Long noncoding RNA LRG modulates Drosophila locomotion by sequestering Synaptotagmin 1 protein.

Apparently, the genomes of many organisms are pervasively transcribed, and long noncoding RNAs (lncRNAs) make up the majority of cellular transcripts. LncRNAs have been reported to play important roles in many biological processes; however, their effects on locomotion are poorly understood. Here, we presented a novel lncRNA, Locomotion Regulatory Gene (LRG), which participates in locomotion by sequestering Synaptotagmin 1 (SYT1). LRG deficiency resulted in higher locomotion speed which could be rescued by pan-neuronal overexpression but not by limited ellipsoid body, motoneuron or muscle-expression of LRG. At the molecular level, the synaptic vesicles (SVs) release and movement-related SYT1 protein was recognized as LRG-interacting protein candidate. Furthermore, LRG had no effects on SYT1 expression. Genetically, the behavioral defects in LRG mutant could be rescued by pan-neuronal knock-down of Syt1. Taken together, all the results suggested LRG exerts regulatory effects on locomotion via sequestering SYT1 thereby blocking its function without affecting its expression. Our work displays a new function of lncRNA and provides insights for revealing the pathogenesis of neurological diseases with motor disorders.

Local membrane source gathering by p62 body drives autophagosome formation.

Autophagosomes are double-membrane vesicles generated intracellularly to encapsulate substrates for lysosomal degradation during autophagy. Phase separated p62 body plays pivotal roles during autophagosome formation, however, the underlying mechanisms are still not fully understood. Here we describe a spatial membrane gathering mode by which p62 body functions in autophagosome formation. Mass spectrometry-based proteomics reveals significant enrichment of vesicle trafficking components within p62 body. Combining cellular experiments and biochemical reconstitution assays, we confirm the gathering of ATG9 and ATG16L1-positive vesicles around p62 body, especially in Atg2ab DKO cells with blocked lipid transfer and vesicle fusion. Interestingly, p62 body also regulates ATG9 and ATG16L vesicle trafficking flux intracellularly. We further determine the lipid contents associated with p62 body via lipidomic profiling. Moreover, with in vitro kinase assay, we uncover the functions of p62 body as a platform to assemble ULK1 complex and invigorate PI3KC3-C1 kinase cascade for PI3P generation. Collectively, our study raises a membrane-based working model for multifaceted p62 body in controlling autophagosome biogenesis, and highlights the interplay between membraneless condensates and membrane vesicles in regulating cellular functions.

In vitro Reconstitution of Phase-separated p62 Bodies on the Arp2/3-derived Actin Network.

In cells, p62/SQSTM1 undergoes liquid-liquid phase separation (LLPS) with poly-ubiquitin chains to form p62 bodies that work as a hub for various cellular events, including selective autophagy. Cytoskeleton components such as Arp2/3-derived branched actin network and motor protein myosin 1D have been shown to actively participate in the formation of phase-separated p62 bodies. Here, we describe a detailed protocol on the purification of p62 and other proteins, the assembly of the branched actin network, and the reconstitution of p62 bodies along with cytoskeletal structures in vitro. This cell-free reconstitution of p62 bodies vividly mimics the phenomenon in which low concentrations of protein in vivo rely on cytoskeleton dynamics to increase the local concentration to reach the threshold for phase separation. This protocol provides an easily implemented and typical model system to study cytoskeleton-involved protein phase separation.

Neurotransmitters Affect Larval Development by Regulating the Activity of Prothoracicotropic Hormone-Releasing Neurons in Drosophila melanogaster.

Ecdysone, an essential insect steroid hormone, promotes larval metamorphosis by coordinating growth and maturation. In Drosophila melanogaster, prothoracicotropic hormone (PTTH)-releasing neurons are considered to be the primary promoting factor in ecdysone biosynthesis. Recently, studies have reported that the regulatory mechanisms of PTTH release in Drosophila larvae are controlled by different neuropeptides, including allatostatin A and corazonin. However, it remains unclear whether neurotransmitters provide input to PTTH neurons and control the metamorphosis in Drosophila larvae. Here, we report that the neurotransmitters acetylcholine (ACh) affect larval development by modulating the activity of PTTH neurons. By downregulating the expression of different subunits of nicotinic ACh receptors in PTTH neurons, pupal volume was significantly increased, whereas pupariation timing was relatively unchanged. We also identified that PTTH neurons were excited by ACh application ex vivo in a dose-dependent manner via ionotropic nicotinic ACh receptors. Moreover, in our Ca2+ imaging experiments, relatively low doses of OA caused increased Ca2+ levels in PTTH neurons, whereas higher doses led to decreased Ca2+ levels. We also demonstrated that a low dose of OA was conveyed through OA ß-type receptors. Additionally, our electrophysiological experiments revealed that PTTH neurons produced spontaneous activity in vivo, which provides the possibility of the bidirectional regulation, coming from neurons upstream of PTTH cells in Drosophila larvae. In summary, our findings indicate that several different neurotransmitters are involved in the regulation of larval metamorphosis by altering the activity of PTTH neurons in Drosophila.

Lamina feedback neurons regulate the bandpass property of the flicker-induced orientation response in Drosophila.

Natural scenes contain complex visual cues with specific features, including color, motion, flicker, and position. It is critical to understand how different visual features are processed at the early stages of visual perception to elicit appropriate cellular responses, and even behavioral output. Here, we studied the visual orientation response induced by flickering stripes in a novel behavioral paradigm in Drosophila melanogaster. We found that free walking flies exhibited bandpass orientation response to flickering stripes of different frequencies. The most sensitive frequency spectrum was confined to low frequencies of 2-4 Hz. Through genetic silencing, we showed that lamina L1 and L2 neurons, which receive visual inputs from R1 to R6 neurons, were the main components in mediating flicker-induced orientation behavior. Moreover, specific blocking of different types of lamina feedback neurons Lawf1, Lawf2, C2, C3, and T1 modulated orientation responses to flickering stripes of particular frequencies, suggesting that bandpass orientation response was generated through cooperative modulation of lamina feedback neurons. Furthermore, we found that lamina feedback neurons Lawf1 were glutamatergic. Thermal activation of Lawf1 neurons could suppress neural activities in L1 and L2 neurons, which could be blocked by the glutamate-gated chloride channel inhibitor picrotoxin (PTX). In summary, lamina monopolar neurons L1 and L2 are the primary components in mediating flicker-induced orientation response. Meanwhile, lamina feedback neurons cooperatively modulate the orientation response in a frequency-dependent way, which might be achieved through modulating neural activities of L1 and L2 neurons.

Long noncoding RNA SMRG regulates Drosophila macrochaetes by antagonizing scute through E(spl)mß.

It is obvious that the majority of cellular transcripts are long noncoding RNAs (lncRNAs). Although studies suggested that lncRNAs participate in many biological processes through diverse mechanisms, however, little is known about their effects on epidermal mechanoreceptors. Here, we identified one novel Drosophila lncRNA, Scutellar Macrochaetes Regulatory Gene (SMRG), which regulates scutellar macrochaetes that act as mechanoreceptors by antagonizing the proneural gene scute (sc), through the repressor Enhancer-of-split mß (E(spl)mß). SMRG deficiency induced supernumerary scutellar macrochaetes and simultaneously a high sc RNA level in the adult thorax. Genetically, sc overexpression enhanced this supernumerary phenotype, while heterozygous sc mutant rescued this phenotype, both of which were mediated by E(spl)mß. At the molecular level, SMRG recruited E(spl)mß to the sc promoter region, which in turn suppressed sc expression. Our work presents a novel function of lncRNA and offers insights into the molecular mechanism underlying mechanoreceptor development.

cGMP-Dependent Protein Kinase Encoded by foraging Regulates Motor Axon Guidance in Drosophila by Suppressing Lola Function.

Correct pathfinding and target recognition of a developing axon are exquisitely regulated processes that require multiple guidance factors. Among these factors, the second messengers, cAMP and cGMP, are known to be involved in establishing the guidance cues for axon growth through different intracellular signaling pathways. However, whether and how cGMP-dependent protein kinase (PKG) regulates axon guidance remains poorly understood. Here, we show that the motor axons of intersegmental nerve b (ISNb) in the Drosophila embryo display targeting defects during axon development in the absence of foraging(for), a gene encoding PKG.In vivo tag expression revealed PKG to be present in the ventral nerve code at late embryonic stages, supporting its function in embryonic axon guidance. Mechanistic studies showed that the transcription factor longitudinal lacking(lola) genetically interacts with for.PKG physically associates with the LolaT isoform via the C-terminal zinc-finger-containing domain. Overexpression of PKG leads to the cytoplasmic retention of LolaT in S2 cells, suggesting a role for PKG in mediating the nucleocytoplasmic trafficking of Lola. Together, these findings reveal a novel function of PKG in regulating the establishment of neuronal connectivity by sequestering Lola in the cytoplasm. SIGNIFICANCE STATEMENT: Axon pathfinding and target recognition are important processes in the formation of specific neuronal connectivity, which rely upon precise coordinated deployment of multiple guidance factors. This paper reveals the role of cGMP-dependent protein kinase (PKG) in regulating the pathfinding and targeting of the developing axons in Drosophila Moreover, our study indicates that PKG regulates the cytoplasmic-nuclear trafficking of the transcription factor LolaT, suggesting a mechanism of PKG in directing motor axon guidance. These findings highlight a new function of PKG in axon guidance by suppressing a transcription factor.

Pontibacter soli sp. nov., isolated from the soil of a Populus rhizosphere in Xinjiang, China.

A Gram-stain negative, rod-shaped, non-motile and pink-pigmented bacterial strain, designated strain HYL7-26(T), was isolated from a soil in the Desert Park of Huyang forest located in Xinjiang, China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain HYL7-26(T) belongs to the genus Pontibacter in the family Cytophagaceae. The 16S rRNA gene sequence similarity between strain HYL7-26(T) and type strains of Pontibacter species ranged from 93.2 to 96.0 %. Strain HYL7-26(T) was found to contain iso-C15:0 (15.9 %), iso-C17:0 3-OH (9.5 %) and summed feature 4 (comprising anteiso-C17:1 B and/or iso-C17:1 I, 21.0 %, as defined by the MIDI system) as the major cellular fatty acids. The major respiratory quinone was identified as MK-7 and the DNA G+C content was determined to be 43.8 mol%. sym-Homospermidine was the major polyamine observed in the cells. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain HYL7-26(T) is considered to represent a novel species of the genus Pontibacter, for which the name Pontibacter soli sp. nov. is proposed. The type strain is HYL7-26(T) (=CCTCC AB 206240(T) = NRRL B-59490(T)).

Pontibacter populi sp. nov., isolated from the soil of a Euphrates poplar (Populus euphratica) forest.

A Gram-negative-staining, rod-shaped, non-motile and pink bacterial strain was isolated from the soil of a Populus euphratica forest located in Xinjiang, China. The strain, designated strain HYL7-15(T), was subjected to a taxonomic analysis using a polyphasic approach. 16S rRNA gene sequence analyses indicated that the isolate belonged to the phylum Bacteroidetes and was related to the genus Pontibacter, with sequence similarities ranging from 93.1 to 95.0% with other species of the genus Pontibacter. Strain HYL7-15(T) contained MK-7 as the predominant menaquinone and its DNA G+C content was 44.9 mol%. The major cellular fatty acids of the novel strain were iso-C(15:0) (16.49%), iso-C(17:0) 3-OH (10.96%) and summed feature 4 (comprising anteiso-C(17:1) B and/or iso-C(17:1) I, 18.46%). The major polar lipids were phosphatidylethanolamine (PE) and phosphatidylglycerol (PG); diphosphatidylglycerol (DPG), three unknown aminophospholipids (APLs) and two unknown phospholipids (PLs) were also detected. On the basis of the evidence presented, it is concluded that strain HYL7-15(T) represents a novel species of the genus Pontibacter, for which the name Pontibacter populi sp. nov. is proposed. The type strain is HYL7-15(T) (=CCTCC AB 206239(T)=NRRL B-59488(T)).

Mucilaginibacter soli sp. nov., isolated from Arctic tundra soil.

A novel pale-pink-coloured strain, designated R9-65(T), was isolated from a tundra soil near Ny-Ålesund, Svalbard Archipelago, Norway (78° N). The cells were facultatively anaerobic, Gram-staining-negative, non-motile and rod-shaped. Growth occurred at 4-32 °C (optimum, 25-28 °C), at pH 5.0-9.0 (optimum, pH 6.0-7.0) and with 0-1.0% (w/v) NaCl (optimum, no NaCl). Flexirubin-type pigments were absent. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain R9-65(T) belonged to the genus Mucilaginibacter in the family Sphingobacteriaceae. The 16S rRNA gene sequence similarity between strain R9-65(T) and type strains of related species ranged from 93.4 to 96.6%. Strain R9-65(T) contained summed feature 3 (C(16:1)ω7c and/or C(16:1)ω6c, 34.3%) and iso-C(15:0) (20.3%) as major cellular fatty acids, MK-7 as the major respiratory quinone, and phosphatidylethanolamine as the main polar lipid. The DNA G+C content of strain R9-65(T) was 47.2 mol%. On the basis of phylogenetic, physiological and chemotaxonomic data, strain R9-65(T) is considered to represent a novel species of the genus Mucilaginibacter, for which the name Mucilaginibacter soli sp. nov. is proposed. The type strain is R9-65(T) (=CCTCC AB 2010331(T)=NRRL B-59458(T)).

Cohnella arctica sp. nov., isolated from Arctic tundra soil.

A psychrotolerant Gram-reaction-negative, rod-shaped and orange-pigmented bacterium, designated strain M9-62T, which was motile by means of peritrichous flagella, was isolated from tundra soil sampled near Ny-Ålesund, Svalbard Islands, Norway (78° N). Growth occurred at 4-30 °C (optimum, 25 °C) and pH 5.0-8.0 (optimum, pH 6.0-7.0). Analysis of the 16S rRNA gene sequence of strain M9-62T placed it in the genus Cohnella; sequence similarities of the isolate with type strains of members of related genera ranged from 92.0 to 96.3 %. Strain M9-62T contained anteiso-C15:0 (51.1 %), iso-C16:0 (7.5 %) and C16:0 (6.1 %) as the major cellular fatty acids and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and lysyl-phosphatidylglycerol as the main polar lipids. The major respiratory quinone was MK-7 and the DNA G+C content was 50.3 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain M9-62T is considered to represent a novel species of the genus Cohnella, for which the name Cohnella arctica sp. nov. is proposed; the type strain is M9-62T (=CCTCC AB 2010228T=NRRL B-59459T).

Sphingomonas xinjiangensis sp. nov., isolated from desert sand.

A Gram-negative, aerobic, motile, Sphingomonas-like rod, strain 10-1-84(T), was isolated from a sand sample collected from the desert of Xinjiang, China. The isolate contained Q-10 as the predominant ubiquinone and C(18 : 1)ω7c, C(16 : 0), C(16 : 1)ω7c and C(14 : 0) 2-OH as the major fatty acids. The polyamine pattern contained predominantly sym-homospermidine. The main polar lipids were sphingoglycolipid, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidyldimethylethanolamine and an unknown polar lipid. The DNA G+C content was 63.3 mol%. 16S rRNA gene sequence similarity between strain 10-1-84(T) and the type strains of species of the genus Sphingomonas ranged from 91.11 to 96.54 %. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain 10-1-84(T) belonged to the genus Sphingomonas. On the basis of phylogenetic analysis and physiological and biochemical characterization, strain 10-1-84(T) represents a novel species of the genus Sphingomonas, for which the name Sphingomonas xinjiangensis sp. nov. is proposed. The type strain is 10-1-84(T) ( = CCTCC AB 208035(T)  = NRRL B-51332(T)).

The effect of protonation on the spectra and stabilities of alkoxyl substituted phthalocyaninatometals.

The protonation abilities of phthalocyaninatometals (MPcs) increase but their stabilities reduce by the introduction of alkoxyl substituents at alpha position. In the toluene, the order of mono-protonation rate for the tetra-alpha-(2, 2, 4-trimethyl-3-pentoxy)phthalocyaninatometals sorts with the center metals is Zn>Co>Cu>Ni>Fe, which is opposite to the order of their wavelength difference between the Q bands and X bands. However, their mono-protonated species can be decomposed easily at the rate order FePc>CoPc>CuPc>NiPc>ZnPc, analogous to their decomposition abilities in the benzoylperoxide (BPO) oxidation. In addition, it is interesting that a more remarkable decomposition is found when partial CuPc was mono-protonated.