Analysis of functional surfaces on the actin nucleation promoting factor Dip1 required for Arp2/3 complex activation and endocytic actin network assembly.

Arp2/3 complex nucleates branched actin filaments that drive processes like endocytosis and lamellipodial protrusion. WISH/DIP/SPIN90 (WDS) proteins form a class of Arp2/3 complex activators or nucleation promoting factors (NPFs) that, unlike WASP family NPFs, activate Arp2/3 complex without requiring preformed actin filaments. Therefore, activation of Arp2/3 complex by WDS proteins is thought to produce the initial actin filaments that seed branching nucleation by WASP-bound Arp2/3 complexes. However, whether activation of Arp2/3 complex by WDS proteins is important for the initiation of branched actin assembly in cells has not been directly tested. Here, we used structure-based point mutations of the Schizosaccharomyces pombe WDS protein Dip1 to test the importance of its Arp2/3-activating activity in cells. Six of thirteen Dip1 mutants caused severe defects in Arp2/3 complex activation in vitro, and we found a strong correlation between the ability of mutants to activate Arp2/3 complex and to rescue endocytic actin assembly defects caused by deleting Dip1. These data support a model in which Dip1 activates Arp2/3 complex to produce actin filaments that initiate branched actin assembly at endocytic sites. Dip1 mutants that synergized with WASP in activating Arp2/3 complex in vitro showed milder defects in cells compared to those that did not, suggesting that in cells the two NPFs may coactivate Arp2/3 complex to initiate actin assembly. Finally, the mutational data reveal important complementary electrostatic contacts at the Dip1-Arp2/3 complex interface and corroborate the previously proposed wedge model, which describes how Dip1 binding triggers structural changes that activate Arp2/3 complex.

Recombinant water stress protein 1 (Re-WSP1) suppresses colon cancer cell growth through the miR-539/ß-catenin signaling pathway.

BACKGROUND: Nostoc commune Vauch. is a nitrogen-fixing blue-green algae that expresses a large number of active molecules with medicinal properties. Our previous study found that a water stress protein (WSP1) from N. commune and its recombinant counterpart (Re-WSP1) exhibited significant anti-colon cancer activity both in vitro and in vivo. This study is to investigate the effects of Re-WSP1 on proliferation of colon cancer cells and to elucidate the relevant mechanisms. METHODS: Real-time quantitative PCR was used to detect the expression of miR-539 in colon cancer HT-29 and DLD1 cells. Colon cancer cells were transfected with miR-539 mimics and negative controls, and cell proliferation were detected by CCK8 and clonogenic assays. The target gene of miR-539 was predicted, and the dual luciferase reporter gene experiment was used to verify the target gene. After colon cancer cells were transfected with miR-539 mimics or inhibitors, the expression of target gene ß-catenin was detected by Western blot. miR-539 inhibitor confirmed cell proliferation. RESULTS: Re-WSP1 inhibited colon cancer cell growth in a dose-dependent manner. Re-WSP1 inhibited the expression of ß-catenin, which was partly reversed by LiCl treatment. Quantitative PCR analysis showed that the expression of miR-539 was significantly upregulated after Re-WSP1 treatment. Moreover, miR-539 negatively regulated the expression of ß-catenin by directly binding to the 3'UTR of ß-catenin mRNA. The cell growth inhibition and the decrease in ß-catenin expression induced by Re-WSP1 were significantly reversed by miR-539 inhibitor. CONCLUSION: Re-WSP1 suppresses colon cancer cell growth via the miR-539/ß-catenin axis.

The RNA editing factor DUA1 is crucial to chloroplast development at low temperature in rice.

Low temperature stress hinders plant growth and chloroplast development and can limit the geographic range of cultivars. In rice, japonica cultivars have greater chilling tolerance than indica cultivars, but the molecular mechanism underlying chilling tolerance is unclear. Here, we report an RNA-binding protein, DUA1, cloned from the indica cultivar Dular, which exhibits a deficiency in chloroplast development at an early stage of development under low-temperature conditions. DUA1 shares high sequence homology with the pentatricopeptide repeat family and functions in plastid RNA editing under low-temperature conditions. Our data suggest that DUA1 can bind to the plastid-encoded rps8-182 transcript and disruption of DUA1 activity impairs editing. The RNA editing cofactor WSP1, a partner of DUA1, also participates in chloroplast development at low temperature. Western blot analysis indicates that WSP1 enhances DUA1 stability under low temperatures. DUA1 sequence analyses of rice core germplasm revealed that three major haplotypes of DUA1 and one haplotype showed substantial differences in chlorophyll content under low-temperature conditions. Variation at DUA1 may play an important role in the adaptation of rice to different growing regions.

Eng2 is a component of a dynamic protein complex required for endocytic uptake in fission yeast.

Eng2 is a glucanase required for spore release, although it is also expressed during vegetative growth, suggesting that it might play other cellular functions. Its homology to the Saccharomyces cerevisiae Acf2 protein, previously shown to promote actin polymerization at endocytic sites in vitro, prompted us to investigate its role in endocytosis. Interestingly, depletion of Eng2 caused profound defects in endocytic uptake, which were not due to the absence of its glucanase activity. Analysis of the dynamics of endocytic proteins by fluorescence microscopy in the eng2Δ strain unveiled a previously undescribed phenotype, in which assembly of the Arp2/3 complex appeared uncoupled from the internalization of the endocytic coat and resulted in a fission defect. Strikingly also, we found that Eng2-GFP dynamics did not match the pattern of other endocytic proteins. Eng2-GFP localized to bright cytosolic spots that moved around the cellular poles and occasionally contacted assembling endocytic patches just before recruitment of Wsp1, the Schizosaccharomyces pombe WASP. Interestingly, Csh3-YFP, a WASP-interacting protein, interacted with Eng2 by co-immunoprecipitation and was recruited to Eng2 in bright cytosolic spots. Altogether, our work defines a novel endocytic functional module, which probably couples the endocytic coat to the actin module.

Synergy between Wsp1 and Dip1 may initiate assembly of endocytic actin networks.

The actin filament nucleator Arp2/3 complex is activated at cortical sites in Schizosaccharomyces pombe to assemble branched actin networks that drive endocytosis. Arp2/3 complex activators Wsp1 and Dip1 are required for proper actin assembly at endocytic sites, but how they coordinately control Arp2/3-mediated actin assembly is unknown. Alone, Dip1 activates Arp2/3 complex without preexisting actin filaments to nucleate 'seed' filaments that activate Wsp1-bound Arp2/3 complex, thereby initiating branched actin network assembly. In contrast, because Wsp1 requires preexisting filaments to activate, it has been assumed to function exclusively in propagating actin networks by stimulating branching from preexisting filaments. Here we show that Wsp1 is important not only for propagation but also for initiation of endocytic actin networks. Using single molecule total internal reflection fluorescence microscopy we show that Wsp1 synergizes with Dip1 to co-activate Arp2/3 complex. Synergistic co-activation does not require preexisting actin filaments, explaining how Wsp1 contributes to actin network initiation in cells.

Vascular Effects of H2S-Donors: Fluorimetric Detection of H2S Generation and Ion Channel Activation in Human Aortic Smooth Muscle Cells.

Hydrogen sulfide (H2S) evokes vascular effects through several mechanisms including in wide part the activation of some ion channels such as ATP-sensitive potassium (KATP) channels and voltage-gated Kv7 potassium channels. Electrophysiological methods are very accurate, but they require high expertise and high specialized equipment. A more manageable fluorimetric technique which allows to record the membrane potential variations by the employment of an anionic bis-oxonol dye named DiBac4(3) with the administration of different blockers of several potassium channels could be useful to discover the targets of H2S-induced vascular hyperpolarization. Coupled with this technique, a fluorimetric detection (by the use of WSP-1 dye) of H2S generation in human vascular smooth muscle cells after H2S-donor administration could confirm the ability of these molecules to evoke the hyperpolarizing effect through the H2S release.

Dip1 Co-opts Features of Branching Nucleation to Create Linear Actin Filaments that Activate WASP-Bound Arp2/3 Complex.

When activated by Wiskott-Aldrich syndrome proteins (WASP), Arp2/3 complex nucleates branched actin filaments important for processes like cellular motility and endocytosis [1]. WASP-mediated activation of Arp2/3 complex requires a preformed actin filament, ensuring that activation by WASP creates branched instead of linear filaments. However, this biochemical requirement also means that assembly of branched actin networks must be primed with an initial seed filament [2-4]. We recently described a class of activators called WISH/DIP/SPIN90 (WDS) proteins, which, unlike WASP, activate Arp2/3 complex without a preformed filament [4]. Although this property may allow WDS proteins to serve as seed filament generators, it is unknown whether actin filaments nucleated by WDS-activated Arp2/3 complex can activate WASP-bound Arp2/3 complex. Further, despite their potential importance as branched actin network initiators, little is known about how WDS proteins turn on Arp2/3 complex. Here, we use two-color single-molecule total internal reflection fluorescence (TIRF) microscopy to show that Dip1, the S. pombe WDS protein [5], co-opts features of branching nucleation to activate Arp2/3 complex. Specifically, it activates Arp2/3 complex to nucleate linear filaments analogous to the branch created by WASP-mediated activation. The barbed ends of Dip1-Arp2/3 nucleated filaments are free to elongate, and their pointed ends remain anchored to Dip1-bound Arp2/3 complex. The linear filaments nucleated by Dip1-bound Arp2/3 complex activate WASP-bound Arp2/3 complex as potently as spontaneously nucleated or branched actin filaments. These observations provide important insights into the regulation of Arp2/3 complex by its activators and the molecular basis for initiation of branched actin networks.