Non-canonical autophagy functions of ATG16L1 in epithelial cells limit lethal infection by influenza A virus.

Influenza A virus (IAV) and SARS-CoV-2 (COVID-19) cause pandemic infections where cytokine storm syndrome and lung inflammation lead to high mortality. Given the high social and economic cost of respiratory viruses, there is an urgent need to understand how the airways defend against virus infection. Here we use mice lacking the WD and linker domains of ATG16L1 to demonstrate that ATG16L1-dependent targeting of LC3 to single-membrane, non-autophagosome compartments - referred to as non-canonical autophagy - protects mice from lethal IAV infection. Mice with systemic loss of non-canonical autophagy are exquisitely sensitive to low-pathogenicity IAV where extensive viral replication throughout the lungs, coupled with cytokine amplification mediated by plasmacytoid dendritic cells, leads to fulminant pneumonia, lung inflammation and high mortality. IAV was controlled within epithelial barriers where non-canonical autophagy reduced IAV fusion with endosomes and activation of interferon signalling. Conditional mouse models and ex vivo analysis showed that protection against IAV infection of lung was independent of phagocytes and other leucocytes. This establishes non-canonical autophagy in airway epithelial cells as a novel innate defence that restricts IAV infection and lethal inflammation at respiratory surfaces.

Vitamin D intakes among women living with and without HIV in Canada.

BACKGROUND: Patterns of vitamin D intake are relatively unexplored among women living with HIV, despite its importance for women's health. We compared vitamin D dietary and supplement intakes in women with HIV and population-based national controls and investigated barriers to intake. METHODS: In this case-control study, women with HIV in the Children and Women: AntiRetrovirals and Markers of Aging (CARMA) cohort were matched with Canadian Multicentre Osteoporosis Study (CaMos) controls. Participants were queried for vitamin D in dairy consumption, supplementation/dosage, and sociodemographic variables. We assessed barriers to supplementation and factors associated with dietary intake by regression modelling. RESULTS: Ninety-five women living with HIV were age-matched to 284 controls. Women with HIV had lower income and bone mineral density and were more likely to smoke, take multiple medications and be non-white. Vitamin D dietary intake was lower in women living with HIV versus controls [0.76 vs. 1.79 µg/day; adjusted odds ratio (aOR) for greater than or equal to median intake 0.29 (0.12-0.61), p = 0.002], but any supplementation was higher [62.2% vs. 44.7%; aOR = 3.44 (95% CI: 1.16-11.00), p = 0.03]. Total vitamin D intake was similar between groups. Smoking was associated with no supplementation; non-white ethnicity and low income were related to lower dietary intake. CONCLUSIONS: Women living with HIV showed lower dietary vitamin D intake but higher supplementation rates, suggesting that care providers are promoting supplementation. Women living with HIV who smoke, have low incomes and are non-white may particularly benefit from targeted efforts to improve vitamin D intake.

Prevalence, causes, and impact of non-visualized pregnancy losses in a recurrent pregnancy loss population.

STUDY QUESTION: Does the occurrence of non-visualized pregnancy loss (NVPL) affect future reproductive outcomes in patients with recurrent pregnancy loss (RPL)? SUMMARY ANSWER: The number of previous NVPLs is a significant predictor of subsequent live birth in patients with RPL. WHAT IS KNOWN ALREADY: The number of preceding miscarriages is a strong indicator for future reproductive outcomes. However, NVPL particularly has been sparsely addressed in previous literature. STUDY DESIGN, SIZE, DURATION: We performed a retrospective cohort study of 1981 patients attending a specialized recurrent pregnancy loss clinic (RPL) from January 2012 to March 2021. A total of 1859 patients met the inclusion criteria of the study and were included in the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients with a history of RPL, defined as ≥2 pregnancy losses before 20 weeks gestation, who attended a specialized RPL clinic in a tertiary care center were included. Patients' evaluation included parental karyotyping, antiphospholipid antibodies screening, uterine cavity assessment with hysterosalpingography (HSG) or hysteroscopy, maternal thyroid stimulating hormone (TSH) testing, and serum hemoglobin A1C testing. Other investigations were performed only when indicated such as testing for inherited thrombophilias, serum prolactin, oral glucose tolerance test, and endometrial biopsy. Patients were divided into three groups; patients who experienced NVPLs only (pure NVPLs group), patients with only visualized pregnancy losses (pure VPLs group), and patients with history of both NVPLs and VPLs (mixed group). Statistical analysis was performed using Wilcoxon rank-sum tests for continuous variables and Fisher's exact tests for categorical variables. Significance was detected when P values <0.05. A logistic regression model was used to determine the impact of NVPLs and VPLs numbers on any live birth subsequent to the initial RPL clinic visit. MAIN RESULTS AND THE ROLE OF CHANCE: The prevalence of patients with pure NVPLs, pure VPLs, and mixed losses was 14.7% (274/1859), 31.8% (591/1859), and 53.5% (994/1859), respectively. The prevalence of acquired and congenital uterine anomalies diagnosed by HSG or hysteroscopy was significantly different between pure NVPLs, pure VPLs, and mixed groups (16.8% versus 23.7% versus. 20.7%, respectively P = 0.05). There were no significant differences in the results of other RPL investigations or baseline demographics between the three groups. A logistic regression model controlling for maternal age at the initial RPL clinic visit and the follow-up duration showed that the numbers of NVPLs (odds ratio (OR): 0.77, CI: 0.68-0.88) and VPLs (OR: 0.75, CI: 0.64-0.86) are strong predictors for subsequent live births after the initial RPL clinic visit (P < 0.001). The odds of having a live birth decreased by 23% and 25% with each additional NVPL and VPL, respectively. LIMITATIONS, REASONS FOR CAUTION: This study may be limited by its retrospective design. Some of our data, including home pregnancy tests and obstetric history, are based on patient self-reporting, which could have overstated the true prevalence of NVPLs. Another limitation is the lack of available live birth data for all patients at the time of the analysis. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this is the first study to examine and analyze the reproductive outcomes of patients with pure NVPLs in a substantial cohort of patients with RPL. NVPLs seem to affect future live births the same way as clinical miscarriages, which supports their inclusion in RPL definitions. STUDY FUNDING/COMPETING INTEREST(S): This study was supported in part by Canadian Institute Heath Grant (CIHR): Reference Number/W11-179912 and Women's Health Research Institute (WHRI), Vancouver, BC, Canada. M.A.B: Research grants from Canadian Institute for Health Research (CIHR) and Ferring Pharmaceutical. M.A.B. is on the advisory board for AbbVie and Baxter. TRIAL REGISTRATION NUMBER: N/A.

The WD40 domain of ATG16L1 is required for its non-canonical role in lipidation of LC3 at single membranes.

A hallmark of macroautophagy is the covalent lipidation of LC3 and insertion into the double-membrane phagophore, which is driven by the ATG16L1/ATG5-ATG12 complex. In contrast, non-canonical autophagy is a pathway through which LC3 is lipidated and inserted into single membranes, particularly endolysosomal vacuoles during cell engulfment events such as LC3-associated phagocytosis. Factors controlling the targeting of ATG16L1 to phagophores are dispensable for non-canonical autophagy, for which the mechanism of ATG16L1 recruitment is unknown. Here we show that the WD repeat-containing C-terminal domain (WD40 CTD) of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Using this strategy to inhibit non-canonical autophagy specifically, we show a reduction of MHC class II antigen presentation in dendritic cells from mice lacking the WD40 CTD Further, we demonstrate activation of non-canonical autophagy dependent on the WD40 CTD during influenza A virus infection. This suggests dependence on WD40 CTD distinguishes between macroautophagy and non-canonical use of autophagy machinery.

Enhancing knowledge, skills, and comfort in providing anesthesia assistance during obstetric general anesthesia for operating room nurses: a prospective observational study.

BACKGROUND: Due to a nationwide shortage of anesthesia assistants, operating room nurses are often recruited to assist with the induction of obstetric general anesthesia (GA). We developed and administered a training program and hypothesized there would be significant improvements in knowledge and skills in anesthesia assistance during obstetric GA by operating room nurses following training with adequate retention at six months. METHODS: Following informed consent, all operating room nurses at our institution were invited to participate in the study. Baseline knowledge of participants was assessed using a 14-item multiple choice questionnaire (MCQ), and skills were assessed using a 12-item checklist scored by direct observation during simulated induction of GA. Next, a 20-min didactic lecture followed by a ten-minute hands-on skills station were delivered. Knowledge and skills were immediately reassessed after training, and again at six weeks and six months. The primary outcomes of this study were adequate knowledge and skills retention at six months, defined as achieving ≥ 80% in MCQ and ≥ 80% in skills checklist scores and analyzed using longitudinal mixed-effects linear regression. RESULTS: A total of 34 nurses completed the study at six months. The mean MCQ score at baseline was 8.9 (95% confidence interval [CI], 8.5 to 9.4) out of 14. The mean skills checklist score was 5.5 (95% CI, 4.9 to 6.1) out of 12. The mean comfort scores for assisting elective and emergency Cesarean deliveries were 3.6 (95% CI, 3.2 to 3.9) and 3.1 (95% CI, 2.7 to 3.5) out of 5, respectively. There was a significant difference in the mean MCQ and skills checklist scores across the different study periods (overall P value < 0.001). Post hoc pairwise tests suggested that, compared with baseline, there were significantly higher mean MCQ scores at all time points after the training program at six weeks (11.9; 95% CI, 11.4 to 12.4; P < 0.001) and at six months (12.0; 95% CI, 11.5 to 12.4; P < 0.001). DISCUSSION: The knowledge and skills of operating room nurses in providing anesthesia assistance during obstetric GA at our institution were low at baseline. Following a single 30-min in-house, anesthesiologist-led, structured training program, scores in both dimensions significantly improved. Although knowledge improvements were adequately retained for up to six months, skills improvements decayed rapidly, suggesting that sessions should be repeated at six-week intervals, at least initially.

RéSUMé: CONTEXTE: En raison d'une pénurie nationale d'assistants en anesthésie, le personnel infirmier de la salle d'opération est souvent sollicité pour aider à l'induction de l'anesthésie générale (AG) obstétricale. Nous avons élaboré et administré un programme de formation et émis l'hypothèse qu'il y aurait des améliorations significatives dans les connaissances et les compétences en matière d'assistance en anesthésie pendant l'anesthésie générale obstétricale par les infirmières de salle d'opération après avoir suivi une formation, avec une rétention adéquate à six mois. MéTHODE: Après avoir obtenu le consentement éclairé, tout le personnel infirmier de salle d'opération de notre établissement a été invité à participer à l'étude. Les connaissances de base des participants ont été évaluées à l'aide d'un questionnaire à choix multiples (QCM) à 14 éléments, et les compétences ont été évaluées à l'aide d'une liste de contrôle de 12 éléments notée par observation directe lors d'une simulation d'induction d'anesthésie générale. Par la suite, un cours didactique de 20 minutes suivi d'une station de compétences pratiques de dix minutes a été donné. Les connaissances et les compétences ont été réévaluées immédiatement après la formation, puis de nouveau à six semaines et six mois. Les critères d'évaluation principaux de cette étude étaient la rétention adéquate des connaissances et des compétences à six mois, définie comme l'atteinte de ≥ 80 % dans les scores du QCM et ≥ 80 % dans les scores de la liste de contrôle des compétences et analysée à l'aide d'une régression linéaire longitudinale à effets mixtes. RéSULTATS: Au total, 34 infirmières ont terminé l'étude à six mois. Au début de l'étude, le score moyen au QCM était de 8,9 (intervalle de confiance [IC] à 95 %, 8,5 à 9,4) sur 14. Le score moyen sur la liste de contrôle des compétences était de 5,5 (IC 95 %, 4,9 à 6,1) sur 12. Les scores moyens d'aisance dans l'assistance pour un accouchement par césarienne programmé et d'urgence étaient de 3,6 (IC 95 %, 3,2 à 3,9) et 3,1 (IC 95 %, 2,7 à 3,5) sur 5, respectivement. Une différence significative a été observée dans les scores moyens au QCM et sur la liste de contrôle des compétences entre les différentes périodes d'étude (valeur globale P < 0,001). Les tests appariés post-hoc ont suggéré que, par rapport aux connaissances évaluées au début de l'étude, les scores moyens au QCM étaient significativement plus élevés à tous les moments après le programme de formation, à six semaines (11,9; IC 95 %, 11,4 à 12,4; P < 0,001) et à six mois (12,0; IC 95 %, 11,5 à 12,4; P < 0,001). DISCUSSION: Les connaissances et les compétences du personnel infirmier de salle d'opération dans la prestation d'une assistance en anesthésie pendant l'AG obstétricale dans notre établissement étaient faibles au commencement de notre étude. Après un seul programme de formation structuré de 30 minutes à l'interne, dirigé par un anesthésiologiste, les scores dans les deux dimensions se sont considérablement améliorés. Bien que les améliorations des connaissances aient été retenues de manière adéquate jusqu'à six mois, les améliorations des compétences se sont rapidement détériorées, ce qui suggère que les séances devraient être répétées à des intervalles de six semaines, au moins au début.

Functional diversification of Arabidopsis SEC1-related SM proteins in cytokinetic and secretory membrane fusion.

Sec1/Munc18 (SM) proteins contribute to membrane fusion by interacting with Qa-SNAREs or nascent trans-SNARE complexes. Gymnosperms and the basal angiosperm Amborella have only a single SEC1 gene related to the KEULE gene in Arabidopsis However, the genomes of most angiosperms including Arabidopsis encode three SEC1-related SM proteins of which only KEULE has been functionally characterized as interacting with the cytokinesis-specific Qa-SNARE KNOLLE during cell-plate formation. Here we analyze the closest paralog of KEULE named SEC1B. In contrast to the cytokinesis defects of keule mutants, sec1b mutants are homozygous viable. However, the keule sec1b double mutant was nearly gametophytically lethal, displaying collapsed pollen grains, which suggests substantial overlap between SEC1B and KEULE functions in secretion-dependent growth. SEC1B had a strong preference for interaction with the evolutionarily ancient Qa-SNARE SYP132 involved in secretion and cytokinesis, whereas KEULE interacted with both KNOLLE and SYP132. This differential interaction with Qa-SNAREs is likely conferred by domains 1 and 2a of the two SM proteins. Comparative analysis of all four possible combinations of the relevant SEC1 Qa-SNARE double mutants revealed that in cytokinesis, the interaction of SEC1B with KNOLLE plays no role, whereas the interaction of KEULE with KNOLLE is prevalent and functionally as important as the interactions of both SEC1B and KEU with SYP132 together. Our results suggest that functional diversification of the two SEC1-related SM proteins during angiosperm evolution resulted in enhanced interaction of SEC1B with Qa-SNARE SYP132, and thus a predominant role of SEC1B in secretion.

Crescentic glomerulonephritis in children.

BACKGROUND: To date, there is insufficient knowledge about crescentic glomerulonephritis (cGN), the most frequent immunologic cause of acute kidney injury in children. METHODS: Over a period of 16 years, we retrospectively analyzed kidney biopsy results, the clinical course, and laboratory data in 60 pediatric patients diagnosed with cGN. RESULTS: The underlying diseases were immune complex GN (n = 45/60, 75%), including IgA nephropathy (n = 19/45, 42%), lupus nephritis (n = 10/45, 22%), Henoch-Schoenlein purpura nephritis (n = 7/45, 16%) and post-infectious GN (n = 7/45, 16%), ANCA-associated pauci-immune GN (n = 10/60, 17%), and anti-glomerular basement-membrane GN (n = 1/60, 2%). Patient CKD stages at time of diagnosis and at a median of 362 days (range 237-425) were CKD I: n = 13/n = 29, CKD II: n = 15/n = 9, CKD III: n = 16/n = 7, CKD IV: n = 3/n = 3, CKD V: n = 13/n = 5. Course of cGN was different according to class of cGN, duration of disease from first clinical signs to diagnosis of cGN by biopsy, percentage of crescentic glomeruli, amount of tubular atrophy/interstitial fibrosis and necrosis on renal biopsy, gender, age, nephrotic syndrome, arterial hypertension, dialysis at presentation, and relapse. Forty-eight/60 children were treated with ≥ 5 (methyl-) prednisolone pulses and 53 patients received oral prednis(ol)one in combination with mycophenolate mofetil (n = 20), cyclosporine A (n = 20), and/or cyclophosphamide (n = 6), rituximab (n = 5), azathioprine (n = 2), tacrolimus (n = 1), and plasmapheresis/immunoadsorption (n = 5). CONCLUSIONS: The treatment success of cGN is dependent on early diagnosis and aggressive therapy, as well as on the percentage of crescentic glomeruli on renal biopsy and on the underlying type of cGN. CsA and MMF seem to be effective alternatives to cyclophosphamide.

[Rapid Progressive Glomerulonephritis in Children]. / Die Rasch Progessive Glomerulonephritis im Kindesalter.

Rapid Progressive Glomerulonephritis (RPGN) is a rare but very severe disease in children that often leads to acute and/or chronic renal failure requiring dialysis therapy. In this review, diagnostic criteria of RPGN including renal biopsy are discussed. An overview on general therapeutic strategies is discussed. Entities/diseases that cause RPGN are then described and special diagnostic and therapeutic features are given.

Cell type-specific transcriptome analysis in the early Arabidopsis thaliana embryo.

In multicellular organisms, cellular differences in gene activity are a prerequisite for differentiation and establishment of cell types. In order to study transcriptome profiles, specific cell types have to be isolated from a given tissue or even the whole organism. However, whole-transcriptome analysis of early embryos in flowering plants has been hampered by their size and inaccessibility. Here, we describe the purification of nuclear RNA from early stage Arabidopsis thaliana embryos using fluorescence-activated nuclear sorting (FANS) to generate expression profiles of early stages of the whole embryo, the proembryo and the suspensor. We validated our datasets of differentially expressed candidate genes by promoter-reporter gene fusions and in situ hybridization. Our study revealed that different classes of genes with respect to biological processes and molecular functions are preferentially expressed either in the proembryo or in the suspensor. This method can be used especially for tissues with a limited cell population and inaccessible tissue types. Furthermore, we provide a valuable resource for research on Arabidopsis early embryogenesis.

Arabidopsis µ-adaptin subunit AP1M of adaptor protein complex 1 mediates late secretory and vacuolar traffic and is required for growth.

Adaptor protein (AP) complexes are the predominant coat proteins of membrane vesicles in post-Golgi trafficking of mammalian cells. Each AP complex contains a specific medium subunit, µ-adaptin, that selects cargo proteins bearing sequence-specific sorting motifs. Much less is known about the AP complexes and their µ subunits in plants. Because of uncertain homology, the µ-adaptins of Arabidopsis have been designated muA through muD [Happel et al. (2004) Plant J 37(5):678-693]. Furthermore, only muD has been assigned to a specific AP complex, AP-3, involved in Golgi-vacuolar trafficking [Niihama et al. (2009) Plant Cell Physiol 50(12):2057-2068, Zwiewka et al. (2011) Cell Res 21(12):1711-1722, and Wolfenstetter et al. (2012) Plant Cell 24(1):215-232]. In contrast, the µ subunit of neither the post-Golgi trafficking AP-1 complex nor the endocytic AP-2 complex has been identified. Here, we report the functional analysis of redundant AP-1 µ-adaptins AP1M1 (also known as muB1) and AP1M2 (also known as muB2). Coimmunoprecipitation revealed that both AP1M2 and its less strongly expressed isoform AP1M1 are complexed with the large subunit γ-adaptin of AP-1. In addition, AP1M2 was localized at or near the trans-Golgi network. Knockout mutations of AP1M2 impaired pollen function and arrested plant growth whereas the ap1m1 ap1m2 double mutant was nearly pollen-lethal. At the cellular level, the absence of AP1M2 entailed inhibition of multiple trafficking pathways from the trans-Golgi network to the vacuole and to the plasma membrane in interphase and to the plane of cell division in cytokinesis. Thus, AP-1 is crucial in post-Golgi trafficking in plant cells and required for cell division and plant growth.

Plant cytokinesis: a tale of membrane traffic and fusion.

Cytokinesis separates the forming daughter cells. Higher plants have lost the ability to constrict the plasma membrane (PM) in the division plane. Instead, trans-Golgi network (TGN)-derived membrane vesicles are targeted to the centre of the division plane and generate, by homotypic fusion, the partitioning membrane named cell plate (CP). The CP expands in a centrifugal fashion until its margin fuses with the PM at the cortical division site. Mutant screens in Arabidopsis have identified a cytokinesis-specific syntaxin named KNOLLE and an interacting Sec1/Munc18 (SM) protein named KEULE both of which are required for vesicle fusion during cytokinesis. KNOLLE is only made during M-phase, targeted to the division plane and degraded in the vacuole at the end of cytokinesis. Here we address mechanisms of KNOLLE trafficking and interaction of KNOLLE with different soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) partners and with SM-protein KEULE, ensuring membrane fusion in cytokinesis.

α6ß1 and α7ß1 integrins are required in Schwann cells to sort axons.

During development, Schwann cells extend lamellipodia-like processes to segregate large- and small-caliber axons during the process of radial sorting. Radial sorting is a prerequisite for myelination and is arrested in human neuropathies because of laminin deficiency. Experiments in mice using targeted mutagenesis have confirmed that laminins 211, 411, and receptors containing the ß1 integrin subunit are required for radial sorting; however, which of the 11 α integrins that can pair with ß1 forms the functional receptor is unknown. Here we conditionally deleted all the α subunits that form predominant laminin-binding ß1 integrins in Schwann cells and show that only α6ß1 and α7ß1 integrins are required and that α7ß1 compensates for the absence of α6ß1 during development. The absence of either α7ß1 or α6ß1 integrin impairs the ability of Schwann cells to spread and to bind laminin 211 or 411, potentially explaining the failure to extend cytoplasmic processes around axons to sort them. However, double α6/α7 integrin mutants show only a subset of the abnormalities found in mutants lacking all ß1 integrins, and a milder phenotype. Double-mutant Schwann cells can properly activate all the major signaling pathways associated with radial sorting and show normal Schwann cell proliferation and survival. Thus, α6ß1 and α7ß1 are the laminin-binding integrins required for axonal sorting, but other Schwann cell ß1 integrins, possibly those that do not bind laminins, may also contribute to radial sorting during peripheral nerve development.

Longitudinal Assessment of Bone Mineral Density in Women Living With and Without HIV Across Reproductive Phases.

BACKGROUND: Women living with HIV commonly experience low areal bone mineral density (BMD), but whether this is affected by low ovarian hormonal states (prolonged amenorrhea or menopause) is unknown. We compared rates of BMD loss between women living with HIV and HIV-negative control women and investigated its association with low ovarian hormonal states. SETTING: Women living with HIV were enrolled from Vancouver Canada and controls from 9 Canadian sites. METHODS: This longitudinal analysis included age-matched women living with HIV in the Children and Women: AntiRetrovirals and Markers of Aging cohort and controls in the population-based Canadian Multicentre Osteoporosis Study. Rate of change/year in BMD at the total hip and lumbar spine (L1-L4) between 3 and 5 years was compared between groups, adjusting for sociodemographic and clinical variables. RESULTS: Ninety-two women living with HIV (median [interquartile range] age: 49.5 [41.6-54.1] years and body mass index: 24.1 [20.7-30.8] kg/m 2 ) and 278 controls (age: 49.0 [43.0-55.0] years and body mass index: 25.8 [22.9-30.6] kg/m 2 ) were included. Total hip BMD loss was associated with HIV (ß: -0.003 [95% CI: -0.006 to -0.0001] g/cm 2 /yr), menopause (ß: -0.007 [-0.01 to -0.005] g/cm 2 /yr), and smoking (ß: -0.003 [-0.006 to -0.0002] g/cm 2 /yr); BMD gain was linked with higher body mass index (ß: 0.0002 [0.0007-0.0004] g/cm 2 /yr). Menopause was associated with losing L1-L4 BMD (ß: -0.01 [-0.01 to -0.006] g/cm 2 /yr). Amenorrhea was not associated with BMD loss. CONCLUSIONS: HIV and menopause negatively influenced total hip BMD. These data suggest women living with HIV require hip BMD monitoring as they age.

Mechanisms of functional specificity among plasma-membrane syntaxins in Arabidopsis.

Syntaxins and interacting SNARE proteins enable membrane fusion in diverse trafficking pathways. The Arabidopsis SYP1 family of plasma membrane-localized syntaxins comprises nine members, of which KNOLLE and PEN1 play specific roles in cytokinesis and innate immunity, respectively. To identify mechanisms conferring specificity of action, we examined one member of each subfamily-KNOLLE/SYP111, PEN1/SYP121 and SYP132-in regard to subcellular localization, dynamic behavior and complementation of knolle and pen1 mutants when expressed from the same promoters. Our results suggest that cytokinesis-specific syntaxin requires high-level accumulation during cell-plate formation, which necessitates de novo synthesis rather than endocytosis of pre-made protein from the plasma membrane. In contrast, syntaxin in innate immunity does not need upregulation of expression but instead requires pathogen-induced and endocytosis-dependent retargeting to the infection site. This feature of PEN1 is not afforded by SYP132. Additionally, PEN1 could not substitute for KNOLLE because of SNARE domain differences, as revealed by protein chimeras. In contrast, SYP132 was able to rescue knolle as did KNOLLE-SYP132 chimeras. Unlike KNOLLE and PEN1, which appear to have evolved to perform specialized functions, SYP132 stably localized at the plasma membrane and thus might play a role in constitutive membrane fusion.

NSF/αSNAP2-mediated cis-SNARE complex disassembly precedes vesicle fusion in Arabidopsis cytokinesis.

Eukaryotic membrane fusion requires trans-SNARE complexes bridging the gap between adjacent membranes1. Fusion between a transport vesicle and its target membrane transforms the trans- into a cis-SNARE complex. The latter interacts with the hexameric AAA+-ATPase N-ethylmaleimide-sensitive factor (NSF) and its co-factor alpha-soluble NSF attachment protein (αSNAP), forming a 20S complex2,3. ATPase activity disassembles the SNAP receptor (SNARE) complex into Qa-SNARE, which folds back onto itself, and its partners4,5. The fusion of identical membranes has a different sequence of events6. The fusion partners each have cis-SNARE complexes to be broken up by NSF and αSNAP. The Qa-SNARE monomers are then stabilized by interaction with Sec1/Munc18-type regulators (SM proteins) to form trans-SNARE complexes, as shown for the yeast vacuole7. Membrane fusion in Arabidopsis cytokinesis is formally akin to vacuolar fusion8. Membrane vesicles fuse with one another to form the partitioning membrane known as the cell plate. Cis-SNARE complexes of cytokinesis-specific Qa-SNARE KNOLLE and its SNARE partners are assembled at the endoplasmic reticulum and delivered by traffic via the Golgi/trans-Golgi network to the cell division plane9. The SM protein KEULE is required for the formation of trans-SNARE complexes between adjacent membrane vesicles10. Here we identify NSF and its adaptor αSNAP2 as necessary for the disassembly of KNOLLE cis-SNARE complexes, which is a prerequisite for KNOLLE-KEULE interaction in cytokinesis. In addition, we show that NSF is required for other trafficking pathways and interacts with the respective Q-SNAREs. The SNARE complex disassembly machinery is conserved in plants and plays a unique essential role in cytokinesis.

Absence of the primary cilia formation gene Talpid3 impairs muscle stem cell function.

Skeletal muscle stem cells (MuSC) are crucial for tissue homoeostasis and repair after injury. Following activation, they proliferate to generate differentiating myoblasts. A proportion of cells self-renew, re-enter the MuSC niche under the basal lamina outside the myofiber and become quiescent. Quiescent MuSC have a primary cilium, which is disassembled upon cell cycle entry. Ex vivo experiments suggest cilia are important for MuSC self-renewal, however, their requirement for muscle regeneration in vivo remains poorly understood. Talpid3 (TA3) is essential for primary cilia formation and Hedgehog (Hh) signalling. Here we use tamoxifen-inducible conditional deletion of TA3 in MuSC (iSC-KO) and show that regeneration is impaired in response to cytotoxic injury. Depletion of MuSC after regeneration suggests impaired self-renewal, also consistent with an exacerbated phenotype in TA3iSC-KO mice after repeat injury. Single cell transcriptomics of MuSC progeny isolated from myofibers identifies components of several signalling pathways, which are deregulated in absence of TA3, including Hh and Wnt. Pharmacological activation of Wnt restores muscle regeneration, while purmorphamine, an activator of the Smoothened (Smo) co-receptor in the Hh pathway, has no effect. Together, our data show that TA3 and primary cilia are important for MuSC self-renewal and pharmacological treatment can efficiently restore muscle regeneration.

Fukutin-related protein alters the deposition of laminin in the eye and brain.

Mutations in fukutin-related protein (FKRP) are responsible for a common group of muscular dystrophies ranging from adult onset limb girdle muscular dystrophies to severe congenital forms with associated structural brain involvement. The defining feature of this group of disorders is the hypoglycosylation of α-dystroglycan and its inability to effectively bind extracellular matrix ligands such as laminin α2. However, α-dystroglycan has the potential to interact with a number of laminin isoforms many of which are basement membrane/tissue specific and developmentally regulated. To further investigate this we evaluated laminin α-chain expression in the cerebral cortex and eye of our FKRP knock-down mouse (FKRP(KD)). These mice showed a marked disturbance in the deposition of laminin α-chains including α1, α2, α4, and α5, although only laminin α1- and γ1-chain mRNA expression was significantly upregulated relative to controls. Moreover, there was a diffuse pattern of laminin deposition below the pial surface which correlated with an abrupt termination of many of the radial glial cells. This along with the pial basement membrane defects, contributed to the abnormal positioning of both early- and late-born neurons. Defects in the inner limiting membrane of the eye were associated with a reduction of laminin α1 demonstrating the involvement of the α-dystroglycan:laminin α1 axis in the disease process. These observations demonstrate for the first time that a reduction in Fkrp influences the ability of tissue-specific forms of α-dystroglycan to direct the deposition of several laminin isoforms in the formation of different basement membranes.

Basement membrane deposition of nidogen 1 but not nidogen 2 requires the nidogen binding module of the laminin gamma1 chain.

The nidogen-laminin interaction is proposed to play a key role in basement membrane (BM) assembly. However, though there are similarities, the phenotypes in mice lacking nidogen 1 and 2 (nidogen double null) differ to those of mice lacking the nidogen binding module (γ1III4) of the laminin γ1 chain. This indicates different cell- and tissue-specific functions for nidogens and their interaction with laminin and poses the question of whether the phenotypes in nidogen double null mice are caused by the loss of the laminin-nidogen interaction or rather by other unknown nidogen functions. To investigate this, we analyzed BMs, in particular those in the skin of mice lacking the nidogen binding module. In contrast to nidogen double null mice, all skin BMs in γ1III4-deficient mice appeared normal. Furthermore, although nidogen 1 deposition was strongly reduced, nidogen 2 appeared unchanged. Mice with additional deletion of the laminin γ3 chain, which contains a γ1-like nidogen binding module, showed a further reduction of nidogen 1 in the dermoepidermal BM; however, this again did not affect nidogen 2. This demonstrates that in vivo only nidogen 1 deposition is critically dependent on the nidogen binding modules of the laminin γ1 and γ3 chains, whereas nidogen 2 is independently recruited either by binding to an alternative site on laminin or to other BM proteins.

Functional anatomy of the Arabidopsis cytokinesis-specific syntaxin KNOLLE.

In plant cytokinesis, Golgi/trans-Golgi network-derived vesicles are targeted to the plane of cell division where they fuse with one another to form the partitioning membrane (cell plate). This membrane fusion requires a specialised syntaxin (Qa-SNARE), named KNOLLE in Arabidopsis. KNOLLE is only made during the M-phase of the cell cycle, targeted to the plane of cell division and degraded in the vacuole at the end of cytokinesis. To identify the parts of KNOLLE required for proper targeting and function in membrane fusion, we generated chimeric syntaxins comprising complementary fragments from KNOLLE and MVB-localized PEP12 (SYP21). Surprisingly, targeting of the chimeric protein was not specified by the C-terminal membrane anchor. Rather the N-terminal region including helix Ha and the adjacent linker to helix Hb appeared to played a critical role. However, deletion of this N-terminal fragment from KNOLLE (KN(Δ1-82) ) had the same effect as its presence in the chimeric protein (KN(1-82) -PEP12(64-279) ), suggesting that targeting to the plane of cell division occurs by default, i.e. when no sorting signal would target the syntaxin to a specific endomembrane compartment. Once the full-length syntaxin accumulated at the plane of division, phenotypic rescue of the knolle mutant only required the SNARE domain plus the adjacent linker connecting helix Hc to the SNARE domain from KNOLLE. Our results suggest that targeting of syntaxin to the plane of cell division occurs without active sorting, whereas syntaxin-mediated membrane fusion requires sequence-specific features.