High-throughput screening of cell-secreted proteins is essential for various biotechnological applications. In this article, we show a microfluidic approach to perform the analysis of cell-secreted proteins in nanoliter droplet arrays by two complementary methods, fluorescence microscopy and mass spectrometry. We analyzed the secretion of the enzyme phytase, a phosphatase used as an animal feed additive, from a low number of yeast cells. Yeast cells were encapsulated in nanoliter volumes by droplet microfluidics and deposited on spatially defined spots on the surface of a glass slide mounted on the motorized stage of an inverted fluorescence microscope. During the following incubation for several hours to produce phytase, the droplets can be monitored by optical microscopy. After addition of a fluorogenic substrate at a defined time, the relative concentration of phytase was determined in every droplet. Moreover, we demonstrate the use of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to monitor the multistep conversion of the native substrate phytic acid by phytase secreted in 7 nL droplets containing 50−100 cells. Our method can be adapted to various other protocols. As the droplets are easily accessible, compounds such as assay reagents or matrix molecules can be added to all or to selected droplets only, or part of the droplet volume could be removed. Hence, this platform is a versatile tool for questions related to cell secretome analysis.
Nucleated cells eliminate lesions induced by bacterial pore-forming toxins, such as pneumolysin via shedding patches of damaged plasmalemma into the extracellular milieu. Recently, we have shown that the majority of shed pneumolysin is present in the form of inactive pre-pores. This finding is surprising considering that shedding is triggered by Ca-influx following membrane perforation and therefore is expected to positively discriminate for active pores versus inactive pre-pores. Here we provide evidence for the existence of plasmalemmal domains that are able to attract pneumolysin at high local concentrations. Within such a domain an immediate plasmalemmal perforation induced by a small number of pneumolysin pores would be capable of triggering the elimination of a large number of not yet active pre-pores/monomers and thus pre-empt more frequent and perilous perforation events. Our findings provide further insights into the functioning of the cellular repair machinery which benefits from an inhomogeneous plasmalemmal distribution of pneumolysin.
Specific interactions of peptides with lipid membranes are essential for cellular communication and constitute a central aspect of the innate host defense against pathogens. A computational method for generating innovative membrane-pore-forming peptides inspired by natural templates is presented. Peptide representation in terms of sequence- and topology-dependent hydrophobic moments is introduced. This design concept proves to be appropriate for the de novo generation of first-in-class membrane-active peptides with the anticipated mode of action. The designed peptides outperform the natural template in terms of their antibacterial activity. They form a kinked helical structure and self-assemble in the membrane by an entropy-driven mechanism to form dynamically growing pores that are dependent on the lipid composition. The results of this study demonstrate the unique potential of natural template-based peptide design for chemical biology and medicinal chemistry.
In the field of bottom-up synthetic biology, lipid membranes are the scaffold to create minimal cells and mimic reactions and processes at or across the membrane. In this context, we employ here a versatile microfluidic platform that enables precise positioning of nanoliter droplets with user-specified lipid compositions and in a defined pattern. Adjacent droplets make contact and form a droplet interface bilayer to simulate cellular membranes. Translocation of molecules across membranes are tailored by the addition of alpha-hemolysin to selected droplets. Moreover, we developed a protocol to analyze the translocation of non-fluorescent molecules between droplets with mass spectrometry. Our method is capable of automated formation of one- and two-dimensional droplet networks, which we demonstrated by connecting droplets containing different compound and enzyme solutions to perform translocation experiments and a multistep enzymatic cascade reaction across the droplet network. Our platform opens doors for creating complex artificial systems for bottom-up synthetic biology.
We investigated the permeation of molecules across lipid membranes on an open microfluidic platform. An array of droplet pairs was created by spotting aqueous droplets, dispersed in a lipid oil solution, onto a plate with cavities surrounded by a hydrophobic substrate. Droplets in two adjacent cavities come in contact and form an artificial lipid bilayer, called a droplet interface bilayer (DIB). The method allows for monitoring permeation of fluorescently tagged compounds from a donor droplet to an acceptor droplet. A mathematical model was applied to describe the kinetics and determine the permeation coefficient. We also demonstrate that permeation kinetics can be followed over a series of droplets, all connected via DIBs. Moreover, by changing the lipid oil composition after spotting donor droplets, we were able to create asymmetric membranes that we used to mimic the asymmetry of the cellular plasma membrane. Finally, we developed a protocol to separate and extract the droplets for label-free analysis of permeating compounds by liquid chromatography–mass spectrometry. Our versatile platform has the potential to become a new tool for the screening of drug membrane permeability in the future.
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