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- ItemDecreased Effective Macromolecular Crowding in Escherichia coli Adapted to Hyperosmotic Stress(Washington, DC : Soc., 2019) Liu, Boqun; Hasrat, Zarief; Poolman, Bert; Boersma, Arnold J.; Mullineaux, Conrad W.Escherichia coli adapts to changing environmental osmolality to survive and maintain growth. It has been shown that the diffusion of green fluorescent protein (GFP) in cells adapted to osmotic upshifts is higher than expected from the increase in biopolymer volume fraction. To better understand the physicochemical state of the cytoplasm in adapted cells, we now follow the macromolecular crowding during adaptation with fluorescence resonance energy transfer (FRET)-based sensors. We apply an osmotic upshift and find that after an initial increase, the apparent crowding decreases over the course of hours to arrive at a value lower than that before the osmotic upshift. Crowding relates to cell volume until cell division ensues, after which a transition in the biochemical organization occurs. Analysis of single cells by microfluidics shows that changes in cell volume, elongation, and division are most likely not the cause for the transition in organization. We further show that the decrease in apparent crowding upon adaptation is similar to the apparent crowding in energy-depleted cells. Based on our findings in combination with literature data, we suggest that adapted cells have indeed an altered biochemical organization of the cytoplasm, possibly due to different effective particle size distributions and concomitant nanoscale heterogeneity. This could potentially be a general response to accommodate higher biopolymer fractions yet retaining crowding homeostasis, and it could apply to other species or conditions as well.IMPORTANCE Bacteria adapt to ever-changing environmental conditions such as osmotic stress and energy limitation. It is not well understood how biomolecules reorganize themselves inside Escherichia coli under these conditions. An altered biochemical organization would affect macromolecular crowding, which could influence reaction rates and diffusion of macromolecules. In cells adapted to osmotic upshift, protein diffusion is indeed faster than expected on the basis of the biopolymer volume fraction. We now probe the effects of macromolecular crowding in cells adapted to osmotic stress or depleted in metabolic energy with a genetically encoded fluorescence-based probe. We find that the effective macromolecular crowding in adapted and energy-depleted cells is lower than in unstressed cells, indicating major alterations in the biochemical organization of the cytoplasm.
- ItemTowards a Biohybrid Lung: Endothelial Cells Promote Oxygen Transfer through Gas Permeable Membranes(New York, NY [u.a.] : Hindawi Publ. Corp., 2017) Menzel, Sarah; Finocchiaro, Nicole; Donay, Christine; Thiebes, Anja Lena; Hesselmann, Felix; Arens, Jutta; Djeljadini, Suzana; Wessling, Matthias; Schmitz-Rode, Thomas; Jockenhoevel, Stefan; Cornelissen, Christian GabrielIn patients with respiratory failure, extracorporeal lung support can ensure the vital gas exchange via gas permeable membranes but its application is restricted by limited long-term stability and hemocompatibility of the gas permeable membranes, which are in contact with the blood. Endothelial cells lining these membranes promise physiological hemocompatibility and should enable prolonged application. However, the endothelial cells increase the diffusion barrier of the blood-gas interface and thus affect gas transfer. In this study, we evaluated how the endothelial cells affect the gas exchange to optimize performance while maintaining an integral cell layer. Human umbilical vein endothelial cells were seeded on gas permeable cell culture membranes and cultivated in a custom-made bioreactor. Oxygen transfer rates of blank and endothelialized membranes in endothelial culture medium were determined. Cell morphology was assessed by microscopy and immunohistochemistry. Both setups provided oxygenation of the test fluid featuring small standard deviations of the measurements. Throughout the measuring range, the endothelial cells seem to promote gas transfer to a certain extent exceeding the blank membranes gas transfer performance by up to 120%. Although the underlying principles hereof still need to be clarified, the results represent a significant step towards the development of a biohybrid lung.
- ItemThe more the merrier: effects of macromolecular crowding on the structure and dynamics of biological membranes(Oxford [u.a.] : Wiley-Blackwell, 2020) Löwe, Maryna; Kalacheva, Milara; Boersma, Arnold J.; Kedrov, AlexejProteins are essential and abundant components of cellular membranes. Being densely packed within the limited surface area, proteins fulfil essential tasks for life, which include transport, signalling and maintenance of cellular homeostasis. The high protein density promotes nonspecific interactions, which affect the dynamics of the membrane-associated processes, but also contribute to higher levels of membrane organization. Here, we provide a comprehensive summary of the most recent findings of diverse effects resulting from high protein densities in both living membranes and reconstituted systems and display why the crowding phenomenon should be considered and assessed when studying cellular pathways. Biochemical, biophysical and computational studies reveal effects of crowding on the translational mobility of proteins and lipids, oligomerization and clustering of integral membrane proteins, and also folding and aggregation of proteins at the lipid membrane interface. The effects of crowding pervade to larger length scales, where interfacial and transmembrane crowding shapes the lipid membrane. Finally, we discuss the design and development of fluorescence-based sensors for macromolecular crowding and the perspectives to use those in application to cellular membranes and suggest some emerging topics in studying crowding at biological interfaces. © 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies