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Joshua Mills, L Johanna Gebhard, Florence Schubotz, Anna Shevchenko, Daan R Speth, Yan Liao, Iain G Duggin, Anita Marchfelder, Susanne Erdmann
Extracellular vesicle formation in Euryarchaeota is driven by a small GTPase.
Proc Natl Acad Sci U.S.A., 121(10) Art. No. e2311321121 (2024)
Open Access PubMed Source   

Since their discovery, extracellular vesicles (EVs) have changed our view on how organisms interact with their extracellular world. EVs are able to traffic a diverse array of molecules across different species and even domains, facilitating numerous functions. In this study, we investigate EV production in Euryarchaeota, using the model organism Haloferax volcanii. We uncover that EVs enclose RNA, with specific transcripts preferentially enriched, including those with regulatory potential, and conclude that EVs can act as an RNA communication system between haloarchaea. We demonstrate the key role of an EV-associated small GTPase for EV formation in H. volcanii that is also present across other diverse evolutionary branches of Archaea. We propose the name, ArvA, for the identified family of archaeal vesiculating GTPases. Additionally, we show that two genes in the same operon with arvA (arvB and arvC) are also involved in EV formation. Both, arvB and arvC, are closely associated with arvA in the majority of other archaea encoding ArvA. Our work demonstrates that small GTPases involved in membrane deformation and vesiculation, ubiquitous in Eukaryotes, are also present in Archaea and are widely distributed across diverse archaeal phyla.
@article{Mills8691,
author={Joshua Mills, L Johanna Gebhard, Florence Schubotz, Anna Shevchenko, Daan R Speth, Yan Liao, Iain G Duggin, Anita Marchfelder, Susanne Erdmann},
title={Extracellular vesicle formation in Euryarchaeota is driven by a small GTPase.},
journal ={Proceedings of the National Academy of Sciences of the United States of America},
volume={121},
issue ={10},
pages={null--null},
year=2024
}

Katarzyna P Adamala#, Marileen Dogterom#, Yuval Elani#, Petra Schwille#, Masahiro Takinoue#, T-Y Dora Tang#
Present and future of synthetic cell development.
Nat Rev Mol Cell Biol, 25(3) 162-167 (2024)
PubMed Source  

@article{Adamala8626,
author={Katarzyna P Adamala, Marileen Dogterom, Yuval Elani, Petra Schwille, Masahiro Takinoue, T-Y Dora Tang},
title={Present and future of synthetic cell development.},
journal ={Nature reviews. Molecular cell biology},
volume={25},
issue ={3},
pages={162--167},
year=2024
}

Mengfei Gao, Dishi Wang, Michaela Wilsch-Bräuninger, Weihua Leng, Jonathan Schulte, Nina Morgner, Dietmar Appelhans, T-Y Dora Tang
Cell Free Expression in Proteinosomes Prepared from Native Protein-PNIPAAm Conjugates.
Macromol Biosci, 24(3) Art. No. e2300464 (2024)
Open Access PubMed Source   

Towards the goal of building synthetic cells from the bottom-up, the establishment of micrometer-sized compartments that contain and support cell free transcription and translation that couple cellular structure to function is of critical importance. Proteinosomes, formed from crosslinked cationized protein-polymer conjugates offer a promising solution to membrane-bound compartmentalization with an open, semi-permeable membrane. Critically, to date, there has been no demonstration of cell free transcription and translation within water-in-water proteinosomes. Herein, a novel approach to generate proteinosomes that can support cell free transcription and translation is presented. This approach generates proteinosomes directly from native protein-polymer (BSA-PNIPAAm) conjugates. These native proteinosomes offer an excellent alternative as a synthetic cell chassis to other membrane bound compartments. Significantly, the native proteinosomes are stable under high salt conditions that enables the ability to support cell free transcription and translation and offer enhanced protein expression compared to proteinosomes prepared from traditional methodologies. Furthermore, the integration of native proteinosomes into higher order synthetic cellular architectures with membrane free compartments such as liposomes is demonstrated. The integration of bioinspired architectural elements with the central dogma is an essential building block for realizing minimal synthetic cells and is key for exploiting artificial cells in real-world applications.
@article{Gao8621,
author={Mengfei Gao, Dishi Wang, Michaela Wilsch-Bräuninger, Weihua Leng, Jonathan Schulte, Nina Morgner, Dietmar Appelhans, T-Y Dora Tang},
title={Cell Free Expression in Proteinosomes Prepared from Native Protein-PNIPAAm Conjugates.},
journal ={Macromolecular bioscience},
volume={24},
issue ={3},
pages={null--null},
year=2024
}

Cedric Landerer, Jonas Pöhls, Agnes Toth-Petroczy
Fitness effects of phenotypic mutations at proteome-scale reveal optimality of translation machinery.
Mol Biol Evol, Art. No. doi: 10.1093/molbev/msae048 (2024)
Open Access PubMed Source Full Text   

Errors in protein translation can lead to non-genetic, phenotypic mutations, including amino acid misincorporations. While phenotypic mutations can increase protein diversity, the systematic characterization of their proteome-wide frequencies and their evolutionary impact has been lacking. Here, we developed a mechanistic model of translation errors to investigate how selection acts on protein populations produced by amino acid misincorporations. We fitted the model to empirical observations of misincorporations obtained from over a hundred mass spectrometry datasets of E. coli and S. cerevisiae. We found that on average 20-23% of proteins synthesized in the cell are expected to harbour at least one amino acid misincorporation, and that deleterious misincorporations are less likely to occur. Combining misincorporation probabilities and the estimated fitness effects of amino acid substitutions in a population genetics framework, we found 74% of mistranslation events in E. coli and 94% in S. cerevisiae to be neutral. We further show that the set of available synonymous tRNAs is subject to evolutionary pressure, as the presence of missing tRNAs would increase codon-anticodon cross-reactivity and misincorporation error rates. Overall, we find that the translation machinery is likely optimal in E. coli and S. cerevisiae and that both local solutions at the level of codons and a global solution such as the tRNA pool can mitigate the impact of translation errors. We provide a framework to study the evolutionary impact of codon specific translation errors and a method for their proteome-wide detection across organisms and conditions.
@article{Landerer8689,
author={Cedric Landerer, Jonas Pöhls, Agnes Toth-Petroczy},
title={Fitness effects of phenotypic mutations at proteome-scale reveal optimality of translation machinery.},
journal ={Molecular biology and evolution},
volume={},
pages={1--1},
year=2024
}

Laura Meißner, Lukas Niese, Irene Schüring, Aniruddha Mitra, Stefan Diez
Human kinesin-5 KIF11 drives the helical motion of anti-parallel and parallel microtubules around each other.
EMBO J, Art. No. doi: 10.1038/s44318-024-00048-x (2024)
Open Access PubMed Source   

During mitosis, motor proteins and microtubule-associated protein organize the spindle apparatus by cross-linking and sliding microtubules. Kinesin-5 plays a vital role in spindle formation and maintenance, potentially inducing twist in the spindle fibers. The off-axis power stroke of kinesin-5 could generate this twist, but its implications in microtubule organization remain unclear. Here, we investigate 3D microtubule-microtubule sliding mediated by the human kinesin-5, KIF11, and found that the motor caused right-handed helical motion of anti-parallel microtubules around each other. The sidestepping ratio increased with reduced ATP concentration, indicating that forward and sideways stepping of the motor are not strictly coupled. Further, the microtubule-microtubule distance (motor extension) during sliding decreased with increasing sliding velocity. Intriguingly, parallel microtubules cross-linked by KIF11 orbited without forward motion, with nearly full motor extension. Altering the length of the neck linker increased the forward velocity and pitch of microtubules in anti-parallel overlaps. Taken together, we suggest that helical motion and orbiting of microtubules, driven by KIF11, contributes to flexible and context-dependent filament organization, as well as torque regulation within the mitotic spindle.
@article{Meißner8692,
author={Laura Meißner, Lukas Niese, Irene Schüring, Aniruddha Mitra, Stefan Diez},
title={Human kinesin-5 KIF11 drives the helical motion of anti-parallel and parallel microtubules around each other.},
journal ={The EMBO journal},
volume={},
pages={1--1},
year=2024
}

Matthew J Bovyn#, Pierre A. Haas#
Shaping epithelial lumina under pressure.
Biochem Soc Trans, 52(1) 331-342 (2024)
Open Access PubMed Source Full Text   

The formation of fluid- or gas-filled lumina surrounded by epithelial cells pervades development and disease. We review the balance between lumen pressure and mechanical forces from the surrounding cells that governs lumen formation. We illustrate the mechanical side of this balance in several examples of increasing complexity, and discuss how recent work is beginning to elucidate how nonlinear and active mechanics and anisotropic biomechanical structures must conspire to overcome the isotropy of pressure to form complex, non-spherical lumina.
@article{Bovyn8690,
author={Matthew J Bovyn, Pierre A. Haas},
title={Shaping epithelial lumina under pressure.},
journal ={Biochemical Society transactions},
volume={52},
issue ={1},
pages={331--342},
year=2024
}

Elena Erben, Weida Liao, Antonio Minopoli, Nicola Maghelli, Eric Lauga, Moritz Kreysing
Opto-fluidically multiplexed assembly and micro-robotics.
Light Sci Appl, 13(1) Art. No. 59 (2024)
Open Access PubMed Source   

Techniques for high-definition micromanipulations, such as optical tweezers, hold substantial interest across a wide range of disciplines. However, their applicability remains constrained by material properties and laser exposure. And while microfluidic manipulations have been suggested as an alternative, their inherent capabilities are limited and further hindered by practical challenges of implementation and control. Here we show that the iterative application of laser-induced, localized flow fields can be used for the relative positioning of multiple micro-particles, irrespectively of their material properties. Compared to the standing theoretical proposal, our method keeps particles mobile, and we show that their precision manipulation is non-linearly accelerated via the multiplexing of temperature stimuli below the heat diffusion limit. The resulting flow fields are topologically rich and mathematically predictable. They represent unprecedented microfluidic control capabilities that are illustrated by the actuation of humanoid micro-robots with up to 30 degrees of freedom, whose motions are sufficiently well-defined to reliably communicate personal characteristics such as gender, happiness and nervousness. Our results constitute high-definition micro-fluidic manipulations with transformative potential for assembly, micro-manufacturing, the life sciences, robotics and opto-hydraulically actuated micro-factories.
@article{Erben8687,
author={Elena Erben, Weida Liao, Antonio Minopoli, Nicola Maghelli, Eric Lauga, Moritz Kreysing},
title={Opto-fluidically multiplexed assembly and micro-robotics.},
journal ={Light, science & applications},
volume={13},
issue ={1},
pages={null--null},
year=2024
}

Stefan Golfier, Thomas Quail, Jan Brugués
Single-Molecule Approaches to Study DNA Condensation.
Methods Mol Biol, 2740 1-19 (2024)
PubMed Source   

Proteins drive genome compartmentalization across different length scales. While the identities of these proteins have been well-studied, the physical mechanisms that drive genome organization have remained largely elusive. Studying these mechanisms is challenging owing to a lack of methodologies to parametrize physical models in cellular contexts. Furthermore, because of the complex, entangled, and dense nature of chromatin, conventional live imaging approaches often lack the spatial resolution to dissect these principles. In this chapter, we will describe how to image the interactions of λ-DNA with proteins under purified and cytoplasmic conditions. First, we will outline how to prepare biotinylated DNA, functionalize coverslips with biotin-conjugated poly-ethylene glycol (PEG), and assemble DNA microchannels compatible for the imaging of protein-DNA interactions using total internal fluorescence microscopy. Then we will describe experimental methods to image protein-DNA interactions in vitro and DNA loop extrusion using Xenopus laevis egg extracts.
@article{Golfier8685,
author={Stefan Golfier, Thomas Quail, Jan Brugués},
title={Single-Molecule Approaches to Study DNA Condensation.},
journal ={Methods in molecular biology (Clifton, N.J.)},
volume={2740},
pages={1--19},
year=2024
}

Thorsten Meyer, Oskar Knittelfelder, Martin Smolnig, Patrick Rockenfeller
Quantifying yeast lipidomics by high-performance thin-layer chromatography (HPTLC) and comparison to mass spectrometry-based shotgun lipidomics.
Microb Cell, 11 57-68 (2024)
Open Access PubMed Source Full Text   

Lipidomic analysis in diverse biological settings has become a frequent tool to increase our understanding of the processes of life. Cellular lipids play important roles not only as being the main components of cellular membranes, but also in the regulation of cell homeostasis as lipid signaling molecules. Yeast has been harnessed for biomedical research based on its good conservation of genetics and fundamental cell organisation principles and molecular pathways. Further application in so-called humanised yeast models have been developed which take advantage of yeast as providing the basics of a living cell with full control over heterologous expression. Here we present evidence that high-performance thin-layer chromatography (HPTLC) represents an effective alternative to replace cost intensive mass spectrometry-based lipidomic analyses. We provide statistical comparison of identical samples by both methods, which support the use of HPTLC for quantitative analysis of the main yeast lipid classes.
@article{Meyer8686,
author={Thorsten Meyer, Oskar Knittelfelder, Martin Smolnig, Patrick Rockenfeller},
title={Quantifying yeast lipidomics by high-performance thin-layer chromatography (HPTLC) and comparison to mass spectrometry-based shotgun lipidomics.},
journal ={Microbial cell (Graz, Austria)},
volume={11},
pages={57--68},
year=2024
}

Landi Sun✳︎#, Jana Meissner✳︎, Jianfeng He, Lihong Cui, Tobias Fürstenhaupt, Xin Liang#
Resolving the In Situ Three-Dimensional Structure of Fly Mechanosensory Organelles Using Serial Section Electron Tomography.
Bio Protoc, 14(4) Art. No. e4940 (2024)
Open Access PubMed Source Full Text   

Mechanosensory organelles (MOs) are specialized subcellular entities where force-sensitive channels and supporting structures (e.g., microtubule cytoskeleton) are organized in an orderly manner. The delicate structure of MOs needs to be resolved to understand the mechanisms by which they detect forces and how they are formed. Here, we describe a protocol that allows obtaining detailed information about the nanoscopic ultrastructure of fly MOs by using serial section electron tomography (SS-ET). To preserve fine structural details, the tissues are cryo-immobilized using a high-pressure freezer followed by freeze-substitution at low temperature and embedding in resin at room temperature. Then, sample sections are prepared and used to acquire the dual-axis tilt series images, which are further processed for tomographic reconstruction. Finally, tomograms of consecutive sections are combined into a single larger volume using microtubules as fiducial markers. Using this protocol, we managed to reconstruct the sensory organelles, which provide novel molecular insights as to how fly mechanosensory organelles work and are formed. Based on our experience, we think that, with minimal modifications, this protocol can be adapted to a wide range of applications using different cell and tissue samples. Key features • Resolving the high-resolution 3D ultrastructure of subcellular organelles using serial section electron tomography (SS-ET). • Compared with single-axis tilt series, dual-axis tilt series provides a much wider coverage of Fourier space, improving resolution and features in the reconstructed tomograms. • The use of high-pressure freezing and freeze-substitution maximally preserves the fine structural details.
@article{Sun8688,
author={Landi Sun, Jana Meissner, Jianfeng He, Lihong Cui, Tobias Fürstenhaupt, Xin Liang},
title={Resolving the In Situ Three-Dimensional Structure of Fly Mechanosensory Organelles Using Serial Section Electron Tomography.},
journal ={Bio-protocol},
volume={14},
issue ={4},
pages={null--null},
year=2024
}


✳︎ joint first authors, # joint corresponding authors
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