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Wilhelm L. and Gruber S.
Methods Mol Biol. (2017), 1624, 117-126.
doi: 10.1007/978-1-4939-7098-8_10.

A Chromosome Co-Entrapment Assay to Study Topological Protein-DNA Interactions.

Chromosome organization, DNA replication, and transcription are only some of the processes relying on dynamic and highly regulated protein-DNA interactions. Here, we describe a biochemical assay to study the molecular details of associations between ring-shaped protein complexes and chromosomes in the context of living cells. Any protein complex embracing chromosomal DNA can be enriched by this method, allowing for the underlying loading mechanisms to be investigated.


Brain region mediates pleasure of eating

Providing the body with food is essential for survival. But even when full, we can still take pleasure in eating. Researchers at the Max Planck Institute of Neurobiology in Martinsried and the Friedrich Miescher Institute in Basel have characterized a type of neuron in the amygdala of the mouse brain that is involved in making eating rewarding. When given the choice, mice choose to activate these amygdala neurons. Artificially activating these neurons increases food intake even when the mice are not hungry. The neurobiologists have identified the neuronal circuitry underlying this behavior, raising the possibility that there could be cells with a similar function in the human brain.

The amygdala in the brain plays a key role in emotional responses, decision-making and association of events with emotions like fear or pleasure. In recent years, it has become apparent that this brain region also plays a role in eating behavior. Researchers at the California Institute of Technology have previously shown that activating a certain type of neurons in the amygdala (known as PKC-delta neurons) causes mice to stop eating. “If the mice eat something which has gone bad, for example, activity of these cells causes them to immediately stop eating,” explains Rüdiger Klein, Director at the Max Planck Institute of Neurobiology. “I found this study on ‘anorexia neurons’ in the amygdala fascinating,” says Klein, “so when three doctoral students with very different methodological backgrounds came to me, I proposed them to work on the amygdala project. Their task was to find out whether there are neurons that are involved in positively regulating food consumption.” With this task in mind, the group focused on a different population of amygdala cells named HTR2a neurons.”

Specializing in behavior, electrophysiology and anatomy, the three doctoral students were able to provide insight into HTR2a cell function from a range of angles. “It was a very collaborative project,” recalls Amelia Douglass, one of the three lead authors of the study, which was published in Nature Neuroscience. “We frequently sat down together, went through the results and then built on them, applying new cutting-edge methods in the process.” Using this approach, the young researchers gradually discovered the role of the previously unstudied HTR2a amygdala cells and identified the neural circuitry involved. “Basically we showed that HTR2a cells have a positive effect on food consumption in mice, and that the mice like it when these cells are active,” says Douglass.

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Using light alone, scientists from the Max Planck Institute of Neurobiology in Martinsried are now able to reveal pairs or chains of functionally connected neurons under the microscope. The new optogenetic method, named Optobow, allows probing the pathways along which information flows by targeted activation of individual neurons and monitoring the responses of neighboring cells. The shape of the cells and their contact points are also revealed – even in dense tissue in which the thin fibers of thousands of cells are interwoven. With Optobow, it is thus possible to discover individual components of functional circuits in the living brain.

Modern methods provide increasingly detailed insights into the structure and functions of the brain. It is now possible to observe under the microscope when and where neurons are active during a particular task, such as sensory perception or behavior. However, it is still largely impossible to establish whether the active cells are connected to each other and to identify the sequence in which they exchange information. To date, such information could only be obtained, in part and with considerable effort, using electrophysiology and electron microscopy methods.

With electrophysiology, the activity of neighboring cells is measured using very thin, hollow needles, which serve as electrodes. These are inserted into the brain through holes in the skin and the skull of the animal. However, it is almost impossible to record activity from very small, densely clustered or deep-lying neurons, and it is also difficult to trace long connection pathways between neurons. Moreover, impulses from only one, or few cells, can be recorded at a time. With modern electron microscopy processes (connectomics), all neurons and their connections in a fixed brain are recorded, layer by layer, by a scanning electron microscope and then reconstructed on a computer. Although this method provides a fantastically detailed snapshot of the neural wiring pattern, the information about the dynamic transmission of nerve impulses in the living brain is lost. Both of these approaches thus have clear limitations. “We were looking for a way to observe the connections and transmission of information between nerve cells in the active brain without killing or disrupting the brain,” explains Dominique Förster. Motivated by this quest, Förster and his colleagues from Herwig Baier’s Department at the Max Planck Institute of Neurobiology developed the Optobow method.

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Understanding brain function generally requires a deep engagement with the matter at least at two separate levels. These are the level of behavioral algorithms on the one hand and the level of neural implementation on the other. These questions can be rephrased as “What exactly is the brain doing?” and “How does it do whatever it is doing?”. Central to this division of labor in the brain sciences is the idea, inspired by David Marr, that the brain implements algorithms, whether for sensory processing, decision making, or motor control, and that these algorithms can be inferred from careful observation of ethologically relevant behaviors. Once a particular algorithm is understood and delineated, we can interrogate its neural implementation by measuring from and manipulating the underlying neural circuits in the context of behavior. Uncovering behavioral algorithms usually does not require modern and cutting edge technologies like optogenetics, wholebrain imaging and genetic editing, it merely requires precise observation, thorough experimental design and, most importantly, rigorous and deep thinking. Such basic scientific qualities are somewhat threatened, and often considered quaint, in the modern era of big data, high throughput and cutting edge technologies. In light of these concerns it comes as a truly pleasant surprise that a study based exclusively on such somewhat antiquated techniques can still find a place in a high impact journal and generate plenty of enthusiasm in the scientific community. The study published recently in Nature by the group of Florian Engert at Harvard in collaboration with the group of Ruben Portugues at the Max Planck Institute of Neurobiology relied entirely on the old-fashioned technique of careful behavioral observations and could have been accomplished in the same form half a century ago. As such it might as well considered “timeless”.

In short, they wanted to know how a larval zebrafish, when placed into a flowing body of water, can detect the presence of the current and then effectively swim against it. More precisely, the group, spearheaded by the team of Pablo Oteiza and Iris Odstrcil, identified the lateral line as one of the main sensory modalities the fish can use to detect that it is drifting with respect to the shore – and more importantly - they could uncover the precise algorithms that translate this sensory input into the appropriate motor commands.

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Pünzeler S, Link S, Wagner G, Keilhauer EC, Kronbeck N, Spitzer RM, Leidescher S, Markaki Y, Mentele E, Regnard C, Schneider K, Takahashi D, Kusakabe M, Vardabasso C, Zink LM, Straub T, Bernstein E, Harata M, Leonhardt H, Mann M, Rupp RA, Hake SB.
EMBO J, 2017, doi: 10.15252/embj.201695757. [Epub ahead of print]

Multivalent binding of PWWP2A to H2A.Z regulates mitosis and neural crest differentiation.

Replacement of canonical histones with specialized histone variants promotes altering of chromatin structure and function. The essential histone variant H2A.Z affects various DNA-based processes via poorly understood mechanisms. Here, we determine the comprehensive interactome of H2A.Z and identify PWWP2A as a novel H2A.Z-nucleosome binder. PWWP2A is a functionally uncharacterized, vertebrate-specific protein that binds very tightly to chromatin through a concerted multivalent binding mode. Two internal protein regions mediate H2A.Z-specificity and nucleosome interaction, whereas the PWWP domain exhibits direct DNA binding. Genome-wide mapping reveals that PWWP2A binds selectively to H2A.Z-containing nucleosomes with strong preference for promoters of highly transcribed genes. In human cells, its depletion affects gene expression and impairs proliferation via a mitotic delay. While PWWP2A does not influence H2A.Z occupancy, the C-terminal tail of H2A.Z is one important mediator to recruit PWWP2A to chromatin. Knockdown of PWWP2A in Xenopus results in severe cranial facial defects, arising from neural crest cell differentiation and migration problems. Thus, PWWP2A is a novel H2A.Z-specific multivalent chromatin binder providing a surprising link between H2A.Z, chromosome segregation, and organ development.


Red, green and blue: normally, no more than three different colours can be detected simultaneously in fluorescence microscopy. Thanks to recent RGB nanotechnology, similar to that used in computer monitors, it is now possible to generate 124 virtual colours under the microscope. The three primary colours are arranged in various mixing ratios on a special DNA lattice. This creates individual colour pixels under the microscope. The new method was developed by scientists at the Max Planck Institute of Biochemistry, Ludwig Maximilian University of Munich, Germany, and the Wyss Institute for Biologically Inspired Engineering at Harvard University in the US. The team’s work was published in the journal Science Advances

Biomedical research has made tremendous strides in recent decades. Using the latest microscopes, scientists are analyzing the function and interactions of molecules in cells with ever greater detail. Now, researchers are looking for methods to image multiple molecules simultaneously.

RGB nanotechnology
A team of scientists from Germany and the US headed by Ralf Jungmann and Peng Yin has now developed substances known as metafluorophors. “The technology can be compared to that of an RGB monitor,” explains Jungmann, Leader of the Molecular Imaging and Bionanotechnology Research Group. To display a wide range of colours on a screen, each colour is mixed from the three primary colours: red, green and blue. “We’ve transferred this approach to the nanometre scale. Instead of a single fluorescence dye molecule, multiple fluorescent molecules are applied to a carrier material, which serves as a kind of experimental board. Depending on the proportion of the three primary colours, they appear in different colours under the microscope, comparable to nanometre-scale colour pixels on a computer screen.“

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Jonak K, Zagoriy I, Oz T, Graf P, Rojas J, Mengoli V, Zachariae W.
Cell Cycle, 2017, [Epub ahead of print].

APC/C-Cdc20 mediates deprotection of centromeric cohesin at meiosis II in yeast.

Cells undergoing meiosis produce haploid gametes through one round of DNA replication followed by 2 rounds of chromosome segregation. This requires that cohesin complexes, which establish sister chromatid cohesion during S phase, are removed in a stepwise manner. At meiosis I, the separase protease triggers the segregation of homologous chromosomes by cleaving cohesin's Rec8 subunit on chromosome arms. Cohesin persists at centromeres because the PP2A phosphatase, recruited by the shugoshin protein, dephosphorylates Rec8 and thereby protects it from cleavage. While chromatids disjoin upon cleavage of centromeric Rec8 at meiosis II, it was unclear how and when centromeric Rec8 is liberated from its protector PP2A. One proposal is that bipolar spindle forces separate PP2A from Rec8 as cells enter metaphase II. We show here that sister centromere biorientation is not sufficient to "deprotect" Rec8 at meiosis II in yeast. Instead, our data suggest that the ubiquitin-ligase APC/CCdc20 removes PP2A from centromeres by targeting for degradation the shugoshin Sgo1 and the kinase Mps1. This implies that Rec8 remains protected until entry into anaphase II when it is phosphorylated concurrently with the activation of separase. Here, we provide further support for this model and speculate on its relevance to mammalian oocytes.


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Lademann CA, Renkawitz J, Pfander B, Jentsch S.
Cell Rep, 2017, 19, 1294-1303.

The INO80 Complex Removes H2A.Z to Promote Presynaptic Filament Formation during Homologous Recombination.

The INO80 complex (INO80-C) is an evolutionarily conserved nucleosome remodeler that acts in transcription, replication, and genome stability. It is required for resistance against genotoxic agents and is involved in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR). However, the causes of the HR defect in INO80-C mutant cells are controversial. Here, we unite previous findings using a system to study HR with high spatial resolution in budding yeast. We find that INO80-C has at least two distinct functions during HR-DNA end resection and presynaptic filament formation. Importantly, the second function is linked to the histone variant H2A.Z. In the absence of H2A.Z, presynaptic filament formation and HR are restored in INO80-C-deficient mutants, suggesting that presynaptic filament formation is the crucial INO80-C function during HR.


Scientists from the Max Planck Institute of Neurobiology in Martinsried have developed a method that allows them to identify nerve cells involved in a specific motor command. For the first time, it is now possible to evoke behaviour of a small fish by artificially activating just a handful of neurons. Understanding the core components of a neural circuit is a key step for deciphering the complex code underlying even elementary brain functions.

Recent years have seen much progress in understanding of the brain’s structure and function. Advances in microscopy and functional imaging enable researchers to monitor the activity of neuronal populations, while an animal perceives sensory stimuli or generates specific behaviours. However, these studies often cannot distinguish cause from consequence of the observed changes in activity. Using the method of optogenetics, scientists can find out which neurons are essential for the chain of events that ultimately lead to behaviour, and which neurons may serve other tasks or are merely by-standers. A particular challenge for this field of research is the staggering degree of "interconnectedness" of neuronal networks. Activating even a single neuron may send ripples through a large part of the nervous system. The new study from Herwig Baier and his team at the Max Planck Institute of Neurobiology has removed both obstacles in one sweep: it is now possible to pinpoint cause and effect to the cellular components of neural circuitry while simultaneously watching how activity propagates through the entire brain network and evokes behaviour.

The Martinsried scientists developed a workflow allowing the 3D photo-stimulation of multiple targeted neurons while, at the same time, imaging network activity in the brain of a zebrafish larva. “The zebrafish with its small, translucent brain is ideal for our new method”, explains Marco dal Maschio, one of the two lead authors of the publication describing the technique.

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Del Toro D*, Ruff T*, Cederfjäll E, Villalba A, Seyit-Bremer G, Borrell V, Klein R.
* co-first-author
Cell, 2017, 169, 621-635.

Regulation of Cerebral Cortex Folding by Controlling Neuronal Migration via FLRT Adhesion Molecules.

The folding of the mammalian cerebral cortex into sulci and gyri is thought to be favored by the amplification of basal progenitor cells and their tangential migration. Here, we provide a molecular mechanism for the role of migration in this process by showing that changes in intercellular adhesion of migrating cortical neurons result in cortical folding. Mice with deletions of FLRT1 and FLRT3 adhesion molecules develop macroscopic sulci with preserved layered organization and radial glial morphology. Cortex folding in these mutants does not require progenitor cell amplification but is dependent on changes in neuron migration. Analyses and simulations suggest that sulcus formation in the absence of FLRT1/3 results from reduced intercellular adhesion, increased neuron migration, and clustering in the cortical plate. Notably, FLRT1/3 expression is low in the human cortex and in future sulcus areas of ferrets, suggesting that intercellular adhesion is a key regulator of cortical folding across species.