Mechano-metabolism of adherent cells in 2D and 3D microenvironments.

Cells regulate their shape and metabolic activity in response to the mechano-chemical properties of their microenvironment. To elucidate the impact of matrix stiffness and ligand density on a cell's bioenergetics, we developed a non-equilibrium, active chemo-mechanical model that accounts for mechanical energy of the cell and matrix, chemical energy from ATP hydrolysis, interfacial energy, and mechano-sensitive regulation of stress fiber assembly through signaling. By integrating the kinetics and energetics of these processes we introduce the concept of the metabolic potential of the cell that, when minimized, gives experimentally testable predictions of the cell contractility, shape, and the ATP consumption. Specifically, we show that MDA-MB-231 breast cancer cells in 3D collagen gels follow a spherical to spindle to spherical change in morphology with increasing matrix stiffness consistent with experimental observations. This biphasic transition in cell shape emerges from a competition between increased contractility accompanied by ATP hydrolysis enabled by mechano-sensitive signaling, which lowers the volumetric contribution to the metabolic potential of elongated cells and the interfacial energy which is lower for spherical shapes. On 2D hydrogels, our model predicts a hemispherical to spindle to disc shape transition with increasing gel stiffness. In both cases, we show that increasing matrix stiffness monotonically increases the cell's contractility as well as ATP consumption. Our model also predicts how the increased energy demand in stiffer microenvironments is met by AMPK activation, which is confirmed through experimental measurement of activated AMPK levels as a function of matrix stiffness carried out here in both 2D and 3D micro-environments. Further, model predictions of increased AMPK activation on stiffer micro-environments are found to correlate strongly with experimentally measured upregulation of mitochondrial potential, glucose uptake and ATP levels. The insights from our model can be used to understand mechanosensitive regulation of metabolism in physiological events such as metastasis and tumor progression during which cells experience dynamic changes in their microenvironment and metabolic state.

In silico analysis shows that dynamic changes in curvature guide cell migration over long distances.

In vitro experiments have shown that cell scale curvatures influence cell migration; cells avoid convex hills and settle in concave valleys. However, it is not known whether dynamic changes in curvature can guide cell migration. This study extends a previous in-silico model to explore the effects over time of changing the substrate curvature on cell migration guidance. By simulating a dynamic surface curvature using traveling wave patterns, we investigate the influence of wave height and speed, and find that long-distance cell migration guidance can be achieved on specific wave patterns. We propose a mechanistic explanation of what we call dynamic curvotaxis and highlight those cellular features that may be involved. Our results open a new area of study for understanding cell mobility in dynamic environments, from single-cell in vitro experiments to multi-cellular in vivo mechanisms.

Response of Osteoblasts on Amine-Based Nanocoatings Correlates with the Amino Group Density.

Increased life expectancy in industrialized countries is causing an increased incidence of osteoporosis and the need for bioactive bone implants. The integration of implants can be improved physically, but mainly by chemical modifications of the material surface. It was recognized that amino-group-containing coatings improved cell attachment and intracellular signaling. The aim of this study was to determine the role of the amino group density in this positive cell behavior by developing controlled amino-rich nanolayers. This work used covalent grafting of polymer-based nanocoatings with different amino group densities. Titanium coated with the positively-charged trimethoxysilylpropyl modified poly(ethyleneimine) (Ti-TMS-PEI), which mostly improved cell area after 30 min, possessed the highest amino group density with an N/C of 32%. Interestingly, changes in adhesion-related genes on Ti-TMS-PEI could be seen after 4 h. The mRNA microarray data showed a premature transition of the MG-63 cells into the beginning differentiation phase after 24 h indicating Ti-TMS-PEI as a supportive factor for osseointegration. This amino-rich nanolayer also induced higher bovine serum albumin protein adsorption and caused the cells to migrate slower on the surface after a more extended period of cell settlement as an indication of a better surface anchorage. In conclusion, the cell spreading on amine-based nanocoatings correlated well with the amino group density (N/C).

Curvature in Biological Systems: Its Quantification, Emergence, and Implications across the Scales.

Surface curvature both emerges from, and influences the behavior of, living objects at length scales ranging from cell membranes to single cells to tissues and organs. The relevance of surface curvature in biology is supported by numerous experimental and theoretical investigations in recent years. In this review, first, a brief introduction to the key ideas of surface curvature in the context of biological systems is given and the challenges that arise when measuring surface curvature are discussed. Giving an overview of the emergence of curvature in biological systems, its significance at different length scales becomes apparent. On the other hand, summarizing current findings also shows that both single cells and entire cell sheets, tissues or organisms respond to curvature by modulating their shape and their migration behavior. Finally, the interplay between the distribution of morphogens or micro-organisms and the emergence of curvature across length scales is addressed with examples demonstrating these key mechanistic principles of morphogenesis. Overall, this review highlights that curved interfaces are not merely a passive by-product of the chemical, biological, and mechanical processes but that curvature acts also as a signal that co-determines these processes.

Rational Design and Characterisation of Novel Mono- and Bimetallic Antibacterial Linde Type A Zeolite Materials.

The development of antimicrobial devices and surfaces requires the setup of suitable materials, able to store and release active principles. In this context, zeolites, which are microporous aluminosilicate minerals, hold great promise, since they are able to serve as a reservoir for metal-ions with antimicrobial properties. Here, we report on the preparation of Linde Type A zeolites, partially exchanged with combinations of metal-ions (Ag+, Cu2+, Zn2+) at different loadings (0.1-11.9 wt.%). We combine X-ray fluorescence, scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray diffraction to monitor the metal-ion contents, distribution, and conservation of the zeolite structure after exchange. Then, we evaluate their antimicrobial activity, using agar dilution and optical-density monitoring of Escherichia coli cultures. The results indicate that silver-loaded materials are at least 70-fold more active than the copper-, zinc-, and non-exchanged ones. Moreover, zeolites loaded with lower Ag+ concentrations remain active down to 0.1 wt.%, and their activities are directly proportional to the total Ag content. Sequential exchanges with two metal ions (Ag+ and either Cu2+, Zn2+) display synergetic or antagonist effects, depending on the quantity of the second metal. Altogether, this work shows that, by combining analytical and quantitative methods, it is possible to fine-tune the composition of bi-metal-exchanged zeolites, in order to maximise their antimicrobial potential, opening new ways for the development of next-generation composite zeolite-containing antimicrobial materials, with potential applications for the design of dental or bone implants, as well as biomedical devices and pharmaceutical products.

Kinetically stable sub-50 nm fluorescent block copolymer nanoparticles via photomediated RAFT dispersion polymerization for cellular imaging.

Self-assembled block copolymer nanoparticles (NPs) have emerged as major potential nanoscale vehicles for fluorescence bioimaging. The preparation of NPs with high yields possessing high kinetic stability to prevent the leakage of fluorophore molecules is crucial to their practical implementation. Here, we report a photomediated RAFT polymerization-induced self-assembly (PISA) yielding uniform and nanosized poly((oligo(ethylene glycol) acrylate)-block-poly(benzyl acrylate) particles (POEGA-b-PBzA) with a concentration of 22 wt%, over 20 times more than with micellization and nanoprecipitation. The spherical diblock copolymer nanoparticles have an average size of 10-50 nm controllable through the degree of polymerization of the stabilizing POEGA block. Subsequent dialysis against water and swelling with Nile red solution led to highly stable fluorescent NPs able to withstand the changes in concentration, ionic strength, pH or temperature. A PBzA/water interfacial tension of 48.6 mN m-1 hinders the exchange between copolymer chains, resulting in the trapping of NPs in a "kinetically frozen" state responsible for high stability. A spectroscopic study combining fluorescence and UV-vis absorption agrees with a preferential distribution of fluorophores in the outer POEGEA shell despite its hydrophobic nature. Nile red-doped POEGA-b-PBzA micelles without initiator residues and unimers but with high structural stability turn out to be noncytotoxic, and can be used for the optical imaging of cells. Real-time confocal fluorescence microscopy shows a fast cellular uptake using C2C12 cell lines in minutes, and a preferential localization in the perinuclear region, in particular in the vesicles.

Size-Dependent Internalization Efficiency of Macrophages from Adsorbed Nanoparticle-Based Monolayers.

Functional coatings based on the assembly of submicrometric or nanoparticles are found in many applications in the biomedical field. However, these nanoparticle-based coatings are particularly fragile since they could be exposed to cells that are able to internalize nanoparticles. Here, we studied the efficiency of RAW 264.7 murine macrophages to internalize physisorbed silica nanoparticles as a function of time and particle size. This cell internalization efficiency was evaluated from the damages induced by the cells in the nanoparticle-based monolayer on the basis of scanning electron microscopy and confocal laser scanning microscopy observations. The internalization efficiency in terms of the percentage of nanoparticles cleared from the substrate is characterized by two size-dependent regimes. Additionally, we highlighted that a delay before internalization occurs, which increases with decreasing adsorbed nanoparticle size. This internalization is characterized by a minimal threshold that corresponds to 35 nm nanoparticles that are not internalized during the 12-h incubation considered in this work.

Topographical curvature is sufficient to control epithelium elongation.

How biophysical cues can control tissue morphogenesis is a central question in biology and for the development of efficient tissue engineering strategies. Recent data suggest that specific topographies such as grooves and ridges can trigger anisotropic tissue growth. However, the specific contribution of biologically relevant topographical features such as cell-scale curvature is still unclear. Here we engineer a series of grooves and ridges model topographies exhibiting specific curvature at the ridge/groove junctions and monitored the growth of epithelial colonies on these surfaces. We observe a striking proportionality between the maximum convex curvature of the ridges and the elongation of the epithelium. This is accompanied by the anisotropic distribution of F-actin and nuclei with partial exclusion of both in convex regions as well as the curvature-dependent reorientation of pluricellular protrusions and mitotic spindles. This demonstrates that curvature itself is sufficient to trigger and modulate the oriented growth of epithelia through the formation of convex "topographical barriers" and establishes curvature as a powerful tuning parameter for tissue engineering and biomimetic biomaterial design.

Synthesis of FAU-Type Zeolite Membranes with Antimicrobial Activity.

In this study, a layer of a pure and dense phase of FAU-type zeolite was synthesized directly on the surface of α-Al2O3 plane macroporous support. Before hydrothermal synthesis, a step of cleaning of the support by an anionic detergent was performed, a roughness surface is created, allowing the anchoring of the zeolite nuclei and then their growth, favoring in this sense the formation of a homogeneous zeolite layer. The obtained membranes were fully characterized using X-ray diffraction analysis (XRD), nitrogen sorption, scanning electron microscopy (SEM), and mercury porosimetry. After 24 h of thermal treatment at 75 °C, a homogeneous zeolite layer composed of bipyramidal crystals of FAU-type zeolite is obtained with a thickness of about 2.5 µm. No obvious defects or cracks can be observed. It was found that the increase in heating temperature could lead to the appearance of an impurity phase, GIS-type zeolite. Then the ideal zeolite membrane was exchanged with Ag+ or Zn2+ cations to studies their antimicrobial properties. Zeolites membranes exchanged with Ag+ showed an agar-diffusive bactericidal activity against gram negative Escherichia coli (E. coli) bacteria. Zn2+ exchanged zeolite membrane presented a bacteriostatic activity that is less diffusive in agar. As expected, non-exchanged zeolite membrane (in its Na+ form) have no effect on bacterial activity. This process is particularly interesting for the synthesis of a good quality FAU-type zeolite membranes with antimicrobial properties.

Surface Texturization of Breast Implants Impacts Extracellular Matrix and Inflammatory Gene Expression in Asymptomatic Capsules.

BACKGROUND: Texturing processes have been designed to improve biocompatibility and mechanical anchoring of breast implants. However, a high degree of texturing has been associated with severe abnormalities. In this study, the authors aimed to determine whether implant surface topography could also affect physiology of asymptomatic capsules. METHODS: The authors collected topographic measurements from 17 different breast implant devices by interferometry and radiographic microtomography. Morphologic structures were analyzed statistically to obtain a robust breast implant surface classification. The authors obtained three topographic categories of textured implants (i.e., "peak and valleys," "open cavities," and "semiopened cavities") based on the cross-sectional aspects. The authors simultaneously collected 31 Baker grade I capsules, sorted them according to the new classification, established their molecular profile, and examined the tissue organization. RESULTS: Each of the categories showed distinct expression patterns of genes associated with the extracellular matrix (Timp and Mmp members) and inflammatory response (Saa1, Tnsf11, and Il8), despite originating from healthy capsules. In addition, slight variations were observed in the organization of capsular tissues at the histologic level. CONCLUSIONS: The authors combined a novel surface implant classification system and gene profiling analysis to show that implant surface topography is a bioactive cue that can trigger gene expression changes in surrounding tissue, even in Baker grade I capsules. The authors' new classification system avoids confusion regarding the word "texture," and could be transposed to implant ranges of every manufacturer. This new classification could prove useful in studies on potential links between specific texturizations and the incidence of certain breast-implant associated complications.

Actomyosin, vimentin and LINC complex pull on osteosarcoma nuclei to deform on micropillar topography.

Cell deformation occurs in many critical biological processes, including cell extravasation during immune response and cancer metastasis. These cells deform the nucleus, their largest and stiffest organelle, while passing through narrow constrictions in vivo and the underlying mechanisms still remain elusive. It is unclear which biochemical actors are responsible and whether the nucleus is pushed or pulled (or both) during deformation. Herein we use an easily-tunable poly-L-lactic acid micropillar topography, mimicking in vivo constrictions to determine the mechanisms responsible for nucleus deformation. Using biochemical tools, we determine that actomyosin contractility, vimentin and nucleo-cytoskeletal connections play essential roles in nuclear deformation, but not A-type lamins. We chemically tune the adhesiveness of the micropillars to show that pulling forces are predominantly responsible for the deformation of the nucleus. We confirm these results using an in silico cell model and propose a comprehensive mechanism for cellular and nuclear deformation during confinement. These results indicate that microstructured biomaterials are extremely versatile tools to understand how forces are exerted in biological systems and can be useful to dissect and mimic complex in vivo behaviour.

A Biophysical Model for Curvature-Guided Cell Migration.

The latest experiments have shown that adherent cells can migrate according to cell-scale curvature variations via a process called curvotaxis. Despite identification of key cellular factors, a clear understanding of the mechanism is lacking. We employ a mechanical model featuring a detailed description of the cytoskeleton filament networks, the viscous cytosol, the cell adhesion dynamics, and the nucleus. We simulate cell adhesion and migration on sinusoidal substrates. We show that cell adhesion on three-dimensional curvatures induces a gradient of pressure inside the cell that triggers the internal motion of the nucleus. We propose that the resulting out-of-equilibrium position of the nucleus alters cell migration directionality, leading to cell motility toward concave regions of the substrate, resulting in lower potential energy states. Altogether, we propose a simple mechanism explaining how intracellular mechanics enable the cells to react to substratum curvature, induce a deterministic cell polarization, and break down cells basic persistent random walk, which correlates with latest experimental evidences.

Dynamic adaptation of mesenchymal stem cell physiology upon exposure to surface micropatterns.

Human mesenchymal stem (hMSCs) are defined as multi-potent colony-forming cells expressing a specific subset of plasma membrane markers when grown on flat tissue culture polystyrene. However, as soon as hMSCs are used for transplantation, they are exposed to a 3D environment, which can strongly impact cell physiology and influence proliferation, differentiation and metabolism. Strategies to control in vivo hMSC behavior, for instance in stem cell transplantation or cancer treatment, are skewed by the un-physiological flatness of the standard well plates. Even though it is common knowledge that cells behave differently in vitro compared to in vivo, only little is known about the underlying adaptation processes. Here, we used micrometer-scale defined surface topographies as a model to describe the phenotype of hMSCs during this adaptation to their new environment. We used well established techniques to compare hMSCs cultured on flat and topographically enhanced polystyreneand observed dramatically changed cell morphologies accompanied by shrinkage of cytoplasm and nucleus, a decreased overall cellular metabolism, and slower cell cycle progression resulting in a lower proliferation rate in cells exposed to surface topographies. We hypothesized that this reduction in proliferation rate effects their sensitivity to certain cancer drugs, which was confirmed by higher survival rate of hMSCs cultured on topographies exposed to paclitaxel. Thus, micro-topographies can be used as a model system to mimic the natural cell micro-environment, and be a powerful tool to optimize cell treatment in vitro.

Curvotaxis directs cell migration through cell-scale curvature landscapes.

Cells have evolved multiple mechanisms to apprehend and adapt finely to their environment. Here we report a new cellular ability, which we term "curvotaxis" that enables the cells to respond to cell-scale curvature variations, a ubiquitous trait of cellular biotopes. We develop ultra-smooth sinusoidal surfaces presenting modulations of curvature in all directions, and monitor cell behavior on these topographic landscapes. We show that adherent cells avoid convex regions during their migration and position themselves in concave valleys. Live imaging combined with functional analysis shows that curvotaxis relies on a dynamic interplay between the nucleus and the cytoskeleton-the nucleus acting as a mechanical sensor that leads the migrating cell toward concave curvatures. Further analyses show that substratum curvature affects focal adhesions organization and dynamics, nuclear shape, and gene expression. Altogether, this work identifies curvotaxis as a new cellular guiding mechanism and promotes cell-scale curvature as an essential physical cue.

Artificial import substrates reveal an omnivorous peroxisomal importomer.

The peroxisome matrix protein importomer has the remarkable ability to transport oligomeric protein substrates across the bilayer. However, the selectivity and relation between import and overall peroxisome homeostasis remain unclear. Here, we microinject artificial import substrates and employ quantitative microscopy to probe limits and capabilities of the importomer. DNA and polysaccharides are "piggyback" imported when noncovalently bound by a peroxisome targeting signal (PTS)-bearing protein. A dimerization domain that can be tuned to systematically vary the binding dissociation constant (Kd ) shows that a Kd in the millimolar range is sufficient to promote piggyback import. Microinjection of import substrate at high levels results in peroxisome growth and a proportional accumulation of peroxisome membrane proteins (PMPs). However, corresponding PMP mRNAs do not accumulate, suggesting that this response is posttranscriptionally regulated. Together, our data show that the importomer can tolerate diverse macromolecular species. Coupling between matrix import and membrane biogenesis suggests that matrix protein expression levels can be sufficient to regulate peroxisome size.

Influence of multiscale and curved structures on the migration of stem cells.

Understanding how topographical cues can control cell behavior is a major fundamental question which is of particular interest for implant design. Recent findings show that cell-scale curvature, as well as nanoscale topography, can affect different aspects of cell migration. However, the correlation between specific curvature radii and cell behavior, as well as the combinatorial effect of nanoscale topography and cell-scale curvature, has not yet been investigated. Herein, the authors employ a new femtosecond laser ablation method to generate multiscale topographical patterns directly on titanium surfaces. The process allows us to produce microgrooves of specific curvature imprinted with oriented nanotopographical features called Laser-Induced Periodic Surface Structures (LIPSS). The authors show that curved grooves stimulate the stem cell migration speed in comparison to flat or linear grooves. The fastest velocities are observed on 75 µm curvature radius, whereas cells migrating on 125 µm curvatures exhibit a lower speed similar to the ones migrating on straight lines. Double replicas of these grooves allow us to mask the LIPSS while keeping identical the cell-scale pattern, therefore permitting to uncouple the effect of nanoscale and microscale topographies. The authors found that the presence of nanoscale topographies improves the reading of microgrooves curvature by cells. Altogether, this work shows that the combination of specific curvatures together with nanopatterning can control the velocity of migrating stem cells and promote the use of femtosecond laser ablation in the context of surface implant design.

Role of the Nucleus as a Sensor of Cell Environment Topography.

The proper integration of biophysical cues from the cell vicinity is crucial for cells to maintain homeostasis, cooperate with other cells within the tissues, and properly fulfill their biological function. It is therefore crucial to fully understand how cells integrate these extracellular signals for tissue engineering and regenerative medicine. Topography has emerged as a prominent component of the cellular microenvironment that has pleiotropic effects on cell behavior. This progress report focuses on the recent advances in the understanding of the topography sensing mechanism with a special emphasis on the role of the nucleus. Here, recent techniques developed for monitoring the nuclear mechanics are reviewed and the impact of various topographies and their consequences on nuclear organization, gene regulation, and stem cell fate is summarized. The role of the cell nucleus as a sensor of cell-scale topography is further discussed.

Cellular Subcompartments through Cytoplasmic Streaming.

Cytoplasmic streaming occurs in diverse cell types, where it generally serves a transport function. Here, we examine streaming in multicellular fungal hyphae and identify an additional function wherein regimented streaming forms distinct cytoplasmic subcompartments. In the hypha, cytoplasm flows directionally from cell to cell through septal pores. Using live-cell imaging and computer simulations, we identify a flow pattern that produces vortices (eddies) on the upstream side of the septum. Nuclei can be immobilized in these microfluidic eddies, where they form multinucleate aggregates and accumulate foci of the HDA-2 histone deacetylase-associated factor, SPA-19. Pores experiencing flow degenerate in the absence of SPA-19, suggesting that eddy-trapped nuclei function to reinforce the septum. Together, our data show that eddies comprise a subcellular niche favoring nuclear differentiation and that subcompartments can be self-organized as a consequence of regimented cytoplasmic streaming.

Hydrophobic handoff for direct delivery of peroxisome tail-anchored proteins.

Tail-anchored (TA) proteins are inserted into membranes post-translationally through a C-terminal transmembrane domain (TMD). The PEX19 protein binds peroxisome TA proteins in the cytoplasm and delivers them to the membrane through the PEX3 receptor protein. An amphipathic segment in PEX19 promotes docking on PEX3. However, how this leads to substrate insertion is unknown. Here we reconstitute peroxisome TA protein biogenesis into two sequential steps of substrate TMD engagement and membrane insertion. We identify a series of previously uncharacterized amphipathic segments in PEX19 and identify one whose hydrophobicity is required for membrane insertion, but not TMD chaperone activity or PEX3 binding. A membrane-proximal hydrophobic surface of PEX3 promotes an unconventional form of membrane intercalation, and is also required for TMD insertion. Together, these data support a mechanism in which hydrophobic moieties in the TMD chaperone and its membrane-associated receptor act in a concerted manner to prompt TMD release and membrane insertion.

Multiple modes for gatekeeping at fungal cell-to-cell channels.

Cell-to-cell channels appear to be indispensable for successful multicellular organization and arose independently in animals, plants and fungi. Most of the fungi obtain nutrients from the environment by growing in an exploratory and invasive manner, and this ability depends on multicellular filaments known as hyphae. These cells grow by tip extension and can be divided into compartments by cell walls that typically retain a central pore that allows intercellular transport and cooperation. In the major clade of filamentous Ascomycota, integrity of this coenocytic organization is maintained by Woronin body organelles, which function as emergency patches of septal pores. In this issue of Molecular Microbiology, Bleichrodt and co-workers show that Woronin bodies can also form tight reversible associations with the pore and further link this to variation in levels of compartmental gene expression. These data define an additional modality of Woronin body-dependent gatekeeping. This commentary focuses on the implications of this work and the potential role of different modes of pore gating in controlling the growth and development of fungal tissues.