Stress Granule Assembly Can Facilitate but Is Not Required for TDP-43 Cytoplasmic Aggregation

Stress granules (SGs) are hypothesized to facilitate TAR DNA-binding protein 43 (TDP-43) cytoplasmic mislocalization and aggregation, which may underly amyotrophic lateral sclerosis pathology. However, much data for this hypothesis is indirect. Additionally, whether P-bodies (PBs; related mRNA-protein granules) affect TDP-43 phenotypes is unclear. Here, we determine that induction of TDP-43 expression in yeast results in the accumulation of SG-like foci that in >90% of cases become the sites where TDP-43 cytoplasmic foci first appear. Later, TDP-43 foci associate less with SGs and more with PBs, though independent TDP-43 foci also accumulate. However, depleting or over-expressing yeast SG and PB proteins reveals no consistent trend between SG or PB assembly and TDP-43 foci formation, toxicity or protein abundance. In human cells, immunostaining endogenous TDP-43 with different TDP-43 antibodies reveals distinct localization and aggregation behaviors. Following acute arsenite stress, all phospho-TDP-43 foci colocalize with SGs. Interestingly, in SG assembly mutant cells (G3BP1/2ΔΔ), TDP-43 is enriched in nucleoli. Finally, formation of TDP-43 cytoplasmic foci following low-dose chronic arsenite stress is impaired, but not completely blocked, in G3BP1/2ΔΔ cells. Collectively, our data suggest that SG and PB assembly may facilitate TDP-43 cytoplasmic localization and aggregation but are likely not essential for these events.

Changes in biochemistry and histochemical characteristics during somatic embryogenesis in Ormosia henryi Prain

ABSTRACTMature embryos were used as an explant for embryogenic callus (EC) induction, and then EC was further developed to form somatic embryos during somatic embryogenesis (SE) of Ormosia henryi Prain; however, some mature embryos could induced non-embryogenic callus (NEC), browning callus (BC) or snowflake callus (SC). These phenomena might be related to the biochemical and histochemical differences during somatic embryo induction. The present study was conducted to analyze the biochemical events and histochemical changes at different SE stages in 0. henryi. The contents of soluble sugar, starch, soluble protein, H2O2, and endogenous hormones and the activities of polyphenoloxidase (PPO), superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD) and catalase (CAT) were measured at different SE stages, such as EC, globular embryo (GE), and cotyledon embryo (CE), and in abnormal tissue, such as NEC, BC, and SC. The results showed that the contents of soluble sugar and starch; the activities of PPO, SOD, APX and POD; and the ratios of indole-3-acetic acid/abscisic acid (IAA/ABA), IAA/gibberellins (IAA/GAs), auxin /GAs (AUX/GAs), and AUX/ABA decreased gradually at different SE stages. In contrast, the contents of soluble protein, H2O2, all endogenous hormones gradually increased. However, CAT activity and the ratios of IAA/cytokinins (IAA/CKs), AUX/CKs, ABA/CKs, and GAs/CKs first increased and then decreased. The high contents of GAs and ABA, high ratios of ABA/CKs and GAs/CKs and low ratios of IAA/ABA, IAA/GAs, AUX/GAs and AUX/ABA were responsible for the inability of the callus to form EC. The low enzyme activities, low contents of energy substances and H2O2 were related to NEC formation. The high contents of soluble sugar, H2O2, AUX, CKs and PPO activity and the low content of soluble protein were the basic causes of BC formation. The high-energy substances contents and low activities of SOD and POD facilitated SC formation. Histochemical observation showed that starch granule staining gradually lightened with SE development, but protein granules were darkly stained. Compared with EC, starch and protein granules were stained darker in SC, and lighter in NEC and BC. These results showed that energy substances were the material basis of SE, which affected enzyme activities, regulated reactive oxygen species (ROS) metabolism, and thus regulated the morphogenesis and development of somatic embryos. In addition, the contents and ratios of endogenous hormones affected the dedifferentiation, dedifferentiation and embryogenesis of somatic cells. To induce EC from mature embryos and further develop their formation into somatic embryos, it is necessary to adjust the energy supply and hormone ratio in the medium.One-sentence summarySomatic embryogenesis and abnormal callus tissues formation of Ormosia henryi Prain were associated with energy substances, reactive oxygen species, enzyme activities and endogenous hormones, as well as histochemical characteristics.

Fusion protein EWS-FLI1 is incorporated into a protein granule in cells

ABSTRACTEwing sarcoma is driven by fusion proteins containing a low complexity (LC) domain that is intrinsically disordered and a powerful transcriptional regulator. The most common fusion protein found in Ewing sarcoma, EWS-FLI1, takes its LC domain from the RNA-binding protein EWSR1 (Ewing Sarcoma RNA-binding protein 1) and a DNA-binding domain from the transcription factor FLI1 (Friend Leukemia Virus Integration 1). EWS-FLI1 binds RNA polymerase II (RNA Pol II) and can self-assemble through a process known as phase separation. The ability of self-oligomerizing RNA-binding proteins like EWSR1 to assemble into ribonucleoprotein granules in cells has received significant attention but the role of phase separation in EWS-FLI1 activity is less understood. We investigated the intersecting roles of EWSR1 and EWS-FLI1 to control gene expression and tumorigenic cell growth in Ewing sarcoma. We also studied interactions among EWS-FLI1, EWSR1, and RNA Pol II. We applied a crosslinking approach to demonstrate the incorporation of EWSR1 and RNA Pol II into protein granules in cells. We also identified protein granules in cells associated with the fusion protein, EWS-FLI1. Interactions through the LC domain, which allow EWS-FLI1 to bind EWSR1 and RNA Pol II, were found to be required for inclusion into the cellular granules observed by TEM. The physical characterization of EWS-FLI1 assemblies reported here offers insight into a large protein assembly that may allow EWS-FLI1 to engage its wide network of protein partners while driving tumorigenesis.

The use of granules of food protein concentrated non hydrolyzed at growing piglets

This article presents a study of the effectiveness of concentrated non-hydrolyzed feed protein granules (CNHPG) on the productivity dynamics and the quality final prod-ucts when included in the diets of suckling pigs and weaned piglets. Concentrated non-hydrolyzed feed protein granules represent a new type of soy protein concentrate free from antibiotics, hormonal remedies and GMOs. This feed supplement can be included either individually to the basic ration or into the composition of mixed feeds as the main highly digestible protein source with the original amino acid composition. The results of the research showed that CNHPG contribute to an increase in piglets average daily gain of 2.0-2.7%, reduce feed costs for obtaining a unit of production by 0.1-0.3 kg, as well as an additional gross gain in body weight from 8.2% to 14.6%