Soil moisture-atmosphere feedback dominates land carbon uptake variability.

Year-to-year changes in carbon uptake by terrestrial ecosystems have an essential role in determining atmospheric carbon dioxide concentrations1. It remains uncertain to what extent temperature and water availability can explain these variations at the global scale2-5. Here we use factorial climate model simulations6 and show that variability in soil moisture drives 90 per cent of the inter-annual variability in global land carbon uptake, mainly through its impact on photosynthesis. We find that most of this ecosystem response occurs indirectly as soil moisture-atmosphere feedback amplifies temperature and humidity anomalies and enhances the direct effects of soil water stress. The strength of this feedback mechanism explains why coupled climate models indicate that soil moisture has a dominant role4, which is not readily apparent from land surface model simulations and observational analyses2,5. These findings highlight the need to account for feedback between soil and atmospheric dryness when estimating the response of the carbon cycle to climatic change globally5,7, as well as when conducting field-scale investigations of the response of the ecosystem to droughts8,9. Our results show that most of the global variability in modelled land carbon uptake is driven by temperature and vapour pressure deficit effects that are controlled by soil moisture.

The three major axes of terrestrial ecosystem function.

The leaf economics spectrum1,2 and the global spectrum of plant forms and functions3 revealed fundamental axes of variation in plant traits, which represent different ecological strategies that are shaped by the evolutionary development of plant species2. Ecosystem functions depend on environmental conditions and the traits of species that comprise the ecological communities4. However, the axes of variation of ecosystem functions are largely unknown, which limits our understanding of how ecosystems respond as a whole to anthropogenic drivers, climate and environmental variability4,5. Here we derive a set of ecosystem functions6 from a dataset of surface gas exchange measurements across major terrestrial biomes. We find that most of the variability within ecosystem functions (71.8%) is captured by three key axes. The first axis reflects maximum ecosystem productivity and is mostly explained by vegetation structure. The second axis reflects ecosystem water-use strategies and is jointly explained by variation in vegetation height and climate. The third axis, which represents ecosystem carbon-use efficiency, features a gradient related to aridity, and is explained primarily by variation in vegetation structure. We show that two state-of-the-art land surface models reproduce the first and most important axis of ecosystem functions. However, the models tend to simulate more strongly correlated functions than those observed, which limits their ability to accurately predict the full range of responses to environmental changes in carbon, water and energy cycling in terrestrial ecosystems7,8.

Bending the curve of terrestrial biodiversity needs an integrated strategy.

Increased efforts are required to prevent further losses to terrestrial biodiversity and the ecosystem services that it  provides1,2. Ambitious targets have been proposed, such as reversing the declining trends in biodiversity3; however, just feeding the growing human population will make this a challenge4. Here we use an ensemble of land-use and biodiversity models to assess whether-and how-humanity can reverse the declines in terrestrial biodiversity caused by habitat conversion, which is a major threat to biodiversity5. We show that immediate efforts, consistent with the broader sustainability agenda but of unprecedented ambition and coordination, could enable the provision of food for the growing human population while reversing the global terrestrial biodiversity trends caused by habitat conversion. If we decide to increase the extent of land under conservation management, restore degraded land and generalize landscape-level conservation planning, biodiversity trends from habitat conversion could become positive by the mid-twenty-first century on average across models (confidence interval, 2042-2061), but this was not the case for all models. Food prices could increase and, on average across models, almost half (confidence interval, 34-50%) of the future biodiversity losses could not be avoided. However, additionally tackling the drivers of land-use change could avoid conflict with affordable food provision and reduces the environmental effects of the food-provision system. Through further sustainable intensification and trade, reduced food waste and more plant-based human diets, more than two thirds of future biodiversity losses are avoided and the biodiversity trends from habitat conversion are reversed by 2050 for almost all of the models. Although limiting further loss will remain challenging in several biodiversity-rich regions, and other threats-such as climate change-must be addressed to truly reverse the declines in biodiversity, our results show that ambitious conservation efforts and food system transformation are central to an effective post-2020 biodiversity strategy.

Broad domains of histone marks in the highly compact Paramecium macronuclear genome.

The unicellular ciliate Paramecium contains a large vegetative macronucleus with several unusual characteristics, including an extremely high coding density and high polyploidy. As macronculear chromatin is devoid of heterochromatin, our study characterizes the functional epigenomic organization necessary for gene regulation and proper Pol II activity. Histone marks (H3K4me3, H3K9ac, H3K27me3) reveal no narrow peaks but broad domains along gene bodies, whereas intergenic regions are devoid of nucleosomes. Our data implicate H3K4me3 levels inside ORFs to be the main factor associated with gene expression, and H3K27me3 appears in association with H3K4me3 in plastic genes. Silent and lowly expressed genes show low nucleosome occupancy, suggesting that gene inactivation does not involve increased nucleosome occupancy and chromatin condensation. Because of a high occupancy of Pol II along highly expressed ORFs, transcriptional elongation appears to be quite different from that of other species. This is supported by missing heptameric repeats in the C-terminal domain of Pol II and a divergent elongation system. Our data imply that unoccupied DNA is the default state, whereas gene activation requires nucleosome recruitment together with broad domains of H3K4me3. In summary, gene activation and silencing in Paramecium run counter to the current understanding of chromatin biology.

Deep learning and process understanding for data-driven Earth system science.

Machine learning approaches are increasingly used to extract patterns and insights from the ever-increasing stream of geospatial data, but current approaches may not be optimal when system behaviour is dominated by spatial or temporal context. Here, rather than amending classical machine learning, we argue that these contextual cues should be used as part of deep learning (an approach that is able to extract spatio-temporal features automatically) to gain further process understanding of Earth system science problems, improving the predictive ability of seasonal forecasting and modelling of long-range spatial connections across multiple timescales, for example. The next step will be a hybrid modelling approach, coupling physical process models with the versatility of data-driven machine learning.

Modelling the probability of meeting IUCN Red List criteria to support reassessments.

Comparative extinction risk analysis-which predicts species extinction risk from correlation with traits or geographical characteristics-has gained research attention as a promising tool to support extinction risk assessment in the IUCN Red List of Threatened Species. However, its uptake has been very limited so far, possibly because existing models only predict a species' Red List category, without indicating which Red List criteria may be triggered. This prevents such approaches to be integrated into Red List assessments. We overcome this implementation gap by developing models that predict the probability of species meeting individual Red List criteria. Using data on the world's birds, we evaluated the predictive performance of our criterion-specific models and compared it with the typical criterion-blind modelling approach. We compiled data on biological traits (e.g. range size, clutch size) and external drivers (e.g. change in canopy cover) often associated with extinction risk. For each specific criterion, we modelled the relationship between extinction risk predictors and species' Red List category under that criterion using ordinal regression models. We found criterion-specific models were better at identifying threatened species compared to a criterion-blind model (higher sensitivity), but less good at identifying not threatened species (lower specificity). As expected, different covariates were important for predicting extinction risk under different criteria. Change in annual temperature was important for criteria related to population trends, while high forest dependency was important for criteria related to restricted area of occupancy or small population size. Our criteria-specific method can support Red List assessors by producing outputs that identify species likely to meet specific criteria, and which are the most important predictors. These species can then be prioritised for re-evaluation. We expect this new approach to increase the uptake of extinction risk models in Red List assessments, bridging a long-standing research-implementation gap.

Co-chaperones of the Human Endoplasmic Reticulum: An Update.

In mammalian cells, the rough endoplasmic reticulum (ER) plays central roles in the biogenesis of extracellular plus organellar proteins and in various signal transduction pathways. For these reasons, the ER comprises molecular chaperones, which are involved in import, folding, assembly, export, plus degradation of polypeptides, and signal transduction components, such as calcium channels, calcium pumps, and UPR transducers plus adenine nucleotide carriers/exchangers in the ER membrane. The calcium- and ATP-dependent ER lumenal Hsp70, termed immunoglobulin heavy-chain-binding protein or BiP, is the central player in all these activities and involves up to nine different Hsp40-type co-chaperones, i.e., ER membrane integrated as well as ER lumenal J-domain proteins, termed ERj or ERdj proteins, two nucleotide exchange factors or NEFs (Grp170 and Sil1), and NEF-antagonists, such as MANF. Here we summarize the current knowledge on the ER-resident BiP/ERj chaperone network and focus on the interaction of BiP with the polypeptide-conducting and calcium-permeable Sec61 channel of the ER membrane as an example for BiP action and how its functional cycle is linked to ER protein import and various calcium-dependent signal transduction pathways.

[How can we ensure the future care for children and adolescents with mental disorders?] / Wie sichern wir in Zukunft die Versorgung von Kindern und Jugendlichen mit psychischen Erkrankungen?

The interdisciplinary care of children and adolescents with mental disorders requires services from various German codes of social law and-within the medical care system-enclosing inpatient and outpatient services. The increasing demand, the increase in severity of disorders, and the general shortage of staff in social services put pressure on the structures of the interdisciplinary service networks resulting in long waiting periods, long distances, and regionally insufficient care. The medical field of child and adolescent psychiatry and psychotherapy (CAPP) plays the central and coordinating role within the cooperative care for children and adolescents with mental disorders. The CAPP is in clear need of reforms; however, these are markedly different from the reform needs of the German somatic medical care system and differ substantially from those of the (adult) psychiatry, psychotherapy, and psychosomatics disciplines. This discussion paper describes the reform requirements, the specifics of the CAPP structures, and suggestions to overcome sectors of service provision, enhance networking, intensify telemedicine, and develop evidence-based prevention and early recognition of child mental disorders.

Tropical dry woodland loss occurs disproportionately in areas of highest conservation value.

Tropical and subtropical dry woodlands are rich in biodiversity and carbon. Yet, many of these woodlands are under high deforestation pressure and remain weakly protected. Here, we assessed how deforestation dynamics relate to areas of woodland protection and to conservation priorities across the world's tropical dry woodlands. Specifically, we characterized different types of deforestation frontier from 2000 to 2020 and compared them to protected areas (PAs), Indigenous Peoples' lands and conservation areas for biodiversity, carbon and water. We found that global conservation priorities were always overrepresented in tropical dry woodlands compared to the rest of the globe (between 4% and 96% more than expected, depending on the type of conservation priority). Moreover, about 41% of all dry woodlands were characterized as deforestation frontiers, and these frontiers have been falling disproportionately in areas with important regional (i.e. tropical dry woodland) conservation assets. While deforestation frontiers were identified within all tropical dry woodland classes of woodland protection, they were lower than the average within protected areas coinciding with Indigenous Peoples' lands (23%), and within other PAs (28%). However, within PAs, deforestation frontiers have also been disproportionately affecting regional conservation assets. Many emerging deforestation frontiers were identified outside but close to PAs, highlighting a growing threat that the conserved areas of dry woodland will become isolated. Understanding how deforestation frontiers coincide with major types of current woodland protection can help target context-specific conservation policies and interventions to tropical dry woodland conservation assets (e.g. PAs in which deforestation is rampant require stronger enforcement, inactive deforestation frontiers could benefit from restoration). Our analyses also identify recurring patterns that can be used to test the transferability of governance approaches and promote learning across social-ecological contexts.

Radiometal Complexes as Pharmacokinetic Modifiers: A Potent 68Ga-Labeled Gastrin-Releasing Peptide Receptor Antagonist Based on the Macrocyclic Metal Chelator NODIA-Me.

The gastrin-releasing peptide receptor (GRPr) is overexpressed in various cancer types including prostate and breast carcinomas, making it an attractive target for molecular imaging and therapy. In this work, we designed a novel GRPr antagonistic probe comprising metal chelator NODIA-Me. This 1,4,7-triazacyclononane-based chelator forms positively charged metal complexes due to its neutral methylimidazole arms. Because a positive charge at the N-terminus of GRPr conjugates is responsible for high receptor affinity as exemplified by the current gold standard DOTA-RM2, we investigated if a positively charged radiometal complex can be used as a pharmacokinetic modifier to also produce high-affinity GRPr conjugates. In this respect, the bioconjugate NODIA-Me-Ahx-JMV594 was prepared by a combination of solid-phase peptide synthesis and solution-based reactions in a 94% yield. Radiolabeling provided the 68Ga-labeled conjugate in radiochemical yields of >95% and radiochemical purities of >98% with mean molar activities of Am ∼17 MBq nmol-1. The competitive GRPr affinity of the metal-free and 69/71Ga-labeled conjugate was determined to be IC50 = 0.41 ± 0.06 and 1.45 ± 0.06 nM, respectively. The metal-free GRPr antagonist DOTA-RM2 and its corresponding 69/71Ga complex had IC50 values of 1.42 ± 0.07 and 0.98 ± 0.19 nM, respectively. Small-animal PET imaging of mice bearing GRPr(+) PC-3 tumors revealed high radioactivity accumulation in the tumors and in the pancreas as an organ with high levels of GRPr expression. These findings were corroborated by the corresponding ex vivo biodistribution data, in which the tumors and the pancreas exhibited the highest radioactivity accumulation. By coinjection of an excess of NODIA-Me-Ahx-JMV594, uptake in the tumors and GRPr(+) organs was significantly reduced, confirming specific receptor-mediated uptake. The estrogen receptor-positive tumor of a female breast cancer patient was clearly visualized by PET imaging using 68Ga-labeled NODIA-Me-Ahx-JMV594. To summarize, the positive charge at the N-terminus of the conjugate induced by the Ga(NODIA-Me) complex resulted in high GRPr affinity comparable to that of the potent antagonist DOTA-RM2. The conjugate NODIA-Me-Ahx-JMV594 is a promising probe for imaging of GRPr tumors that warrants further evaluation in larger patient cohorts as well as in combination with other radiometals.

Prioritizing the reassessment of data-deficient species on the IUCN Red List.

Despite being central to the implementation of conservation policies, the usefulness of the International Union for Conservation of Nature (IUCN) Red List of Threatened Species is hampered by the 14% of species classified as data-deficient (DD) because information to evaluate these species' extinction risk was lacking when they were last assessed or because assessors did not appropriately account for uncertainty. Robust methods are needed to identify which DD species are more likely to be reclassified in one of the data-sufficient IUCN Red List categories. We devised a reproducible method to help red-list assessors prioritize reassessment of DD species and tested it with 6887 DD species of mammals, reptiles, amphibians, fishes, and Odonata (dragonflies and damselflies). For each DD species in these groups, we calculated its probability of being classified in a data-sufficient category if reassessed today from covariates measuring available knowledge (e.g., number of occurrence records or published articles available), knowledge proxies (e.g., remoteness of the range), and species characteristics (e.g., nocturnality); calculated change in such probability since last assessment from the increase in available knowledge (e.g., new occurrence records); and determined whether the species might qualify as threatened based on recent rate of habitat loss determined from global land-cover maps. We identified 1907 species with a probability of being reassessed in a data-sufficient category of >0.5; 624 species for which this probability increased by >0.25 since last assessment; and 77 species that could be reassessed as near threatened or threatened based on habitat loss. Combining these 3 elements, our results provided a list of species likely to be data-sufficient such that the comprehensiveness and representativeness of the IUCN Red List can be improved.

Priorización de la reevaluación de las especies con datos deficientes en la Lista Roja de la UICN Resumen No obstante que es fundamental para la implementación de políticas de conservación, la utilidad de la Lista Roja de Especies Amenazadas de la Unión Internacional para la Conservación de la Naturaleza (UICN) está limitada por el 14% de especies clasificadas con datos deficientes (DD) debido a que la información para evaluar el riesgo de extinción de estas especies no existía cuando fueron evaluadas la última vez o porque los evaluadores no consideraron la incertidumbre apropiadamente. Se requieren métodos robustos para identificar las especies DD con mayor probabilidad de ser reclasificadas en alguna de las categorías en la Lista Roja UICN con datos suficientes. Diseñamos un método reproducible para ayudar a que los evaluadores de la lista roja prioricen la reevaluación de especies DD y lo probamos con 6,887 especies DD de mamíferos, reptiles, anfibios, peces y Odonata (libélulas y caballitos del diablo). Para cada una de las especies DD en estos grupos, calculamos la probabilidad de ser clasificadas en una categoría con datos suficientes si fuera reevaluada hoy a partir de covariables que miden el conocimiento disponible (e.g., número de registros de ocurrencia o artículos publicados disponibles), sustitutos de conocimiento (e.g., extensión del rango de distribución) y características de la especie ((e.g., nocturnidad); calculamos el cambio en tal probabilidad desde la última reevaluación a partir del incremento en el conocimiento disponible (e.g., registros de ocurrencia nuevos); y determinamos si las especies podrían calificar como amenazadas con base en pérdidas de hábitat recientes a partir de mapas globales de cobertura de suelo recientes. Identificamos 1,907 especies con una probabilidad >0.5 de ser reclasificados en una categoría con datos suficientes; 624 especies cuya probabilidad aumentó en >0.25 desde la última evaluación, y 77 especies que podrían ser reclasificadas como casi en peligro con base en la pérdida de hábitat. Combinando estos 3 elementos, nuestros resultados proporcionaron una lista de especies probablemente con datos suficientes de tal modo que la exhaustividad y la representatividad de la Lista Roja de la UICN pueden ser mejoradas.

Compensatory water effects link yearly global land CO2 sink changes to temperature.

Large interannual variations in the measured growth rate of atmospheric carbon dioxide (CO2) originate primarily from fluctuations in carbon uptake by land ecosystems. It remains uncertain, however, to what extent temperature and water availability control the carbon balance of land ecosystems across spatial and temporal scales. Here we use empirical models based on eddy covariance data and process-based models to investigate the effect of changes in temperature and water availability on gross primary productivity (GPP), terrestrial ecosystem respiration (TER) and net ecosystem exchange (NEE) at local and global scales. We find that water availability is the dominant driver of the local interannual variability in GPP and TER. To a lesser extent this is true also for NEE at the local scale, but when integrated globally, temporal NEE variability is mostly driven by temperature fluctuations. We suggest that this apparent paradox can be explained by two compensatory water effects. Temporal water-driven GPP and TER variations compensate locally, dampening water-driven NEE variability. Spatial water availability anomalies also compensate, leaving a dominant temperature signal in the year-to-year fluctuations of the land carbon sink. These findings help to reconcile seemingly contradictory reports regarding the importance of temperature and water in controlling the interannual variability of the terrestrial carbon balance. Our study indicates that spatial climate covariation drives the global carbon cycle response.

Quantitative Mass Spectrometry Characterizes Client Spectra of Components for Targeting of Membrane Proteins to and Their Insertion into the Membrane of the Human ER.

To elucidate the redundancy in the components for the targeting of membrane proteins to the endoplasmic reticulum (ER) and/or their insertion into the ER membrane under physiological conditions, we previously analyzed different human cells by label-free quantitative mass spectrometry. The HeLa and HEK293 cells had been depleted of a certain component by siRNA or CRISPR/Cas9 treatment or were deficient patient fibroblasts and compared to the respective control cells by differential protein abundance analysis. In addition to clients of the SRP and Sec61 complex, we identified membrane protein clients of components of the TRC/GET, SND, and PEX3 pathways for ER targeting, and Sec62, Sec63, TRAM1, and TRAP as putative auxiliary components of the Sec61 complex. Here, a comprehensive evaluation of these previously described differential protein abundance analyses, as well as similar analyses on the Sec61-co-operating EMC and the characteristics of the topogenic sequences of the various membrane protein clients, i.e., the client spectra of the components, are reported. As expected, the analysis characterized membrane protein precursors with cleavable amino-terminal signal peptides or amino-terminal transmembrane helices as predominant clients of SRP, as well as the Sec61 complex, while precursors with more central or even carboxy-terminal ones were found to dominate the client spectra of the SND and TRC/GET pathways for membrane targeting. For membrane protein insertion, the auxiliary Sec61 channel components indeed share the client spectra of the Sec61 complex to a large extent. However, we also detected some unexpected differences, particularly related to EMC, TRAP, and TRAM1. The possible mechanistic implications for membrane protein biogenesis at the human ER are discussed and can be expected to eventually advance our understanding of the mechanisms that are involved in the so-called Sec61-channelopathies, resulting from deficient ER protein import.

Immunohistochemistry Reveals TRPC Channels in the Human Hearing Organ-A Novel CT-Guided Approach to the Cochlea.

TRPC channels are critical players in cochlear hair cells and sensory neurons, as demonstrated in animal experiments. However, evidence for TRPC expression in the human cochlea is still lacking. This reflects the logistic and practical difficulties in obtaining human cochleae. The purpose of this study was to detect TRPC6, TRPC5 and TRPC3 in the human cochlea. Temporal bone pairs were excised from ten body donors, and the inner ear was first assessed based on computed tomography scans. Decalcification was then performed using 20% EDTA solutions. Immunohistochemistry with knockout-tested antibodies followed. The organ of Corti, the stria vascularis, the spiral lamina, spiral ganglion neurons and cochlear nerves were specifically stained. This unique report of TRPC channels in the human cochlea supports the hypothesis of the potentially critical role of TRPC channels in human cochlear health and disease which has been suggested in previous rodent experiments.

The 3q Oncogene SEC62 Predicts Response to Neoadjuvant Chemotherapy and Regulates Tumor Cell Migration in Triple Negative Breast Cancer.

In the absence of targeted treatment options, neoadjuvant chemotherapy (NACT) is applied widely for triple-negative breast cancer (TNBC). Response to NACT is an important parameter predictive of oncological outcomes (progression-free and overall survival). An approach to the evaluation of predictive markers enabling therapy individualization is the identification of tumor driver genetic mutations. This study was conducted to investigate the role of SEC62, harbored at 3q26 and identified as a driver of breast cancer pathogenesis, in TNBC. We analyzed SEC62 expression in The Cancer Genome Atlas database, and immunohistologically investigated SEC62 expression in pre- and post-NACT tissue samples from 64 patients with TNBC treated at the Department of Gynecology and Obstetrics/Saarland University Hospital/Homburg between January 2010 and December 2018 and compared the effect of SEC62 on tumor cell migration and proliferation in functional assays. SEC62 expression dynamics correlated positively with the response to NACT (p ≤ 0.01) and oncological outcomes (p ≤ 0.01). SEC62 expression stimulated tumor cell migration (p ≤ 0.01). The study findings indicate that SEC62 is overexpressed in TNBC and serves as a predictive marker for the response to NACT, a prognostic marker for oncological outcomes, and a migration-stimulating oncogene in TNBC.

The cytoprotective protein MANF promotes neuronal survival independently from its role as a GRP78 cofactor.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-stress-regulated protein exhibiting cytoprotective properties through a poorly understood mechanism in various in vitro and in vivo models of neuronal and non-neuronal damage. Although initially characterized as a secreted neurotrophic factor for midbrain dopamine neurons, MANF has recently gained more interest for its intracellular role in regulating the ER homeostasis, including serving as a cofactor of the chaperone glucose-regulated protein 78 (GRP78). We aimed for a better understanding of the neuroprotective mechanisms of MANF. Here we show for the first time that MANF promotes the survival of ER-stressed neurons in vitro as a general unfolded protein response (UPR) regulator, affecting several UPR pathways simultaneously. Interestingly, MANF does not affect naïve neurons. We hypothesize that MANF regulates UPR signaling toward a mode more compatible with neuronal survival. Screening of MANF interacting proteins from two mammalian cell lines revealed a conserved interactome of 15 proteins including several ER chaperones such as GRP78, GRP170, protein disulfide isomerase family A member 1, and protein disulfide isomerase family A member 6. Further characterization confirmed previously published finding that MANF is a cofactor of GRP78 interacting with its nucleotide binding domain. Using microscale thermophoresis and nuclear magnetic resonance spectroscopy, we discovered that MANF is an ATP binding protein and that ATP blocks the MANF-GRP78 interaction. Interestingly, functional analysis of the antiapoptotic properties of MANF mutants in cultured neurons revealed divergent roles of MANF as a GRP78 cofactor and as an antiapoptotic regulator of UPR. We conclude that the co-factor type interaction with GRP78 is dispensable for the survival-promoting activity of MANF in neurons.

Defective cell adhesion function of solute transporter, SLC4A11, in endothelial corneal dystrophies.

Corneal endothelial cell (CEnC) loss is often associated with blinding endothelial corneal dystrophies: dominantly inherited, common (5%) Fuchs endothelial corneal dystrophy (FECD) and recessive, rare congenital hereditary endothelial dystrophy (CHED). Mutations of SLC4A11, an abundant corneal solute transporter, cause CHED and some cases of FECD. The link between defective SLC4A11 solute transport function and CEnC loss is, however, unclear. Cell adhesion assays using SLC4A11-transfected HEK293 cells and primary human CEnC revealed that SLC4A11 promotes adhesion to components of Descemet's membrane (DM), the basement membrane layer to which CEnC bind. An antibody against SLC4A11 extracellular loop 3 (EL3) suppressed cell adhesion, identifying EL3 as the DM-binding site. Earlier studies showed that some SLC4A11 mutations cause FECD and CHED by impairing solute transport activity or cell surface trafficking. Without affecting these functions, FECD-causing mutations in SLC4A11-EL3 compromised cell adhesion capacity. In an energy-minimized SLC4A11-EL3 three-dimensional model, these mutations cluster and are buried within the EL3 structure. A GST fusion protein of SLC4A11-EL3 interacts with principal DM protein, COL8A2, as identified by mass spectrometry. Engineered SLC4A11-EL3-containing protein, STIC (SLC4A11-EL3 Transmembrane-GPA Integrated Chimera), promotes cell adhesion in transfected HEK293 cells and primary human CEnC, confirming the cell adhesion role of EL3. Taken together, the data suggest that SLC4A11 directly binds DM to serve as a cell adhesion molecule (CAM). These data further suggest that cell adhesion defects contribute to FECD and CHED pathology. Observations with STIC point toward a new therapeutic direction in these diseases: replacement of lost cell adhesion capacity.

The SND proteins constitute an alternative targeting route to the endoplasmic reticulum.

In eukaryotes, up to one-third of cellular proteins are targeted to the endoplasmic reticulum, where they undergo folding, processing, sorting and trafficking to subsequent endomembrane compartments. Targeting to the endoplasmic reticulum has been shown to occur co-translationally by the signal recognition particle (SRP) pathway or post-translationally by the mammalian transmembrane recognition complex of 40 kDa (TRC40) and homologous yeast guided entry of tail-anchored proteins (GET) pathways. Despite the range of proteins that can be catered for by these two pathways, many proteins are still known to be independent of both SRP and GET, so there seems to be a critical need for an additional dedicated pathway for endoplasmic reticulum relay. We set out to uncover additional targeting proteins using unbiased high-content screening approaches. To this end, we performed a systematic visual screen using the yeast Saccharomyces cerevisiae, and uncovered three uncharacterized proteins whose loss affected targeting. We suggest that these proteins work together and demonstrate that they function in parallel with SRP and GET to target a broad range of substrates to the endoplasmic reticulum. The three proteins, which we name Snd1, Snd2 and Snd3 (for SRP-independent targeting), can synthetically compensate for the loss of both the SRP and GET pathways, and act as a backup targeting system. This explains why it has previously been difficult to demonstrate complete loss of targeting for some substrates. Our discovery thus puts in place an essential piece of the endoplasmic reticulum targeting puzzle, highlighting how the targeting apparatus of the eukaryotic cell is robust, interlinked and flexible.

Ciliary Proteins Repurposed by the Synaptic Ribbon: Trafficking Myristoylated Proteins at Rod Photoreceptor Synapses.

The Unc119 protein mediates transport of myristoylated proteins to the photoreceptor outer segment, a specialized primary cilium. This transport activity is regulated by the GTPase Arl3 as well as by Arl13b and Rp2 that control Arl3 activation/inactivation. Interestingly, Unc119 is also enriched in photoreceptor synapses and can bind to RIBEYE, the main component of synaptic ribbons. In the present study, we analyzed whether the known regulatory proteins, that control the Unc119-dependent myristoylated protein transport at the primary cilium, are also present at the photoreceptor synaptic ribbon complex by using high-resolution immunofluorescence and immunogold electron microscopy. We found Arl3 and Arl13b to be enriched at the synaptic ribbon whereas Rp2 was predominantly found on vesicles distributed within the entire terminal. These findings indicate that the synaptic ribbon could be involved in the discharge of Unc119-bound lipid-modified proteins. In agreement with this hypothesis, we found Nphp3 (Nephrocystin-3), a myristoylated, Unc119-dependent cargo protein enriched at the basal portion of the ribbon in close vicinity to the active zone. Mutations in Nphp3 are known to be associated with Senior-Løken Syndrome 3 (SLS3). Visual impairment and blindness in SLS3 might thus not only result from ciliary dysfunctions but also from malfunctions of the photoreceptor synapse.

Two Piwis with Ago-like functions silence somatic genes at the chromatin level.

Most sRNA biogenesis mechanisms involve either RNAse III cleavage or ping-pong amplification by different Piwi proteins harbouring slicer activity. Here, we follow the question why the mechanism of transgene-induced silencing in the ciliate Paramecium needs both Dicer activity and two Ptiwi proteins. This pathway involves primary siRNAs produced from non-translatable transgenes and secondary siRNAs from targeted endogenous loci. Our data does not indicate any signatures from ping-pong amplification but Dicer cleavage of long dsRNA. Ptiwi13 and 14 prefer different sub-cellular localizations and different preferences for primary and secondary siRNAs but do not load them mutually exclusive. Both Piwis enrich for antisense RNAs and show a general preference for uridine-rich sRNAs along the entire sRNA length. In addition, Ptiwi14-loaded siRNAs show a 5´-U signature. Our data indicates both Ptiwis and 2´-O-methylation contributing to strand selection of Dicer cleaved siRNAs. This unexpected function of the two distinct vegetative Piwis extends the increasing knowledge of the diversity of Piwi functions in diverse silencing pathways. We describe an unusual mode of action of Piwi proteins extending not only the great variety of Piwi-associated RNAi pathways but moreover raising the question whether this could have been the primordial one.