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Smart biomaterials are increasingly being used to control stem cell fate in vitro by the recapitulation of the native niche microenvironment. By integrating experimental measurements with numerical models, we show that in mesenchymal stem cells grown inside a 3D synthetic niche both nuclear transport of a myogenic factor and the passive nuclear diffusion of a smaller inert protein are reduced. Our results also suggest that cell morphology modulates nuclear proteins import through a partition of the nuclear envelope surface, which is a thin but extremely permeable annular portion in cells cultured on 2D substrates. Therefore, our results support the hypothesis that in stem cell differentiation, the nuclear import of gene-regulating transcription factors is controlled by a strain-dependent nuclear envelope permeability, probably related to the reorganization of stretch-activated nuclear pore complexes.

Three-dimensional culture systems and suitable substrates topographies demonstrated to drive stem cell fate in vitro by mechanical conditioning. For example, the Nichoid 3D scaffold remodels stem cells and shapes nuclei, thus promoting stem cell expansion and stemness maintenance. However, the mechanisms involved in force transmission and in biochemical signaling at the basis of fate determination are not yet clear. Among the available investigation systems, confocal fluorescence microscopy using fluorescent dyes enables the observation of cell function and shape at the subcellular scale in vital and fixed conditions. Contrarily, nonlinear optical microscopy techniques, which exploit multi-photon processes, allow to study cell behavior in vital and unlabeled conditions. We apply confocal fluorescence microscopy, coherent anti-Stokes Raman scattering (CARS), and second harmonic generation (SHG) microscopy to characterize the phenotypic expression of mesenchymal stem cells (MSCs) towards adipogenic and chondrogenic differentiation inside Nichoid scaffolds, in terms of nuclear morphology and specific phenotypic products, by comparing these techniques. We demonstrate that the Nichoid maintains a rounded nuclei during expansion and differentiation, promoting MSCs adipogenic differentiation while inhibiting chondrogenesis. We show that CARS and SHG techniques are suitable for specific estimation of the lipid and collagenous content, thus overcoming the limitations of using unspecific fluorescent probes.

Despite intracellular molecular dynamics being fundamental to understand pathological, biomechanical or biochemical events, several processes are still not clear because of the difficulty of monitoring and measuring these phenomena. To engineer an effective fluorescent tool useful to improve protein intracellular tracking studies, we fused a supernegative green fluorescent protein, (−30)GFP, to a myogenic transcription factor, MyoD. The (−30)GFP-MyoD was able to pass the plasma membrane when complexed with cationic lipids. Fluorescence confocal microscopy showed the protein delivery in just 3 hours with high levels of protein transduction efficiency. Confocal acquisitions also confirmed the maintenance of the MyoD nuclear localization. To examine how the supernegative GFP influenced MyoD activity, we did gene expression analyses, which showed an inhibitory effect of (−30)GFP on transcription factor function. This negative effect was possibly due to a charge-driven interference mechanism, as suggested by further investigations by molecular dynamics simulations. Summarizing these results, despite the functional limitations related to the charge structural characteristics that specifically affected MyoD function, we found (−30)GFP is a suitable fluorescent label for improving protein intracellular tracking studies, such as nucleocytoplasmic transport in mechanotransduction.

Cell fate is correlated to mechanotransduction, in which forces transmitted by the cytoskeleton filaments alter the nuclear shape, affecting transcription factor import/export, cells transcription activity and chromatin distribution. There is in fact evidence that stem cells cultured in 3D environments mimicking the native niche are able to maintain their stemness or modulate their cellular function. However, the molecular and biophysical mechanisms underlying cellular mechanosensing are still largely unclear. The propagation of mechanical stimuli via a direct pathway from cell membrane integrins to SUN proteins residing in the nuclear envelop has been demonstrated, but we suggest that the cells' fate is mainly affected by the force distribution at the nuclear envelope level, where the SUN protein transmits the stimuli via its mechanical connection to several cell structures such as chromatin, lamina and the nuclear pore complex (NPC). In this review, we analyze the NPC structure and organization, which have not as yet been fully investigated, and its plausible involvement in cell fate. NPC is a multiprotein complex that spans the nuclear envelope, and is involved in several key cellular processes such as bidirectional nucleocytoplasmic exchange, cell cycle regulation, kinetochore organization, and regulation of gene expression. As several connections between the NPC and the nuclear envelope, chromatin and other transmembrane proteins have been identified, it is reasonable to suppose that nuclear deformations can alter the NPC structure. We provide evidence that the transmission of mechanical forces may significantly affects the basket conformation via the Nup153-SUN1 connection, both altering the passage of molecules through it and influencing the state of chromatin packing. Finally, we review the known correlations between a pathological NPC structure and diseases such as cancer, autoimmune disease, aging and laminopathies.

Mesenchymal stem cells (MSCs) have immune-modulatory and tissue-regenerative properties that make them a suitable and promising tool for cell-based therapy application. Since the bio-chemo-mechanical environment influences MSC fate and behavior, the understanding of the mechanosensors involved in the transduction of mechanical inputs into chemical signals could be pivotal. In this context, the nuclear pore complex is a molecular machinery that is believed to have a key role in force transmission and in nucleocytoplasmic shuttling regulation. To fully understand the nuclear pore complex role and the nucleocytoplasmic transport dynamics, recent advancements in fluorescence microscopy provided the possibility to study passive and facilitated nuclear transports also in mechanically stimulated cell culture conditions. Here, we review the current available methods for the investigation of nucleocytoplasmic shuttling, including photo-perturbation-based approaches, fluorescence correlation spectroscopy, and single-particle tracking techniques. For each method, we analyze the advantages, disadvantages, and technical limitations. Finally, we summarize the recent knowledge on mechanical regulation of nucleocytoplasmic translocation in MSC, the relevant progresses made so far, and the future perspectives in the field.

Graphical abstractDisplay Omitted We engineered micropatterned PDMS membranes to guide Schwann cell migration.Migration occurs along PDMS microgratings for single cells and upon wound healing.We identified topographies that might be exploited for nerve regeneration. We engineered anisotropic microstructured membranes for nerve repair applications by exploiting electron beam lithography on silicon substrates and soft lithography techniques. Our substrates were patterned with polydimethylsiloxane (PDMS) gratings (alternating lines of grooves and ridges) of varying ridge/groove width and depth. These gratings were used as scaffolds to study rat Schwann cell contact interaction for cell migration. We observed that cell motion was affected by the presence of the grating and by its periodicity, while cells on flat substrates showed random spatial migration. Our results allow the identification of specific topographical elements that may be exploited for the production of new devices for enhancing nerve regeneration by promoting Schwann cell invasion, proliferation, and terminal differentiation.

Atomic force microscopy (AFM) proved to be able to obtain high-resolution three-dimensional images of single-membrane proteins, isolated, crystallized, or included in reconstructed model membranes. The extension of this technique to native systems, such as the protein immersed in a cell membrane, needs a careful manipulation of the biological sample to meet the experimental constraints for high-resolution AFM imaging. In this article, a general protocol for sample preparation is presented, based on the mechanical stretch of the cell membrane. The effectiveness for AFM imaging has been tested on the basis of an integrated optical and AFM approach and the proposed method has been applied to cells expressing cystic fibrosis transmembrane conductance regulator, a channel involved in cystic fibrosis, showing the possibility to identify and analyze single proteins in the plasma membrane. Microsc. Res. Tech. 76:723–732, 2013. © 2013 Wiley Periodicals, Inc.

The arsenal of fluorescent probes tailored to functional imaging of cells is rapidly growing and benefits from recent developments in imaging strategies. Here, we present a new molecular rotor, which displays strong absorption in the green region of the spectrum, very little solvatochromism, and strong emission sensitivity to local viscosity. The emission increase is paralleled by an increase in emission lifetime. Owing to its concentration-independent nature, fluorescence lifetime is particularly suitable to image environmental properties, such as viscosity, at the intracellular level. Accordingly, we demonstrate that intracellular viscosity measurements can be efficiently carried out by lifetime imaging with our probe and phasor analysis, an efficient method for measuring lifetime-related properties (e.g., bionalyte concentration or local physicochemical features) in living cells. Notably, we show that it is possible to monitor the partition of our probe into different intracellular regions/organelles and to follow mitochondrial de-energization upon oxidative stress.

By combining spectroscopic measurements under high pressure with molecular dynamics simulations we investigate how sub-angstrom structural perturbations are able to tune protein function. We monitored the variations in fluorescence output of two Green Fluorescent Protein mutants (termed Mut2 and Mut2Y, the latter containing the key T203Y mutation) subjected to pressures up to 600 MPa, at various temperatures in the 280-320 K range. By performing ∼100ns molecular dynamics simulations of the protein structures at various pressures, we evidenced subtle changes in conformation and dynamics around the light-absorbing chromophore. Such changes explain the measured spectral tuning, especially in the case of the sizable 120 cm-1 red-shift observed for pressurized Mut2Y, but absent in Mut2. Previous work by Barstow et al [1] on pressure effects on GFP also involved a T203Y mutant. Using cryocooling X-ray crystallography at high pressure they linked the observed, pressure-induced, fluorescence blue shift at low temperature (77 K) to key changes in relative conformation of the chromophore and Y203 phenol ring. At room temperature, however, they observe a red shift at high pressure, analogous to the one in Mut2Y. Our investigation of structural variations in compressed Mut2Y also explains their result, bridging the gap between low-temperature and room-temperature high-pressure effects.[1] Barstow, B., Ando, N., Kim, C. U., & Gruner, S. M. (2008). Alteration of citrine structure by hydrostatic pressure explains the accompanying spectral shift. Proc Natl Acad Sci USA, 105(36), 13362-13366.

A polysaccharide-flavonoid conjugate was developend and proposed for the treatment of pancreatic ductal adenocarcinoma (PDAC).The conjugate was synthesized by free radical grafting reaction between catechin and dextran. The chemical characterization of the conjugate was obtained by UV-Vis, 1H-NMR, FT-IR and GPC analyses, while the functionalization degree was determined by the Folin-Ciocalteu assay. The biological activity of the catechin-dextran conjugate was tested on two different cell lines derived from human pancreatic cancer (MIA PaCa-2 and PL45 cells), and the toxicity towards human pancreatic nestin-expressing cells evaluated.Both the cancer cell lines are killed when exposed to the conjugate, and undergo apoptosis after the incubation with catechin-dextran which resulted more effective in killing pancreatic tumor cells compared to the catechin alone. Moreover, our experimental data indicate that the conjugate was less cytotoxic to human pancreatic nestin-expressing cells which are considered a good model of non-neoplastic pancreatic cells.The suitability of newly synthesized Dextran-Catechin conjugate in the treatment of PDAC was proved confirming the high potential application of the proposed macromolecula system in the cancer therapy.

In this study, we present a microdevice coated with titanium dioxide for cells and particles separation and handling. The microsystem consists of a pair of planar interdigitated gold micro-electrode arrays on a quartz substrate able to generate a traveling electric completed with a microfabricated three-dimensional glass structure for cell confinement. Dielectrophoretic forces were exploited for both vertical and lateral cell motions. In order to provide a biocompatible passivation layer to the electrodes a highly biocompatible nanostructured titanium dioxide film was deposited by supersonic cluster beam deposition (SCBD) on the electrode array. The dielectrophoretic effects of the chip were initially tested using polystyrene beads. To test the biocompatibility and capability of dielectrophoretic cell movement of the device, four cell lines (NIH3T3, SH-SY5Y, MDCK, and PC12) were used. Separation of beads from SH-SY5Y cells was also obtained.

The arsenal of fluorescent probes tailored to functional imaging of cells is rapidly growing and benefits from recent developments in imaging strategies. We here present two new molecular rotors characterized by a push-pull hyperconjugated structure and which absorb in the blue-green region of the spectrum. These probes display significant emission and lifetime increase by increasing local microviscosity. Owing to its concentration-independent nature, fluorescence lifetime is particularly suitable to report on environmental properties, such as viscosity, in the cellular context, i.e. where the fluorescent probe concentration cannot be controlled. Remarkably, we demonstrate that our probes allow for efficient intracellular viscosity measurements by means of the phasor approach to fluorescence lifetime imaging, a fit-free method that overcome the main drawbacks of conventional lifetime imaging (1). More specifically, we show that one probe can be used to monitor membrane organization (i.e. the fractions of liquid ordered and disordered phases) and membrane viscosity, whereas the other reports on the local viscosity in plasma membrane, lysosomes, and mitochondria (2). Notably, measurements were carried out in both physiological and non-physiological conditions, in view of correlating dysfunctional states of the cell with viscosity changes in intracellular organelles.1. Digman, M.A., Caiolfa, V.R., Zamai, M., and Gratton, E. (2008) Biophys J 94, L14-16.2. Battisti, A., Panettieri, S., Abbandonato, G., Jacchetti, E., Cardarelli, F., Signore, G., Beltram, F., and Bizzarri, R. (2013) Anal. Bioanal. Chem. 405, 6223-6233.

Understanding how the substrate topography acts on human bone marrow-derived mesenchymal stromal cells (MSCs) can help the rational design of scaffolds for improving bone regeneration protocols. MSCs are highly sensitive to the extracellular physical properties and can be successfully manipulated by simple contact interaction with supporting substrates. To this end, some polymeric materials were introduced, but polyethylene terephthalate (PET), a thermoplastic polymer approved by the US Food and Drug Administration for clinical use and very attractive in terms of biocompatibil- ity and mechanical properties, has not been tested yet in terms of cell mechanotransduction. Here, we propose PET nanogratings (alternating lines of submicron ridges and grooves) as scaffolds for stimulating mechanotransduction mechanisms. Low-temperature hot embossing is exploited as fabrication method, and standard oxygen plasma activation as functionalization to improve cell adhesion and spreading. We show that the substrate directionality stimulus is opti- mally delivered to the MSCs, which in turn elongate and align to the nanograting lines. Finally, we verify that this polari- zation occurs also at level of cytoskeleton fibers and, though to a lesser extent, of nuclei.

Inhalation of quartz induces silicosis, a lung disease where alveolar macrophages release inflammatory mediators, including prostaglandin-E2 (PGE2) and tumor necrosis factor ? (TNF-?). Here we report the pivotal role of abscisic acid (ABA), a recently discovered human inflammatory hormone, in silica-induced activation of murine RAW264.7 macrophages and of rat alveolar macrophages (AMs). Stimulation of both RAW264.7 cells and AMs with quartz induced a significant increase of ABA release (5- and 10-fold, respectively), compared to untreated cells. In RAW264.7 cells, autocrine ABA released after quartz stimulation sequentially activates the plasma membrane receptor LANCL2 and NADPH oxidase, generating a Ca2+ influx resulting in NF? B nuclear translocation and PGE2 and TNF-? release (3-, 2-, and 3.5-fold increase, respectively, compared to control, unstimulated cells). Quartz-stimulated RAW264.7 cells silenced for LANCL2 or preincubated with a monoclonal antibody against ABA show an almost complete inhibition of NF? B nuclear translocation and PGE2 and TNF-? release compared to controls electroporated with a scramble oligonucleotide or preincubated with an unrelated antibody. AMs showed similar early and late ABA-induced responses as RAW264.7 cells. These findings identify ABA and LANCL2 as key mediators in quartz-induced inflammation, providing possible new targets for antisilicotic therapy.--Magnone, M., Sturla, L., Jacchetti, E., Scarfì, S., Bruzzone, S., Usai, C., Guida, L., Salis, A., Damonte, G., De Flora, A., Zocchi, E. Autocrine abscisic acid plays a key role in quartz-induced macrophage activation.

J. Neurochem. (2010) 113, 489–501. Abstract The effect of GABA on glutamate release from astrocytes has been studied in healthy mice and in a murine transgenic model of amyotrophic lateral sclerosis (ALS), using mouse spinal cord gliosomes labeled with [3H]d-aspartate ([3H]d-ASP). GABA concentration-dependently evoked the release of [3H]d-ASP. The effect of GABA was not mimicked by GABAA or GABAB receptor agonists or counteracted by antagonists, excluding receptor involvement. However, it was prevented by the GABA transport inhibitor N-(4,4-phenyl-3-butenyl)-nipecotic acid (SKF 89976A), suggesting participation of GABA transporters type 1 (GAT1) placed on glutamate-releasing astrocyte-derived gliosomes. Accordingly, GAT1 co-expressed with glutamate–aspartate transporter (GLAST) and glutamate transporter type 1 (GLT1) in the majority of glial particles. [3H]d-aspartate release was Ca2+-independent and not blocked by the glutamate uptake inhibitor dl-threo-b-benzyloxyaspartic acid (dl-TBOA); instead, it was abrogated by the anion channel blockers niflumic acid and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). The GAT1-mediated release of [3H]d-ASP was significantly enhanced in spinal cord gliosomes from the mouse model of ALS. This excessive [3H]d-ASP release was very precocious, largely preceding the onset of the disease symptoms. These data indicate that GAT1, GLAST and GLT1 coexist on the same gliosome in mouse spinal cord and that activation of GAT1 transporters elicits glutamate release by anion channel opening. This phenomenon might have pathological relevance, because [3H]d-ASP release is enhanced in experimental ALS.

Abscisic acid (ABA) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. Recently, ABA was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells, and by human and murine pancreatic beta cells. ABA autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin-sensitive receptor/G-protein complex, cAMP, CD38-produced cADP-ribose and intracellular calcium. Here we show that the lanthionine synthetase C-like protein LANCL2 is required for ABA binding on the membrane of human granulocytes and that LANCL2 is necessary for transduction of the ABA signal into the cell-specific functional responses in granulocytes and in rat insulinoma cells. Co-expression of LANCL2 and CD38 in the human HeLa cell line reproduces the ABA-signaling pathway. Results obtained with granulocytes and CD38(+)/LANCL2(+) HeLa transfected with a chimeric G-protein (G alpha(q/i)) suggest that the pertussis toxin-sensitive G-protein coupled to LANCL2 is a G(i). Identification of LANCL2 as a critical component of the ABA-sensing protein complex will enable the screening of synthetic ABA antagonists as prospective new anti-inflammatory and anti-diabetic agents.

Background Poly(amidoamine)s (PAAs) are synthetic polymers endowed with many biologically interesting properties, being highly biocompatible, non toxic and biodegradable. Hydrogels based on PAAs can be easily modified during the synthesis by the introduction of functional co-monomers. Aim of this work is the development and testing of novel amphoteric nanosized poly(amidoamine) hydrogel film incorporating 4-aminobutylguanidine (agmatine) moieties to create RGD-mimicking repeating units for promoting cell adhesion. Results A systematic comparative study of the response of an epithelial cell line was performed on hydrogels with agmatine and on non-functionalized amphoteric poly(amidoamine) hydrogels and tissue culture plastic substrates. The cell adhesion on the agmatine containing substrates was comparable to that on plastic substrates and significantly enhanced with respect to the non-functionalized controls. Interestingly, spreading and proliferation on the functionalized supports are slower than on plastic exhibiting the possibility of an easier control of the cell growth kinetics. In order to favor the handling of the samples, a procedure for the production of bi-layered constructs was also developed by means the deposition via spin coating of a thin layer of hydrogel on a pre-treated cover slip. Conclusion The obtained results reveal that PAAs hydrogels can be profitably functionalized and, in general, undergo physical and chemical modifications to meet specific requirements. In particular the incorporation of agmatine warrants good potential in the field of cell culturing and the development of supported functionalized hydrogels on cover glass are very promising substrates for applications in cell screening devices.