Development of mechanosensitive synthetic cells for biomedical applications.

The ability of cells to sense and respond to their physical environment plays a fundamental role in a broad spectrum of biological processes. As one of the most essential molecular force sensors and transducers found in cell membranes, mechanosensitive (MS) ion channels can convert mechanical inputs into biochemical or electrical signals to mediate a variety of sensations. The bottom-up construction of cell-sized compartments displaying cell-like organization, behaviors, and complexity, also known as synthetic cells, has gained popularity as an experimental platform to characterize biological functions in isolation. By reconstituting MS channels in the synthetic lipid bilayers, we envision using mechanosensitive synthetic cells for several medical applications. Here, we describe three different concepts for using ultrasound, shear stress, and compressive stress as mechanical stimuli to activate drug release from mechanosensitive synthetic cells for disease treatments.

Calcium-triggered DNA-mediated membrane fusion in synthetic cells.

In cells, membrane fusion is mediated by SNARE proteins, whose activities are calcium-dependent. While several non-native membrane fusion mechanisms have been demonstrated, few can respond to external stimuli. Here, we develop a calcium-triggered DNA-mediated membrane fusion strategy where fusion is regulated using surface-bound PEG chains that are cleavable by the calcium-activated protease calpain-1.

Calcium-triggered DNA-mediated membrane fusion in synthetic cells.

In cells, membrane fusion is mediated by SNARE proteins, whose activities are calcium-dependent. While several non-native membrane fusion mechanisms have been demonstrated, few can respond to external stimuli. Here, we develop a calcium-triggered DNA-mediated membrane fusion strategy where fusion is regulated using surface-bound PEG chains that are cleavable by the calcium-activated protease calpain-1.

Mechanosensitive Channel-Based Optical Membrane Tension Reporter.

Plasma membrane tension functions as a global physical organizer of cellular activities. Technical limitations of current membrane tension measurement techniques have hampered in-depth investigation of cellular membrane biophysics and the role of plasma membrane tension in regulating cellular processes. Here, we develop an optical membrane tension reporter by repurposing an E. coli mechanosensitive channel via insertion of circularly permuted GFP (cpGFP), which undergoes a large conformational rearrangement associated with channel activation and thus fluorescence intensity changes under increased membrane tension.

Membrane stretching activates calcium permeability of a putative channel Pkd2 during fission yeast cytokinesis.

Pkd2 is the fission yeast homologue of polycystins. This putative ion channel localizes to the plasma membrane. It is required for the expansion of cell volume during interphase growth and cytokinesis, the last step of cell division. However, the channel activity of Pkd2 remains untested. Here, we examined the calcium permeability and mechanosensitivity of Pkd2 through in vitro reconstitution and calcium imaging of pkd2 mutant cells. Pkd2 was translated and inserted into the lipid bilayers of giant unilamellar vesicles using a cell-free expression system. The reconstituted Pkd2 permeated calcium when the membrane was stretched via hypoosmotic shock. In vivo, inactivation of Pkd2 through a temperature-sensitive mutation pkd2-B42 reduced the average intracellular calcium level by 34%. Compared with the wild type, the hypomorphic mutation pkd2-81KD reduced the amplitude of hypoosmotic shock-triggered calcium spikes by 59%. During cytokinesis, mutations of pkd2 reduced the calcium spikes, accompanying cell separation and the ensuing membrane stretching, by 60%. We concluded that fission yeast polycystin Pkd2 allows calcium influx when activated by membrane stretching, representing a likely mechanosensitive channel that contributes to the cytokinetic calcium spikes.

In Vitro Synthesis and Reconstitution Using Mammalian Cell-Free Lysates Enables the Systematic Study of the Regulation of LINC Complex Assembly.

Understanding the structure and structure-function relationships of membrane proteins is a fundamental problem in biomedical research. Given the difficulties inherent to performing mechanistic biochemical and biophysical studies of membrane proteins in vitro, we previously developed a facile HeLa cell-based cell-free expression (CFE) system that enables the efficient reconstitution of full-length (FL) functional inner nuclear membrane Sad1/UNC-84 (SUN) proteins (i.e., SUN1 and SUN2) in supported lipid bilayers. Here, we provide evidence that suggests that the reconstitution of CFE-synthesized FL membrane proteins in supported lipid bilayers occurs primarily through the fusion of endoplasmic reticulum-derived microsomes present within our CFE reactions with our supported lipid bilayers. In addition, we demonstrate the ease with which our synthetic biology platform can be used to investigate the impact of the chemical environment on the ability of CFE-synthesized FL SUN proteins reconstituted in supported lipid bilayers to interact with the luminal domain of the KASH protein nesprin-2. Moreover, we use our platform to study the molecular requirements for the homo- and heterotypic interactions between SUN1 and SUN2. Finally, we show that our platform can be used to simultaneously reconstitute three different CFE-synthesized FL membrane proteins in a single supported lipid bilayer. Overall, these results establish our HeLa cell-based CFE and supported lipid bilayer reconstitution platform as a powerful tool for performing mechanistic dissections of the oligomerization and function of FL membrane proteins in vitro. While our platform is not a substitute for cell-based studies, it does provide important mechanistic insights into the biology of difficult-to-study membrane proteins.

In Vitro Reconstitution Platforms of Mammalian Cell-Free Expressed Membrane Proteins.

Membrane proteins are essential components in cell membranes and enable cells to communicate with their outside environment and to carry out intracellular signaling. Functional reconstitution of complex membrane proteins using cell-free expression (CFE) systems has been proved to be challenging mainly due to the lack of necessary machinery for proper folding and translocation of nascent membrane proteins and their delivery to the supplied synthetic bilayers. Here, we provide protocols for detergent-free, cell-free reconstitution of functional membrane proteins using HeLa-based CFE system and outline assays for studying their membrane insertion, topology, and their orientation upon incorporation into the supported lipid bilayers or bilayers of giant unilamellar vesicles as well as methods to isolate functional translocated cell-free produced membrane proteins.

Rhombic-Shaped Channel Stent with Enhanced Drug Capacity and Fatigue Life.

A drug-eluting stent with rhombic-shaped drug reservoirs is proposed, aimed at providing long-term drug delivery and enhanced fatigue life. Unique rhombic-shaped reservoirs or channels on the stent struts can increase the total drug capacity and improve the stress distribution for longer fatigue life, without compromising other important clinical attributes. Our rhombic-shaped channel stent increases the total drug capacity by multiple times. Its fatigue safety factor, even with the large rhombic cutouts on the stent struts, could be 50% higher than that of the conventional drug-eluting stent. A pulsed fiber-optic laser and a series of expansions and heat treatments were used to make the first prototype of our rhombic-shaped channel stent. This new concept may open up a wide variety of new treatment options and opportunities for the medical industry in the future.