One of the best-studied mechanosensitive channels is the mechanosensitive channel of large conductance (MscL). MscL senses tension in the membrane evoked by an osmotic down shock and directly couples it to large conformational changes leading to the opening of the channel. Spectroscopic techniques offer unique possibilities to monitor these conformational changes if it were possible to generate tension in the lipid bilayer, the native environment of MscL, during the measurements. To this end, asymmetric insertion of l-α-lysophosphatidylcholine (LPC) into the lipid bilayer has been effective; however, how LPC activates MscL is not fully understood. Here, the effects of LPC on tension-sensitive mutants of a bacterial MscL and on MscL homologs with different tension sensitivities are reported, leading to the conclusion that the mode of action of LPC is different from that of applied tension. Our results imply that LPC shifts the free energy of gating by interfering with MscL-membrane coupling. Furthermore, we demonstrate that the fine-tuned addition of LPC can be used for controlled activation of MscL in spectroscopic studies.

the orientation of elongated gel fragments. The patterning of hundreds to tens of thousands of elongated microgel shapes in programmed orientations remains challenging. The various existing 3D-printing techniques, including microextrusion printing, [11] weaving, [12] inkjet printing, [13] laser-assisted printing, [14] and water-in-oil droplet printing, [15] cannot order elongated microgel shapes as repeating units.In this work, we present an innovative approach that uses a modified dropletbased microfluidics system to generate and print gelatin methacrylate (GelMa) [16,17] and Matrigel [18] microrods. Continuous-flow lithography can generate rigid elongated gel shapes but the setup is costly and challenging to integrate with 3D printing. [19] Droplet-based microfluidics has been used to fabricate rigid gel microrods, [20] but the published approaches were subject to channel clogging and unable to handle soft gel shapes, such as Matrigel microrods. Moreover, mm-length hydrogel microrods of high aspect ratio containing living cells have not been produced previously.In the present work, microrods were produced with a droplet-based microfluidics tubing (DMT) system that allowed high-throughput formation, transport and gelling of droplet plugs in polytetrafluoroethylene (PTFE) tubing. GelMa and Matrigel were gelled at 8 and 37 °C, respectively. Increased thermostability of the printed GelMa networks was achieved by photo-crosslinking. Cells in the microrods showed ≈90% viability after printing, and assumed expected morphologies. For example, human myofibroblasts cultured in GelMa rods underwent proliferation and elongation. Stromal cells in the Matrigel rods migrated to the periphery of the rods, forming an annular distribution. We patterned GelMa microrods in predefined geometries by using a micromanipulator or a rotating recipient container. Structures were stabilized by rapid fusion of the incompletely gelled rod-rod interfaces immediately after printing. GelMa and Matrigel microrods containing two or more segregated materials, Janus and ternary microrods, [21] were also produced. Finally, by using orientation and patterning, we also printed a microrod tube with luminal dimensions (≈2 cm in diameter) comparable to the human small intestine. Results Generation of GelMa MicrorodsOur DMT printing system allows the fabrication of gel microrods in microfluidics tubing and the synchronous 3D patterning Large scale 3D ordering of anisotropic gel objects, such as gel microrods, both rigid and soft, is in demand for the engineering of replica tissues but has not yet been achieved. Here, monodisperse gel microrods of gelatin methacrylate (GelMa) or Matrigel are generated by a droplet-based microfluidics tubing system. The microrods are 50-300 µm wide and 1-3 mm long; the GelMa versions are produced at up to 50 s −1 while the more fragile Matrigel versions are produced at up to 10 s −1 followed by 1 h of gelation. Upon ejection from the tubing, the rods can be printed into robust 3D structures of centimeter dimensions in w...

Membrane proteins are prime drug targets as they control the transit of information, ions, and solutes across membranes. Here, we present a membrane-on-nanopore platform to analyze nonelectrogenic channels and transporters that are typically not accessible by electrophysiological methods in a multiplexed manner. The silicon chip contains 250 000 femtoliter cavities, closed by a silicon dioxide top layer with defined nanopores. Lipid vesicles containing membrane proteins of interest are spread onto the nanopore-chip surface. Transport events of ligand-gated channels were recorded at single-molecule resolution by high-parallel fluorescence decoding.

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Single molecule studies on membrane proteins embedded in their native environment are hampered by the intrinsic difficulty of immobilizing elastic and sensitive biological membranes without interfering with protein activity. Here, we present hydrogels composed of nano-scaled fibers as a generally applicable tool to immobilize biological membrane vesicles of various size and lipid composition. Importantly, membrane proteins immobilized in the hydrogel as well as soluble proteins are fully active. The triggered opening of the mechanosensitive channel of large conductance (MscL) reconstituted in giant unilamellar vesicles (GUVs) was followed in time on single GUVs. Thus, kinetic studies of vectorial transport processes across biological membranes can be assessed on single, hydrogel immobilized, GUVs. Furthermore, protein translocation activity by the membrane embedded protein conducting channel of bacteria, SecYEG, in association with the soluble motor protein SecA was quantitatively assessed in bulk and at the single vesicle level in the hydrogel. This technique provides a new way to investigate membrane proteins in their native environment at the single molecule level by means of fluorescence microscopy.

recapitulate the structure, cellular organization, and functions of their native counterparts. Though the size is usually up to three orders of magnitude smaller than the native organs, the miniaturized organ model retrieves the multicellular and cellmatrix interactions, and holds the promise to recapitulate some critical functions of native organs. [8] On the other hand, shortage of organs available for transplantation, and the risk of infection and organ rejection coupled with allotransplantation-associated immunosuppression call for the development of engineered organs based on acceptor's genetic information and tissue material (like patient-induced pluripotent stem cell derived differentiated cell lines). [11,12] Modular assembly of microtissues, [13] either in vitro followed by culture and transplantation, or in vivo, such as in situ printing, [14] has become a promising approach to engineer artificial organs across a wide range of scales for regeneration or repair purposes.Hydrogels are a class of polymeric materials that hold large amounts of water in the 3D networks, and have been widely used as the artificial biomaterial to mimic the cellular environment supporting the living tissue networks. [15][16][17] Hydrogels can be either natural or synthetic. [17] Here, we will primarily introduce the microfluidics-based fabrication of hydrogel microtissues in the form of modular structures and microfibers, with a particular focus on one specific class of materials that provides a more native environment to cells, the extracellular matrix (ECM) or ECM-like materials. [18] Microfluidics is a revolutionary platform that manipulates fluids across a wide range of viscosity, [19,20] and has emerged as a powerful technique to miniaturize fluids into microscale and manipulate the fluids online, including mixing, merging, splitting and reaction, etc. [20][21][22] The continuous flow manner can produce various microfibers, [23] whereas droplet-based microfluidics which carriers the hydrogel precursor in discrete manners allows the high-throughput production of monodisperse modular microstructures variable in size, shape, and composition. [24] Microfluidics Fabrication of Soft Microtissues and the Bottom-Up AssemblyMicrotissues are cell-laden solid microstructures gelled from hydrogel precursor solutions, adopting and maintaining identical shapes before and after gelation. Normally, cells are loaded into Soft microtissues comprising living cells and supportive matrices have attracted the attention of researchers for their potential as in vitro organ models that represent the patient's tissue heterogeneity, as well as building blocks for artificial organs or regenerative tissues. Microfluidics is known for its capability to produce monodisperse microstructures in high-throughput. This review summarizes the progress on microfluidics fabrication of hydrogel-based microstructures and soft microtissues, and the challenges faced by this field. It mainly focuses on the strengths and limitations of microfluidics fabrication of ex...

A microfluidics‐tubing‐based 3D‐printing system is presented by Hagan Bayley and co‐workers in article number https://doi.org/10.1002/adbi.201700075. It produces and synchronously patterns monodisperse microrods, which can be tailored in size, shape and composition, into tubular 3D architectures of centimeter dimensions.

Mechanosensitive Channel of Large Conductance (MscL) allows bacteria to respond to osmotic stress in the environment. It senses the increase in the lateral pressure in the membrane due to sudden hypo-osmotic shock and acts as a safety valve. MscL has one of the largest pores in nature; in its open state it allows the passage of ions and small molecules upto 6.5 kDa. MscL has been used in this study as an externally controlled valve i.e. the opening of the channel is controlled by external stimuli. Several techniques like patch clamp, EPR spectroscopy has been applied towards elucidating the gating mechanism of MscL. EPR is effective in tracking the initial conformational changes that the protein may undergo during gating. The main challenge in using spectroscopy is that, unlike patch clamp technique, tension cannot be applied directly for opening the channel. L-a-lysophosphatidylcholine, a reported activator of MscL was studied in this work to trigger opening of the channel in a controlled way. In our work we provide evidence that LPC mimics tension in opening the channel. Our findings also clearly show that LPC can be used for phenotypic characterization of MscL mutants, in a much simpler experiment than patch clamp. A clear differentiation in activity between GOF, LOF and Wt Ec MscL is observed at 4 mM LPC. In conclusion, we characterized an activator with which the mechanism of channel gating can be studied in a controlled way.