Neuronal connectome of a visual eye circuit in Platynereis dumerilii

Visual navigation is widespread in animals, but how the light stimulus is translated into the coordinated motor output is poorly understood. In my thesis I reconstruct the neuronal connectome of the visual eyes in the marine annelid Platynereis using serial-section transmission electron microscopy. In the eye circuit, photoreceptor cells connect via three layers of interneurons to the motor neurons, which innervate trunk muscles and ciliated cells. By combining laser ablation and selective eye illumination experiments I could identify a visual eye circuit, which is able to detect spatial light information to perform either positive or negative phototaxis. I also showed a strong reciprocal interneuron motif, which modulates the contrast during visual navigation

Spectral Tuning of Phototaxis by a Go-Opsin in the Rhabdomeric Eyes of Platynereis

SummaryPhototaxis is characteristic of the pelagic larval stage of most bottom-dwelling marine invertebrates [1]. Larval phototaxis is mediated by simple eyes that can express various types of light-sensitive G-protein-coupled receptors known as opsins [2–8]. Since opsins diversified early during metazoan evolution in the marine environment [9], understanding underwater light detection could elucidate this diversification. Opsins have been classified into three major families, the r-opsins, the c-opsins, and the Go/RGR opsins, a family uniting Go-opsins, retinochromes, RGR opsins, and neuropsins [10, 11]. The Go-opsins form an ancient and poorly characterized group retained only in marine invertebrate genomes. Here, we characterize a Go-opsin from the marine annelid Platynereis dumerilii [3–5, 12–15]. We found Go-opsin1 coexpressed with two r-opsins in depolarizing rhabdomeric photoreceptor cells in the pigmented eyes of Platynereis larvae. We purified recombinant Go-opsin1 and found that it absorbs in the blue-cyan range of the light spectrum. To characterize the function of Go-opsin1, we generated a Go-opsin1 knockout Platynereis line by zinc-finger-nuclease-mediated genome engineering. Go-opsin1 knockout larvae were phototactic but showed reduced efficiency of phototaxis to wavelengths matching the in vitro Go-opsin1 spectrum. Our results highlight spectral tuning of phototaxis as a potential mechanism contributing to opsin diversity

The Nereid on the rise: Platynereis as a model system.

The Nereid Platynereis dumerilii (Audouin and Milne Edwards (Annales des Sciences Naturelles 1:195-269, 1833) is a marine annelid that belongs to the Nereididae, a family of errant polychaete worms. The Nereid shows a pelago-benthic life cycle: as a general characteristic for the superphylum of Lophotrochozoa/Spiralia, it has spirally cleaving embryos developing into swimming trochophore larvae. The larvae then metamorphose into benthic worms living in self-spun tubes on macroalgae. Platynereis is used as a model for genetics, regeneration, reproduction biology, development, evolution, chronobiology, neurobiology, ecology, ecotoxicology, and most recently also for connectomics and single-cell genomics. Research on the Nereid started with studies on eye development and spiralian embryogenesis in the nineteenth and early twentieth centuries. Transitioning into the molecular era, Platynereis research focused on posterior growth and regeneration, neuroendocrinology, circadian and lunar cycles, fertilization, and oocyte maturation. Other work covered segmentation, photoreceptors and other sensory cells, nephridia, and population dynamics. Most recently, the unique advantages of the Nereid young worm for whole-body volume electron microscopy and single-cell sequencing became apparent, enabling the tracing of all neurons in its rope-ladder-like central nervous system, and the construction of multimodal cellular atlases. Here, we provide an overview of current topics and methodologies for P. dumerilii, with the aim of stimulating further interest into our unique model and expanding the active and vibrant Platynereis community

BigStitcher: reconstructing high-resolution image datasets of cleared and expanded samples.

Light-sheet imaging of cleared and expanded samples creates terabyte-sized datasets that consist of many unaligned three-dimensional image tiles, which must be reconstructed before analysis. We developed the BigStitcher software to address this challenge. BigStitcher enables interactive visualization, fast and precise alignment, spatially resolved quality estimation, real-time fusion and deconvolution of dual-illumination, multitile, multiview datasets. The software also compensates for optical effects, thereby improving accuracy and enabling subsequent biological analysis

State of the climate in 2018

In 2018, the dominant greenhouse gases released into Earth’s atmosphere—carbon dioxide, methane, and nitrous oxide—continued their increase. The annual global average carbon dioxide concentration at Earth’s surface was 407.4 ± 0.1 ppm, the highest in the modern instrumental record and in ice core records dating back 800 000 years. Combined, greenhouse gases and several halogenated gases contribute just over 3 W m−2 to radiative forcing and represent a nearly 43% increase since 1990. Carbon dioxide is responsible for about 65% of this radiative forcing. With a weak La Niña in early 2018 transitioning to a weak El Niño by the year’s end, the global surface (land and ocean) temperature was the fourth highest on record, with only 2015 through 2017 being warmer. Several European countries reported record high annual temperatures. There were also more high, and fewer low, temperature extremes than in nearly all of the 68-year extremes record. Madagascar recorded a record daily temperature of 40.5°C in Morondava in March, while South Korea set its record high of 41.0°C in August in Hongcheon. Nawabshah, Pakistan, recorded its highest temperature of 50.2°C, which may be a new daily world record for April. Globally, the annual lower troposphere temperature was third to seventh highest, depending on the dataset analyzed. The lower stratospheric temperature was approximately fifth lowest. The 2018 Arctic land surface temperature was 1.2°C above the 1981–2010 average, tying for third highest in the 118-year record, following 2016 and 2017. June’s Arctic snow cover extent was almost half of what it was 35 years ago. Across Greenland, however, regional summer temperatures were generally below or near average. Additionally, a satellite survey of 47 glaciers in Greenland indicated a net increase in area for the first time since records began in 1999. Increasing permafrost temperatures were reported at most observation sites in the Arctic, with the overall increase of 0.1°–0.2°C between 2017 and 2018 being comparable to the highest rate of warming ever observed in the region. On 17 March, Arctic sea ice extent marked the second smallest annual maximum in the 38-year record, larger than only 2017. The minimum extent in 2018 was reached on 19 September and again on 23 September, tying 2008 and 2010 for the sixth lowest extent on record. The 23 September date tied 1997 as the latest sea ice minimum date on record. First-year ice now dominates the ice cover, comprising 77% of the March 2018 ice pack compared to 55% during the 1980s. Because thinner, younger ice is more vulnerable to melting out in summer, this shift in sea ice age has contributed to the decreasing trend in minimum ice extent. Regionally, Bering Sea ice extent was at record lows for almost the entire 2017/18 ice season. For the Antarctic continent as a whole, 2018 was warmer than average. On the highest points of the Antarctic Plateau, the automatic weather station Relay (74°S) broke or tied six monthly temperature records throughout the year, with August breaking its record by nearly 8°C. However, cool conditions in the western Bellingshausen Sea and Amundsen Sea sector contributed to a low melt season overall for 2017/18. High SSTs contributed to low summer sea ice extent in the Ross and Weddell Seas in 2018, underpinning the second lowest Antarctic summer minimum sea ice extent on record. Despite conducive conditions for its formation, the ozone hole at its maximum extent in September was near the 2000–18 mean, likely due to an ongoing slow decline in stratospheric chlorine monoxide concentration. Across the oceans, globally averaged SST decreased slightly since the record El Niño year of 2016 but was still far above the climatological mean. On average, SST is increasing at a rate of 0.10° ± 0.01°C decade−1 since 1950. The warming appeared largest in the tropical Indian Ocean and smallest in the North Pacific. The deeper ocean continues to warm year after year. For the seventh consecutive year, global annual mean sea level became the highest in the 26-year record, rising to 81 mm above the 1993 average. As anticipated in a warming climate, the hydrological cycle over the ocean is accelerating: dry regions are becoming drier and wet regions rainier. Closer to the equator, 95 named tropical storms were observed during 2018, well above the 1981–2010 average of 82. Eleven tropical cyclones reached Saffir–Simpson scale Category 5 intensity. North Atlantic Major Hurricane Michael’s landfall intensity of 140 kt was the fourth strongest for any continental U.S. hurricane landfall in the 168-year record. Michael caused more than 30 fatalities and $25 billion (U.S. dollars) in damages. In the western North Pacific, Super Typhoon Mangkhut led to 160 fatalities and$6 billion (U.S. dollars) in damages across the Philippines, Hong Kong, Macau, mainland China, Guam, and the Northern Mariana Islands. Tropical Storm Son-Tinh was responsible for 170 fatalities in Vietnam and Laos. Nearly all the islands of Micronesia experienced at least moderate impacts from various tropical cyclones. Across land, many areas around the globe received copious precipitation, notable at different time scales. Rodrigues and Réunion Island near southern Africa each reported their third wettest year on record. In Hawaii, 1262 mm precipitation at Waipā Gardens (Kauai) on 14–15 April set a new U.S. record for 24-h precipitation. In Brazil, the city of Belo Horizonte received nearly 75 mm of rain in just 20 minutes, nearly half its monthly average. Globally, fire activity during 2018 was the lowest since the start of the record in 1997, with a combined burned area of about 500 million hectares. This reinforced the long-term downward trend in fire emissions driven by changes in land use in frequently burning savannas. However, wildfires burned 3.5 million hectares across the United States, well above the 2000–10 average of 2.7 million hectares. Combined, U.S. wildfire damages for the 2017 and 2018 wildfire seasons exceeded $40 billion (U.S. dollars)

Biological investigations on the "lower acyclic terpene utilization (atu) pathway" in Pseudomonas aeruginosa and chemical synthesis of important intermediates of this pathway

Der Abbau von Citronellol und verwandten azyklischen Terpenoiden ist aufgrund einer Methylgruppe in beta-Position schwierig und nicht weit verbreitet. Die Organismen P. aeruginosa und P. citronellolis umgehen die Blockierung der beta-Oxidation durch Carboxylierung dieser Methylgruppe auf Stufe des CoenzymA-Esters 19 (Geranyl-CoA). Durch Wasseranlagerung an die Doppelbindung wird die Voraussetzung für die Abspaltung der Acetat-Funktionalität geschaffen, wobei die resultierende Verbindung 23 (7-Methyl-3-oxo-6-octenoyl-CoA) folgend weiter über beta-Oxidation abgebaut werden kann. •In Rohextrakten von Citronellsäure (Cs)-gewachsenen P. aeruginosa PAO1 Zellen wurde eine spezifische Geranyl-CoA Carboxylase (GCase) Aktivität von 11 ± 6 mU/mg und eine spezifische Methylcrotonyl-CoA Carboxylase (MCase) Aktivität von 47 ± 11 mU/mg bestimmt. Rohextrakte von Isovaleriansäure (Iso)-gewachsenen Zellen enthielten 54 ± 22 mU/mg MCase Aktivität. •Eine über Avidin-Chromatographie gereinigte Präparation an Biotin-Carboxylasen wies MCase Aktivität in Höhe von 4600 ± 966 mU/mg (Iso-Zellen) bzw. von 1900 ± 1200 mU/mg (Cs-Zellen) auf. GCase Aktivität konnte nicht nachgewiesen werden. •Die Untereinheiten der MCase und GCase wurden rekombinant in E. coli synthetisiert und partiell gereinigt. Eine Bestimmung der Aktivität mit den selbsthergestellten CoA-Substraten konnte aber nur mit MCoA für die MCase erfolgen (2700 ± 126 mU/mg). Mit der rekombinanten GCase und GCoA 19 bzw. MCoA 29 konnte dagegen keine Aktivität bestimmt werden. •Neben den Carboxylasen wurden auch AtuD, AtuE und AtuA rekombinant exprimiert und als His-Tag Fusionsproteine aus E. coli Rohextrakten gereinigt. •Für AtuD konnte Citronellyl-CoA Dehydrogenase Aktivität sowohl über einen optischen Enzymassay (464 ± 9,9 mU/mg) als auch über den Nachweis des Reaktionsproduktes mittels HPLC-(ESI)-MS gezeigt werden. Eine Isolierung des so bereitgestellten cis-Geranyl-CoAs 19a aus dem Reaktionsansatz war jedoch nicht möglich. Auch eine Kopplung des Assays mit dem Carboxylase Assay und somit ein möglicher direkter Umsatz des gebildeten cis-GCoAs 19a über die Geranyl-CoA Carboxylase war nicht erfolgreich. •Die gereinigten rekombinanten Proteine AtuE (IHG-CoA Hydratase) und AtuA (mögliche HHG-CoA Lyase) wurden in erste Kristallisationsversuche eingesetzt. Für AtuE konnte kürzlich die Struktur mit 2,3 Å Auflösung gelöst werden. Der geplanten biochemischen Analyse beider Enzyme stand die Nichtverfügbarkeit der entsprechenden Substrate Isohexenyl-glutaconyl-CoA (IHG-CoA, 20) und 3-Hydroxy-3-isohexenyl-glutaryl-CoA (HHG-CoA, 21) entgegen. Der Versuch das Substrat der Hydratase enzymatisch über die Reaktion der vorangestellten GCase zu generieren scheiterte. Im Verlauf der chemischen Arbeiten gelang es allerdings eine Vorstufe des AtuE-Substrates 94 darzustellen. Der notwendige CoA-Ester 20 wurde synthetisiert. Eine Aktivität von nativem und rekombinantem AtuE als Hydratase konnte allerdings noch nicht gezeigt werden. Eine Funktion von AtuA als vermutliche HHG-CoA Lyase konnte in dieser Arbeit aufgrund des fehlenden Substrates 21 nicht untersucht werden. •Eine Trennung des cis/trans Isomerengemisches der Geranylsäure 8 war erst nach Überführung dieser in die jeweiligen Methylester 115a bzw. 115b möglich. Beide Methylester können nun als Referenzen für folgende Metabolit-Analysen dienen. Eine Verseifungsreaktion lieferte die isomerenreinen Säuren (8a, 8b) und nach der CoA-Ester Synthese auch cis- und trans-Geranyl-CoA (19a, 19b). •Unter Verwendung des Geranylsäuremethylesters 115 konnte erstmals eine Vorstufe des Hydratase-Substrates 125 in hoher Ausbeute dargestellt werden. Eine Trennung der beiden Doppelbindungsisomere (125a, 125b) gelang über semi-präparative HPLC. Diese Methylester stehen nun ebenfalls als Referenzverbindungen zur Verfügung. Durch Verseifung konnte die racemische Dicarbonsäure 94 und nach Synthese des CoA-Esters erstmalig auch das racemische Hydratase-Substrat 20 erhalten werden. Eine mögliche stereospezifische Hydroxylierung durch das Enzym Isohexenyl-glutaconyl-CoA Hydratase (AtuE) konnte nicht untersucht werden, da die hier dargestellten Isomere der Vorstufe (125a, 125b) nicht in die CoA-Ester Synthese eingesetzt werden konnten. •Versuche eine Vorstufe des Hydratase-Produktes/Lyase-Substrates darzustellen, waren nicht erfolgreich, jedoch konnten Daten erhalten werden, welche auf eine Instabilität der Verbindung 104 als eine Vorstufe des HHG-CoAs 21 hindeuten. Dies könnte bedeuten, dass auch das Substrat 21 der Lyase selbst nicht stabil ist und somit in vivo nur als kurzlebiges Intermediat vorliegt. Das 3-Hydroxy-3-isohexenyl-glutaryl-CoA (HHG-CoA, 21) ist für weitere Untersuchungen somit bislang nicht verfügbar. Im Gegensatz dazu gelang es erstmalig eine Vorstufe des Lyase-Produktes darzustellen. Dieser Methylester 133 steht nun ebenfalls als Referenzverbindung für anschließende Untersuchungen zur Verfügung.The degradation of citronellol and related acyclic terpenoid molecules is difficult due to a methyl group in beta-position and not widely spread. P. aeruginosa and P. citronellolis circumvent the blocking of the beta-oxidation by modifying this methyl group. During the “lower atu pathway” the methyl group of the coenzyme A ester 19 (geranyl-CoA) is first carboxylated. By hydration of the double bound the conditions for the cleavage of the acetate functionality are established. The resulting compound 23 (7-methyl-3-oxo-6-octenoyl-CoA) can then be further degraded by beta-oxidation. •In crude extracts of sodium citronellate (Cs)-grown P. aeruginosa PAO1 cells a specific geranyl-CoA carboxylase (GCase) activity of 11 ± 6 mU/mg and a specific methylcrotonyl-CoA carboxylase (MCase) activity of 47 ± 11 mU/mg was detected. Crude extracts of isovaleric acid (Iso)-grown cells showed MCase activity of 54 ± 22 mU/mg. •A by avidin-chromatography purified preparation of biotin-carboxylases showed MCase activity in an amount of 4600 ± 966 mU/mg (Iso cells) and of 1900 ± 1200 mU/mg (Cs cells). GCase activity could not be detected. •The subunits of the MCase and GCase were synthesized recombinantly in E. coli and partially purified. A determination of the activity of both carboxylases with the self-made CoA-substrates could be done only with MCoA for the MCase (2700 mU/mg ± 126 mU/mg). In contrast with the recombinant GCase and GCoA 19 or MCoA 29 no activity could be determined. •In addition to the carboxylases AtuD, AtuE and AtuA were recombinantly expressed as his-tag fusion proteins and purified from E. coli crude extracts. •For AtuD the citronellyl-CoA dehydrogenase activity could be shown by an optical enzyme assay (464 ± 9,9 mU/mg) and also by detecting the reaction product by HPLC-(ESI)-MS. An isolation of the so provided cis-geranyl-CoA 19a from the reaction mixture was not possible. Also a coupling of the assay with the carboxylase assay, and thus a possible direct turnover of the resulting cis-GCoA 19a by the geranyl-CoA carboxylase was not successful. •The purified recombinant proteins AtuE (isohexenyl-glutaconyl-CoA hydratase) and AtuA (putative 3-hydroxy-3-isohexenyl-glutaryl-CoA:acetate lyase) were used in crystallization experiments. Recently, for AtuE the structure was resolved at 2.3 Å. For AtuA are currently still no usable crystals available. The planned biochemical analysis of both enzymes was finally opposed by the non-availability of the appropriate substrates isohexenyl-glutaconyl-CoA (IHG-CoA, 20) and 3-hydroxy-3-isohexenyl-glutaryl-CoA (HHG-CoA, 21). The attempt to generate the substrate of the hydratase enzymatically by the reaction of the preceding GCase failed. In the course of the chemical works a precursor of the AtuE substrate 94 was successfully obtained. The necessary CoA ester 20 was synthesised. However, an activity of native and recombinant AtuE as IHG-CoA hydratase could not be shown yet under the chosen conditions. The function of AtuA presumed HHG-CoA lyase could not be studied in this work due to the lack of substrate 21. •A separation of the cis/trans isomers of geranic acid 8 was possible only after the conversion of these into the respective methyl ester 115a and 115a. Both methyl esters can now serve as references for the following metabolite analysis. Saponification resulted in the isomerically pure acids (8a, 8b) and after the CoA ester synthesis the cis and trans geranyl-CoA (19a, 19b) were available. •Using the geranic acid methyl ester 115 a precursor of the hydratase substrate 125 could be shown in high yield for the first time. A separation of the two double bond isomers (125a, 125b) was carried out by semi-preparative HPLC. These methyl esters are now available as reference compounds. Through saponification the racemic dicarboxylic acid 94 and after the synthesis of the CoA ester the racemic hydratase substrate 20 were obtained for the first time. A possible stereospecific hydroxylation by the enzyme isohexenyl-glutaconyl-CoA hydratase (AtuE) couldn´t be investigated, because the shown isomers of the precursor (125a, 125b) couldn´t be used in the CoA ester synthesis. •Attempts to synthesise a precursor of the hydratase product/lyase substrate were made but not successful. Data that indicate an instability of the compound 104 as a precursor of the HHG-CoA 21 were obtained. This could imply that the substrat 21 of the lyase itself is not stable in vivo and thus is present only as a short-lived intermediate. Therefore the 3-hydroxy-3-isohexenyl-glutaryl-CoA (HHG-CoA, 21) is currently not available for further investigations. In contrast a precursor of the lyase product 133 was synthesised for the first time. This methyl ester 133 is now also available as a reference compound for subsequent investigations

Data from: Neuronal connectome of a sensory-motor circuit for visual navigation

No description availabl

Phototaxis ImageJ Macro

ImageJ macro used to score phototaxis videos