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High-grade serous carcinomas (HGSC) make up the majority of ovarian cancer cases. Unfortunately, they have the lowest survival rates. HGSC is a tumor type that occurs primarily in the ovaries and spreads throughout the abdominal cavity. Most patients are diagnosed with late-stage disease that has already spread. Until recently, therapy has been limited to surgery and traditional chemotherapeutic agents. A systematic examination of the tumor and surrounding tissue — particularly normal cells called fibroblasts — has revealed a new treatment target that could potentially prevent the rapid dissemination and poor prognosis associated with high-grade serous carcinoma.

In close collaboration with Fabian Coscia and Matthias Mann, from the Max Planck Institute of Biochemistry in Munich and University of Copenhagen, researchers profiled the expression of more than 5,000 proteins in both normal and cancerous tissues derived from minute amounts of patient biobank material. “When we then got our first data, we were fascinated to find that the metastatic stroma was characterized by a highly conserved protein signature, as opposed to the cancer cells”, adds Fabian Coscia, postdoctoral researcher in Matthias Mann’s group and one of the two first-authors of the study. As these metastatic changes were seen in all of their analyzed patients, the team then went on to understand its functional role during metastasis with the ultimate goal to find novel therapeutic targets.

Indeed, they discovered a metabolic enzyme, nicotinamide N-methyltransferase (NNMT), highly expressed in the stroma surrounding metastatic cancer cells. The researchers found that NNMT causes widespread gene expression changes in the tumor stroma, converting normal fibroblasts to cancer-associated fibroblasts that support and accelerate tumor growth. Stromal NNMT expression encouraged ovarian cancer migration, proliferation, growth and metastasis. It was associated with poor clinical outcomes in patients.

They also found that inhibition of NNMT activity may be able to reduce or even reverse many of the tumor-promoting effects of cancer-associated fibroblasts. This suggests, they note, that the stroma should be explored as a new treatment target. Coscia, co-first author on the manuscript who led the proteomics analysis, added that “this method may be used to discover other proteins that are important for metastasis and to identify early changes during disease development.”

“When we put it all together,” Lengyel added, “it gave us exciting results. We have linked high-end technology including proteomics and metabolomics to functional analysis to improve our understanding of the stroma."

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In general, a nerve cell receives input from a number of presynaptic cells, processes the signals, and passes its output to downstream cells. In the cell CT1, however, each of the approximately 1400 cell areas works like a separate neuron. This allows CT1 to access information from all facets of the fly's complex eye and to contribute locally to the calculation of motion direction. Using a computer model of the cell, Alexander Borst and Matthias Meier from the Max Planck Institute of Neurobiology show that CT1 is reaching biophysical limits.

"That’s an amazing cell!" This was the first impression of Alexander Borst, as Matthias Meier showed him the results. Together, the two neurobiologists have demonstrated what is also suspected for amacrine cells in the mammalian retina: It is possible that numerous isolated microcircuits exist in a single nerve cell.

Borst and Meier investigate the visual system of fruit flies, whose complex eyes each consist of about 700 facets. CT1 contacts each of the cell columns that connect to these facets in the brain. In addition, the synapses of CT1 reach into two different brain regions, responsible for the processing of light or dark edges. Thus, CT1 connects to about 1400 areas in the fly brain. This, however, should corrupt the whole system. Each cell column processes changes in light perceived by “their” facet. If the signals of the columns were mixed, the entire image information for downstream cells would be lost.

As flies see very well, a loss of image information does not seem to be an issue. The two neurobiologists could show that each contact area of CT1 is an electrically isolated, independent functional unit. Each of these units receives input from its associated column and returns its output to the same column. Calcium measurements and computer modelling show that essentially, there is no cross-talk between neighboring units or with the cell body.

For the cell units to be electrically isolated from each other, their connections should be thin and long, which increases the electrical resistance. CT1 achieves this with connections of merely 100 nanometers in diameter. In addition, the "connection cables" often form loops. In this way, the connections between neighboring units are about ten times longer than needed to bridge the distance. "It wouldn’t be possible for the connections to get much thinner or longer in the fly brain," says Borst. Why CT1 is so different from most other cells is still a mystery. "It saves cell bodies, but that is certainly not the only reason”, muses Matthias Meier. “If that was the case, such huge amacrine cells wouldn’t be so rare." So far, only very few cells are known with such a structure. Amongst them, CT1 is an extreme example, of which only two cells exist in a fly brain, one per hemisphere.

The scientists are also not yet sure about the exact functions of CT1. The output of the CT1 subunits goes to T4 or T5 cells, depending on their location. These calculate the direction of images moving in front of the fly’s eye. Interestingly, CT1 cells specifically target the motion-sensitive T4 and T5 cells only on one-half of their dendrites. How CT1 thereby affects motion vision is one of the next questions the Max Planck neurobiologists want to investigate.

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Hopfler, M., Kern, M.J., Straub, T., Prytuliak, R., Habermann, B.H., Pfander, B., and Jentsch, S.
EMBO J, 2019, [Epub ahead of print].
(IMPRS-LS students are in bold)
doi: 10.15252/embj.2018100368

Slx5/Slx8-dependent ubiquitin hotspots on chromatin contribute to stress tolerance

Chromatin is a highly regulated environment, and protein association with chromatin is often controlled by post-translational modifications and the corresponding enzymatic machinery. Specifically, SUMO-targeted ubiquitin ligases (STUbLs) have emerged as key players in nuclear quality control, genome maintenance, and transcription. However, how STUbLs select specific substrates among myriads of SUMOylated proteins on chromatin remains unclear. Here, we reveal a remarkable co-localization of the budding yeast STUbL Slx5/Slx8 and ubiquitin at seven genomic loci that we term "ubiquitin hotspots". Ubiquitylation at these sites depends on Slx5/Slx8 and protein turnover on the Cdc48 segregase. We identify the transcription factor-like Ymr111c/Euc1 to associate with these sites and to be a critical determinant of ubiquitylation. Euc1 specifically targets Slx5/Slx8 to ubiquitin hotspots via bipartite binding of Slx5 that involves the Slx5 SUMO-interacting motifs and an additional, novel substrate recognition domain. Interestingly, the Euc1-ubiquitin hotspot pathway acts redundantly with chromatin modifiers of the H2A.Z and Rpd3L pathways in specific stress responses. Thus, our data suggest that STUbL-dependent ubiquitin hotspots shape chromatin during stress adaptation.


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Burgold, J.*, Schulz-Trieglaff, E.K.*, Voelkl, K., Gutierrez-Angel, S., Bader, J.M., Hosp, F., Mann, M., Arzberger, T., Klein, R., Liebscher, S., and Dudanova, I.
Sci Rep, 2019, 9, 6634.
* authors contributed equally
(IMPRS-LS students and IMPRS-LS alumni are in bold)
doi: 10.1038/s41598-019-43024-w

Cortical circuit alterations precede motor impairments in Huntington's disease mice

Huntington's disease (HD) is a devastating hereditary movement disorder, characterized by degeneration of neurons in the striatum and cortex. Studies in human patients and mouse HD models suggest that disturbances of neuronal function in the neocortex play an important role in disease onset and progression. However, the precise nature and time course of cortical alterations in HD have remained elusive. Here, we use chronic in vivo two-photon calcium imaging to longitudinally monitor the activity of identified single neurons in layer 2/3 of the primary motor cortex in awake, behaving R6/2 transgenic HD mice and wildtype littermates. R6/2 mice show age-dependent changes in cortical network function, with an increase in activity that affects a large fraction of cells and occurs rather abruptly within one week, preceeding the onset of motor defects. Furthermore, quantitative proteomics demonstrate a pronounced downregulation of synaptic proteins in the cortex, and histological analyses in R6/2 mice and human HD autopsy cases reveal a reduction in perisomatic inhibitory synaptic contacts on layer 2/3 pyramidal cells. Taken together, our study provides a time-resolved description of cortical network dysfunction in behaving HD mice and points to disturbed excitation/inhibition balance as an important pathomechanism in HD.


A collaboration between Georgia Tech and the Max Planck Institute of Neurobiology (MPIN) has received a grant of $750,000 over three years from the Human Frontier Science Program (HFSP). The award will allow research on the molecular and genetic encoding of complex behaviors.

The team is led by Georgia Tech’s J. Todd Streelman and MPIN’s Herwig Baier. Streelman is a professor in, and the chair of, the Georgia Tech School of Biological Sciences. Baier is professor and director at the Max Planck Institute of Neurobiology. “It remains incredibly difficult to identify the cellular basis and the genetic variants underlying complex behavior,” Streelman says. “Understanding how behavior is encoded requires solving a dual problem involving neurodevelopment and circuit function.”

To find answers, Streelman and Baier will develop a model system to chart the complex path from genome to brain to behavior in cichlid fish from Lake Malawi. Male cichlid fish build bowers to attract females for mating. The bowers are either pits, which are depressions in the sand, or castles, which look like volcanoes. Each type corresponds to a specific behavior encoded in a fish strain.

When the two strains mate, their male offspring display a remarkable behavior: First they construct a pit then a castle. This behavior indicates that a single brain containing two genomes can produce each behavior in succession. Moreover, gene expression in the brain is biased toward the pit variant of the genome – or pit allele -- when the fish are digging pits and toward the castle allele when they are building castles. “This phenomenon offers the chance to identify both the genome regulatory logic and the neural circuitry underlying complex behavior in one sweep,” Baier says.

Streelman’s group will use single-cell RNA sequencing to pinpoint the cell populations that mediate context-dependent, allele-specific expression in male bower builders. Baier’s team will use genome editing and optogenetic tools to manipulate particular neurons in the brains of behaving bower builders.

The award is one of only 25 made from a total of 654 letters of intent HFSP received from around the world. HFSP provides funding for frontier research in the life sciences. The highly competitive program is implemented by the International Human Frontier Science Program Organization, based in Strasbourg, France.

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graduation

Congratulations on your PhD!

Sara Gutiérrez Ángel
Mutant Huntingtin toxicity modifiers revealed by a spatiotemporal proteomic profiling
RG: Rüdiger Klein

Ryan Sherrard
Post-transcriptional regulation of the central apoptotic pathway by microRNAs and RNA-binding proteins during C. elegans development
RG: Barbara Conradt


 

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Lemke, S.B., Weidemann, T., Cost, A.L., Grashoff, C., and Schnorrer, F.
PLoS Biol, 2019, 17, e3000057.
doi: 10.1371/journal.pbio.3000057

A small proportion of Talin molecules transmit forces at developing muscle attachments in vivo.

Cells in developing organisms are subjected to particular mechanical forces that shape tissues and instruct cell fate decisions. How these forces are sensed and transmitted at the molecular level is therefore an important question, one that has mainly been investigated in cultured cells in vitro. Here, we elucidate how mechanical forces are transmitted in an intact organism. We studied Drosophila muscle attachment sites, which experience high mechanical forces during development and require integrin-mediated adhesion for stable attachment to tendons. Therefore, we quantified molecular forces across the essential integrin-binding protein Talin, which links integrin to the actin cytoskeleton. Generating flies expressing 3 Förster resonance energy transfer (FRET)-based Talin tension sensors reporting different force levels between 1 and 11 piconewton (pN) enabled us to quantify physiologically relevant molecular forces. By measuring primary Drosophila muscle cells, we demonstrate that Drosophila Talin experiences mechanical forces in cell culture that are similar to those previously reported for Talin in mammalian cell lines. However, in vivo force measurements at developing flight muscle attachment sites revealed that average forces across Talin are comparatively low and decrease even further while attachments mature and tissue-level tension remains high. Concomitantly, the Talin concentration at attachment sites increases 5-fold as quantified by fluorescence correlation spectroscopy (FCS), suggesting that only a small proportion of Talin molecules are mechanically engaged at any given time. Reducing Talin levels at late stages of muscle development results in muscle-tendon rupture in the adult fly, likely as a result of active muscle contractions. We therefore propose that a large pool of adhesion molecules is required to share high tissue forces. As a result, less than 15% of the molecules experience detectable forces at developing muscle attachment sites at the same time. Our findings define an important new concept of how cells can adapt to changes in tissue mechanics to prevent mechanical failure in vivo.


In everyday life, blinking lights can send signals – for example, that a car is going to turn. Now, researchers have engineered tiny “blinkers” that reveal single molecules of RNA or protein inside cells based on the duration and frequency of each flash. The research, published in the ACS journal Nano Letters, could allow scientists to see the locations of many different biomolecules in a cell simultaneously, possibly leading to better diagnostics and treatments.

Recently, scientists have developed super-resolution microscopes that can image single molecules that are only a few nanometers in size. To discriminate a specific nucleic acid or protein, they typically add a fluorescent probe that binds to that molecule and emits a certain wavelength of light. However, because the emission wavelengths of different fluorescent probes can overlap, researchers can usually only detect three or four unique proteins or nucleic acids at a time, instead of the thousands that exist in cells. Ralf Jungmann and colleagues from the Max Planck Institute of Biochemistry in Martinsried wondered if they could use fluorescent probes that blink with light at a variable duration and frequency to detect dozens of biomolecules at once. That way, they could use a single fluorophore to image many different molecules.

The researchers based their system on complementary sequences of DNA that come together to link a fluorophore with a target biomolecule and then fall apart again, generating a blinking fluorescent signal. By varying the length and number of DNA sequences bound to the target, the researchers could adjust how long the blink lasted, as well as how often blinks occurred. To test their approach in cells, the researchers imaged two different RNA molecules and two proteins. Then, they used three fluorescent probes to image 124 distinct DNA structures that contained different numbers of target DNAs so that they blinked at different frequencies. The procedure took only a few minutes and had an accuracy of 97.6 percent, the researchers say.

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graduation

Congratulations on your PhD!

Leonie Mönkemeyer
Structural and Functional Studies on the Eukaryotic Chaperonin TRiC and its Cooperating Chaperone Hgh1
RG: F.-Ulrich Hartl

Yan Xiao
GroEL Ring Separation and Exchange in the Chaperonin Reaction
RG: F.-Ulrich Hartl


 

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Monkemeyer, L., Klaips, C.L., Balchin, D., Korner, R., Hartl, F.U., and Bracher, A.
Mol Cell, 2019, [Epub ahead of print].
doi: 10.1016/j.molcel.2019.01.034

Chaperone Function of Hgh1 in the Biogenesis of Eukaryotic Elongation Factor 2

Eukaryotic elongation factor 2 (eEF2) is an abundant and essential component of the translation machinery. The biogenesis of this 93 kDa multi-domain protein is assisted by the chaperonin TRiC/CCT. Here, we show in yeast cells that the highly conserved protein Hgh1 (FAM203 in humans) is a chaperone that cooperates with TRiC in eEF2 folding. In the absence of Hgh1, a substantial fraction of newly synthesized eEF2 is degraded or aggregates. We solved the crystal structure of Hgh1 and analyzed the interaction of wild-type and mutant Hgh1 with eEF2. These experiments revealed that Hgh1 is an armadillo repeat protein that binds to the dynamic central domain III of eEF2 via a bipartite interface. Hgh1 binding recruits TRiC to the C-terminal eEF2 module and prevents unproductive interactions of domain III, allowing efficient folding of the N-terminal GTPase module. eEF2 folding is completed upon dissociation of TRiC and Hgh1.