Research Spotlight: How the Benton lab studies the evolution of behavior using fruit flies

Lukas Breitzler (Group Larsch) writes about research from colleagues in the Benton lab.

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On the third floor of the Genopode, many doors open to fruit flies. A first-time visitor might walk past rows of incubators and mistake them for the usual industrial-sized refrigerators and freezers that line the hallways of the CIG. But behind their doors are hundreds of plastic tubes teeming with numberless small fruit flies. Another door opens to a room with two large utility shelves stacked with more boxes of the same tubes. Foam plugs prevent their inhabitants from escaping, while food at the bottom allows them to feed ad libitum. At first glance, the flies look identical. But handwritten notes on labelling tape indicate that the tubes house several different species and genetic strains. Unassuming as they initially appear, these flies are central to the Benton lab’s efforts to answer a fundamental question in biology: how does behavior evolve?

“As a geneticist, I am interested in relating gene changes to neural circuits and behavior,” says Richard Benton, principal investigator of the Benton lab. Richard occupies one of the corner offices on the floor with a view of Lac Léman. Next to his desk, a table holds more boxes of tubes filled with flies. Richard explains that in the past 10 to 20 years, many labs have made impressive advances explaining how genes and neural circuits influence behavior in Drosophila melanogaster—the most commonly studied species of fruit fly. Researchers typically manipulate genes to alter neuronal processes and observe their impact on behavior. “We’re taking a slightly different approach, which is looking at natural variation in behavior to learn how circuits function and how they’re built,” says Richard. Animal genomes contain tens of thousands of genes, some of which influence behaviors. Pinpointing a single gene’s role often involves a fair amount of educated guesswork; not only do you have to pick a gene, but you also have to know what behavior to look for. Richard’s group prefers to approach this problem by working backward: identify differences in behavior first, then investigate the “nuts and bolts of what’s changing genetically.” That’s precisely the approach the lab took in their latest study, published in Nature, which revealed how changes in gene regulation enabled Drosophila to adapt their behavior to varying day lengths. A key aspect to this approach was examining the behavior of not only D. melanogaster but also that of closely related Drosophila species.

One of these species will be familiar to those who have joined the Benton lab’s progress reports: Drosophila sechellia; a close cousin to D. melanogaster. The two species are so closely related, in fact, that they are capable of producing hybrid offspring. But despite their genetic similarities, their habitats are strikingly different. D. sechellia feeds and lays eggs exclusively on Noni fruit, a plant toxic to other Drosophila species. (The fruit also emits a horrendous odor once it reaches a certain stage of ripeness, earning it the nickname ‘vomit fruit’ in regions where it grows. The Benton lab keeps a small, airtight incubator filled with them.) Additionally, while D. melanogaster can be found virtually all over the world—including in northern latitudes, where they experience highly variable day lengths throughout the year—D. sechellia live exclusively on the Seychelles islands near the equator, where the sun rises and sets at nearly the same time year-round. Previously, the group detailed some of the genetic differences that enabled D. sechellia’s specialization to noni fruit. However, whether the pronounced differences in seasonal daylight drove evolutionary adaptation had never been investigated—until Michael Shahandeh, the recent study’s first author, decided to take a look.

Indeed Michael and collaborators conducted experiments demonstrating that D. sechellia barely adjusted their behavior to longer photoperiods. The flies were most active around the same time every day, regardless of whether days were 12 or 20 hours long. D. Melanogaster on the other hand adjusted their activity without problem. Importantly, other species closely related to D. sechellia also adjusted their behavior without problem, suggesting that D. sechellia lost its ability to regulate daily rhythms through evolution as it adapted to a stable environment.

With a behavioral phenotype in hand, the difficulty now lay in identifying the genetic mechanism. Fortunately, the team had decades of research on the circadian biology of D. melanogaster to draw on. This allowed them to identify a list of candidate genes to test.

To find the gene relevant for regulating daily activity, “Michael devised a clever approach,” says Richard. By taking D. melanogaster strains with mutations for the different candidate genes and interbreeding them with D. sechellia, the lab created hybrid offspring that carried one gene copy from each species. In most cases the D. sechellia allele compensated for the missing D. melanogaster gene and the hybrids behaved normally. But when one specific gene—called Pdf—was disrupted, the sechellia allele failed to make up for the loss; the hybrids lost their ability to shift activity under longer days. This indicated that Pdf, which encodes a neuropeptide that transmits signals generated by core molecular timekeeping mechanisms in Drosophila, regulated behavioral plasticity.

Follow-up experiments found that Pdf expression was weaker and less variable in neurons of D. sechellia compared to D. melanogaster and pinpointed those differences to changes in the gene’s regulatory region. However, the lab needed to establish that those differences in gene regulation actually caused the behavior they observed. Using genetic engineering, they therefore cloned the Pdf regulatory sequences of both species and inserted them into mutant flies lacking their own functional Pdf regulatory sequence. And indeed, flies with the D. sechellia sequence showed reduced plasticity compared to flies with the melanogaster sequence. This final experiment completed the picture, showing how a subtle gene regulatory change could alter neuronal function and, ultimately, the way the flies behave.

It is exceedingly difficult to understand how evolution links genetic changes to their effects on neurons and behavior. The Benton lab achieves this by asking the right questions in the right system. “It’s really Drosophila as a whole and Drosophila sechellia as a specialized species. And it pays off to have an ecological framework,” says Richard. Genetic mechanisms can shape behavior through subtle and unexpected pathways. The Benton lab’s latest study is one of several examples from their research demonstrating how this framework not only deepens our understanding of evolution, but also uncovers mechanisms that might otherwise remain hidden.

Nat Metab co-auth.: B.Thorens

Autophagy regulator ATG5 preserves cerebellar function by safeguarding its glycolytic activity

Janine Tutas 1 2Marianna Tolve 1 2Ebru Özer-Yildiz 1 2Lotte Ickert 1 2Ines Klein 3Quinn Silverman 3Filip Liebsch 4Frederik Dethloff 5Patrick Giavalisco 5Heike Endepols 6 7 8Theodoros Georgomanolis 1Bernd Neumaier 7 8Alexander Drzezga 7 9 10Guenter Schwarz 4 11Bernard Thorens 12Graziana Gatto 3Christian Frezza 1 13Natalia L Kononenko 14 15 16 17

Affiliations Expand

Abstract

Dysfunctions in autophagy, a cellular mechanism for breaking down components within lysosomes, often lead to neurodegeneration. The specific mechanisms underlying neuronal vulnerability due to autophagy dysfunction remain elusive. Here we show that autophagy contributes to cerebellar Purkinje cell (PC) survival by safeguarding their glycolytic activity. Outside the conventional housekeeping role, autophagy is also involved in the ATG5-mediated regulation of glucose transporter 2 (GLUT2) levels during cerebellar maturation. Autophagy-deficient PCs exhibit GLUT2 accumulation on the plasma membrane, along with increased glucose uptake and alterations in glycolysis. We identify lysophosphatidic acid and serine as glycolytic intermediates that trigger PC death and demonstrate that the deletion of GLUT2 in ATG5-deficient mice mitigates PC neurodegeneration and rescues their ataxic gait. Taken together, this work reveals a mechanism for regulating GLUT2 levels in neurons and provides insights into the neuroprotective role of autophagy by controlling glucose homeostasis in the brain.

Nat Commun auth.: group Fajas

CDK4 inactivation inhibits apoptosis via mitochondria-ER contact remodeling in triple-negative breast cancer

Dorian V Ziegler 1Kanishka Parashar 1Lucia Leal-Esteban 1Jaime López-Alcalá 1 2Wilson Castro 3Nadège Zanou 4Laia Martinez-Carreres 1Katharina Huber 1Xavier Pascal Berney 1María M Malagón 2 5Catherine Roger 1Marie-Agnès Berger 6Yves Gouriou 6Giulia Paone 1Hector Gallart-Ayala 7George Sflomos 8Carlos Ronchi 8Julijana Ivanisevic 7Cathrin Brisken 8 9Jennifer Rieusset 6Melita Irving 3Lluis Fajas 10 11

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Abstract

The energetic demands of proliferating cells during tumorigenesis require close coordination between the cell cycle and metabolism. While CDK4 is known for its role in cell proliferation, its metabolic function in cancer, particularly in triple-negative breast cancer (TNBC), remains unclear. Our study, using genetic and pharmacological approaches, reveals that CDK4 inactivation only modestly impacts TNBC cell proliferation and tumor formation. Notably, CDK4 depletion or long-term CDK4/6 inhibition confers resistance to apoptosis in TNBC cells. Mechanistically, CDK4 enhances mitochondria-endoplasmic reticulum contact (MERCs) formation, promoting mitochondrial fission and ER-mitochondrial calcium signaling, which are crucial for TNBC metabolic flexibility. Phosphoproteomic analysis identified CDK4’s role in regulating PKA activity at MERCs. In this work, we highlight CDK4’s role in mitochondrial apoptosis inhibition and suggest that targeting MERCs-associated metabolic shifts could enhance TNBC therapy.

Commun Biol, co-auth.:M.Quadroni

Arabidopsis conditional photosynthesis mutants abc1k1 and var2 accumulate partially processed thylakoid preproteins and are defective in chloroplast biogenesis

Joy Collombat 1Manfredo Quadroni 2Véronique Douet 1Rosa Pipitone 3Fiamma Longoni 1Felix Kessler 4

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Abstract

Photosynthetic activity is established during chloroplast biogenesis. In this study we used 680 nm red light to overexcite Photosystem II and disrupt photosynthesis in two conditional mutants (var2 and abc1k1) which reversibly arrested chloroplast biogenesis. During biogenesis, chloroplasts import most proteins associated with photosynthesis. Some of these must be inserted in or transported across the thylakoid membrane into the thylakoid lumen. They are synthesized in the cytoplasm with cleavable targeting sequences and the lumenal ones have bi-partite targeting sequences (first for the chloroplast envelope, second for the thylakoid membrane). Cleavage of these peptides is required to establish photosynthesis and a critical step of chloroplast biogenesis. We employ a combination of Western blotting and mass spectrometry to analyze proteins in var2 and abc1k1. Under red light, var2 and abc1k1 accumulated incompletely cleaved processing intermediates of thylakoid proteins. These findings correlated with colorless cotyledons, and defects in both chloroplast morphology and photosynthesis. Together the results provide evidence for the requirement of active photosynthesis for processing of photosystem-associated thylakoid proteins and concomitantly progression of chloroplast biogenesis.

Cell Mol Life Sci: co-auth.: W.Wahli

Palmitate potentiates the SMAD3-PAI-1 pathway by reducing nuclear GDF15 levels

Marta Montori-Grau 1 2 3 4Emma Barroso 1 2 3 4Javier Jurado-Aguilar 1 2 3 4Mona Peyman 1 2 3 4Walter Wahli 5 6 7Xavier Palomer 1 2 3 4Manuel Vázquez-Carrera 8 9 10 11

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Abstract

Nuclear growth differentiation factor 15 (GDF15) reduces the binding of the mothers’ against decapentaplegic homolog (SMAD) complex to its DNA-binding elements. However, the stimuli that control this process are unknown. Here, we examined whether saturated fatty acids (FA), particularly palmitate, regulate nuclear GDF15 levels and the activation of the SMAD3 pathway in human skeletal myotubes and mouse skeletal muscle, where most insulin-stimulated glucose use occurs in the whole organism. Human LHCN-M2 myotubes and skeletal muscle from wild-type and Gdf15-/- mice fed a standard (STD) or a high-fat (HFD) diet were subjected to a series of studies to investigate the involvement of lipids in nuclear GDF15 levels and the activation of the SMAD3 pathway. The saturated FA palmitate, but not the monounsaturated FA oleate, increased the expression of GDF15 in human myotubes and, unexpectedly, decreased its nuclear levels. This reduction was prevented by the nuclear export inhibitor leptomycin B. The decrease in nuclear GDF15 levels caused by palmitate was accompanied by increases in SMAD3 protein levels and in the expression of its target gene SERPINE1, which encodes plasminogen activator inhibitor 1 (PAI-1). HFD-fed Gdf15-/- mice displayed aggravated glucose intolerance compared to HFD-fed WT mice, with increased levels of SMAD3 and PAI-1 in the skeletal muscle. The increased PAI-1 levels in the skeletal muscle of HFD-fed Gdf15-/- mice were accompanied by a reduction in one of its targets, hepatocyte growth factor (HGF)α, a cytokine involved in glucose metabolism. Interestingly, PAI-1 acts as a ligand of signal transducer and activator of transcription 3 (STAT3) and the phosphorylation of this transcription factor was exacerbated in HFD-fed Gdf15-/- mice compared to HFD-fed WT mice. At the same time, the protein levels of insulin receptor substrate 1 (IRS-1) were reduced. These findings uncover a potential novel mechanism through which palmitate induces the SMAD3-PAI-1 pathway to promote insulin resistance in skeletal muscle by reducing nuclear GDF15 levels.

EMBO J. auth.: group Gatfield

MCTS2 and distinct eIF2D roles in uORF-dependent translation regulation revealed by in vitro re-initiation assays

Romane Meurs 1Mara De Matos 1Adrian Bothe 2Nicolas Guex 3Tobias Weber 4Aurelio A Teleman 4Nenad Ban 2David Gatfield 5

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Free article

Abstract

Ribosomes scanning from the mRNA 5′ cap to the start codon may initiate at upstream open reading frames (uORFs), decreasing protein biosynthesis. Termination at a uORF can lead to re-initiation, where 40S subunits resume scanning and initiate another translation event downstream. The noncanonical translation factors MCTS1-DENR participate in re-initiation at specific uORFs, but knowledge of other trans-acting factors or uORF features influencing re-initiation is limited. Here, we establish a cell-free re-initiation assay using HeLa lysates to address this question. Comparing in vivo and in vitro re-initiation on uORF-containing reporters, we validate MCTS1-DENR-dependent re-initiation in vitro. Using this system and ribosome profiling in cells, we found that knockdown of the MCTS1-DENR homolog eIF2D causes widespread gene deregulation unrelated to uORF translation, and thus distinct to MCTS1-DENR-dependent re-initiation regulation. Additionally, we identified MCTS2, encoded by an Mcts1 retrogene, as a DENR partner promoting re-initiation in vitro, providing a plausible explanation for clinical differences associated with DENR vs. MCTS1 mutations in humans.