Tat Activity.

The Tat protein of HIV-1 has several well-known properties, such as nucleocytoplasmic trafficking, transactivation of transcription, interaction with tubulin, regulation of mitotic progression, and induction of apoptosis. Previous studies have identified a couple of lysine residues in Tat that are essential for its functions. In order to analyze the functions of all the lysine residues in Tat, we mutated them individually to alanine, glutamine, and arginine. Through systematic analysis of the lysine mutants, we discovered several previously unidentified characteristics of Tat. We found that lysine acetylation could modulate the subcellular localization of Tat, in addition to the regulation of its transactivation activity. Our data also revealed that lysine mutations had distinct effects on microtubule assembly and Tat binding to bromodomain proteins. By correlation analysis, we further found that the effects of Tat on apoptosis and mitotic progression were not entirely attributed to its effect on microtubule assembly. Our findings suggest that Tat may regulate diverse cellular activities through binding to different proteins and that the acetylation of distinct lysine residues in Tat may modulate its interaction with various partners

Patronin-mediated minus end growth is required for dendritic microtubule polarity.

Microtubule minus ends are thought to be stable in cells. Surprisingly, in Drosophila and zebrafish neurons, we observed persistent minus end growth, with runs lasting over 10 min. In Drosophila, extended minus end growth depended on Patronin, and Patronin reduction disrupted dendritic minus-end-out polarity. In fly dendrites, microtubule nucleation sites localize at dendrite branch points. Therefore, we hypothesized minus end growth might be particularly important beyond branch points. Distal dendrites have mixed polarity, and reduction of Patronin lowered the number of minus-end-out microtubules. More strikingly, extra Patronin made terminal dendrites almost completely minus-end-out, indicating low Patronin normally limits minus-end-out microtubules. To determine whether minus end growth populated new dendrites with microtubules, we analyzed dendrite development and regeneration. Minus ends extended into growing dendrites in the presence of Patronin. In sum, our data suggest that Patronin facilitates sustained microtubule minus end growth, which is critical for populating dendrites with minus-end-out microtubules

Structure-function analysis of ceTIR-1/hSARM1 explains the lack of Wallerian axonal degeneration in C. elegans

Summary: Wallerian axonal degeneration (WD) does not occur in the nematode C. elegans, in contrast to other model animals. However, WD depends on the NADase activity of SARM1, a protein that is also expressed in C. elegans (ceSARM/ceTIR-1). We hypothesized that differences in SARM between species might exist and account for the divergence in WD. We first show that expression of the human (h)SARM1, but not ceTIR-1, in C. elegans neurons is sufficient to confer axon degeneration after nerve injury. Next, we determined the cryoelectron microscopy structure of ceTIR-1 and found that, unlike hSARM1, which exists as an auto-inhibited ring octamer, ceTIR-1 forms a readily active 9-mer. Enzymatically, the NADase activity of ceTIR-1 is substantially weaker (10-fold higher Km) than that of hSARM1, and even when fully active, it falls short of consuming all cellular NAD+. Our experiments provide insight into the molecular mechanisms and evolution of SARM orthologs and WD across species

Identification of Proteins Required for Precise Positioning of Apc2 in Dendrites

In Drosophila neurons, uniform minus-end-out polarity in dendrites is maintained in part by kinesin-2-mediated steering of growing microtubules at branch points. Apc links the kinesin motor to growing microtubule plus ends and Apc2 recruits Apc to branch points where it functions. Because Apc2 acts to concentrate other steering proteins to branch points, we wished to understand how Apc2 is targeted. From an initial broad candidate RNAi screen, we found Miro (a mitochondrial transport protein), Ank2, Axin, spastin and Rac1 were required to position Apc2-GFP at dendrite branch points. YFP-Ank2-L8, Axin-GFP and mitochondria also localized to branch points suggesting the screen identified relevant proteins. By performing secondary screens, we found that energy production by mitochondria was key for Apc2-GFP positioning and spastin acted upstream of mitochondria. Ank2 seems to act independently from other players, except its membrane partner, Neuroglian (Nrg). Rac1 likely acts through Arp2/3 to generate branched actin to help recruit Apc2-GFP. Axin can function in a variety of wnt signaling pathways, one of which includes heterotrimeric G proteins and Frizzleds. Knockdown of Gαs, Gαo, Fz and Fz2, reduced targeting of Apc2 and Axin to branch points. Overall our data suggest that mitochondrial energy production, Nrg/Ank2, branched actin generated by Arp2/3 and Fz/G proteins/Axin function as four modules that control localization of the microtubule regulator Apc2 to its site of action in dendrite branch points

Analyses on the Infection Process of Rice Virus and the Spatiotemporal Expression Pattern of Host Defense Genes Based on a Determined-Part Inoculation Approach

Rice viral diseases adversely affect crop yield and quality. Most rice viruses are transmitted through insect vectors. However, the traditional whole-plant inoculation method cannot control the initial inoculation site in rice plants because the insect feeding sites in plants are random. To solve this problem, we established a determined-part inoculation approach in this study that restricted the insect feeding sites to specific parts of the rice plant. Rice stripe virus (RSV) was used as the model virus and was inoculated at the bottom of the stem using our method. Quantitative real-time PCR and Western blot analyses detected RSV only present at the bottom of the Nipponbare (NPB) stem at 1 day post-inoculation (dpi), indicating that our method successfully controlled the inoculation site. With time, RSV gradually moved from the bottom of the stem to the leaf in NPB rice plants, indicating that systemic viral spread can also be monitored using this method. In addition, a cultivar resistant to RSV, Zhendao 88 (ZD88), was inoculated using this method. We found that RSV accumulation in ZD88 was significantly lower than in NPB. Additionally, the expression level of the resistant gene STV11 in ZD88 was highly induced at the initial invasion stage of RSV (1 dpi) at the inoculation site, whereas it remained relatively stable at non-inoculated sites. This finding indicated that STV11 directly responded to RSV invasion to inhibit virus accumulation at the invasion site. We also proved that this approach is suitable for other rice viruses, such as Rice black-streaked dwarf virus (RBSDV). Interestingly, we determined that systemic infection with RSV was faster than that with RBSDV in NPB, which was consistent with findings in field trails. In summary, this approach is suitable for characterizing the viral infection process in rice plants, comparing the local viral accumulation and spread among different cultivars, analyzing the spatiotemporal expression pattern of resistance-associated genes, and monitoring the infection rate for different viruses

Bilaterian Giant Ankyrins Have a Common Evolutionary Origin and Play a Conserved Role in Patterning the Axon Initial Segment

<div><p>In vertebrate neurons, the axon initial segment (AIS) is specialized for action potential initiation. It is organized by a giant 480 Kd variant of ankyrin G (AnkG) that serves as an anchor for ion channels and is required for a plasma membrane diffusion barrier that excludes somatodendritic proteins from the axon. An unusually long exon required to encode this 480Kd variant is thought to have been inserted only recently during vertebrate evolution, so the giant ankyrin-based AIS scaffold has been viewed as a vertebrate adaptation for fast, precise signaling. We re-examined AIS evolution through phylogenomic analysis of ankyrins and by testing the role of ankyrins in proximal axon organization in a model multipolar <i>Drosophila</i> neuron (ddaE). We find giant isoforms of ankyrin in all major bilaterian phyla, and present evidence in favor of a single common origin for giant ankyrins and the corresponding long exon in a bilaterian ancestor. This finding raises the question of whether giant ankyrin isoforms play a conserved role in AIS organization throughout the Bilateria. We examined this possibility by looking for conserved ankyrin-dependent AIS features in <i>Drosophila</i> ddaE neurons via live imaging. We found that ddaE neurons have an axonal diffusion barrier proximal to the cell body that requires a giant isoform of the neuronal ankyrin Ank2. Furthermore, the potassium channel shal concentrates in the proximal axon in an Ank2-dependent manner. Our results indicate that the giant ankyrin-based cytoskeleton of the AIS may have evolved prior to the radiation of extant bilaterian lineages, much earlier than previously thought.</p></div