VAMP724 and VAMP726 are involved in autophagosome formation in Arabidopsis thaliana.

Macroautophagy/autophagy, an evolutionarily conserved degradative process essential for cell homeostasis and development in eukaryotes, involves autophagosome formation and fusion with a lysosome/vacuole. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play important roles in regulating autophagy in mammals and yeast, but relatively little is known about SNARE function in plant autophagy. Here we identified and characterized two Arabidopsis SNAREs, AT4G15780/VAMP724 and AT1G04760/VAMP726, involved in plant autophagy. Phenotypic analysis showed that mutants of VAMP724 and VAMP726 are sensitive to nutrient-starved conditions. Live-cell imaging on mutants of VAMP724 and VAMP726 expressing YFP-ATG8e showed the formation of abnormal autophagic structures outside the vacuoles and compromised autophagic flux. Further immunogold transmission electron microscopy and electron tomography (ET) analysis demonstrated a direct connection between the tubular autophagic structures and the endoplasmic reticulum (ER) in vamp724-1 vamp726-1 double mutants. Further transient co-expression, co-immunoprecipitation and double immunogold TEM analysis showed that ATG9 (autophagy related 9) interacts and colocalizes with VAMP724 and VAMP726 in ATG9-positive vesicles during autophagosome formation. Taken together, VAMP724 and VAMP726 regulate autophagosome formation likely working together with ATG9 in Arabidopsis.Abbreviations: ATG, autophagy related; BTH, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester; Conc A, concanamycin A; EM, electron microscopy; ER, endoplasmic reticulum; FRET, Förster/fluorescence resonance energy transfer; MS, Murashige and Skoog; MVB, multivesicular body; PAS, phagophore assembly site; PM, plasma membrane; PVC, prevacuolar compartment; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TEM, transmission electron microscopy; TGN, trans-Golgi network; WT, wild-type.

Structural insights into how vacuolar sorting receptors recognize the sorting determinants of seed storage proteins.

In Arabidopsis, vacuolar sorting receptor isoform 1 (VSR1) sorts 12S globulins to the protein storage vacuoles during seed development. Vacuolar sorting is mediated by specific protein-protein interactions between VSR1 and the vacuolar sorting determinant located at the C terminus (ctVSD) on the cargo proteins. Here, we determined the crystal structure of the protease-associated domain of VSR1 (VSR1-PA) in complex with the C-terminal pentapeptide (468RVAAA472) of cruciferin 1, an isoform of 12S globulins. The 468RVA470 motif forms a parallel ß-sheet with the switch III residues (127TMD129) of VSR1-PA, and the 471AA472 motif docks to a cradle formed by the cargo-binding loop (95RGDCYF100), making a hydrophobic interaction with Tyr99. The C-terminal carboxyl group of the ctVSD is recognized by forming salt bridges with Arg95. The C-terminal sequences of cruciferin 1 and vicilin-like storage protein 22 were sufficient to redirect the secretory red fluorescent protein (spRFP) to the vacuoles in Arabidopsis protoplasts. Adding a proline residue to the C terminus of the ctVSD and R95M substitution of VSR1 disrupted receptor-cargo interactions in vitro and led to increased secretion of spRFP in Arabidopsis protoplasts. How VSR1-PA recognizes ctVSDs of other storage proteins was modeled. The last three residues of ctVSD prefer hydrophobic residues because they form a hydrophobic cluster with Tyr99 of VSR1-PA. Due to charge-charge interactions, conserved acidic residues, Asp129 and Glu132, around the cargo-binding site should prefer basic residues over acidic ones in the ctVSD. The structural insights gained may be useful in targeting recombinant proteins to the protein storage vacuoles in seeds.

Relationships between Severity of Internet Gaming Disorder, Severity of Problematic Social Media Use, Sleep Quality and Psychological Distress.

Internet gaming and social media use are prevalent and integral to many people's lives. However, excessive engagement in either could lead to negative health impacts. This study aimed to investigate relationships between severities of internet gaming disorder (IGD) and problematic social media use (operationalized as social media addiction; SMA) with sleep quality and psychological distress among young adults. A cross-sectional study with snowball sampling was conducted among Hong Kong university students in 2019. All participants (n = 300; mean (SD) age = 20.89 (1.48); 122 males (40.67%)) responded to an online survey that included Chinese versions of the Internet Gaming Disorder Scale-Short Form (IGDS9-SF), Bergen Social Media Addiction Scale (BSMAS), Pittsburgh Sleep Quality Index (PSQI), and Depression Anxiety Stress Scales (DASS-21). Multiple linear regressions demonstrated that IGDS-SF9 scores demonstrated associations with psychological distress measures (standardized coefficient (ß) = 0.295 for depression, 0.325 for anxiety, 0.339 for stress, all p < 0.001). BSMAS scores showed similar albeit numerically less robust associations (ß = 0.235 for depression, p < 0.001; 0.219 for anxiety, p = 0.001; 0.262 for stress, p < 0.001). BSMAS scores demonstrated associations with poorer sleep quality (ß = 0.292; p < 0.001) and IGDS9-SF scores (ß = 0.157; p = 0.024) showed a significantly less robust association (p = 0.01 for comparing the two ßs). These findings suggest that both severities of IGD and SMA associate with more psychological distress and poorer sleep quality, although the strengths of associations may differ.

A whole-cell electron tomography model of vacuole biogenesis in Arabidopsis root cells.

Plant vacuoles are dynamic organelles that play essential roles in regulating growth and development. Two distinct models of vacuole biogenesis have been proposed: separate vacuoles are formed by the fusion of endosomes, or the single interconnected vacuole is derived from the endoplasmic reticulum. These two models are based on studies of two-dimensional (2D) transmission electron microscopy and 3D confocal imaging, respectively. Here, we performed 3D electron tomography at nanometre resolution to illustrate vacuole biogenesis in Arabidopsis root cells. The whole-cell electron tomography analysis first identified unique small vacuoles (SVs; 400-1,000 nm in diameter) as nascent vacuoles in early developmental cortical cells. These SVs contained intraluminal vesicles and were mainly derived/matured from multivesicular body (MVB) fusion. The whole-cell vacuole models and statistical analysis on wild-type root cells of different vacuole developmental stages demonstrated that central vacuoles were derived from MVB-to-SV transition and subsequent fusions of SVs. Further electron tomography analysis on mutants defective in MVB formation/maturation or vacuole fusion demonstrated that central vacuole formation required functional MVBs and membrane fusion machineries.