High-viscosity driven modulation of biomechanical properties of human mesenchymal stem cells promotes osteogenic lineage.

Biomechanical cues could effectively govern cell gene expression to direct the differentiation of specific stem cell lineage. Recently, the medium viscosity has emerged as a significant mechanical stimulator that regulates the cellular mechanical properties and various physiological functions. However, whether the medium viscosity can regulate the mechanical properties of human mesenchymal stem cells (hMSCs) to effectively trigger osteogenic differentiation remains uncertain. The mechanism by which cells sense and respond to changes in medium viscosity, and regulate cell mechanical properties to promote osteogenic lineage, remains elusive. In this study, we demonstrated that hMSCs, cultured in a high-viscosity medium, exhibited larger cell spreading area and higher intracellular tension, correlated with elevated formation of actin stress fibers and focal adhesion maturation. Furthermore, these changes observed in hMSCs were associated with activation of TRPV4 (transient receptor potential vanilloid sub-type 4) channels on the cell membrane. This feedback loop among TRPV4 activation, cell spreading and intracellular tension results in calcium influx, which subsequently promotes the nuclear localization of NFATc1 (nuclear factor of activated T cells 1). Concomitantly, the elevated intracellular tension induced nuclear deformation and promoted the nuclear localization of YAP (YES-associated protein). The concurrent activation of NFATc1 and YAP significantly enhanced alkaline phosphatase (ALP) for pre-osteogenic activity. Taken together, these findings provide a more comprehensive view of how viscosity-induced alterations in biomechanical properties of MSCs impact the expression of osteogenesis-related genes, and ultimately promote osteogenic lineage.

Snail Augments Nuclear Deformability to Promote Lymph Node Metastasis of Head and Neck Squamous Cell Carcinoma.

Up to 50% of head and neck squamous cell carcinoma (HNSCC) patients have lymph node (LN) metastasis, resulting in poor survival rate. Numerous studies have supported the notion that the alterations of gene expression and mechanical properties of cancer cells play an important role in cancer metastasis. However, which genes and how they regulate the biomechanical properties of HNSCC cells to promote LN metastasis remains elusive. In this study, we used an LN-metastatic mouse model in vivo to generate an LN-metastatic head and neck squamous cell carcinoma cell line and compared the differences in the biomolecular and biomechanical properties of LN-metastatic and non-metastatic cells. Our results showed that LN-metastatic cells had a higher level of Snail expression compared to non-LN-metastatic cells. The higher Snail expression promoted the cellular invasion capability in confined environments, mainly by increasing the longitudinal strain of the cell nuclei, which could be attributed to the stronger cell traction force and softer nuclear stiffness. These two biomechanical changes were correlated, respectively, to a larger amount of focal adhesion and less amount of nuclear lamins. Taken together, our works revealed not only the biomechanical profiles of LN-metastatic cells but also the corresponding biomolecular expressions to pinpoint the key process in LN metastasis.

Nucleated transcriptional condensates amplify gene expression.

Membraneless organelles or condensates form through liquid-liquid phase separation1-4, which is thought to underlie gene transcription through condensation of the large-scale nucleolus5-7 or in smaller assemblies known as transcriptional condensates8-11. Transcriptional condensates have been hypothesized to phase separate at particular genomic loci and locally promote the biomolecular interactions underlying gene expression. However, there have been few quantitative biophysical tests of this model in living cells, and phase separation has not yet been directly linked with dynamic transcriptional outputs12,13. Here, we apply an optogenetic approach to show that FET-family transcriptional regulators exhibit a strong tendency to phase separate within living cells, a process that can drive localized RNA transcription. We find that TAF15 has a unique charge distribution among the FET family members that enhances its interactions with the C-terminal domain of RNA polymerase II. Nascent C-terminal domain clusters at primed genomic loci lower the energetic barrier for nucleation of TAF15 condensates, which in turn further recruit RNA polymerase II to drive transcriptional output. These results suggest that positive feedback between interacting transcriptional components drives localized phase separation to amplify gene expression.

Composition-dependent thermodynamics of intracellular phase separation.

Intracellular bodies such as nucleoli, Cajal bodies and various signalling assemblies represent membraneless organelles, or condensates, that form via liquid-liquid phase separation (LLPS)1,2. Biomolecular interactions-particularly homotypic interactions mediated by self-associating intrinsically disordered protein regions-are thought to underlie the thermodynamic driving forces for LLPS, forming condensates that can facilitate the assembly and processing of biochemically active complexes, such as ribosomal subunits within the nucleolus. Simplified model systems3-6 have led to the concept that a single fixed saturation concentration is a defining feature of endogenous LLPS7-9, and has been suggested as a mechanism for intracellular concentration buffering2,7,8,10. However, the assumption of a fixed saturation concentration remains largely untested within living cells, in which the richly multicomponent nature of condensates could complicate this simple picture. Here we show that heterotypic multicomponent interactions dominate endogenous LLPS, and give rise to nucleoli and other condensates that do not exhibit a fixed saturation concentration. As the concentration of individual components is varied, their partition coefficients change in a manner that can be used to determine the thermodynamic free energies that underlie LLPS. We find that heterotypic interactions among protein and RNA components stabilize various archetypal intracellular condensates-including the nucleolus, Cajal bodies, stress granules and P-bodies-implying that the composition of condensates is finely tuned by the thermodynamics of the underlying biomolecular interaction network. In the context of RNA-processing condensates such as the nucleolus, this manifests in the selective exclusion of fully assembled ribonucleoprotein complexes, providing a thermodynamic basis for vectorial ribosomal RNA flux out of the nucleolus. This methodology is conceptually straightforward and readily implemented, and can be broadly used to extract thermodynamic parameters from microscopy images. These approaches pave the way for a deeper understanding of the thermodynamics of multicomponent intracellular phase behaviour and its interplay with the nonequilibrium activity that is characteristic of endogenous condensates.

Competing Protein-RNA Interaction Networks Control Multiphase Intracellular Organization.

Liquid-liquid phase separation (LLPS) mediates formation of membraneless condensates such as those associated with RNA processing, but the rules that dictate their assembly, substructure, and coexistence with other liquid-like compartments remain elusive. Here, we address the biophysical mechanism of this multiphase organization using quantitative reconstitution of cytoplasmic stress granules (SGs) with attached P-bodies in human cells. Protein-interaction networks can be viewed as interconnected complexes (nodes) of RNA-binding domains (RBDs), whose integrated RNA-binding capacity determines whether LLPS occurs upon RNA influx. Surprisingly, both RBD-RNA specificity and disordered segments of key proteins are non-essential, but modulate multiphase condensation. Instead, stoichiometry-dependent competition between protein networks for connecting nodes determines SG and P-body composition and miscibility, while competitive binding of unconnected proteins disengages networks and prevents LLPS. Inspired by patchy colloid theory, we propose a general framework by which competing networks give rise to compositionally specific and tunable condensates, while relative linkage between nodes underlies multiphase organization.

Intracellular nonequilibrium fluctuating stresses indicate how nonlinear cellular mechanical properties adapt to microenvironmental rigidity.

Living cells are known to be in thermodynamically nonequilibrium, which is largely brought about by intracellular molecular motors. The motors consume chemical energies to generate stresses and reorganize the cytoskeleton for the cell to move and divide. However, since there has been a lack of direct measurements characterizing intracellular stresses, questions remained unanswered on the intricacies of how cells use such stresses to regulate their internal mechanical integrity in different microenvironments. This report describes a new experimental approach by which we reveal an environmental rigidity-dependent intracellular stiffness that increases with intracellular stress - a revelation obtained, surprisingly, from a correlation between the fluctuations in cellular stiffness and that of intracellular stresses. More surprisingly, by varying two distinct parameters, environmental rigidity and motor protein activities, we observe that the stiffness-stress relationship follows the same curve. This finding provides some insight into the intricacies by suggesting that cells can regulate their responses to their mechanical microenvironment by adjusting their intracellular stress.

Fibroblast Promotes Head and Neck Squamous Cell Carcinoma Cell Invasion through Mechanical Barriers in 3D Collagen Microenvironments.

Cancer metastasis involves not only cancer cells but also fibroblasts and the surrounding collagen matrices. Previous studies have reported that in tumor tissues, cancer cells and fibroblasts surrounded by dense collagen are often associated with a high risk of cancer metastasis. However, the mechanism of the interaction between the cancer cells, fibroblasts, and the surrounding collagen matrices in vivo to promote cancer cell invasion in different collagen concentration environments remains unclear. To address this issue, we cocultured head and neck squamous cell carcinoma (OECM-1 cells) and human dermal fibroblasts (HDFs) to form 3D spheroids, embedded in collagen gel with different concentrations to delineate their roles and their interactions in cancer cell invasion. We showed that in single-species spheroids, the OECM-1 cells could not remodel the high-concentration (8 mg/mL) collagen matrices to invade into the surrounding collagen. In contrast, in the coculture spheroids, the HDF cells could remodel the collagen matrices, via MMP-meditated collagen degradation, to increase the invasion capability of OECM-1 cells. In the case of low-concentration (2 mg/mL) collagen matrices, both HDF and OECM-1 cells in the coculture spheroids could independently invade into the surrounding collagen via force remodeling of collagen. Our results revealed that the assistance of HDFs was critical for OECM-1 cell invasion into the surrounding extracellular matrix with high collagen concentration, high storage modulus, and small pore sizes. These insightful results shed light on the possible optimal invasion strategy of cancer tumors in vivo in response to different storage moduli of surrounding collagen matrices.

Quantifying Dynamics in Phase-Separated Condensates Using Fluorescence Recovery after Photobleaching.

Cells contain numerous membraneless organelles that assemble by intracellular liquid-liquid phase separation. The viscous properties and associated biomolecular mobility within these condensed phase droplets, or condensates, are increasingly recognized as important for cellular function and also dysfunction, for example, in protein aggregation pathologies. Fluorescence recovery after photobleaching (FRAP) is widely used to assess condensate fluidity and to estimate protein diffusion coefficients. However, the models and assumptions utilized in FRAP analysis of protein condensates are often not carefully considered. Here, we combine FRAP experiments on both in vitro reconstituted droplets and intracellular condensates with systematic examination of different models that can be used to fit the data and evaluate the impact of model choice on measured values. A key finding is that model boundary conditions can give rise to widely divergent measured values. This has important implications, for example, in experiments that bleach subregions versus the entire condensate, two commonly employed experimental approaches. We suggest guidelines for determining the appropriate modeling framework and highlight emerging questions about the molecular dynamics at the droplet interface. The ability to accurately determine biomolecular mobility both in the condensate interior and at the interface is important for obtaining quantitative insights into condensate function, a key area for future research.

Controlling the material properties and rRNA processing function of the nucleolus using light.

The nucleolus is a prominent nuclear condensate that plays a central role in ribosome biogenesis by facilitating the transcription and processing of nascent ribosomal RNA (rRNA). A number of studies have highlighted the active viscoelastic nature of the nucleolus, whose material properties and phase behavior are a consequence of underlying molecular interactions. However, the ways in which the material properties of the nucleolus impact its function in rRNA biogenesis are not understood. Here we utilize the Cry2olig optogenetic system to modulate the viscoelastic properties of the nucleolus. We show that above a threshold concentration of Cry2olig protein, the nucleolus can be gelled into a tightly linked, low mobility meshwork. Gelled nucleoli no longer coalesce and relax into spheres but nonetheless permit continued internal molecular mobility of small proteins. These changes in nucleolar material properties manifest in specific alterations in rRNA processing steps, including a buildup of larger rRNA precursors and a depletion of smaller rRNA precursors. We propose that the flux of processed rRNA may be actively tuned by the cell through modulating nucleolar material properties, which suggests the potential of materials-based approaches for therapeutic intervention in ribosomopathies.

Regulating substrate mechanics to achieve odontogenic differentiation for dental pulp stem cells on TiO2 filled and unfilled polyisoprene.

We have shown that materials other than hydrogels commonly used in tissue engineering can be effective in enabling differentiation of dental pulp stem cells (DPSC). Here we demonstrate that a hydrophobic elastomer, polyisoprene (PI), a component of Gutta-percha, normally used to obturate the tooth canal, can also be used to initiate differentiation of the pulp. We showed that PI substrates without additional coating promote cell adhesion and differentiation, while their moduli can be easily adjusted either by varying the coating thickness or incorporation of inorganic particles. DPSC plated on those PI substrates were shown, using SPM and hysitron indentation, to adjust their moduli to conform to differentially small changes in the substrate modulus. In addition, optical tweezers were used to separately measure the membrane and cytoplasm moduli of DPSC, with and without Rho kinase inhibitor. The results indicated that the changes in modulus were attributed predominantly to changes within the cytoplasm, rather than the cell membrane. CLSM was used to identify cell morphology. Differentiation, as determined by qRT-PCR, of the upregulation of OCN, and COL1α1 as well as biomineralization, characterized by SEM/EDAX, was observed on hard PI substrates in the absence of induction factors, i.e. dexamethasone, with moduli 3-4 MPa, regardless of preparation. SEM showed that even though biomineralization was deposited on both spun cast thin PI and filled thick PI substrates, the minerals were aggregated into large clusters on thin PI, and uniformly distributed on filled thick PI, where it was templated within banded collagen fibers. STATEMENT OF SIGNIFICANCE: This manuscript demonstrates the potential of polyisoprene (PI), an elastomeric polymer, for use in tissue engineering. We show how dental pulp stem cells adjust their moduli continuously to match infinitesimally small changes in substrate mechanics, till a critical threshold is reached when they will differentiate. The lineage of differentiation then becomes a sensitive function of both mechanics and morphology for a given chemical composition. Since PI is a major component of Gutta-percha, the FDA approved material commonly used for obturating the root canal, this work suggests that it can easily be adapted for in vivo use in dental regeneration.

Early stage mechanical remodeling of collagen surrounding head and neck squamous cell carcinoma spheroids correlates strongly with their invasion capability.

Mechanical remodeling of stromal collagen, such as reorientation and deformation of collagen matrix, generated by invading cancer cells, plays an important role in the progression of cancer invasion and metastasis. In this study, we applied time-lapse microscopy in conjunction with particle displacement mapping to analyze time-dependent contraction and expansion deformations of collagen surrounding individual spheroids of head and neck squamous cell carcinoma cells (HNSCC), OECM-1 & SAS, as the cancer cells detached from the spheroid and invaded into the surrounding 3D collagen matrix. Our results revealed that highly-invasive HNSCC spheroids, stimulated by epidermal growth factor (EGF), generated a strong contraction deformation of the surrounding collagen in the very early stage, and aligned the collagen fibers radially with respect to the center of the spheroid. This initial collagen contraction deformation generated by the HNSCC spheroid bears a strong positive correlation with the overall extent of subsequent cancer cells invasion; hence, it may serve as an early indicator of the invasion capability of the HNSCC spheroids. STATEMENT OF SIGNIFICANCE: Mechanical remodeling of extracellular matrix (ECM) generated by cancer cells plays an important role in the progression of cancer invasion and metastasis. We observed that the extent of initial contraction deformation of collagen surrounding a head and neck squamous cell carcinoma cell (HNSCC) spheroid played an indispensable role in early stage to promote cancer cells invasion into the surrounding ECM. Our results revealed that more invasive HNSCC spheroids generated a larger extent of initial collagen contraction to align the surrounding collagen and to promote cancer cells invasion. This initial collagen contraction deformation generated by the HNSCC spheroids bears a strong positive correlation with the overall extent of cancer cells invasion; hence, it may serve as an early indicator of the invasion capability of the HNSCC spheroids.

Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds.

Liquid-liquid phase separation plays a key role in the assembly of diverse intracellular structures. However, the biophysical principles by which phase separation can be precisely localized within subregions of the cell are still largely unclear, particularly for low-abundance proteins. Here, we introduce an oligomerizing biomimetic system, "Corelets," and utilize its rapid and quantitative light-controlled tunability to map full intracellular phase diagrams, which dictate the concentrations at which phase separation occurs and the transition mechanism, in a protein sequence dependent manner. Surprisingly, both experiments and simulations show that while intracellular concentrations may be insufficient for global phase separation, sequestering protein ligands to slowly diffusing nucleation centers can move the cell into a different region of the phase diagram, resulting in localized phase separation. This diffusive capture mechanism liberates the cell from the constraints of global protein abundance and is likely exploited to pattern condensates associated with diverse biological processes. VIDEO ABSTRACT.

Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles.

Many intracellular membraneless organelles form via phase separation of intrinsically disordered proteins (IDPs) or regions (IDRs). These include the Caenorhabditis elegans protein LAF-1, which forms P granule-like droplets in vitro. However, the role of protein disorder in phase separation and the macromolecular organization within droplets remain elusive. Here, we utilize a novel technique, ultrafast-scanning fluorescence correlation spectroscopy, to measure the molecular interactions and full coexistence curves (binodals), which quantify the protein concentration within LAF-1 droplets. The binodals of LAF-1 and its IDR display a number of unusual features, including 'high concentration' binodal arms that correspond to remarkably dilute droplets. We find that LAF-1 and other in vitro and intracellular droplets are characterized by an effective mesh size of ∼3-8 nm, which determines the size scale at which droplet properties impact molecular diffusion and permeability. These findings reveal how specific IDPs can phase separate to form permeable, low-density (semi-dilute) liquids, whose structural features are likely to strongly impact biological function.

Lateral optical binding between two colloidal particles.

An optical binding force between two nearby colloidal particles trapped by two coherent laser beams is measured by phase-sensitive detection. The binding force is long-range and spatially oscillatory. For identical linearly-polarized incident beams, the oscillation period is equal to the optical wavelength. For mutually perpendicular polarizations, a new force appears with half-wavelength periodicity, caused by double inter-particle scattering. This force is observable only with cross-polarized incident beams, for which the stronger single-scattering forces are forbidden by parity.

Exposure to TiO2 nanoparticles increases Staphylococcus aureus infection of HeLa cells.

BACKGROUND: Titanium dioxide (TiO2) is one of the most common nanoparticles found in industry ranging from food additives to energy generation. Approximately four million tons of TiO2 particles are produced worldwide each year with approximately 3000 tons being produced in nanoparticulate form, hence exposure to these particles is almost certain. RESULTS: Even though TiO2 is also used as an anti-bacterial agent in combination with UV, we have found that, in the absence of UV, exposure of HeLa cells to TiO2 nanoparticles significantly increased their risk of bacterial invasion. HeLa cells cultured with 0.1 mg/ml rutile and anatase TiO2 nanoparticles for 24 h prior to exposure to bacteria had 350 and 250 % respectively more bacteria per cell. The increase was attributed to bacterial polysaccharides absorption on TiO2 NPs, increased extracellular LDH, and changes in the mechanical response of the cell membrane. On the other hand, macrophages exposed to TiO2 particles ingested 40 % fewer bacteria, further increasing the risk of infection. CONCLUSIONS: In combination, these two factors raise serious concerns regarding the impact of exposure to TiO2 nanoparticles on the ability of organisms to resist bacterial infection.

Low-frequency dielectrophoretic response of a single particle in aqueous suspensions.

We use optical tweezers-based dielectrophoresis (DEP) force spectroscopy to investigate the roles of the electrical double layer in the AC dielectric response of an individual colloidal particle in an aqueous medium. Specifically, we measure the DEP crossover frequency as a function of particles size, medium viscosity, and temperature. Experimental results were compared to low frequency relaxation mechanisms predicted by Schwarz, demonstrating the dielectrophoretic responses in the frequency range between 10 kHz and 1 MHz were dominated by counterion diffusion within the electric double layer.

Formation of contractile networks and fibers in the medial cell cortex through myosin-II turnover, contraction, and stress-stabilization.

The morphology of adhered cells depends crucially on the formation of a contractile meshwork of parallel and cross-linked fibers along the contacting surface. The motor activity and minifilament assembly of non-muscle myosin-II is an important component of cortical cytoskeletal remodeling during mechanosensing. We used experiments and computational modeling to study cortical myosin-II dynamics in adhered cells. Confocal microscopy was used to image the medial cell cortex of HeLa cells stably expressing myosin regulatory light chain tagged with GFP (MRLC-GFP). The distribution of MRLC-GFP fibers and focal adhesions was classified into three types of network morphologies. Time-lapse movies show: myosin foci appearance and disappearance; aligning and contraction; stabilization upon alignment. Addition of blebbistatin, which perturbs myosin motor activity, leads to a reorganization of the cortical networks and to a reduction of contractile motions. We quantified the kinetics of contraction, disassembly and reassembly of myosin networks using spatio-temporal image correlation spectroscopy (STICS). Coarse-grained numerical simulations include bipolar minifilaments that contract and align through specified interactions as basic elements. After assuming that minifilament turnover decreases with increasing contractile stress, the simulations reproduce stress-dependent fiber formation in between focal adhesions above a threshold myosin concentration. The STICS correlation function in simulations matches the function measured in experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness.

The effect of enterohemorrhagic E. coli infection on the cell mechanics of host cells.

Enterohaemorrhagic E. coli (EHEC) is a type of human pathogenic bacteria. The main virulence characteristics of EHEC include the formation of attaching and effacing lesions (A/E lesions) and the production of one or more Shiga-like toxins, which may induce human uremic complications. When EHEC infects host cells, it releases translocated intimin receptor (Tir) and effector proteins inside the host cells, inducing the rearrangement and accumulation of the F-actin cytoskeleton, a phenotype leading to the formation of pedestals in the apical cell surface, and the growth of stress fibers at the base of the cells. To examine the effect of EHEC infection on cell mechanics, we carried out a series of experiments to examine HeLa cells with and without EHEC infection to quantify the changes in (1) focal adhesion area, visualized by anti-vinculin staining; (2) the distribution and orientation of stress fibers; and (3) the intracellular viscoelasticity, via directional video particle tracking microrheology. Our results indicated that in EHEC-infected HeLa cells, the focal adhesion area increased and the actin stress fibers became thicker and more aligned. The cytoskeletal reorganization induced by EHEC infection mediated a dramatic increase in the cytoplasmic elastic shear modulus of the infected cells, and a transition in the viscoelastic behavior of the cells from viscous-like to elastic-like. These changes in mechanobiological characteristics might modulate the attachments between EHEC and the host cell to withstand exfoliation, and between the host cell and the extracellular matrix, and might also alter epithelial integrity.

Intracellular viscoelasticity of HeLa cells during cell division studied by video particle-tracking microrheology.

Cell division plays an important role in regulating cell proliferation and differentiation. It is managed by a complex sequence of cytoskeleton alteration that induces dividing cells to change their morphology to facilitate their division. The change in cytoskeleton structure is expected to affect the intracellular viscoelasticity, which may also contribute to cellular dynamic deformation during cell division. However, the intracellular viscoelasticity during cell division is not yet well understood. In this study, we injected 100-nm (diameter) carboxylated polystyrene beads into the cytoplasm of HeLa cells and applied video particle tracking microrheology to measure their intracellular viscoelasticity at different phases during cell division. The Brownian motion of the intracellular nanoprobes was analyzed to compute the viscoelasticity of HeLa cells in terms of the elastic modulus and viscous modulus as a function of frequency. Our experimental results indicate that during the course of cell division, both intracellular elasticity and viscosity increase in the transition from the metaphase to the anaphase, plausibly due to the remodeling of cytoskeleton and redistributions of molecular motors, but remain approximately the same from the anaphase to the telophase.