Seeing beyond the blot: A critical look at assumptions and raw data interpretation in Western blotting.

Rapid advancements in technology refine our understanding of intricate biological processes, but a crucial emphasis remains on understanding the assumptions and sources of uncertainty underlying biological measurements. This is particularly critical in cell signaling research, where a quantitative understanding of the fundamental mechanisms governing these transient events is essential for drug development, given their importance in both homeostatic and pathogenic processes. Western blotting, a technique developed decades ago, remains an indispensable tool for investigating cell signaling, protein expression, and protein-protein interactions. While improvements in statistical analysis and methodology reporting have undoubtedly enhanced data quality, understanding the underlying assumptions and limitations of visual inspection in Western blotting can provide valuable additional information for evaluating experimental conclusions. Using the example of agonist-induced receptor post-translational modification, we highlight the theoretical and experimental assumptions associated with Western blotting and demonstrate how raw blot data can offer clues to experimental variability that may not be fully captured by statistical analyses and reported methodologies. This article is not intended as a comprehensive technical review of Western blotting. Instead, we leverage an illustrative example to demonstrate how assumptions about experimental design and data normalization can be revealed within raw data and subsequently influence data interpretation.

A Mammalian-Based Synthetic Biology Toolbox to Engineer Membrane-Membrane Interfaces.

Intercellular membrane-membrane interfaces are compartments with specialized functions and unique biophysical properties that are essential in numerous cellular processes including cell signaling, development, and immunity. Using synthetic biology to engineer or to create novel cellular functions in the intercellular regions has led to an increasing need for a platform that allows generation of functionalized intercellular membrane-membrane interfaces. Here, we present a synthetic biology platform to engineer functional membrane-membrane interfaces using a pair of dimerizing proteins in both cell-free and cellular environments. We envisage this platform to be a helpful tool for synthetic biologists who wish to engineer novel intercellular signaling and communication systems.

Cellular mechanotransduction of human osteoblasts in microgravity.

Astronauts experience significant and rapid bone loss as a result of an extended stay in space, making the International Space Station (ISS) the perfect laboratory for studying osteoporosis due to the accelerated nature of bone loss on the ISS. This prompts the question, how does the lack of load due to zero-gravity propagate to bone-forming cells, human fetal osteoblasts (hFOBs), altering their maturation to mineralization? Here, we aim to study the mechanotransduction mechanisms by which bone loss occurs in microgravity. Two automated experiments, 4 microfluidic chips capable of measuring single-cell mechanics of hFOBs via aspiration and cell spheroids incubated in pressure-controlled chambers, were each integrated into a CubeLab deployed to the ISS National Laboratory. For the first experiment, we report protrusion measurements of aspirated cells after exposure to microgravity at the ISS and compare these results to ground control conducted inside the CubeLab. Our analysis revealed slightly elongated protrusions for space samples compared to ground samples indicating softening of hFOB cells in microgravity. In the second experiment, we encapsulated osteoblast spheroids in collagen gel and incubated the samples in pressure-controlled chambers. We found that microgravity significantly reduced filamentous actin levels in the hFOB spheroids. When subjected to pressure, the spheroids exhibited increased pSMAD1/5/9 expression, regardless of the microgravity condition. Moreover, microgravity reduced YAP expression, while pressure increased YAP levels, thus restoring YAP expression for spheroids in microgravity. Our study provides insights into the influence of microgravity on the mechanical properties of bone cells and the impact of compressive pressure on cell behavior and signaling in space.

Cellular mechanotransduction of human osteoblasts in microgravity.

Astronauts experience significant and rapid bone loss as a result of an extended stay in space, making the International Space Station (ISS) the perfect laboratory for studying osteoporosis due to the accelerated nature of bone loss on the ISS. This prompts the question, how does the lack of load due to zero-gravity propagate to bone-forming cells, human fetal osteoblasts (hFOBs), altering their maturation to mineralization? Here, we aim to study the mechanotransduction mechanisms by which bone loss occurs in microgravity. Two automated experiments, microfluidic chips capable of measuring single-cell mechanics via aspiration and cell spheroids incubated in pressure-controlled chambers, were each integrated into a CubeLab deployed to the ISS National Laboratory. For the first experiment, we report protrusion measurements of aspirated cells after exposure to microgravity at the ISS and compare these results to ground control conducted inside the CubeLab. We found slightly elongated protrusions for space samples compared to ground samples indicating softening of hFOB cells in microgravity. In the second experiment, we encapsulated osteoblast spheroids in collagen gel and incubated the samples in pressure-controlled chambers. We found that microgravity significantly reduced filamentous actin levels in the hFOB spheroids. When subjected to pressure, the spheroids exhibited increased pSMAD1/5/9 expression, regardless of the microgravity condition. Moreover, microgravity reduced YAP expression, while pressure increased YAP levels, thus restoring YAP expression for spheroids in microgravity. Our study provides insights into the influence of microgravity on the mechanical properties of bone cells and the impact of compressive pressure on cell signaling in space.

Matrix confinement modulates 3D spheroid sorting and burst-like collective migration.

While it is known that cells with differential adhesion tend to segregate and preferentially sort, the physical forces governing sorting and invasion in heterogeneous tumors remain poorly understood. To investigate this, we tune matrix confinement, mimicking changes in the stiffness and confinement of the tumor microenvironment, to explore how physical confinement influences individual and collective cell migration in 3D spheroids. High levels of confinement lead to cell sorting while reducing matrix confinement triggers the collective fluidization of cell motion. Cell sorting, which depends on cell-cell adhesion, is crucial to this phenomenon. Burst-like migration does not occur for spheroids that have not undergone sorting, regardless of the degree of matrix confinement. Using computational Self-Propelled Voronoi modeling, we show that spheroid sorting and invasion into the matrix depend on the balance between cell-generated forces and matrix resistance. The findings support a model where matrix confinement modulates 3D spheroid sorting and unjamming in an adhesion-dependent manner, providing insights into the mechanisms of cell sorting and migration in the primary tumor and toward distant metastatic sites. STATEMENT OF SIGNIFICANCE: The mechanical properties of the tumor microenvironment significantly influence cancer cell migration within the primary tumor, yet how these properties affect intercellular interactions in heterogeneous tumors is not well understood. By utilizing calcium and calcium chelators, we dynamically alter collagen-alginate hydrogel stiffness and investigate tumor cell behavior within co-culture spheroids in response to varying degrees of matrix confinement. High confinement is found to trigger cell sorting while reducing confinement for sorted spheroids facilitates collective cell invasion. Notably, without prior sorting, spheroids do not exhibit burst-like migration, regardless of confinement levels. This work establishes that matrix confinement and intercellular adhesion regulate 3D spheroid dynamics, offering insights into cellular organization and migration within the primary tumor.

Building Synthetic Cells─From the Technology Infrastructure to Cellular Entities.

The de novo construction of a living organism is a compelling vision. Despite the astonishing technologies developed to modify living cells, building a functioning cell "from scratch" has yet to be accomplished. The pursuit of this goal alone has─and will─yield scientific insights affecting fields as diverse as cell biology, biotechnology, medicine, and astrobiology. Multiple approaches have aimed to create biochemical systems manifesting common characteristics of life, such as compartmentalization, metabolism, and replication and the derived features, evolution, responsiveness to stimuli, and directed movement. Significant achievements in synthesizing each of these criteria have been made, individually and in limited combinations. Here, we review these efforts, distinguish different approaches, and highlight bottlenecks in the current research. We look ahead at what work remains to be accomplished and propose a "roadmap" with key milestones to achieve the vision of building cells from molecular parts.

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System.

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

Evaluating the metropolitan public health preparedness for pandemics using entropy-TOPSIS-IF.

Introduction: Metropolitan governance's efficacy is regularly gauged by its capability for public health preparedness, a critical component, particularly in the post-pandemic climate, as global cities reassess their mitigation abilities. This process has broader implications, curbing mortality rates and amplifying sustainability. Current methodologies for preparedness assessment lean primarily on either Subjective Evaluation-Based Assessment (SBA), predicated on experts' input on various capacity indicators, or they opt for Data-Based quantitative Assessments (DBA), chiefly utilizing public statistic data. Methods: The manuscript discusses an urgent need for integrating both SBA and DBA to adequately measure Metropolitan Public Health Pandemics Preparedness (MPHPP), thus proposing a novel entropy-TOPSIS-IF model for comprehensive evaluation of MPHPP. Within this proposed model, experts' subjective communication is transformed into quantitative data via the aggregation of fuzzy decisions, while objective data is collected from public statistics sites. Shannon's entropy and TOPSIS methods are enacted on these data sets to ascertain the optimal performer after normalization and data isotropy. Results and discussion: The core contribution of the entropy-TOPSIS-IF model lies in its assessment flexibility, making it universally applicable across various contexts, regardless of the availability of expert decisions or quantitative data. To illustrate the efficacy of the entropy-TOPSIS-IF model, a numerical application is presented, examining three Chinese metropolises through chosen criteria according to the evaluations of three experts. A sensitivity analysis is provided to further affirm the stability and robustness of the suggested MPHPP evaluation model.

Rearrangement of GUV-confined actin networks in response to micropipette aspiration.

Although diverse actin network architectures found inside the cell have been individually reconstituted outside of the cell, how different types of actin architectures reorganize under applied forces is not entirely understood. Recently, bottom-up reconstitution has enabled studies where dynamic and phenotypic characteristics of various actin networks can be recreated in an isolated cell-like environment. Here, by creating a giant unilamellar vesicle (GUV)-based cell model encapsulating actin networks, we investigate how actin networks rearrange in response to localized stresses applied by micropipette aspiration. We reconstitute actin bundles and branched bundles in GUVs separately and mechanically perturb them. Interestingly, we find that, when aspirated, protrusive actin bundles that are otherwise randomly oriented in the GUV lumen collapse and align along the axis of the micropipette. However, when branched bundles are aspirated, the network remains intact and outside of the pipette while the GUV membrane is aspirated into the micropipette. These results reveal distinct responses in the rearrangement of actin networks in a network architecture-dependent manner when subjected to physical forces.

Impaired Hepatic Very Low-Density Lipoprotein Secretion Promotes Tumorigenesis and Is Accelerated with Fabp1 Deletion.

Genetic polymorphisms that impair very low-density lipoprotein (VLDL) secretion are linked to hepatic steatosis, fibrosis, and hepatocellular cancer. Liver-specific deletion of microsomal triglyceride transfer protein (Mttp-LKO) impairs VLDL assembly, promoting hepatic steatosis and fibrosis, which are attenuated in Mttp-LKO X Fabp1-null [Fabp1/Mttp double knockout (DKO)] mice. The current study examined the impact of impaired VLDL secretion in Mttp-LKO mice on hepatocellular cancer incidence and progression in comparison to Fabp1/Mttp DKO mice. Diethylnitrosamine-treated Mttp-LKO mice exhibited steatosis with increased tumor burden compared with flox controls, whereas diethylnitrosamine-treated Fabp1/Mttp DKO mice exhibited a paradoxical increase in tumor burden and >50% mortality by 50 weeks. Serum high-density lipoprotein cholesterol was elevated in both Mttp-LKO and Fabp1/Mttp DKO mice, with increased intratumoral expression of apolipoprotein A1 and apolipoprotein E. Lipidomic surveys revealed progressive enrichment in distinct triglyceride species in livers from Mttp-LKO mice with further enrichment in Fabp1/Mttp DKO mice. RNA sequencing revealed mRNA changes suggesting altered monocarboxylic acid use and increased aerobic glycolysis, whereas hepatocytes from Fabp1/Mttp DKO mice exhibited increased capacity to use glucose and glutamine. These metabolic shifts were accompanied by reduced expression of HNF1 homeobox A (HNF1a), which correlated with tumor burden. Taken together, these findings demonstrate that hepatic tumorigenesis is increased in mice with impaired VLDL secretion and further accelerated via pathways including altered fatty acid compartmentalization and shifts in hepatic energy use.

Visualization and Experimental Characterization of Wrapping Layer Using Planar Laser-Induced Fluorescence.

Droplets on nanotextured oil-impregnated surfaces have high mobility due to record-low contact angle hysteresis (∼1-3°), attributed to the absence of solid-liquid contact. Past studies have utilized the ultralow droplet adhesion on these surfaces to improve condensation, reduce hydrodynamic drag, and inhibit biofouling. Despite their promising utility, oil-impregnated surfaces are not fully embraced by industry because of the concern for lubricant depletion, the source of which has not been adequately studied. Here, we use planar laser-induced fluorescence (PLIF) to not only visualize the oil layer encapsulating the droplet (aka wrapping layer) but also measure its thickness since the wrapping layer contributes to lubricant depletion. Our PLIF visualization and experiments show that (a) due to the imbalance of interfacial forces at the three-phase contact line, silicone oil forms a wrapping layer on the outer surface of water droplets, (b) the thickness of the wrapping layer is nonuniform both in space and time, and (c) the time-average thickness of the wrapping layer is ∼50 ± 10 nm, a result that compares favorably with our scaling analysis (∼50 nm), which balances the curvature-induced capillary force with the intermolecular van der Waals forces. Our experiments show that, unlike silicone oil, mineral oil does not form a wrapping layer, an observation that can be exploited to mitigate oil depletion of nanotextured oil-impregnated surfaces. Besides advancing our mechanistic understanding of the wrapping oil layer dynamics, the insights gained from this work can be used to quantify the lubricant depletion rate by pendant droplets in dropwise condensation and water harvesting.

Light-based juxtacrine signaling between synthetic cells.

Cell signaling through direct physical cell-cell contacts plays vital roles in biology during development, angiogenesis, and immune response. Intercellular communication mechanisms between synthetic cells constructed from the bottom up are majorly reliant on diffusible chemical signals, thus limiting the range of responses in receiver cells. Engineering contact-dependent signaling between synthetic cells promises to unlock more complicated signaling schemes with different types of responses. Here, we design and demonstrate a light-activated contact-dependent communication tool for synthetic cells. We utilize a split bioluminescent protein to limit signal generation exclusively to contact interfaces of synthetic cells, driving the recruitment of a photoswitchable protein in receiver cells, akin to juxtacrine signaling in living cells. Our modular design not only demonstrates contact-dependent communication between synthetic cells but also provides a platform for engineering orthogonal contact-dependent signaling mechanisms.

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.

Matrix confinement modulates 3D spheroid sorting and burst-like collective migration.

While it is known that cells with differential adhesion tend to segregate and preferentially sort, the physical forces governing sorting and invasion in heterogeneous tumors remain poorly understood. To investigate this, we tune matrix confinement, mimicking changes in the stiffness and confinement of the tumor microenvironment, to explore how physical confinement influences individual and collective cell migration in 3D spheroids. High levels of confinement lead to cell sorting while reducing matrix confinement triggers the collective fluidization of cell motion. Cell sorting, which depends on cell-cell adhesion, is crucial to this phenomenon. Burst-like migration does not occur for spheroids that have not undergone sorting, regardless of the degree of matrix confinement. Using computational Self-Propelled Voronoi modeling, we show that spheroid sorting and invasion into the matrix depend on the balance between cell-generated forces and matrix resistance. The findings support a model where matrix confinement modulates 3D spheroid sorting and unjamming in an adhesion-dependent manner, providing insights into the mechanisms of cell sorting and migration in the primary tumor and toward distant metastatic sites.

Hybrid Vesicles Enable Mechano-Responsive Hydrogel Degradation.

Stimuli-responsive hydrogels are intriguing biomimetic materials. Previous efforts to develop mechano-responsive hydrogels have mostly relied on chemical modifications of the hydrogel structures. Here, we present a simple, generalizable strategy that confers mechano-responsive behavior on hydrogels. Our approach involves embedding hybrid vesicles, composed of phospholipids and amphiphilic block copolymers, within the hydrogel matrix to act as signal transducers. Under mechanical stress, these vesicles undergo deformation and rupture, releasing encapsulated compounds that can control the hydrogel network. To demonstrate this concept, we embedded vesicles containing ethylene glycol tetraacetic acid (EGTA), a calcium chelator, into a calcium-crosslinked alginate hydrogel. When compressed, the released EGTA sequesters calcium ions and degrades the hydrogel. This study provides a novel method for engineering mechano-responsive hydrogels that may be useful in various biomedical applications.

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.

Methods to mechanically perturb and characterize GUV-based minimal cell models.

Cells shield organelles and the cytosol via an active boundary predominantly made of phospholipids and membrane proteins, yet allowing communication between the intracellular and extracellular environment. Micron-sized liposome compartments commonly known as giant unilamellar vesicles (GUVs) are used to model the cell membrane and encapsulate biological materials and processes in a cell-like confinement. In the field of bottom-up synthetic biology, many have utilized GUVs as substrates to study various biological processes such as protein-lipid interactions, cytoskeletal assembly, and dynamics of protein synthesis. Like cells, it is ideal that GUVs are also mechanically durable and able to stay intact when the inner and outer environment changes. As a result, studies have demonstrated approaches to tune the mechanical properties of GUVs by modulating membrane composition and lumenal material property. In this context, there have been many different methods developed to test the mechanical properties of GUVs. In this review, we will survey various perturbation techniques employed to mechanically characterize GUVs.