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.

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.

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.

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.

Differential regulation of GUV mechanics via actin network architectures.

Actin networks polymerize and depolymerize to construct highly organized structures, thereby endowing the mechanical phenotypes found in a cell. It is generally believed that the amount of filamentous actin and actin network architecture determine cytoplasmic viscoelasticity of the whole cell. However, the intrinsic complexity of a cell and the presence of endogenous cellular components make it difficult to study the differential roles of distinct actin networks in regulating cell mechanics. Here, we model a cell by using giant unilamellar vesicles (GUVs) encapsulating actin filaments and networks assembled by various actin cross-linker proteins. Perturbation of these cytoskeletal vesicles using alternating current electric fields revealed that deformability depends on actin network architecture. While actin-free vesicles exhibited large electromechanical deformations, deformations of GUVs encapsulating actin filaments were significantly dampened. The suppression of electrodeformation of actin-GUVs can be similarly recapitulated by using aqueous poly(ethylene glycol) 8000 solutions at different concentrations to modulate solution viscoelasticity. Furthermore, networks cross-linked by alpha actinin resulted in decreased GUV deformability compared with actin-filament-encapsulating GUVs, and membrane-associated actin networks, through the formation of the dendritic actin cortex, greatly dampened electrodeformation of GUVs. These results highlight that the organization of actin networks regulates the mechanics of GUVs and shed insights into the origin of differential deformability of cells.

Simulating microgravity using a random positioning machine for inducing cellular responses to mechanotransduction in human osteoblasts.

The mechanotransduction pathways that mediate cellular responses to contact forces are better understood than those that mediate response to distance forces, especially the force of gravity. Removing or reducing gravity for significant periods of time involves either sending samples to space, inducing diamagnetic levitation with high magnetic fields, or continually reorienting samples for a period, all in a manner that supports cell culturing. Undesired secondary effects due to high magnetic fields or shear forces associated with fluid flow while reorienting must be considered in the design of ground-based devices. We have developed a lab-friendly and compact random positioning machine (RPM) that fits in a standard tissue culture incubator. Using a two-axis gimbal, it continually reorients samples in a manner that produces an equal likelihood that all possible orientations are visited. We contribute a new control algorithm by which the distribution of probabilities over all possible orientations is completely uniform. Rather than randomly varying gimbal axis speed and/or direction as in previous algorithms (which produces non-uniform probability distributions of orientation), we use inverse kinematics to follow a trajectory with a probability distribution of orientations that is uniform by construction. Over a time period of 6 h of operation using our RPM, the average gravity is within 0.001 23% of the gravity of Earth. Shear forces are minimized by limiting the angular speed of both gimbal motors to under 42 °/s. We demonstrate the utility of our RPM by investigating the effects of simulated microgravity on adherent human osteoblasts immediately after retrieving samples from our RPM. Cytoskeletal disruption and cell shape changes were observed relative to samples cultured in a 1 g environment. We also found that subjecting human osteoblasts in suspension to simulated microgravity resulted in less filamentous actin and lower cell stiffness.

Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles.

Giant unilamellar vesicles (GUVs) are frequently used as models of biological membranes and thus are a great tool to study membrane-related cellular processes in vitro. In recent years, encapsulation within GUVs has proven to be a helpful approach for reconstitution experiments in cell biology and related fields. It better mimics confinement conditions inside living cells, as opposed to conventional biochemical reconstitution. Methods for encapsulation inside GUVs are often not easy to implement, and success rates can differ significantly from lab to lab. One technique that has proven to be successful for encapsulating more complex protein systems is called continuous droplet interface crossing encapsulation (cDICE). Here, a cDICE-based method is presented for rapidly encapsulating cytoskeletal proteins in GUVs with high encapsulation efficiency. In this method, first, lipid-monolayer droplets are generated by emulsifying a protein solution of interest in a lipid/oil mixture. After being added into a rotating 3D-printed chamber, these lipid-monolayered droplets then pass through a second lipid monolayer at a water/oil interface inside the chamber to form GUVs that contain the protein system. This method simplifies the overall procedure of encapsulation within GUVs and speeds up the process, and thus allows us to confine and observe the dynamic evolution of network assembly inside lipid bilayer vesicles. This platform is handy for studying the mechanics of cytoskeleton-membrane interactions in confinement.

Physiologic biomechanics enhance reproducible contractile development in a stem cell derived cardiac muscle platform.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) allow investigations in a human cardiac model system, but disorganized mechanics and immaturity of hPSC-CMs on standard two-dimensional surfaces have been hurdles. Here, we developed a platform of micron-scale cardiac muscle bundles to control biomechanics in arrays of thousands of purified, independently contracting cardiac muscle strips on two-dimensional elastomer substrates with far greater throughput than single cell methods. By defining geometry and workload in this reductionist platform, we show that myofibrillar alignment and auxotonic contractions at physiologic workload drive maturation of contractile function, calcium handling, and electrophysiology. Using transcriptomics, reporter hPSC-CMs, and quantitative immunofluorescence, these cardiac muscle bundles can be used to parse orthogonal cues in early development, including contractile force, calcium load, and metabolic signals. Additionally, the resultant organized biomechanics facilitates automated extraction of contractile kinetics from brightfield microscopy imaging, increasing the accessibility, reproducibility, and throughput of pharmacologic testing and cardiomyopathy disease modeling.

Fascin-induced actin protrusions are suppressed by dendritic networks in giant unilamellar vesicles.

The interactions between actin networks and cell membrane are immensely important for eukaryotic cell functions including cell shape changes, motility, polarity establishment, and adhesion. Actin-binding proteins are known to compete and cooperate using a finite amount of actin monomers to form distinct actin networks. How actin-bundling protein fascin and actin-branching protein Arp2/3 complex compete to remodel membranes is not entirely clear. To investigate fascin- and Arp2/3-mediated actin network remodeling, we applied a reconstitution approach encapsulating bundled and dendritic actin networks inside giant unilamellar vesicles (GUVs). Independently reconstituted, membrane-bound Arp2/3 nucleation forms an actin cortex in GUVs, whereas fascin mediates formation of actin bundles that protrude out of GUVs. Coencapsulating both fascin and Arp2/3 complex leads to polarized dendritic aggregates and significantly reduces membrane protrusions, irrespective of whether the dendritic network is membrane bound or not. However, reducing Arp2/3 complex while increasing fascin restores membrane protrusion. Such changes in network assembly and the subsequent interplay with membrane can be attributed to competition between fascin and Arp2/3 complex to utilize a finite pool of actin.

Confinement Geometry Tunes Fascin-Actin Bundle Structures and Consequently the Shape of a Lipid Bilayer Vesicle.

Depending on the physical and biochemical properties of actin-binding proteins, actin networks form different types of membrane protrusions at the cell periphery. Actin crosslinkers, which facilitate the interaction of actin filaments with one another, are pivotal in determining the mechanical properties and protrusive behavior of actin networks. Short crosslinkers such as fascin bundle F-actin to form rigid spiky filopodial protrusions. By encapsulation of fascin and actin in giant unilamellar vesicles (GUVs), we show that fascin-actin bundles cause various GUV shape changes by forming bundle networks or straight single bundles depending on GUV size and fascin concentration. We also show that the presence of a long crosslinker, α-actinin, impacts fascin-induced GUV shape changes and significantly impairs the formation of filopodia-like protrusions. Actin bundle-induced GUV shape changes are confirmed by light-induced disassembly of actin bundles leading to the reversal of GUV shape. Our study contributes to advancing the design of shape-changing minimal cells for better characterization of the interaction between lipid bilayer membranes and actin cytoskeleton.