Microtubules under mechanical pressure can breach dense actin networks.

The crosstalk between the actin network and microtubules is essential for cell polarity. It orchestrates microtubule organization within the cell, driven by the asymmetry of actin architecture along the cell periphery. The physical intertwining of these networks regulates spatial organization and force distribution in the microtubule network. Although their biochemical interactions are becoming clearer, the mechanical aspects remain less understood. To explore this mechanical interplay, we developed an in vitro reconstitution assay to investigate how dynamic microtubules interact with various actin filament structures. Our findings revealed that microtubules can align and move along linear actin filament bundles through polymerization force. However, they are unable to pass through when encountering dense branched actin meshworks, similar to those present in the lamellipodium along the periphery of the cell. Interestingly, immobilizing microtubules through crosslinking with actin or other means allow the buildup of pressure, enabling them to breach these dense actin barriers. This mechanism offers insights into microtubule progression towards the cell periphery, with them overcoming obstacles within the denser parts of the actin network and ultimately contributing to cell polarity establishment.

Shp2 contributes to the regulation of nuclear shape and cellular viscoelasticity in response to substrate spatial cues.

Cell polarization can be guided by substrate topology through space constraints and adhesion induction, which are part of cellular mechanosensing pathways. Here, we demonstrated that protein tyrosine phosphatase Shp2 plays a crucial role in mediating the response of cells to substrate spatial cues. When compared to cells spreading on surfaces coated uniformly with fibronectin (FN), cells attached to 10 µm-width FN-strip micropattern (MP), which provides spatial cues for uniaxial spreading, exhibited elongated focal adhesions (FAs) and aligned stress fibers in the direction of the MP. As a result of uniaxial cell spreading, nuclei became elongated, dependent on ROCK-mediated actomyosin contractility. Additionally, intracellular viscoelasticity also increased. Shp2-deficient cells did not display elongated FAs mediated by MP, well-aligned stress fibers, or changes in nuclear shape and intracellular viscoelasticity. Overall, our data suggest that Shp2 is involved in regulating FAs and the actin cytoskeleton to modulate nuclear shape and intracellular physical properties in response to substrate spatial cues.

Micropattern Fabricated by Acropetal Migration Controlled through Sequential Photo and Thermal Polymerization.

Bottom-up patterning technology plays a significant role in both nature and synthetic materials, owing to its inherent advantages such as ease of implementation, spontaneity, and noncontact attributes, etc. However, constrained by the uncontrollability of molecular movement, energy interaction, and stress, obtained micropatterns tend to exhibit an inevitable arched outline, resulting in the limitation of applicability. Herein, inspired by auxin's action mode in apical dominance, a versatile strategy is proposed for fabricating precision self-organizing micropatterns with impressive height based on polymerization-induced acropetal migration. The copolymer containing fluorocarbon chains (low surface energy) and tertiary amine (coinitiator) is designed to self-assemble on the surface of the photo-curing system. The selective exposure under a photomask establishes a photocuring boundary and the radicals would be generated on the surface, which is pivotal in generating a vertical concentration difference of monomer. Subsequent heating treatment activates the material continuously transfers from the unexposed area to the exposed area and is accompanied by the obviously vertical upward mass transfer, resulting in the manufacture of a rectilinear profile micropattern. This strategy significantly broadens the applicability of self-organizing patterns, offering the potential to mitigate the complexity and time-consuming limitations associated with top-down methods.

Micropatterned Polymer Nanoarrays with Distinct Superwettability for a Highly Efficient Sweat Collection and Sensing Patch.

Wearable sweat sensor offers a promising means for noninvasive real-time health monitoring, but the efficient collection and accurate analysis of sweat remains challenging. One of the obstacles is to precisely modulate the surface wettability of the microfluidics to achieve efficient sweat collection. Here a facile initiated chemical vapor deposition (iCVD) method is presented to grow and pattern polymer nanocone arrays with distinct superwettability on polydimethylsiloxane microfluidics, which facilitate highly efficient sweat transportation and collection. The nanoarray is synthesized by manipulating monomer supersaturation during iCVD to induce controlled nucleation and preferential vertical growth of fluorinated polymer. Subsequent selective vapor deposition of a conformal hydrogel nanolayer results in superhydrophilic nanoarray floor and walls within the microchannel that provide a large capillary force and a superhydrophobic ceiling that drastically reduces flow friction, enabling rapid sweat transport along varied flow directions. A carbon/hydrogel/enzyme nanocomposite electrode is then fabricated by sequential deposition of highly porous carbon nanoparticles and hydrogel nanocoating to achieve sensitive and stable sweat detection. Further encapsulation of the assembled sweatsensing patch with superhydrophobic nanoarray imparts self-cleaning and water-proof capability. Finally, the sweat sensing patch demonstrates selective and sensitive glucose and lactate detection during the on-body test.

Deep learning for quantifying spatial patterning and formation process of early differentiated human-induced pluripotent stem cells with micropattern images.

Micropatterning is reliable method for quantifying pluripotency of human-induced pluripotent stem cells (hiPSCs) that differentiate to form a spatial pattern of sorted, ordered and nonoverlapped three germ layers on the micropattern. In this study, we propose a deep learning method to quantify spatial patterning of the germ layers in the early differentiation stage of hiPSCs using micropattern images. We propose decoding and encoding U-net structures learning labelled Hoechst (DNA-stained) hiPSC regions with corresponding Hoechst and bright-field micropattern images to segment hiPSCs on Hoechst or bright-field images. We also propose a U-net structure to extract extraembryonic regions on a micropattern, and an algorithm to compares intensities of the fluorescence images staining respective germ-layer cells and extract their regions. The proposed method thus can quantify the pluripotency of a hiPSC line with spatial patterning including cell numbers, areas and distributions of germ-layer and extraembryonic cells on a micropattern, and reveal the formation process of hiPSCs and germ layers in the early differentiation stage by segmenting live-cell bright-field images. In our assay, the cell-number accuracy achieved 86% and 85%, and the cell region accuracy 89% and 81% for segmenting Hoechst and bright-field micropattern images, respectively. Applications to micropattern images of multiple hiPSC lines, micropattern sizes, groups of markers, living and fixed cells show the proposed method can be expected to be a useful protocol and tool to quantify pluripotency of a new hiPSC line before providing it to the scientific community.

On the adhesion-velocity relation and length adaptation of motile cells on stepped fibronectin lanes.

The biphasic adhesion-velocity relation is a universal observation in mesenchymal cell motility. It has been explained by adhesion-promoted forces pushing the front and resisting motion at the rear. Yet, there is little quantitative understanding of how these forces control cell velocity. We study motion of MDA-MB-231 cells on microlanes with fields of alternating Fibronectin densities to address this topic and derive a mathematical model from the leading-edge force balance and the force-dependent polymerization rate. It reproduces quantitatively our measured adhesion-velocity relation and results with keratocytes, PtK1 cells, and CHO cells. Our results confirm that the force pushing the leading-edge membrane drives lamellipodial retrograde flow. Forces resisting motion originate along the whole cell length. All motion-related forces are controlled by adhesion and velocity, which allows motion, even with higher Fibronectin density at the rear than at the front. We find the pathway from Fibronectin density to adhesion structures to involve strong positive feedbacks. Suppressing myosin activity reduces the positive feedback. At transitions between different Fibronectin densities, steady motion is perturbed and leads to changes of cell length and front and rear velocity. Cells exhibit an intrinsic length set by adhesion strength, which, together with the length dynamics, suggests a spring-like front-rear interaction force. We provide a quantitative mechanistic picture of the adhesion-velocity relation and cell response to adhesion changes integrating force-dependent polymerization, retrograde flow, positive feedback from integrin to adhesion structures, and spring-like front-rear interaction.

Performance Studies of Aluminium-Based Gas Electron Multiplier Detector.

In this paper, we report on a systematic study of a soft X-ray Gas Electron Multiplier (GEM) detector built with aluminium-clad kapton GEM foils. The primary objective of this research is to comprehend the performance of this type of detector when irradiated with soft energy photons. The results are analysed and discussed with a particular focus on the long-term detector stability, as well as its gas gain and energy resolution uniformity across the detector area. Presented results lead us to the conclusion that the aluminium based GEM detector is a promising device to suppress the X-ray Fluorescence (XRF) background, simultaneously providing very good stability during long-term measurement campaigns.

Advances in Regulating Cellular Behavior Using Micropatterns.

Micropatterns, characterized as distinct physical microstructures or chemical adhesion matrices on substance surfaces, have emerged as a powerful tool for manipulating cellular activity. By creating specific extracellular matrix microenvironments, micropatterns can influence various cell behaviors, including orientation, proliferation, migration, and differentiation. This review provides a comprehensive overview of the latest advancements in the use of micropatterns for cell behavior regulation. It discusses the influence of micropattern morphology and coating on cell behavior and the underlying mechanisms. It also highlights future research directions in this field, aiming to inspire new investigations in materials medicine, regenerative medicine, and tissue engineering. The review underscores the potential of micropatterns as a novel approach for controlling cell behavior, which could pave the way for breakthroughs in various biomedical applications.

Miro proteins coordinate microtubule- and actin-dependent mitochondrial transport and distribution.

In the current model of mitochondrial trafficking, Miro1 and Miro2 Rho-GTPases regulate mitochondrial transport along microtubules by linking mitochondria to kinesin and dynein motors. By generating Miro1/2 double-knockout mouse embryos and single- and double-knockout embryonic fibroblasts, we demonstrate the essential and non-redundant roles of Miro proteins for embryonic development and subcellular mitochondrial distribution. Unexpectedly, the TRAK1 and TRAK2 motor protein adaptors can still localise to the outer mitochondrial membrane to drive anterograde mitochondrial motility in Miro1/2 double-knockout cells. In contrast, we show that TRAK2-mediated retrograde mitochondrial transport is Miro1-dependent. Interestingly, we find that Miro is critical for recruiting and stabilising the mitochondrial myosin Myo19 on the mitochondria for coupling mitochondria to the actin cytoskeleton. Moreover, Miro depletion during PINK1/Parkin-dependent mitophagy can also drive a loss of mitochondrial Myo19 upon mitochondrial damage. Finally, aberrant positioning of mitochondria in Miro1/2 double-knockout cells leads to disruption of correct mitochondrial segregation during mitosis. Thus, Miro proteins can fine-tune actin- and tubulin-dependent mitochondrial motility and positioning, to regulate key cellular functions such as cell proliferation.

Single cell morphological metrics and cytoskeletal alignment regulate VCAM-1 protein expression.

In the initial stages of atherosclerosis, vascular adhesion molecule-1 (VCAM-1) is a surface protein that mediates leukocyte adhesion to the endothelium's luminal surface. VCAM-1 expression is upregulated on endothelial cells (ECs) under pro-inflammatory conditions and is known to be modulated by fluid shear stress (FSS). High, pulsatile FSS induces endothelial elongation and cytoskeletal alignment and downregulates pro-inflammatory induced VCAM-1 expression, which is associated with an athero-protective EC phenotype. In contrast, athero-prone ECs under low, oscillatory FSS fail to elongate and maintain a cobblestone morphology with random cytoskeletal alignment, while VCAM-1 expression is upregulated. Whether EC shape and cytoskeletal alignment play a role in the regulation of VCAM-1 protein expression independent of FSS has not been previously determined. The goal of this study was to determine the effect of EC morphology, specifically cell elongation and alignment, and cytoskeletal alignment on VCAM-1 protein expression using topographical micropatterning of an endothelial monolayer and single cell image analysis techniques. Elongated ECs with an aligned cytoskeleton significantly downregulated VCAM-1 protein expression in the absence of FSS compared to planar controls. In addition, linear correlations between morphological metrics and protein expression showed that actin alignment had a significantly stronger effect on VCAM-1 expression than cell elongation. Functionally, monocytic U937 cells statically adhered less on micropatterns compared to planar substrates, in a VCAM-1 dependent manner. Therefore, endothelial cellular elongation and alignment as well as cytoskeletal alignment regulate VCAM-1 protein expression and immunogenic functions to produce a less inflammatory phenotype in the absence of hemodynamic effects.

Application of Factorisation Methods to Analysis of Elemental Distribution Maps Acquired with a Full-Field XRF Imaging Spectrometer.

The goal of the work was to investigate the possible application of factor analysis methods for processing X-ray Fluorescence (XRF) data acquired with a full-field XRF spectrometer employing a position-sensitive and energy-dispersive Gas Electron Multiplier (GEM) detector, which provides only limited energy resolution at a level of 18% Full Width at Half Maximum (FWHM) at 5.9 keV. In this article, we present the design and performance of the full-field imaging spectrometer and the results of case studies performed using the developed instrument. The XRF imaging data collected for two historical paintings are presented along with the procedures applied to data calibration and analysis. The maps of elemental distributions were built using three different analysis methods: Region of Interest (ROI), Non-Negative Matrix Factorisation (NMF), and Principal Component Analysis (PCA). The results obtained for these paintings show that the factor analysis methods NMF and PCA provide significant enhancement of selectivity of the elemental analysis in case of limited energy resolution of the spectrometer.

Chemical Vapor Deposition of Ionic Liquids for the Fabrication of Ionogel Films and Patterns.

Film deposition and high-resolution patterning of ionic liquids (ILs) remain a challenge, despite a broad range of applications that would benefit from this type of processing. Here, we demonstrate for the first time the chemical vapor deposition (CVD) of ILs. The IL-CVD method is based on the formation of a non-volatile IL through the reaction of two vaporized precursors. Ionogel micropatterns can be easily obtained via the combination of IL-CVD and standard photolithography, and the resulting microdrop arrays can be used as microreactors. The IL-CVD approach will facilitate leveraging the properties of ILs in a range of applications and microfabricated devices.

Investigation of Copper-Less Gas Electron Multiplier Detectors Responses to Soft X-rays.

In this paper, we report on the systematic study of different variants of X-ray detectors based on GEM technology using modified GEM foils with greatly reduced amount of copper. The main goal of this study was understanding the performance of such detectors applied in X-Ray Fluorescence (XRF) elemental analysis. Reduction of the amount of copper in the detector structure is crucial for suppression of XRF background from copper, but one has to ensure that key detector parameters are not affected by such modification. The tested detector variants include detectors with different types of copper-less GEM foils, which have been manufactured starting from standard copper-clad foils and removing partially the copper layer in additional post-processing steps. The results are analyzed and discussed with a particular focus on the energy resolution, uniformity of gas gain and energy resolution across the detector area, and on the long-term stability of the gas gain. Long-term stability tests performed for selected detectors do not indicate for any accelerated aging of the copper-less detectors compared to standard detectors using copper-clad GEM foils. The presented results lead us to conclude that the copper-less GEM detectors are promising devices to suppress the XRF background.

One-dimensional parallax-free position-sensitive detector for diffraction measurements based on a home-made thin THGEM.

A large parallax-free gas diffraction meter based on a thinner-THGEM (thick gaseous electron multiplier) has been developed at the Beijing Synchrotron Radiation Facility (BSRF). A thinner-THGEM of thickness 200 µm is adopted, which can be shaped into a curve to eliminate parallax-error effects. The detector is designed to have a 48° open angle positioned 20 cm from the powder samples. A front-end electronics board with 128 channels direct-current mode was adapted for the 8 keV BSRF beamline with 0.2 ns/100 ns stable duty cycle. Two powder samples, TiO2 and SnO2, were tested separately. The measured spectra with an angular resolution of 0.148 ± 0.081° are consistent with the data from the powder diffraction file. Combining the gas gain of the thinner-THGEM with the electronic circuit dynamic range, a very broad dynamic range of about 107 could be obtained.

Micropatterned substrates with physiological stiffness promote cell maturation and Pompe disease phenotype in human induced pluripotent stem cell-derived skeletal myocytes.

Recent advances in bioengineering have enabled cell culture systems that more closely mimic the native cellular environment. Here, we demonstrated that human induced pluripotent stem cell (iPSC)-derived myogenic progenitors formed highly-aligned myotubes and contracted when seeded on two-dimensional micropatterned platforms. The differentiated cells showed clear nuclear alignment and formed elongated myotubes dependent on the width of the micropatterned lanes. Topographical cues from micropatterning and physiological substrate stiffness improved the formation of well-aligned and multinucleated myotubes similar to myofibers. These aligned myotubes exhibited spontaneous contractions specifically along the long axis of the pattern. Notably, the micropatterned platforms developed bundle-like myotubes using patient-derived iPSCs with a background of Pompe disease (glycogen storage disease type II) and even enhanced the disease phenotype as shown through the specific pathology of abnormal lysosome accumulations. A highly-aligned formation of matured myotubes holds great potential in further understanding the process of human muscle development, as well as advancing in vitro pharmacological studies for skeletal muscle diseases.

Coding cell micropatterns through peptide inkjet printing for arbitrary biomineralized architectures.

Well-designed micropatterns present in native tissues and organs involve changes in extracellular matrix compositions, cell types and mechanical properties to reflect complex biological functions. However, the design and fabrication of these micropatterns in vitro to meet task-specific biomedical applications remains a challenge. A de novo design strategy to code and synthesize functional micropatterns is presented to engineer cell alignment through the integration of aqueous-peptide inkjet printing and site-specific biomineralization. The inkjet printing provides direct writing of macroscopic biosilica selective peptide-R5 patterns with micrometer-scale resolution on the surface of a biopolymer (silk) hydrogel. This is combined with in situ biomineralization of the R5 peptide for site-specific growth of silica nanoparticles on the micropatterns, avoiding the use of harsh chemicals or complex processing. The functional micropatterned systems are used to align human mesenchymal stem cells and bovine serum albumin. This combination of peptide printing and site-specific biomineralization provides a new route for developing cost-effective micropatterns, with implications for broader materials designs. Coding cell micropatterns through peptide inkjet printing for arbitrary biomineralized architectures is demonstrated here. The functional micropatterned systems are used to align human mesenchymal stem cells and bovine serum albumin in vitro, avoiding the use of harsh chemicals or complex processing, while providing potential applications in developing cost-effective micropatterns to meet task-specific biomedical applications.

Reverse Reconstruction and Bioprinting of Bacterial Cellulose-Based Functional Total Intervertebral Disc for Therapeutic Implantation.

The degradation of intervertebral discs (IVD), a typical hierarchical structured tissue, causes serious neck and back pain. The current methods cannot fully reconstitute the unique structure and function of native IVD. In this study, by reverse reconstruction of the structure of native IVD and bioprinting bacterial cellulose (BC) nanofibers with a high-throughput optimized micropattern screening microchip, a total IVD is created that contained type II collagen-based nucleus pulposus (NP) and hierarchically organized and micropatterned BC-based annulus fibrosus (AF), mimicking native IVD tissue. The artificial NP contains rat NP cells, whereas the AF contains concentrically arranged BC layers with aligned micropatterns and attached AF cells in +/-30° alternate directions between adjacent layers. Long-term (3 months) implantation experiments on rats demonstrate the excellent structural (shape maintenance, hydration, tissue integration) and functional (mechanical support and flexibility) performance of the artificial IVD. This study provides a novel strategy for creating highly sophisticated artificial tissues.

Interaction between the Staphylococcus aureus extracellular adherence protein Eap and its subdomains with platelets.

S. aureus associated bacteremia can lead to severe infections with high risk of mortality (e.g. sepsis, infective endocarditis). Many virulence factors and adhesins of S. aureus are known to directly interact with platelets. Extracellular adherence protein, Eap, one of the most important virulence factors in S. aureus mediated infections is a multi-tandem domain protein and has been shown to interact with almost all cell types in the human circulatory system. By using amine reactive fluorescent N-hydroxysuccinimidyl (NHS)-ester dyes and by direct detection with primary fluorescently conjugated anti-histidine (His-tag) antibodies against detect N-terminal His6, we show Eap subdomain Eap D3D4 specifically interacts and rapidly activates human platelets. Furthermore, we validate our finding by using site directed directional immobilization of Eap D3D4 through N-terminal His6 on nickel (II)-nitrilotriacetic acid (Ni-NTA) functionalized bacteriomimetic microbead arrays to visualize real-time platelet activation through calcium release assay. These methods offer an easily adoptable protocols for screening of S.aureus derived virulence factors and adhesins with platelets.

An integrated enhancement and reconstruction strategy for the quantitative extraction of actin stress fibers from fluorescence micrographs.

BACKGROUND: The stress fibers are prominent organization of actin filaments that perform important functions in cellular processes such as migration, polarization, and traction force generation, and whose collective organization reflects the physiological and mechanical activities of the cells. Easily visualized by fluorescence microscopy, the stress fibers are widely used as qualitative descriptors of cell phenotypes. However, due to the complexity of the stress fibers and the presence of other actin-containing cellular features, images of stress fibers are relatively challenging to quantitatively analyze using previously developed approaches, requiring significant user intervention. This poses a challenge for the automation of their detection, segmentation, and quantitative analysis. RESULT: Here we describe an open-source software package, SFEX (Stress Fiber Extractor), which is geared for efficient enhancement, segmentation, and analysis of actin stress fibers in adherent tissue culture cells. Our method made use of a carefully chosen image filtering technique to enhance filamentous structures, effectively facilitating the detection and segmentation of stress fibers by binary thresholding. We subdivided the skeletons of stress fiber traces into piecewise-linear fragments, and used a set of geometric criteria to reconstruct the stress fiber networks by pairing appropriate fiber fragments. Our strategy enables the trajectory of a majority of stress fibers within the cells to be comprehensively extracted. We also present a method for quantifying the dimensions of the stress fibers using an image gradient-based approach. We determine the optimal parameter space using sensitivity analysis, and demonstrate the utility of our approach by analyzing actin stress fibers in cells cultured on various micropattern substrates. CONCLUSION: We present an open-source graphically-interfaced computational tool for the extraction and quantification of stress fibers in adherent cells with minimal user input. This facilitates the automated extraction of actin stress fibers from fluorescence images. We highlight their potential uses by analyzing images of cells with shapes constrained by fibronectin micropatterns. The method we reported here could serve as the first step in the detection and characterization of the spatial properties of actin stress fibers to enable further detailed morphological analysis.

Micropatterning of nanocomposite polymer scaffolds using sacrificial phosphate glass fibers for tendon tissue engineering applications.

This study presents a simple and reproducible method of micropatterning the novel nanocomposite polymer (POSS-PCU) using a sacrificial phosphate glass fiber template for tendon tissue engineering applications. The diameters of the patterned scaffolds produced were dependent on the diameter of the glass fibers (15 µm) used. Scaffolds were tested for their physical properties and reproducibility using various microscopy techniques. For the first time, we show that POSS-PCU supports growth of human tenocytes cells. Furthermore, we show that cellular alignment, their biological function and expression of various tendon related proteins such as scleraxis, collagen I and III, tenascin-C are significantly elevated on the micropatterned polymer surfaces compared to flat samples. This study demonstrated a simple, reproducible method of micropatterning POSS-PCU nanocomposite polymer for novel tendon repair applications, which when provided with physical cues could help mimic the microenvironment of tenocytes cells.