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Cyclic electron flow around photosystem I (PSI) is a mechanism by which photosynthetic organisms balance the levels of ATP and NADPH necessary for efficient photosynthesis 1 , 2 . NAD(P)H dehydrogenase-like complex (NDH) is a key component of this pathway in most oxygenic photosynthetic organisms 3 , 4 and is the last large photosynthetic membrane-protein complex for which the structure remains unknown. Related to the respiratory NADH dehydrogenase complex (complex I), NDH transfers electrons originating from PSI to the plastoquinone pool while pumping protons across the thylakoid membrane, thereby increasing the amount of ATP produced per NADP + molecule reduced 4 , 5 . NDH possesses 11 of the 14 core complex I subunits, as well as several oxygenic-photosynthesis-specific (OPS) subunits that are conserved from cyanobacteria to plants 3 , 6 . However, the three core complex I subunits that are involved in accepting electrons from NAD(P)H are notably absent in NDH 3 , 5 , 6 , and it is therefore not clear how NDH acquires and transfers electrons to plastoquinone. It is proposed that the OPS subunits—specifically NdhS—enable NDH to accept electrons from its electron donor, ferredoxin 3 – 5 , 7 . Here we report a 3.1 Å structure of the 0.42-MDa NDH complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, obtained by single-particle cryo-electron microscopy. Our maps reveal the structure and arrangement of the principal OPS subunits in the NDH complex, as well as an unexpected cofactor close to the plastoquinone-binding site in the peripheral arm. The location of the OPS subunits supports a role in electron transfer and defines two potential ferredoxin-binding sites at the apex of the peripheral arm. These results suggest that NDH could possess several electron transfer routes, which would serve to maximize plastoquinone reduction and avoid deleterious off-target chemistry of the semi-plastoquinone radical. The structure of NDH, a photosynthetic membrane-protein complex that is related to respiratory complex I, is obtained by single-particle cryo-electron microscopy.

NAD(P)H dehydrogenase-like (NDH) complex NDH-1L of cyanobacteria plays a crucial role in cyclic electron flow (CEF) around photosystem I and respiration processes. NDH-1L couples the electron transport from ferredoxin (Fd) to plastoquinone (PQ) and proton pumping from cytoplasm to the lumen that drives the ATP production. NDH-1L-dependent CEF increases the ATP/NADPH ratio, and is therefore pivotal for oxygenic phototrophs to function under stress. Here we report two structures of NDH-1L from Thermosynechococcus elongatus BP-1, in complex with one Fd and an endogenous PQ, respectively. Our structures represent the complete model of cyanobacterial NDH-1L, revealing the binding manner of NDH-1L with Fd and PQ, as well as the structural elements crucial for proper functioning of the NDH-1L complex. Together, our data provides deep insights into the electron transport from Fd to PQ, and its coupling with proton translocation in NDH-1L. NAD(P)H dehydrogenase-like complex NDH-1L couples the electron transport from ferredoxin (Fd) to plastoquinone (PQ) and proton pumping from cytoplasm to the lumen. Here authors report two structures of NDH-1L from Thermosynechococcus elongatus BP-1, in complex with one Fd and an endogenous PQ, respectively.

The nicotinamide adenine dinucleotide phosphate (NADPH) dehydrogenase (NDH) complex is crucial for photosynthetic cyclic electron flow and respiration, transferring electrons from ferredoxin to plastoquinone while transporting H + across the chloroplast membrane. This process boosts adenosine triphosphate production, regardless of NADPH levels. In flowering plants, NDH forms a supercomplex with photosystem I, enhancing its stability under high light. We report the cryo-electron microscopy structure of the NDH supercomplex in Spinacia oleracea at a resolution of 3.0–3.3 Å. The supercomplex consists of 41 protein subunits, 154 chlorophylls and 38 carotenoids. Subunit interactions are reinforced by 46 distinct lipids. The structure of NDH resembles that of mitochondrial complex I closely, including the quinol-binding site and an extensive internal aqueous passage for proton translocation. A well-resolved catalytic plastoquinone (PQ) occupies the PQ channel. The pronounced structural similarity to complex I sheds light on electron transfer and proton translocation within the NDH supercomplex. Here, the authors report the structure of the nicotinamide adenine dinucleotide phosphate dehydrogenase–photosystem I–light-harvesting complex I supercomplex, providing insights into subunit interactions and the role of cofactors.

Lipid droplets (LDs) have emerged as dynamic and interactive organelles with important roles in lipid metabolism and membrane biogenesis. Here, we report that Saccharomyces cerevisiae Env9 is a novel conserved oxidoreductase involved in LD morphology. Microscopic and biochemical studies confirm localization of tagged Env9 to LDs and implicate its C-terminal hydrophobic domain (aa241–265) in its membrane association and stability. Confocal studies reveal a role for Env9 in LD morphology. Env9 positively affects both formation of large LDs upon overexpression and LD proliferation under poor carbon source. In silico bioinformatic and modeling approaches establish that ENV9 is a widely conserved member of the short-chain dehydrogenase (SDR) superfamily. Bayesian phylogenetic studies strongly support ENV9 as an ortholog of human SDR retinol dehydrogenase 12 ( RDH12 ). Dehydrogenase activity of Env9 was confirmed by in vitro oxidoreductase assays. RDH12 mutations have been linked to Leber Congenital Amaurosis. Similar site-directed point mutations in the predicted Env9 oxidoreductase active site (N146L) or cofactor-binding site (G23–24A) abolished its reductase activity in vitro, consistent with those reported in other retinol dehydrogenases. The same residues were essential for affecting LD size and number in vivo. Taken together, our results implicate oxidoreductase activity of Env9 in its cellular role in LD morphology.

Filopodia are finger-like protrusive structures, containing actin bundles. By incubating frog egg extracts with supported lipid bilayers containing phosphatidylinositol 4,5 bisphosphate, we have reconstituted the assembly of filopodia-like structures (FLSs). The actin assembles into parallel bundles, and known filopodial components localize to the tip and shaft. The filopodia tip complexes self-organize--they are not templated by preexisting membrane microdomains. The F-BAR domain protein toca-1 recruits N-WASP, followed by the Arp2/3 complex and actin. Elongation proteins, Diaphanous-related formin, VASP, and fascin are recruited subsequently. Although the Arp2/3 complex is required for FLS initiation, it is not essential for elongation, which involves formins. We propose that filopodia form via clustering of Arp2/3 complex activators, self-assembly of filopodial tip complexes on the membrane, and outgrowth of actin bundles.

Piceatannol is a natural plant-derived compound with protective effects against cardiovascular diseases. However, its effect on cerebral ischaemia–reperfusion injury (CIRI) induced by oxidative stress remains unclear. This study aimed to investigate piceatannol’s antioxidation in CIRI. An in vitro oxygen–glucose deprivation followed by reoxygenation model was used and cell viability was measured. A middle cerebral artery occlusion followed by reperfusion model was used in vivo. Neurological function, encephalisation quotient, oedema, and volume of the cerebral infarction were then evaluated. The effects of piceatannol on histopathological findings, as well as the ultrastructure of the cortex, were analysed. The activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and lactate dehydrogenase (LDH) and the malondialdehyde (MDA) content was measured both in vitro and in vivo. Finally, the expression of nuclear factor erythroid-2-related factor 2 (Nrf2), hemeoxygenase-1 (HO-1), and nicotinamide adenine dinucleotide phosphate quinone oxidoreductase 1 (NQO1) in cerebral tissue was detected using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting. Our results demonstrated that cell viability in the piceatannol groups was increased. The SOD, GSH-Px activities were increased as LDH activity and MDA content decreased in the piceatannol groups both in vitro and in vivo, reflecting a decrease in oxidative stress. The neurological severity score and infarction volume in the piceatannol groups at doses of 10 and 20 mg/kg were lower than those of the model group. Furthermore, the damage seen on histopathological examination was partially attenuated by piceatannol. RT-qPCR and western blot analysis indicated that the expression of Nrf2, HO-1, and NQO1 were significantly increased by piceatannol. The results of the study demonstrate that piceatannol exerts a protective effect against CIRI.

Extension of neurites from a cell body is essential to form a functional nervous system; however, the mechanisms underlying neuritogenesis are poorly understood. Ena/VASP proteins regulate actin dynamics and modulate elaboration of cellular protrusions. We recently reported that cortical axon-tract formation is lost in Ena/VASP-null mice and Ena/VASP-null cortical neurons lack filopodia and fail to elaborate neurites. Here, we report that neuritogenesis in Ena/VASP-null neurons can be rescued by restoring filopodia formation through ectopic expression of the actin nucleating protein mDia2. Conversely, wild-type neurons in which filopodia formation is blocked fail to elaborate neurites. We also report that laminin, which promotes the formation of filopodia-like actin-rich protrusions, rescues neuritogenesis in Ena/VASP-deficient neurons. Therefore, filopodia formation is a key prerequisite for neuritogenesis in cortical neurons. Neurite initiation also requires microtubule extension into filopodia, suggesting that interactions between actin-filament bundles and dynamic microtubules within filopodia are crucial for neuritogenesis.

Hydrogen peroxide is a major by-product of peroxisomal metabolism and has the potential to cause critical oxidative damage. In all eukaryotes, catalase is thought to be instrumental in removing this H₂O₂. However, plants also contain a peroxisomal membrane-associated ascorbate-dependent electron transfer system, using ascorbate peroxidase and monodehydroascorbate reductase (MDAR). Here, I report that the conditional seedling-lethal sugar-dependent2 mutant of Arabidopsis thaliana is deficient in the peroxisomal membrane isoform of MDAR (MDAR4). Following germination, Arabidopsis seeds rely on storage oil breakdown to supply carbon skeletons and energy for early seedling growth, and massive amounts of H₂O₂ are generated within the peroxisome as a by-product of fatty acid β-oxidation. My data suggest that the membrane-bound MDAR4 component of the ascorbate-dependent electron transfer system is necessary to detoxify H₂O₂, which escapes the peroxisome. This function appears to be critical to protect oil bodies that are in close proximity to peroxisomes from incurring oxidative damage, which otherwise inactivates the triacylglycerol lipase SUGAR-DEPENDENT1 and cuts off the supply of carbon for seedling establishment.

Background: Metformin, a commonly prescribed medication for managing diabetes, has garnered increasing interest as a potential therapeutic option for combating cancer and aging. Methods: The current study investigated the effects of metformin treatment on human meibomian gland epithelial cells (hMGECs) at morphological, molecular, and electron microscopy levels. HMGECs were stimulated in vitro with 1 mM, 5 mM, and 10 mM metformin for 24, 48, and 72 h. The assessed outcomes were cell proliferation assays, lipid production, ultrastructural changes, levels of IGF-1, Nrf2, HO-1, apoptosis-inducing factor 1 (AIF1) at the protein level, and the expression of oxidative stress factors (matrix metallopeptidase 9, activating transcription factor 3, CYBB, or NADPH oxidase 2, xanthine dehydrogenase). Results: Morphological studies showed increased lipid production, the differentiation of hMGECs after stimulation with metformin, and the differentiation effects of undifferentiated hMGECs. Proliferation tests showed a reduction in cell proliferation with increasing concentrations over time. AIF1 apoptosis levels were not significantly regulated, but morphologically, the dying cells at a higher concentration of 5-10 mM showed a rupture and permeabilization of the plasma membrane, a swelling of the cytoplasm, and vacuolization after more than 48 h. The IGF-1 ELISA showed an irregular expression, which mostly decreased over time. Only at 72 h and 10 mM did we have a significant increase. Mitochondrial metabolic markers such as Nrf2 significantly increased over time, while HO-1 decreased partially. The RT-PCR showed a significant increase in MMP9, CYBB, XDH, and ATF with increasing time and metformin concentrations, indicating cell stress. Conclusions: Our results using a cell line suggest that metformin affects the cellular physiology of meibomian gland epithelial cells and induces cell stress in a dose- and duration-dependent manner, causing changes in their morphology and ultrastructure.

We previously demonstrated that a mutation in the 5' untranslated region of Diaphanous homolog 3 (DIAPH3) results in 2 to 3-fold overexpression of the gene, leading to a form of delayed onset, progressive human deafness known as AUNA1 (auditory neuropathy, nonsyndromic, autosomal dominant, 1). To investigate the mechanism of deafness, we generated two lines of transgenic mice overexpressing Diap3, the murine ortholog of DIAPH3, on an FVB/NJ background. Line 771 exhibits a relatively mild 20 dB hearing loss at 12 kHz at 4 and 8 weeks of age, progressing to 40 dB and 60 dB losses at 16 and 24 weeks, respectively, at 12 and 24 kHz. Line 924 shows no hearing loss at 4 or 8 weeks, but manifests 35 and 50 dB threshold shifts at 16 and 24 weeks, respectively, at both 12 and 24 kHz. Notably, mice from the two transgenic lines retain distortion product otoacoustic emissions, indicative of normal cochlear outer hair cell (OHC) function despite elevation of auditory thresholds. Scanning electron microscopy of the organ of Corti demonstrates striking anomalies of the inner hair cell (IHC) stereocilia, while OHCs are essentially intact. Over time, IHCs of both lines develop elongated stereocilia that appear fused with neighboring stereocilia, in parallel to the time course of hearing loss in each line. Furthermore, we observe significant reduction in the number of IHC ribbon synapses over 24 weeks in both lines, although this reduction does not correlate temporally with onset and progression of hearing loss or stereociliary anomalies. In summary, overexpression of wild-type Diap3 in two lines of transgenic mice results in hearing loss that recapitulates human AUNA1 deafness. These findings suggest an essential role of Diap3 in regulating assembly and/or maintenance of actin filaments in IHC stereocilia, as well as a potential role at the IHC ribbon synapse.