Oxygen Imaging of hypoxic pockets in the mouse cerebral cortex

Felix R. M. Beinlich*†, Antonios Asiminas†, Verena Untiet, Zuzanna Bojarowska, Virginia Plá, Björn Sigurdsson, Vincenco Timmel, Lukas Gehrig, Michael H Graber, Hajime Hirase, Maiken Nedergaard

*These authors contributed equally to this work.

Science 383, 1471-1478

Open Access Link

Consciousness is lost within seconds upon cessation of cerebral blood flow. The brain cannot store oxygen, and interruption of oxidative phosphorylation is fatal within minutes. Yet only rudimentary knowledge exists regarding cortical partial oxygen tension (Po2) dynamics under physiological conditions. Here we introduce Green enhanced Nano-lantern (GeNL), a genetically encoded bioluminescent oxygen indicator for Po2 imaging. In awake behaving mice, we uncover the existence of spontaneous, spatially defined “hypoxic pockets” and demonstrate their linkage to the abrogation of local capillary flow. Exercise reduced the burden of hypoxic pockets by 52% compared with rest. The study provides insight into cortical oxygen dynamics in awake behaving animals and concurrently establishes a tool to delineate the importance of oxygen tension in physiological processes and neurological diseases.


A mesothelium divides the subarachnoid space into functional compartments

Kjeld Møllgård*, Felix R. M. Beinlich*, Peter Kusk*, Leo M. Miyakoshi*, Christine Delle, Virginia Plá, Natalie L. Hauglund, Tina Esmail, Martin K. Rasmussen, Rysyard S. Gomolka, Yuki Mori, Maiken Nedergaard

*These authors contributed equally to this work.

Science 379, 84-88

The central nervous system is lined by meninges, classically known as dura, arachnoid, and pia mater. We show the existence of a fourth meningeal layer that compartmentalizes the subarachnoid space in the mouse and human brain, designated the subarachnoid lymphatic-like membrane (SLYM). SLYM is morpho- and immunophenotypically similar to the mesothelial membrane lining of peripheral organs and body cavities, and it encases blood vessels and harbors immune cells. Functionally, the close apposition of SLYM with the endothelial lining of the meningeal venous sinus permits direct exchange of small solutes between cerebrospinal fluid and venous blood, thus representing the mouse equivalent of the arachnoid granulations. The functional characterization of SLYM provides fundamental insights into brain immune barriers and fluid transport.

Transcranial bioluminescence imaging for the study of molecule exchange between venous blood and CSF in live mice

Tina Esmail, Maiken Nedergaard, Felix R.M. Beinlich

STAR Protocols 4, 102652 j.xpro.2023.102652

In bioluminescence imaging (BLI), the biochemical reaction between a substrate and enzyme triggers light emission upon convergence. Unlike fluorescence imaging, BLI does not require excitation. In this protocol, we utilize the high signal-to-background ratio of the reaction between luciferase and its substrate to study the exchange of molecules between blood and cerebrospinal fluid. We outline steps for skull window thinning, cisterna magna infusion, intravascular retroorbital injection, and imaging.

Astrocytic chloride is brain state dependent and modulates inhibitory neurotransmission in mice

Verena Untiet, Felix R. M. Beinlich, Peter Kusk, Ning Kang, Antonio Ladrón-de-Guevara, Wei Song, Celia Kjaerby, Mie Andersen, Natalie Hauglund, Zuzanna Bojarowska, Björn Sigurdsson, Saiyue Deng, Hajime Hirase, Nicolas C. Petersen, Alexei Verkhratsky, Maiken Nedergaard

Nature Communications 14:1871

Information transfer within neuronal circuits depends on the balance and recurrent activity of excitatory and inhibitory neurotransmission. Chloride (Cl−) is the major central nervous system (CNS) anion mediating inhibitory neurotransmission. Astrocytes are key homoeostatic glial cells populating the CNS, although the role of these cells in regulating excitatory-inhibitory balance remains unexplored. Here we show that astrocytes act as a dynamic Cl− reservoir regulating Cl− homoeostasis in the CNS. We found that intracellular chloride concentration ([Cl−]i) in astrocytes is high and stable during sleep. In awake mice astrocytic [Cl−]i is lower and exhibits large fluctuation in response to both sensory input and motor activity. Optogenetic manipulation of astrocytic [Cl−]i directly modulates neuronal activity during locomotion or whisker stimulation. Astrocytes thus serve as a dynamic source of extracellular Cl− available for GABAergic transmission in awake mice, which represents a mechanism for modulation of the inhibitory tone during sustained neuronal activity.

Glymphatic influx and clearance are accelerated by neurovascular couping

Stephanie Holstein-Rønsbo, Yiming Gan, Michael J. Giannetto, Martin Kaag Rasmussen, Björn Sigurdsson, Felix Ralf Michael Beinlich, Laura Rose, Verena Untiet, Lauren M. Hablitz, Douglas H. Kelley, Maiken Nedergaard

Nature Neuroscience 26, 1042-1053

Functional hyperemia, also known as neurovascular coupling, is a phenomenon that occurs when neural activity increases local cerebral blood flow. Because all biological activity produces metabolic waste, we here sought to investigate the relationship between functional hyperemia and waste clearance via the glymphatic system. The analysis showed that whisker stimulation increased both glymphatic influx and clearance in the mouse somatosensory cortex with a 1.6-fold increase in periarterial cerebrospinal fluid (CSF) influx velocity in the activated hemisphere. Particle tracking velocimetry revealed a direct coupling between arterial dilation/ constriction and periarterial CSF flow velocity. Optogenetic manipulation of vascular smooth muscle cells enhanced glymphatic influx in the absence of neural activation. We propose that impedance pumping allows arterial pulsatility to drive CSF in the same direction as blood flow, and we present a simulation that supports this idea. Thus, functional hyperemia boosts not only the supply of metabolites but also the removal of metabolic waste.

Lactate biosensors for spectrally and spatially multiplexed fluorescence imaging

Yusuke Nasu, Abhi Aggarwal, Giang N.T. Le, Camilla Trang Vo, Yuki Kambe, Xinxing Wang, Felix R. M. Beinlich, Ashley Bomin Lee, Tina R. Ram, Fangying Wang, Kelsea A. Gorzo, Yuki Kamijo, Marc Boisvert, Suguru Nishinami, Genki Kawamura, Takeaki Ozawa, Hirofumi Toda, Grant R. Gordon, Shaoyu Ge, Hajime Hirase, Maiken Nedergaard, Marie-Eve Paquet, Mikhail Drobizhev, Kaspar Podgorski, Robert E. Campbell

Nature Communications 14:6598

L-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. However, investigations of the inter- and intracellular dynamics of L-lactate are currently hampered by the limited selection and performance of L-lactate-specific genetically encoded biosensors. Here we now report a spectrally and functionally orthogonal pair of high-performance genetically encoded biosensors: a green fluorescent extracellular L-lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular L-lactate biosensor, designated R-iLACCO1. eLACCO2.1 exhibits excellent membrane localization and robust fluorescence response. To the best of our knowledge, R-iLACCO1 and its affinity variants exhibit larger fluorescence responses than any previously reported intracellular L-lactate biosensor. We demonstrate spectrally and spatially multiplexed imaging of L-lactate dynamics by coexpression of eLACCO2.1 and R-iLACCO1 in cultured cells, and in vivo imaging of extracellular and intracellular L-lactate dynamics in mice.

Development and Characterization of Flavin-Binding Fluorescent Proteins, Part II: Advanced Characterization

Nora Lisa Bitzenhofer, Fabienne Hilgers, Gabriela N. Bosio, Joaquim Torra , Giorgia Casini, Felix R. M. Beinlich, Esther Knieps-Gru ̈ nhagen, Valentin Gordeliy, Karl-Erich Jaeger, Santi Nonell, Ulrich Krauss, Thomas Gensch, Thomas Drepper

Mayank Sharma (ed.), Fluorescent Proteins: Methods and Protocols, Methods in Molecular Biology, vol 2564

Flavin-based fluorescent proteins (FbFPs), a class of small fluorescent proteins derived from light-oxygenvoltage (LOV) domains, bind ubiquitous endogenous flavins as chromophores. Due to their unique properties, they can be used as versatile in vivo reporter proteins under aerobic and anaerobic conditions. This chapter presents methodologies for in-depth characterization of the biochemical, spectroscopic, photophysical, and photochemical properties of FbFPs.


Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Timo Appelhans, Felix R.M. Beinlich, Christian P. Richter, Rainer Kurre, Karin B. Busch

JoVE 136:e57690

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15–27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of selflabeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.


Determining crystal structures through crowdsourcing and coursework

Scott Horowitz, Brian Koepnick, Raoul Martin, Agnes Tymieniecki, Amanda A. Winburn, Seth Cooper, Jeff Flatten, David S. Rogawski, Nicole M. Koropatkin, Tsinatkeab T. Hailu, Neha Jain, Philipp Koldewey, Logan S. Ahlstrom, Matthew R. Chapman, Andrew P. Sikkema, Meredith A. Skiba, Finn P. Maloney, Felix R.M. Beinlich, Foldit Players, University of Michigan students, Zoran Popovic, David Baker, Firas Khatib, James C.A. Bardwell

Nature Communications 7:12549

DOI: 10.1038/ncomms12549

We show here that computer game players can build high-quality crystal structures. Introduction of a new feature into the computer game Foldit allows players to build and realspace refine structures into electron density maps. To assess the usefulness of this feature, we held a crystallographic model-building competition between trained crystallographers, undergraduate students, Foldit players and automatic model-building algorithms. After removal of disordered residues, a team of Foldit players achieved the most accurate structure. Analysing the target protein of the competition, YPL067C, uncovered a new family of histidine triad proteins apparently involved in the prevention of amyloid toxicity. From this study, we conclude that crystallographers can utilize crowdsourcing to interpret electron density information and to produce structure solutions of the highest quality.


Shuttling of PINK1 between Mitochondrial Microcompartments Resolved by Triple-Color Superresolution Microscopy

Felix R. M. Beinlich, Christoph Drees, Jacob Piehler, Karin B. Busch

ACS Chemical Biology 10, 1970-1976

DOI: 10.1021/acschembio.5b00295.

The cytosolic phosphatase and tensin homologue Pten-kinase PINK1 involved in mitochondrial quality control undergoes a proteolytic process inside mitochondria. It has been suggested that the protein is not fully imported into mitochondria during this maturation. Here, we have established live cell triple-color superresolution microscopy by combining FPALM and tracking and localization microscopy (TALM) in order to unravel the spatiotemporal organization of the C-terminal kinase domain of PINK1 during this process. We find that the kinase domain is imported into active mitochondria and colocalizes with respiratory complex I at the inner mitochondrial membrane. When the processing step inside mitochondria is inhibited or mitochondria are de-energized, full length PINK1 distributes between the outer and the inner mitochondrial membranes, indicating a holdup of import. These findings give the molecular base for a dual role of PINK1inside energized mitochondria and outside of de-energized mitochondria.

Life-history trade-offs mediate ‘personality’ variation in two colour morphs of the pea aphid, Acyrthosiphon pisum

Wiebke Schuett, Sasha R. X. Dall, Michaela H. Kloesener, Jana Baeumer, Felix Beinlich and Till Eggers

Journal of Animal Ecology 84, 90-101

DOI: 10.1021/acschembio.5b00295.

  1. Life-history trade-offs are considered a major driving force in the emergence of consistent behavioural differences (personality variation); but empirical tests are scarce.
  2. We investigated links between a personality trait (escape response), life-history and state variables (growth rate, size and age at first reproduction, age-dependent reproductive rates, lifetime reproductive success, life span) in red and green colour morphs of clonal pea aphids, Acyrthosiphon pisum. Escape response (dropping/non-dropping off a plant upon a predatory attack) was measured repeatedly to classify individuals as consistent droppers, consistent nondroppers or inconsistents.
  3. Red morphs experienced stronger trade-offs between early reproduction and life span than green morphs; and red consistent (non)droppers had highest lifetime reproductive success. Red droppers followed a risk-averse life-history strategy (high late reproduction), red nondroppers a risk-prone strategy (high early reproduction), while reproductive rates were equivalent for all green behavioural types and red inconsistents.
  4. This suggests that red morphs suffer the highest costs of dropping (they are most conspicuous to predators), which ‘equivalates’ fitness payoffs to both risk-takers (red non-droppers) and risk-averse red droppers. The strong trade-off also means that committing to a particular lifestyle (being consistent) maximises fitness.
  5. Our study suggests that life-history trade-offs likely mediate personality variation but effects might depend on interactions with other organismal characteristics (here: colour morph).


“Personality” Variation in a Clonal Insect: The Pea Aphid, Acyrthosiphon pisum

Wiebke Schuett, Sasha R. X. Dall, Jana Baeumer, Michaela H. Kloesener, Shinichi Nakagawa, Felix Beinlich and Till Eggers

Developmental Psychobiology 53: 631-640

DOI 10.1002/dev.20538

Individuals are often consistent in their behavior but vary from each other in the level of behavior shown. Despite burgeoning interest in such animal personality variation, studies on invertebrates are scarce, and studies on clonal invertebrates nonexistent. This is surprising given the obvious advantages of using invertebrates/clones to tackle the crucial question why such consistent behavioral differences exist. Here we show that individuals of clonal pea aphids exhibit consistent behavioral differences in their escape responses to a predator attack (dropping vs. nondropping off a plant). However, behavior was not repeatable at the clonal level. Genetically identical clones expressed various phenotypes but different clones produced different proportions of each phenotype (dropper, nondropper, and inconsistent). Manipulations of early environmental conditions had little qualitative impact on such patterns. We discuss the importance of our findings for future studies of the evolutionary and ecological consequences of personality variation.