Levitation and controlled motion of matter in air have a wealth of potential applications ranging from materials processing to biochemistry and pharmaceuticals. We present a unique acoustophoretic ...concept for the contactless transport and handling of matter in air. Spatiotemporal modulation of the levitation acoustic field allows continuous planar transport and processing of multiple objects, from near-spherical (volume of 0.1–10 μL) to wire-like, without being limited by the acoustic wavelength. The independence of the handling principle from special material properties (magnetic, optical, or electrical) is illustrated with a wide palette of application experiments, such as contactless droplet coalescence and mixing, solid–liquid encapsulation, absorption, dissolution, and DNA transfection. More than a century after the pioneering work of Lord Rayleigh on acoustic radiation pressure, a path-breaking concept is proposed to harvest the significant benefits of acoustic levitation in air.
Abstract Understanding and accelerating the mechanisms of endothelial wound healing is of fundamental interest for biotechnology and of significant medical utility in repairing pathologic changes to ...the vasculature induced by invasive medical interventions. We report the fundamental mechanisms that determine the influence of substrate topography and flow on the efficiency of endothelial regeneration. We exposed endothelial monolayers, grown on topographically engineered substrates (gratings), to controlled levels of flow-induced shear stress. The wound healing dynamics were recorded and analyzed in various configurations, defined by the relative orientation of an inflicted wound, the topography and the flow direction. Under flow perpendicular to the wound, the speed of endothelial regeneration was significantly increased on substrates with gratings oriented in the direction of the flow when compared to flat substrates. This behavior is linked to the dynamic state of cell-to-cell adhesions in the monolayer. In particular, interactions with the substrate topography counteract Vascular Endothelial Cadherin phosphorylation induced by the flow and the wounding. This effect contributes to modulating the mechanical connection between migrating cells to an optimal level, increasing their coordination and resulting in coherent cell motility and preservation of the monolayer integrity, thus accelerating wound healing. We further demonstrate that the reduction of vascular endothelial cadherin phosphorylation, through specific inhibition of Src activity, enhances endothelial wound healing in flows over flat substrates.
IQGAP1 is a conserved modular protein overexpressed in cancer and involved in organizing actin and microtubules in motile processes such as adhesion, migration, and cytokinesis. A variety of proteins ...have been shown to interact with IQGAP1, including the small G proteins Rac1 and Cdc42, actin, calmodulin, β-catenin, the microtubule plus end-binding proteins CLIP170 (cytoplasmic linker protein) and adenomatous polyposis coli. However, the molecular mechanism by which IQGAP1 controls actin dynamics in cell motility is not understood. Quantitative co-localization analysis and down-regulation of IQGAP1 revealed that IQGAP1 controls the co-localization of N-WASP with the Arp2/3 complex in lamellipodia. Co-immunoprecipitation supports an in vivo link between IQGAP1 and N-WASP. Pull-down experiments and kinetic assays of branched actin polymerization with N-WASP and Arp2/3 complex demonstrated that the C-terminal half of IQGAP1 activates N-WASP by interacting with its BR-CRIB domain in a Cdc42-like manner, whereas the N-terminal half of IQGAP1 antagonizes this activation by association with a C-terminal region of IQGAP1. We propose that signal-induced relief of the autoinhibited fold of IQGAP1 allows activation of N-WASP to stimulate Arp2/3-dependent actin assembly.
Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) undergo specific assembly steps in Cajal bodies (CBs), nonmembrane-bound compartments within cell nuclei. An example is the U4/U6 ...di-snRNP, assembled from U4 and U6 monomers. These snRNPs can also assemble in the nucleoplasm when cells lack CBs. Here, we address the hypothesis that snRNP concentration in CBs facilitates assembly, by comparing the predicted rates of U4 and U6 snRNP association in nuclei with and without CBs. This was accomplished by a random walk-and-capture simulation applied to a three-dimensional model of the HeLa cell nucleus, derived from measurements of living cells. Results of the simulations indicated that snRNP capture is optimal when nuclei contain three to four CBs. Interestingly, this is the observed number of CBs in most cells. Microinjection experiments showed that U4 snRNA targeting to CBs was U6 snRNP independent and that snRNA concentration in CBs is approximately 20-fold higher than in nucleoplasm. Finally, combination of the simulation with calculated association rates predicted that the presence of CBs enhances U4 and U6 snRNP association by up to 11-fold, largely owing to this concentration difference. This provides a chemical foundation for the proposal that these and other cellular compartments promote molecular interactions, by increasing the local concentration of individual components.
Background information
The mitogenic pathway, composed of RAF kinases, mitogen‐activated protein kinase kinases (MEK) and extracellular signal‐regulated kinases (ERK), promotes cell proliferation and ...migration and is upregulated in many tumours. DiRas3 (ARHI, Noey2), a mainly GTP‐bound Ras‐like protein with an unusual N‐terminal extension, is predominantly lost in ovarian and breast cancers. Its re‐expression in these tissues impairs cell proliferation, autophagy, apoptosis and cell migration. Further, loss of DiRas3 correlates with an increase in growth factor‐induced ERK phosphorylation. Therefore, DIRAS3 proves to be a curious gene with remarkable tumour suppressing capabilities. However, how DiRas3 interferes with ERK phosphorylation, has remained unknown.
Results
We demonstrate that DiRas3 associates in vivo with C‐RAF and directly binds in vitro to C‐RAF, which is upstream of MEK and ERK. Direct binding of DiRas3 to C‐RAF is nucleotide independent, and DiRas3's N‐terminal extension alone is not sufficient for binding C‐RAF. DiRas3 expression inhibits the activating phosphorylations of MEK and ERK. Serum‐induced recruitment of DiRas3 to the plasma membrane depends mainly on its N‐terminal extension and less on its C‐terminus, bound nucleotide or the presence of Ras–GTP. Correspondingly, removal of the N‐terminal extension strongly decreases DiRas3's inhibition of MEK and ERK phosphorylations. Tyrosyl‐phosphatases do not contribute significantly to reduction of ERK‐phosphorylation byDiRas3. Consistently, downregulation of DiRas3 results in a small but significant and persistent increase in MEK and ERK phosphorylation, but does not increase phosphorylation of P38, AKT and c‐Jun NH2‐terminal kinase. Finally, downregulation of DiRas3 causes increased cell migration, through a mechanism that is MEK dependent.
Conclusions
These results support a model in which serum signals induce the recruitment of DiRas3 to the plasma membrane, where it is tethered via its N‐ and C‐termini. At the plasma membrane, DiRas3 interacts with C‐RAF to specifically suppress the activating phosphorylations on MEK and ERK, thus restricting migration of non‐cancer cells. This effect is relatively small, but it is also persistent, suggesting that it contributes to the maintenance of the non‐migratory phenotype of non‐cancerous tissues, in which DiRas3 is expressed.
Upon extracellular serum signals, the low expressed tumour suppressor DiRas3, which is a Ras‐related GTP‐binding protein that is often absent in ovarian and breast cancers, becomes tethered via its N‐terminal extension and its C‐terminus to the plasma membrane. It can directly interact with C‐RAF, dampens the activating MEK and ERK phosphorylations and thereby restricts cell migration in non‐cancerous cells. The effects are small but significant and persistent, pointing at DiRas3 as a potent long‐term inhibitor of metastases.
Background
Topographical modifications of the surface influence several cell functions and can be exploited to modulate cellular activities such as adhesion, migration and proliferation. These ...complex interactions are cell-type specific, therefore engineered substrates featuring patterns of two or more different topographies may be used to obtain the selective separation of different cell lineages. This process has the potential to enhance the performance of biomedical devices promoting, for example, the local coverage with functional tissues while demoting the onset of inflammatory reactions.
Findings & Conclusions
Here we present a computational tool, based on Monte Carlo simulation, which decouples the contribution of cell proliferation and migration and predicts the cell-separation performance of topographically engineered substrates. Additionally, we propose an optimization procedure to shape the topographically engineered areas of a substrate and obtain maximal cell separation.
The spliceosomal small nuclear RNAs (snRNAs) are distributed throughout the nucleoplasm and concentrated in nuclear inclusions termed Cajal bodies (CBs). A role for CBs in the metabolism of snRNPs ...has been proposed but is not well understood. The SART3/p110 protein interacts transiently with the U6 and U4/U6 snRNPs and promotes the reassembly of U4/U6 snRNPs after splicing in vitro. Here we report that SART3/p110 is enriched in CBs but not in gems or residual CBs lacking coilin. The U6 snRNP Sm-like (LSm) proteins, also involved in U4/U6 snRNP assembly, were localized to CBs as well. The levels of SART3/p110 and LSm proteins in CBs were reduced upon treatment with the transcription inhibitor α-amanitin, suggesting that CB localization reflects active processes dependent on transcription/splicing. The NH2-terminal HAT domain of SART3/p110 was necessary and sufficient for specific protein targeting to CBs. Overexpression of truncation mutants containing the HAT domain had dominant negative effects on U6 snRNP localization to CBs, indicating that endogenous SART3/p110 plays a role in targeting the U6 snRNP to CBs. We propose that U4 and U6 snRNPs accumulate in CBs for the purpose of assembly into U4/U6 snRNPs by SART3/p110.
This paper reports the development, modeling, and testing of an original microfluidic chip capable of generating both time-evolving and spatially varying gradients in standard Petri dishes. It ...consists of three sets of five independently controlled parallel channels, and its architecture allows the generation of complex gradient profiles that can be flexibly positioned and dynamically altered in an open cell-chamber environment. A detailed fabrication protocol for the production of these chips using multilayer soft lithography is reported. A comprehensive computational model is also presented based on COMSOL Multiphysics software that includes both diffusion and advection of the fluid as it exits the microchannels. The results of the simulation are successfully applied to model single-channel experiments. The chip is then tested in multi-channel mode, and its ability to produce complex spatially varied concentration profiles is demonstrated. The achievement of steady state of the gradient profile in less than 5 min also allows for the dynamic variation of the profile. Finally, we apply the present chip architecture to investigate the migration of mouse neutrophils in an Interleukin-8 gradient. We report quantitatively on cell migration driven by Interleukin-8 gradient and provide migration speed distribution.
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