Matter to Life Fall Days

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Raphid diatoms are among the few eukaryotes capable of rapid gliding motility, allowing for near-instantaneous directional reversals. An actomyosin system has been proposed to generate the force for this movement, but direct in vivo data on actin and myosin dynamics in diatoms have been lacking. Here, we show that the raphe-associated actin bundles essential for diatom movement do not display directional subunit turnover, indicating they do not directly contribute to force generation. Through phylogenomic analysis, we identified four raphid diatom-specific myosins (CaMyoA-D) in Craspedostauros australis and examined their localization and dynamics via GFP-tagging. Of these, only CaMyoB-D exhibited coordinated movement during gliding, consistent with a role in force production. Additionally, we characterized the complex trajectories of diatom gliding. Although the cell body and raphe remain static, the paths dynamically change shape. We observed correlations between velocity, path curvature, and offset angle, with diatoms switching between one-raphe gliding (high curvature) and two-raphe gliding (low curvature). We propose that these path shapes result from local raphe curvature and differential raphe activation. In summary, our identification of diatom-specific myosins and analysis of complex motility patterns provide new insights into the molecular mechanisms driving diatom gliding motility.

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Cargo delivery systems play a pivotal role in the efficient administration of therapeutic agents, ensuring their transport to target sites. These systems encompass a broad spectrum of delivery strategies, all designed to enhance therapeutic efficacy, reduce side effects, and improve patient adherence. Despite significant advancements, challenges such as uncontrolled release and limited cargo encapsulation remain prevalent. This study introduces a novel "smart" lipid vesicle system, composed of giant unilamellar vesicles (GUVs) and gold nanorods (GNRs), designed to overcome these challenges. The objective of this study is to develop a “smart” biocompatible, trigger-responsive cargo delivery platform that can encapsulate various types of cargo and release them on demand when illuminated with near-infrared (NIR) light. Due to their size and preparation method, GUVs allow for the encapsulation of diverse cargo types and sizes. Gold nanorods, decorated with PEG chains and attached to GUVs via cholesterol moieties, enable NIR light-triggered cargo release. A novel NIR LED setup was employed for illumination, providing a cost-effective and user-friendly alternative to conventional lasers. The cargo delivery system demonstrated superior performance with 10 minutes of illumination yielding better release results compared to 5 minutes. GNRs functionalized with 3.2% Cholesterol-PEG-SH achieved the best release efficiency of around 60%. Control experiments confirmed that the GUVs remained unaffected by NIR light alone, ensuring the system's stability and specificity in response to NIR light. The system was tested with GUVs encapsulating ampicillin, and the illuminated samples showed a 30% decrease in bacterial numbers and reduced bacterial growth, demonstrating its promising potential for cargo delivery applications. This smart GUV-GNR system offers a versatile and highly controlled delivery platform, capable of encapsulating and releasing various types of cargo on demand. The biocompatibility and stability of the system, combined with its efficient release capabilities, make it a promising tool for targeted therapeutics and diagnostics. Future research will explore the optimization of this system and its further application in various biomedical fields.

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Cells organize many of their biochemical reactions by formation and dissolution of non-membrane-bound compartments. Recent experiments show that one common mechanism for such biochemical organization is phase separation of disordered proteins to form liquid-like compartments. These compartments can subsequently harden to form compartments with new material properties such as gels and glasses. These compartments can be described by principles elucidated from condensed-matter physics and are therefore termed biomolecular condensates. I will discuss potential roles of phase separation in organization and robustness of cellular biochemistry.

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Kinetochores provide chromosomes with points of attachment to spindle microtubules during cell division, and are therefore essential for genome inheritance and the propagation of life. In addition to binding microtubules, kinetochores control mitotic surveillance mechanisms that promote chromosome bi-orientation (the error correction mechanism) and prevent premature mitotic exit in presence of incomplete or incorrect microtubule attachments (spindle assembly checkpoint, SAC). Elimination of the NDC80 complex, the main microtubule receptor of kinetochores, causes a SAC deficiency, identifying this complex as a crucial regulatory focus for checkpoint function. In recent years, there has been considerable progress in understanding how the SAC effector, known as the mitotic checkpoint complex (MCC), assembles from its individual components to inhibit its target, the anaphase promoting complex/cyclosome (APC/C). Conversely, how microtubule attachment to kinetochores regulates the SAC remains incompletely understood. From a molecular perspective, answering this question implies investigating the mechanisms that promote targeting of the SAC proteins to unattached kinetochores, and suppress it upon microtubule binding and biorientation. In our recent work, we have combined biochemical reconstitutions, structural biology/modelling, and cell biology to gain insights into this fundamental biological question.

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The chitin shell of the C. elegans embryo largely determines it’s shape and is secreted for 10-15 min right after fertilization, followed by the secretion of proteoglycans during meiosis, and the subsequent internalisation of the chitin producing synthase within ~3 min. We investigate the governing mechanism of chitin synthase dynamics in the early embryo, and their time evolution, ending in the internalisation of chitin synthase. Here, we use ex utero spinning disc microscopy of the oocyte-to-embryo transition to quantify chitin synthase dynamics and its internalization process. We find that chitin synthesis leads to chitin synthase movement of constant velocity. In addition, we find the internalisation dynamics follow the secretion of Cortical Granule Exocytosis cargo, and the interplay of the cargo and interactor proteins at the interface of the membrane and the shell are essential for the remodeling of the early embryo. Using rapid imaging modalities that allow for the quantification of the dynamics driving chitin shell synthesis and the subsequent internalisation of chitin synthase, we highlight how chitin synthase dynamics couple to cortical granule exocytosis and chitin synthase internalization in order to drive this essential mechanism for embryo development.

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One of the early phases of cancer metastasis involves the interstitial migration of cells toward nearby tissues through lymph or blood vessels. In this study, we focus on examining the impact of mechanical stress and confinement on cell heterogeneity and invasiveness. A 3D-1D system was established to mimic the conditions sensed by cancer cells and to investigate their migratory behavior from a tumor-like microenvironment to an invasive milieu. To this end, MDA-MB-231 breast cancer cells were encapsulated for 7 days in 3D alginate-gelatin microcapsules with an elastic modulus of 23 kPa. Cells were then released onto migration platforms, consisting of 1D micropatterned matrix proteins. Under the mechanical stimuli provided by 3D microcapsules, cells exhibit strong heterogeneity and altered morphological characteristics, such as increased sizes. Notely, we observed the presence of cells with increased number of nuclei, often with the formation of micronuclei and cytoplasmic DNA, considered to be involved in tumor growth and metastasis Furthermore, encapsulated MDA cells stably transfected with the Fucci cell cycle reporter revealed a cell cycle arrest in the S phase after 7 days and a decreased cellular division. On 1D platforms, cells derived from the 3D microcapsules display a faster migration speed compared to cells migrating on matrix-coated surfaces. Although further studies are needed to distinguish the specific behaviors of the polynucleated cells, our approach enables a comprehensive understanding of various processes implicated in cancer malignancy, including tumorigenicity, invasion, intravasation, and the generation of heterogeneous populations.

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To be announced

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Inside prokaryotic cells, passive translational diffusion typically limits the rates with which cytoplasmic proteins can reach their locations. Diffusion is thus fundamental to most cellular processes, but the understanding of protein mobility in the highly crowded and non-homogeneous environment of a bacterial cell is still limited. Here we investigated the mobility of a large set of proteins in the cytoplasm of Escherichia coli, by employing fluorescence correlation spectroscopy (FCS) combined with simulations and theoretical modeling. We conclude that cytoplasmic protein mobility could be well described by Brownian diffusion in the confined geometry of the bacterial cell and at the high viscosity imposed by macromolecular crowding. We observed similar size dependence of protein diffusion for the majority of tested proteins, whether native or foreign to E. coli. For the faster-diffusing proteins, this size dependence is well consistent with the Stokes-Einstein relation once taking into account the specific dumbbell shape of protein fusions. Pronounced subdiffusion and hindered mobility are only observed for proteins with extensive interactions within the cytoplasm. Finally, while protein diffusion becomes markedly faster in actively growing cells, at high temperature, or upon treatment with rifampicin, and slower at high osmolarity, all of these perturbations affect proteins of different sizes in the same proportions, which could thus be described as changes of a well-defined cytoplasmic viscosity.

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Sehyeong Jung 1,2, Susanne Braun 1,2, Catalina Molano1,2, Andrij Pich 1,2 1 DWI, Leibniz Institute for Interactive Materials e.V., Aachen, Germany 2 Laboratory of Functional and Interactive Polymers, Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Aachen, Germany Presenting author’s e-mail: pich@dwi.rwth-aachen.de Biological tissues are frequently composed of macromolecular and low-molecular components, containing covalent and non-covalent crosslinks, and are assembled in a modular way exhibiting different hierarchy levels.1 Such hydrogel-like materials often exhibit hysteresis effects and non-linearity of their properties, which is essential to functions like adaptability and time-programming. At present, the performance of synthetic hydrogel materials is not on par with these advanced biological hydrogels. This contribution will focus on chemical design of stimuli-responsive microgels exhibiting non-covalent dynamic crosslinks based on host-guest complexes, ionic bonds or hydrogen bonds.2-9 The development of new synthesis methods that allow controlled integration of supramolecular functionalities into microgels opens new ways to generate functional polymer materials and systems with unique functions like stimuli-responsiveness, re-shaping, and triggered disassembly. A new synthesis method to obtain aqueous supramolecular temperature-responsive colloidal gels using tannic acid as multifunctional physical cross-linker was developed recently.7,8 The precipitation polymerization of N-vinylcaprolactam in the presence of tannic acid leads to the formation of well-defined stimuli-responsive nanogels cross-linked by hydrogen bonds. Alternatively, using microfluidics large microgels can be sysnthesized by crosslinking of amphiphilic polymer chains using tannic acid in aqueous droplets. Obtained colloidal gels equipped with non-covalent crosslinks exhibit unique properties like on-demand degradation, controlled release of ions and small molecules and mechanical re-enforcement.9 Keywords: microgels, supramolecular crosslinks, soft materials References [1] R. Eelkema, A. Pich, Advanced Materials 2019, 1906012. [2] D. Schmitz, A. Pich, Polymer Chemistry 2016, 7, 5687. [3] S.-H. Jung, S. Schneider, F. Plamper, A. Pich, Macromolecules 2020, 53, 1043-1053. [4] R. Schröder, W. Richtering, I. Potemkin, A. Pich, Macromolecules 2018, 51, 17, 6707-6716. [5] P. Saha, M. Kather, S. Banerjee, N. Singha, A. Pich, European Polymer Journal 2019, 118, 195-204. [6] P. Saha, M. Santi, M. Frenken, A. Palanisamy, R. Ganguly, N. Singha, A. Pich, ACS Macro Letters 2020, 9, 895-901. [7] C. Molano, A. Pich, Macromolecular Rapid Communications 2018, 39, 1700808. [8] S. Jung, S. Bulut, L. P. B. Guerzoni, L. De Laporte, A. Pich, J Colloid Interface Sci, 2022, 617, 409-421. [9] E. Izak-Nau, S. Braun, A. Pich, R. Göstl, Advanced Science, 2022, 2104004.

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Focal adhesions are membrane-associated protein complexes crucial for cell migration, adhesion, and signalling. Formation of focal adhesions requires the connection between the actin cytoskeleton and nascent adhesions, which cluster integrins and integrin-associated proteins on the membrane. While recent reconstitution assays suggest that nascent adhesions are formed by the liquid-liquid phase separation induced by protein interaction with charged lipid bilayers (Hsu C-P., PNAS, 2023), how the actin interacts with the nascent integrin complexes remains an open question. Here, we reconstitute protein condensates with a minimal set of nascent adhesion proteins on a solid-supported lipid membrane that can localise G-actin and serve as a nucleation site for actin filaments. Our result shows that including zyxin and VASP in the protein complex is necessary to initialise actin polymerisation out of the condensates and promote the bundling of actin filaments. These in vitro findings suggest a minimal set of proteins to rebuild functional nascent adhesion sites coupled to filamentous actin.

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Organoids have become valuable model systems for developmental biology and biomedical research. One of the most successful examples are intestinal organoids, which recapitulate key characteristics of the development of the gut. In particular, with growth and differentiation they undergo a morphological transition from a spherical epithelial shell to a budded shape. Apico-basal forces from the actomyosin system have been proposed to facilitate this structure formation. Here we introduce a fully three-dimensional (3D) non-invasive force inference technique to indirectly measure such forces. We reconstruct the 3D tissue shape of intestinal organoids and infer individual tensions from microscopy data, assuming a foam-like description of the tissue. Inference is verified by in-silico organoid data simulated with a bubbly vertex model. From the experimental data for the intestinal organoids, we find an increased global apical tension in budded morphologies, consistent with apical contractile forces effecting the observed budded shapes. In the future, our force inference technique could also be applied to study the relation between tissue geometry and force generation in other organoid systems.