The HEAT repeat protein HPO-27 is a lysosome fission factor.

Lysosomes are degradation and signalling centres crucial for homeostasis, development and ageing1. To meet diverse cellular demands, lysosomes remodel their morphology and function through constant fusion and fission2,3. Little is known about the molecular basis of fission. Here we identify HPO-27, a conserved HEAT repeat protein, as a lysosome scission factor in Caenorhabditis elegans. Loss of HPO-27 impairs lysosome fission and leads to an excessive tubular network that ultimately collapses. HPO-27 and its human homologue MROH1 are recruited to lysosomes by RAB-7 and enriched at scission sites. Super-resolution imaging, negative-staining electron microscopy and in vitro reconstitution assays reveal that HPO-27 and MROH1 self-assemble to mediate the constriction and scission of lysosomal tubules in worms and mammalian cells, respectively, and assemble to sever supported membrane tubes in vitro. Loss of HPO-27 affects lysosomal morphology, integrity and degradation activity, which impairs animal development and longevity. Thus, HPO-27 and MROH1 act as self-assembling scission factors to maintain lysosomal homeostasis and function.

Structural insights into the molecular effects of the anthelmintics monepantel and betaine on the Caenorhabditis elegans acetylcholine receptor ACR-23.

Anthelmintics are drugs used for controlling pathogenic helminths in animals and plants. The natural compound betaine and the recently developed synthetic compound monepantel are both anthelmintics that target the acetylcholine receptor ACR-23 and its homologs in nematodes. Here, we present cryo-electron microscopy structures of ACR-23 in apo, betaine-bound, and betaine- and monepantel-bound states. We show that ACR-23 forms a homo-pentameric channel, similar to some other pentameric ligand-gated ion channels (pLGICs). While betaine molecules are bound to the classical neurotransmitter sites in the inter-subunit interfaces in the extracellular domain, monepantel molecules are bound to allosteric sites formed in the inter-subunit interfaces in the transmembrane domain of the receptor. Although the pore remains closed in betaine-bound state, monepantel binding results in an open channel by wedging into the cleft between the transmembrane domains of two neighboring subunits, which causes dilation of the ion conduction pore. By combining structural analyses with site-directed mutagenesis, electrophysiology and in vivo locomotion assays, we provide insights into the mechanism of action of the anthelmintics monepantel and betaine.

The Metabolite Saccharopine Impairs Neuronal Development by Inhibiting the Neurotrophic Function of Glucose-6-Phosphate Isomerase.

Mutations in the Aminoadipate-Semialdehyde Synthase (AASS) gene encoding α-aminoadipic semialdehyde synthase lead to hyperlysinemia-I, a benign metabolic variant without clinical significance, and hyperlysinemia-II with developmental delay and intellectual disability. Although both forms of hyperlysinemia display biochemical phenotypes of questionable clinical significance, an association between neurologic disorder and a pronounced biochemical abnormality remains a challenging clinical question. Here, we report that Aass mutant male and female mice carrying the R65Q mutation in α-ketoglutarate reductase (LKR) domain have an elevated cerebral lysine level and a normal brain development, whereas the Aass mutant mice carrying the G489E mutation in saccharopine dehydrogenase (SDH) domain exhibit elevations of both cerebral lysine and saccharopine levels and a smaller brain with defective neuronal development. Mechanistically, the accumulated saccharopine, but not lysine, leads to impaired neuronal development by inhibiting the neurotrophic effect of glucose-6-phosphate isomerase (GPI). While extracellular supplementation of GPI restores defective neuronal development caused by G498E mutation in SDH of Aass. Altogether, our findings not only unravel the requirement for saccharopine degradation in neuronal development, but also provide the mechanistic insights for understanding the neurometabolic disorder of hyperlysinemia-II.SIGNIFICANCE STATEMENT The association between neurologic disorder and a pronounced biochemical abnormality in hyperlysinemia remains a challenging clinical question. Here, we report that mice carrying the R65Q mutation in lysine α-ketoglutarate reductase (LKR) domain of aminoadipate-semialdehyde synthase (AASS) have an elevated cerebral lysine levels and a normal brain development, whereas those carrying the G489E mutation in saccharopine dehydrogenase (SDH) domain of AASS exhibit an elevation of both cerebral lysine and saccharopine and a small brain with defective neuronal development. Furthermore, saccharopine impairs neuronal development by inhibiting the neurotrophic effect of glucose-6-phosphate isomerase (GPI). These findings demonstrate saccharopine degradation is essential for neuronal development.

Loss of RNA-Binding Protein HuR Leads to Defective Ependymal Cells and Hydrocephalus.

Multiciliated ependymal cells line the ventricle wall and generate CSF flow through ciliary beating. Defects in ependymal cells cause hydrocephalus; however, there are still significant gaps in our understanding the molecular, cellular and developmental mechanisms involved in the pathogenesis of hydrocephalus. Here, we demonstrate that specific deletion of RNA-binding protein (RBP) Hu antigen R (HuR) in the mouse brain results in hydrocephalus and causes postnatal death. HuR deficiency leads to impaired ependymal cell development with defective motile ciliogenesis in both female and male mice. Transcriptome-wide analysis reveals that HuR binds to mRNA transcripts related to ciliogenesis, including cilia and flagella associated protein 52 (Cfap52), the effector gene of Foxj-1 and Rfx transcriptional factors. HuR deficiency accelerates the degradation of Cfap52 mRNA, while overexpression of Cfap52 is able to promote the development of HuR-deficient ependymal cells. Taken together, our results unravel the important role of HuR in posttranscriptional regulation of ependymal cell development by stabilizing Cfap52 mRNA.SIGNIFICANCE STATEMENT This study identifies Hu antigen R (HuR) as a genetic factor involved in the pathogenesis of hydrocephalus. Mechanistically, HuR regulates ependymal cell differentiation and ciliogenesis through stabilizing Cfap52 mRNA, the effector gene of Foxj-1 and Rfx transcriptional factors.

Anatomic reconstruction of the lateral ligaments using allograft tendon and suspensory fixation for chronic lateral ankle instability with poor remnant quality: results and complications.

PURPOSE: Treatment of chronic lateral ankle instability (CLAI) with poor remnant quality is challenging. The aim of the present study was to evaluate clinical results and complications of anatomic reconstruction of the lateral ligaments using allograft tendon and suspensory fixation in the treatment of such patients. METHODS: One hundred and eight patients with CLAI, who were treated surgically using anatomic reconstruction with allograft tendon and suspensory fixation between April 2016 and January 2018 at our hospital, were retrospectively analysed. None of the patients had sufficient ligament remnants for the modified Broström procedure during the intraoperative evaluation. Eighteen patients were excluded. Seventeen patients were lost to follow-up and 73 patients completed the study. The mean duration of instability symptoms was 39.1 months (range, 6-480 months). The mean follow-up time was 57.5 months (range, 48-69 months). Clinical results were evaluated using the Karlsson scoring scale, American Orthopaedic Foot and Ankle Society-Ankle and Hindfoot (AOFAS-AH) score, visual analogue scale (VAS), patients' subjective satisfaction, and incidence of complications. Mechanical stability was evaluated using the varus talar tilt angle (TTA) and anterior talar displacement (ATD). RESULTS: The AOFAS-AH scores significantly improved from 67.7 ± 8.5 points to 89.8 ± 9.5 (p < 0.001). The Karlsson scoring scales evolved from 58.8 ± 16.5 to 88.4 ± 11.2 (p < 0.001). VAS scores significantly decreased from 2.9 ± 1.3 to 1.1 ± 1.0 (p < 0.001). On stress radiographs, TTA decreased from 15.1 ± 2.5 degrees to 5.8 ± 2.1 degrees (p < 0.001), whereas ATD reduced from 13.4 ± 2.9 mm to 5.7 ± 1.5 mm (p < 0.001). Patients' subjective satisfaction indicated 46 excellent, 20 good, 5 fair, and 2 bad results. Postoperatively, 15 cases (20.5%) did not achieve complete relief of discomfort or swelling, 9 cases (12.3%) experienced joint stiffness or decreased range of motion, and 6 cases (8.2%) had soft tissue irritation. Residual instability and reoperation are rare. Allograft rejection or wound infection was not observed. CONCLUSION: For the CLAI patients with poor remnant quality, anatomic reconstruction of the lateral ligaments using allograft tendon and suspensory fixation is an effective procedure, while the top three complications in incidence were residual discomfort, joint stiffness, and soft tissue irritation. LEVELS OF EVIDENCE: Level IV, retrospective case series.

Comparison of prone-supine versus supine position for the treatment of pilon fractures via modified posteromedial approach combined with anterolateral approach.

BACKGROUND: Most of modified posteromedial approaches require prone position for the treatment of pilon fractures. We describe the technique of modified posteromedial approach under supine position. The goal of the study was to compare the radiographic and clinical outcomes of prone-supine versus supine position for the treatment of pilon fractures via modified posteromedial approach combined with anterolateral approach. METHODS: A total of 50 retrospectively consecutive pilon fractures that underwent open reduction internal fixation via modified posteromedial approach combined with anterolateral approach from 2016 to 2019 were reviewed at least a two-year follow up. The positions of patients were divided into two groups: prone-supine versus supine position (26 vs 24, respectively). The operation time, radiographic outcomes including bone union time and ratio of congruent articular reduction were evaluated. The post-operative function was evaluated using the Manchester Oxford score (MOXFQ) and the visual analogue score (VAS). The motion of ankle joint and complications and were also compared. RESULTS: The mean follow-up was 42.2(24.7-73.0) months in the prone-supine group and 42.7(37.3-56.5) months in the supine group (P = .87). The mean operation time was 141.9 ± 10.1 min in the prone-supine group and 107.5 ± 18.9 min in the supine group (P = .00). There was no significant difference in the bone union time and ratio of congruent articular reduction between the two groups. There was no significant difference in the final MOXFQ score, VAS score, and the mean range of ankle motion between the two groups (P > .05). The total incidence of complications was 11.5% in the prone-supine group and 16.6% in the supine group (P = .66). CONCLUSION: The patient in the prone-supine position versus supine position for pilon fractures via modified posteromedial approach combined with anterolateral approach contributed comparable quality of reduction, bone union time functional outcomes and complications. The supine technique was significantly shorter in terms of operation time.

WDR81 regulates adult hippocampal neurogenesis through endosomal SARA-TGFß signaling.

Adult hippocampal neurogenesis, a process considered important for hippocampal function, is regulated at multiple molecular levels. Mutations in the gene encoding the WD40 repeat-containing protein WDR81 are associated with neurological disorders, including cerebellar ataxia, mental retardation, quadrupedal locomotion syndrome (CAMRQ2), and microcephaly. In this study, we show that ablation of WDR81 in adult neural progenitor cells (aNPCs) markedly reduced adult hippocampal neurogenesis and impaired hippocampus-dependent learning. WDR81 suppresses endosomal PtdIns3P synthesis, likely by inhibiting the assembly of the PI3K-III complex. In the absence of WDR81, endosomal PtdIns3P levels are greatly elevated, leading to endosomal persistence of the PtdIns3P-binding protein SARA and consequently hyperactivation of SARA-dependent TGFß signaling. Inhibition of PI3K-III activity or suppression of SARA-dependent TGFß signaling markedly ameliorated the defective adult neurogenesis in WDR81-deficient mice. Taken together, these findings not only uncover the requirement for the WDR81-SARA-TGFß axis in adult hippocampal neurogenesis, but also suggest that defective adult hippocampal neurogenesis contributes to the etiology of WDR81-related neurological diseases.

Gain of circBRAF Represses Glioma Progression by Regulating miR-1290/FBXW7 Axis.

Dysregulated circular RNAs (circRNAs) have been confirmed to partake in the modulation of the glioma progression. Here, we intended to explore the role of circBRAF in glioma and the possible action mechanism. The expression levels of circBRAF, microRNA (miR)-1290 and F-box and WD repeat domain containing 7 (FBXW7) were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) or western blot. Cell viability was assessed by 3-(4, 5)-dimethylthiazole-2-y1)-2, 5-biphenyl tetrazolium bromide (MTT) assay. Cell cycle distribution was determined by flow cytometry. Cell migration and invasion were evaluated through Trans-well assay. Related protein levels were detected by western blot. Targeted relation among circBRAF, miR-1290 and FBXW7 was validated by dual-luciferase reporter, RNA immunoprecipitation (RIP) and pull-down assays. Xenograft model was constructed to explore the function of circBRAF in vivo. Expression of circBRAF and FBXW7 was decreased in glioma tissues and cells. Upregulation of circBRAF inhibited glioma cell proliferation and metastasis in vitro. MiR-1290 was upregulated in glioma, which was sponged by circBRAF. Besides, circBRAF elevated FBXW7 expression by targeting miR-1290. Introduction of miR-1290 or FBXW7 knockdown could counteract the inhibitory effects of circBRAF upregulation on the malignant phenotypes of glioma cells. Overexpression of circBRAF repressed the tumor growth in vivo. Upregulation of circBRAF suppressed glioma evolvement in vitro and in vivo by regulating miR-1290/FBXW7 axis, broadening the cognition of glioma progression.

Function and regulation of the Caenorhabditis elegans Rab32 family member GLO-1 in lysosome-related organelle biogenesis.

Cell type-specific modifications of conventional endosomal trafficking pathways lead to the formation of lysosome-related organelles (LROs). C. elegans gut granules are intestinally restricted LROs that coexist with conventional degradative lysosomes. The formation of gut granules requires the Rab32 family member GLO-1. We show that the loss of glo-1 leads to the mistrafficking of gut granule proteins but does not significantly alter conventional endolysosome biogenesis. GLO-3 directly binds to CCZ-1 and they both function to promote the gut granule association of GLO-1, strongly suggesting that together, GLO-3 and CCZ-1 activate GLO-1. We found that a point mutation in GLO-1 predicted to spontaneously activate, and function independently of it guanine nucleotide exchange factor (GEF), localizes to gut granules and partially restores gut granule protein localization in ccz-1(-) and glo-3(-) mutants. CCZ-1 forms a heterodimeric complex with SAND-1(MON1), which does not function in gut granule formation, to activate RAB-7 in trafficking pathways to conventional lysosomes. Therefore, our data suggest a model whereby the function of a Rab GEF can be altered by subunit exchange. glo-3(-) mutants, which retain low levels of GLO-3 activity, generate gut granules that lack GLO-1 and improperly accumulate RAB-7 in a SAND-1 dependent process. We show that GLO-1 and GLO-3 restrict the distribution of RAB-7 to conventional endolysosomes, providing insights into the segregation of pathways leading to conventional lysosomes and LROs.

Cell biology in China: Focusing on the lysosome.

The view that lysosomes are merely the recycling bins of the cell has changed greatly during recent years. Lysosomes are now known to play a central role in signal transduction, cellular adaptation, plasma membrane repair, immune responses and many other fundamental cellular processes. In conjunction with the seminal discoveries made by international colleagues, many important questions regarding lysosomes are being addressed by Chinese scientists. In this review, we briefly summarize recent exciting findings in China on lysosomal signaling, biogenesis, integrity and physiological functions.

A non-canonical GTPase interaction enables ORP1L-Rab7-RILP complex formation and late endosome positioning.

Endosomal transport represents the primary mode for intracellular trafficking and signaling transduction and thus has to be tightly controlled. The molecular processes controlling the endosomal positioning utilize several large protein complexes, one of which contains the small GTPase Rab7, Rab-interacting lysosomal protein (RILP), and oxysterol-binding protein-related protein 1 (ORP1L). Rab7 is known to interact with RILP through a canonical binding site termed the effector-interacting switch region, but it is not clear how Rab7 interacts with ORP1L, limiting our understanding of the overall process. Here, we report structural and biochemical investigation of the Rab7-ORP1L interaction. We found that, contrary to prior studies, the interaction between Rab7 and the N-terminal ankyrin repeat domain (ARDN) of ORP1L is independent of Rab7's GTP- or GDP-binding state. Moreover, we show that Rab7 interacts with ORP1L ARDN via a unique region consisting of helix3 (α3) and 310-helix 2 (η2). This architecture leaves the canonical effector-interacting switch regions available for RILP binding and thus allows formation of the ORP1L-Rab7-RILP tripartite complex. Mutational disruption of the interacting interface between ORP1L and Rab7 compromised the ability of ORP1L-Rab7-RILP to regulate the late endosome positioning. Collectively, our results again manifested the versatility in the interaction between GTPase and its effector.

Programmed cell death and clearance of cell corpses in Caenorhabditis elegans.

Programmed cell death is critical to the development of diverse animal species from C. elegans to humans. In C. elegans, the cell death program has three genetically distinguishable phases. During the cell suicide phase, the core cell death machinery is activated through a protein interaction cascade. This activates the caspase CED-3, which promotes numerous pro-apoptotic activities including DNA degradation and exposure of the phosphatidylserine "eat me" signal on the cell corpse surface. Specification of the cell death fate involves transcriptional activation of the cell death initiator EGL-1 or the caspase CED-3 by coordinated actions of specific transcription factors in distinct cell types. In the cell corpse clearance stage, recognition of cell corpses by phagocytes triggers several signaling pathways to induce phagocytosis of apoptotic cell corpses. Cell corpse-enclosing phagosomes ultimately fuse with lysosomes for digestion of phagosomal contents. This article summarizes our current knowledge about programmed cell death and clearance of cell corpses in C. elegans.

Optimization of hindfoot alignment radiography.

Background Cobey and Buck described methods to evaluate hindfoot alignment, but it is still unclear which method is better and easier to perform in clinical practice. Purpose To evaluate the optimal method for radiography of hindfoot alignment. Material and Methods We randomly selected 50 patients visiting the foot and ankle surgery outpatient department who underwent hindfoot alignment radiography between 1 July and 31 August 2015. Radiographs were taken using both Cobey's and Buck's methods. The patients were divided into three groups by calcaneal inclination angle. We assessed the calcaneotibial angle, calcaneovertical angle, and the distance from the bottom of the calcaneus to the mid-tibial axis. A comparative analysis was performed separately using the t-test. Results One hundred pairs of data for Cobey's and Buck's methods were obtained. The angles were analyzed separately in valgus, normal, and varus situations. The results showed no significant difference between Cobey's method and Buck's method regardless of any situation ( P > 0.05). Regarding the distance between the bottom of the calcaneus and the mid-tibial axis, the average result of Buck's method was about 1 mm larger than that of Cobey's method in the valgus and normal cases ( P < 0.05), except for varus cases ( P > 0.05). Conclusion Cobey's and Buck's techniques are the classic and popular hindfoot alignment assessment methods. The use of Buck's technique resulted in a better image with a less technical procedure and less time requirement. It is worthy of being popularized and used routinely for hindfoot radiography.

Clathrin and AP2 are required for phagocytic receptor-mediated apoptotic cell clearance in Caenorhabditis elegans.

Clathrin and the multi-subunit adaptor protein complex AP2 are central players in clathrin-mediated endocytosis by which the cell selectively internalizes surface materials. Here, we report the essential role of clathrin and AP2 in phagocytosis of apoptotic cells. In Caenorhabditis elegans, depletion of the clathrin heavy chain CHC-1 and individual components of AP2 led to a significant accumulation of germ cell corpses, which resulted from defects in both cell corpse engulfment and phagosome maturation required for corpse removal. CHC-1 and AP2 components associate with phagosomes in an inter-dependent manner. Importantly, we found that the phagocytic receptor CED-1 interacts with the α subunit of AP2, while the CED-6/Gulp adaptor forms a complex with both CHC-1 and the AP2 complex, which likely mediates the rearrangement of the actin cytoskeleton required for cell corpse engulfment triggered by the CED-1 signaling pathway. In addition, CHC-1 and AP2 promote the phagosomal association of LST-4/Snx9/18/33 and DYN-1/dynamin by forming a complex with them, thereby facilitating the maturation of phagosomes necessary for corpse degradation. These findings reveal a non-classical role of clathrin and AP2 and establish them as indispensable regulators in phagocytic receptor-mediated apoptotic cell clearance.

C. elegans mitochondrial factor WAH-1 promotes phosphatidylserine externalization in apoptotic cells through phospholipid scramblase SCRM-1.

Externalization of phosphatidylserine, which is normally restricted to the inner leaflet of plasma membrane, is a hallmark of mammalian apoptosis. It is not known what activates and mediates the phosphatidylserine externalization process in apoptotic cells. Here, we report the development of an annexin V-based phosphatidylserine labelling method and show that a majority of apoptotic germ cells in Caenorhabditis elegans have surface-exposed phosphatidylserine, indicating that phosphatidylserine externalization is a conserved apoptotic event in worms. Importantly, inactivation of the gene encoding either the C. elegans apoptosis-inducing factor (AIF) homologue (WAH-1), a mitochondrial apoptogenic factor, or the C. elegans phospholipid scramblase 1 (SCRM-1), a plasma membrane protein, reduces phosphatidylserine exposure on the surface of apoptotic germ cells and compromises cell-corpse engulfment. WAH-1 associates with SCRM-1 and activates its phospholipid scrambling activity in vitro. Thus WAH-1, after its release from mitochondria during apoptosis, promotes plasma membrane phosphatidylserine externalization through its downstream effector, SCRM-1.

Exploring lysosomal biology: current approaches and methods.

Lysosomes are the degradation centers and signaling hubs in the cell. Lysosomes undergo adaptation to maintain cell homeostasis in response to a wide variety of cues. Dysfunction of lysosomes leads to aging and severe diseases including lysosomal storage diseases (LSDs), neurodegenerative disorders, and cancer. To understand the complexity of lysosome biology, many research approaches and tools have been developed to investigate lysosomal functions and regulatory mechanisms in diverse experimental systems. This review summarizes the current approaches and tools adopted for studying lysosomes, and aims to provide a methodological overview of lysosomal research and related fields.

Daphmacrimines A-K, Daphniphyllum alkaloids from Daphniphyllum macropodum Miq.

Daphmacrimines A-K (1-11) were isolated from the leaves and stems of Daphniphyllum macropodum Miq. Their structures and stereochemistries were determined by extensive techniques, including HRESIMS, NMR, ECD, IR, and single-crystal X-ray crystallography. Daphmacrimines A-D (1-4) are unprecedented Daphniphyllum alkaloids with a 2-oxazolidinone ring. Daphmacrimine I (9) contains a nitrile group, which is relatively rare in naturally occurring alkaloids. The abilities of daphmacrimines A-D and daphmacrimines G-K to enhance lysosomal biogenesis were evaluated through LysoTracker Red staining. Daphmacrimine K (11) can induce lysosomal biogenesis and promote autophagic flux.

Caenorhabditis elegans protein arginine methyltransferase PRMT-5 negatively regulates DNA damage-induced apoptosis.

Arginine methylation of histone and non-histone proteins is involved in transcription regulation and many other cellular processes. Nevertheless, whether such protein modification plays a regulatory role during apoptosis remains largely unknown. Here we report that the Caenorhabditis elegans homolog of mammalian type II arginine methyltransferase PRMT5 negatively regulates DNA damage-induced apoptosis. We show that inactivation of C. elegans prmt-5 leads to excessive apoptosis in germline following ionizing irradiation, which is due to a CEP-1/p53-dependent up-regulation of the cell death initiator EGL-1. Moreover, we provide evidence that CBP-1, the worm ortholog of human p300/CBP, functions as a cofactor of CEP-1. PRMT-5 forms a complex with both CEP-1 and CBP-1 and can methylate the latter. Importantly, down-regulation of cbp-1 significantly suppresses DNA damage-induced egl-1 expression and apoptosis in prmt-5 mutant worms. These findings suggest that PRMT-5 likely represses CEP-1 transcriptional activity through CBP-1, which represents a novel regulatory mechanism of p53-dependent apoptosis.

WDR91 specifies the endosomal retrieval subdomain for retromer-dependent recycling.

Retromer-dependent endosomal recycling of membrane receptors requires Rab7, sorting nexin (SNX)-retromer, and factors that regulate endosomal actin organization. It is not fully understood how these factors cooperate to form endosomal subdomains for cargo retrieval and recycling. Here, we report that WDR91, a Rab7 effector, is the key factor that specifies the endosomal retrieval subdomain. Loss of WDR91 causes defective recycling of both intracellular and cell surface receptors. WDR91 interacts with SNXs through their PX domain, and with VPS35, thus promoting their interaction with Rab7. WDR91 also interacts with the WASH subunit FAM21. In WDR91-deficient cells, Rab7, SNX-retromer, and FAM21 fail to localize to endosomal subdomains, and endosomal actin organization is impaired. Re-expression of WDR91 enables Rab7, SNX-retromer, and FAM21 to concentrate at WDR91-specific endosomal subdomains, where retromer-mediated membrane tubulation and release occur. Thus, WDR91 coordinates Rab7 with SNX-retromer and WASH to establish the endosomal retrieval subdomains required for retromer-mediated endosomal recycling.