Alignment Of Actin Filaments Research Articles

Spatial confinement modulates endothelial cell behavior and traction force in 3D hydrogel microgrooves

The mechanical environment of vascular endothelial cells (ECs) encompasses a wide range of curvatures due to variations in blood vessel diameters. Integrins, key mediators of cell-matrix interactions, establish connections between the extracellular matrix and the actin cytoskeleton, influencing diverse cellular behaviors. In this study, we explored the impact of spatial confinement on human umbilical vein ECs (HUVECs) cultured within three-dimensional hydrogel microgrooves of varying curvatures and the underlying role of integrins in mediating cellular responses. Employing maskless lithography, we successfully fabricated precise and wall curvatures-controlled hydrogel microgrooves, conferring spatial constraints on the cells. Our investigations revealed substantial alterations in HUVEC behavior within the hydrogel microgrooves with varying sidewall curvatures, marked by reduced cell size, enhanced orientation, and increased apoptosis. Interestingly, microgroove curvature emerged as a crucial factor influencing cell orientation and apoptosis, with rectangular microgrooves eliciting distinct changes in cell orientation, while ring-form microgrooves exhibited higher apoptosis rates. The side-wall effect in the 20 μm region near the microgroove wall had the greatest influence on cell orientation and apoptosis. HUVECs within the microgrooves exhibited elevated integrin expression, and inhibition of αV-integrin by cilengitide significantly curtailed cell apoptosis without affecting proliferation. Additionally, integrin-mediated cell traction force closely correlated with the spatial confinement effect. Cilengitide not only reduced integrin and focal adhesion expression but also attenuated cell traction force and cytoskeletal actin filament alignment. Overall, our findings elucidate the spatial confinement of ECs in hydrogel microgrooves and underscores the pivotal role of integrins, particularly αV-integrin, in mediating cell traction force and apoptosis within this microenvironment.

How Hydrogel Stiffness Affects Adipogenic Differentiation of Mesenchymal Stem Cells under Controlled Morphology.

Matrix stiffness has been disclosed as an essential regulator of cell fate. However, it is barely studied how the matrix stiffness affects stem cell functions when cell morphology changes. Thus, in this study, the effect of hydrogel stiffness on adipogenic differentiation of human bone-marrow-derived mesenchymal stem cells (hMSCs) with controlled morphology was investigated. Micropatterns of different size and elongation were prepared by a photolithographical micropatterning technique. The hMSCs were cultured on the micropatterns and showed a different spreading area and elongation following the geometry of the underlying micropatterns. The cells with controlled morphology were embedded in agarose hydrogels of different stiffnesses. The cells showed a different level of adipogenic differentiation that was dependent on both hydrogel stiffness and cell morphology. Adipogenic differentiation became strong when the cell spreading area decreased and hydrogel stiffness increased. Adipogenic differentiation did not change with cell elongation. Therefore, cell spreading area and hydrogel stiffness could synergistically affect adipogenic differentiation of hMSCs, while cell elongation did not affect adipogenic differentiation. A change of cell morphology and hydrogel stiffness was accompanied by actin filament alignment that was strongly related to adipogenic differentiation. The results indicated that cell morphology could affect cellular sensitivity to hydrogel stiffness. The results will provide useful information for the elucidation of the interaction of stem cells and their microenvironmental biomechanical cues.

Decoding Natural Astrocyte Rhythms: Dynamic Actin Waves Result from Environmental Sensing by Primary Rodent Astrocytes.

Astrocytes are key regulators of brain homeostasis, equilibrating ion, water, and neurotransmitter concentrations and maintaining essential conditions for proper cognitive function. Recently, it has been shown that the excitability of the actin cytoskeleton manifests in second-scale dynamic fluctuations and acts as a sensor of chemophysical environmental cues. However, it is not known whether the cytoskeleton is excitable in astrocytes and how the homeostatic function of astrocytes is linked to the dynamics of the cytoskeleton. Here it is shown that homeostatic regulation involves the excitable dynamics of actin in certain subcellular regions of astrocytes, especially near the cell boundary. The results further indicate that actin dynamics concentrate into "hotspot" regions that selectively respond to certain chemophysical stimuli, specifically the homeostatic challenges of ion or water concentration increases. Substrate topography makes the actin dynamics of astrocytes weaker. Super-resolution images demonstrate that surface topography is also associated with the predominant perpendicular alignment of actin filaments near the cell boundary, whereas flat substrates result in an actin cortex mainly parallel to the cell boundary. Additionally, coculture with neurons increases both the probability of actin dynamics and the strength of hotspots. The excitable systems character of actin thus makes astrocytes direct participants in neural cell network dynamics.

A mechanistic motor-clutch model that explains cell shape dynamics to cyclic stretch.

Many cells in the body experience cyclic mechanical loading, which can impact cellular processes and morphology. In vitro studies often report that cells reorient in response to cyclic stretch of their substrate. To explore cellular mechanisms involved in this reorientation, a computational model was developed by adapting previous computational models of the actin–myosin–integrin motor-clutch system developed by others. The computational model predicts that under most conditions, actin bundles align perpendicular to the direction of applied cyclic stretch, but under specific conditions, such as low substrate stiffness, actin bundles align parallel to the direction of stretch. The model also predicts that stretch frequency impacts the rate of reorientation and that proper myosin function is critical in the reorientation response. These computational predictions are consistent with reports from the literature and new experimental results presented here. The model suggests that the impact of different stretching conditions (stretch type, amplitude, frequency, substrate stiffness, etc.) on the direction of cell alignment can largely be understood by considering their impact on cell–substrate detachment events, specifically whether detachments preferentially occur during stretching or relaxing of the substrate.

Rapid Prototypable Biomimetic Peristalsis Bioreactor Capable of Concurrent Shear and Multi-Axial Strain

Peristalsis is a nuanced mechanical stimulus comprised of multi-axial strain (radial and axial strain) and shear stress. Forces associated with peristalsis regulate diverse biological functions including digestion, reproductive function, and urine dynamics. Given the central role peristalsis plays in physiology and pathophysiology, we were motivated to design a bioreactor capable of holistically mimicking peristalsis. We engineered a novel rotating screw-drive based design combined with a peristaltic pump, in order to deliver multi-axial strain and concurrent shear stress to a biocompatible polydimethylsiloxane (PDMS) membrane “wall.” Radial indentation and rotation of the screw drive against the wall demonstrated multi-axial strain evaluated via finite element modeling. Experimental measurements of strain using piezoelectric strain resistors were in close alignment with model-predicted values (15.9 ± 4.2% vs. 15.2% predicted). Modeling of shear stress on the “wall” indicated a uniform velocity profile and a moderate shear stress of 0.4 Pa. Human mesenchymal stem cells (hMSCs) seeded on the PDMS “wall” and stimulated with peristalsis demonstrated dramatic changes in actin filament alignment, proliferation, and nuclear morphology compared to static controls, perfusion, or strain, indicating that hMSCs sensed and responded to peristalsis uniquely. Lastly, significant differences were observed in gene expression patterns of calponin, caldesmon, smooth muscle actin, and transgelin, corroborating the propensity of hMSCs toward myogenic differentiation in response to peristalsis. Collectively, our data suggest that the peristalsis bioreactor is capable of generating concurrent multi-axial strain and shear stress on a “wall.” hMSCs experience peristalsis differently than perfusion or strain, resulting in changes in proliferation, actin fiber organization, smooth muscle actin expression, and genetic markers of differentiation. The peristalsis bioreactor device has broad utility in the study of development and disease in several organ systems.

Actin filament alignment causes mechanical hysteresis in cross-linked networks.

Cells dynamically control their material properties through remodeling of the actin cytoskeleton, an assembly of cross-linked networks and bundles formed from the biopolymer actin. We recently found that cross-linked networks of actin filaments reconstituted in vitro can exhibit adaptive behavior and thus serve as a model system to understand the underlying mechanisms of mechanical adaptation of the cytoskeleton. In these networks, training, in the form of applied shear stress, can induce asymmetry in the nonlinear elasticity. Here, we explore control over this mechanical hysteresis by tuning the concentration and mechanical properties of cross-linking proteins in both experimental and simulated networks. We find that this effect depends on two conditions: the initial network must exhibit nonlinear strain stiffening, and filaments in the network must be able to reorient during training. Hysteresis depends strongly and non-monotonically on cross-linker concentration, with a peak at moderate concentrations. In contrast, at low concentrations, where the network does not strain stiffen, or at high concentrations, where filaments are less able to rearrange, there is little response to training. Additionally, we investigate the effect of changing cross-linker properties and find that longer or more flexible cross-linkers enhance hysteresis. Remarkably plotting hysteresis against alignment after training yields a single curve regardless of the physical properties or concentration of the cross-linkers.

Collective and contractile filament motions in the myosin motility assay.

Cells require mechanical forces for their physiological functions. The forces are generated mainly from molecular interactions between actin filaments, cross-linking proteins, and myosin motors in the actin cytoskeleton. To better understand the molecular interactions, many studies employed myosin motility assays with actin filaments propelled by myosin heads fixed on a surface. Various interesting behaviors of actin filaments have been observed in the motility assay experiments. Despite the popularity of the motility assays, there were only a few computational models designed for simulating the motility assay systems. Most of the previous models have limitations which precluded full understanding of molecular origins for behaviors of actin filaments. In this study, we used an agent-based computational model based on Brownian dynamics for simulating the motility assay system. Our model rigorously describes the mechanics, dynamics, and interactions of actin filaments, cross-linking proteins, and molecular motors. Using the model, we first investigated how properties of actin filaments and motors affect gliding motions of actin filaments without volume-exclusion effects as a base study. We found that actin filaments can continuously glide at relative fast speed only when they are sufficiently longer than the average spacing between neighboring motors and that the gliding speed of F-actins shows a biphasic dependence on processivity of motors. Then, we showed that volume-exclusion effects between actin filaments can induce diverse collective movements and alignment of actin filaments, thus creating thick bundles and ring-like structures in the absence of cross-linking proteins. Lastly, we demonstrated that cross-linking proteins can lead to distinct contractile behaviors of actin networks depending on the density and kinetics of the cross-linking proteins. Results from our study show the ability of our model to simulate the motility assay system under various conditions and provide insights into understanding of different behaviors of actin filaments.

New CRISPR/Cas9 mouse model confirms association of phosphorylation of ACTN4 at serine 159 with proteinuria and focal segmental glomerulosclerosis

Mutations in α‐actinin‐4 (ACTN4)—an important cytoskeleton protein that binds actin—cause a form of kidney injury in humans called familial focal segmental glomerulosclerosis (FSGS). FSGS is characterized by proteinuria and eventual progression to renal failure. All of the known disease‐causing mutations result in increased ACTN4 binding affinity to actin. We previously showed that this increased actin binding was associated with biomechanical changes that left podocytes vulnerable to mechanical stress and eventual detachment. We also previously detected with mass spectrometry that ACTN4 is phosphorylated at the serine 159 site (S159) in cultured human podocytes. Moreover, phosphomimetic S159D ACTN4 protein (which mimics the effect of phosphorylation at S159) confers similar increased actin binding affinity as disease‐causing mutant ACTN4. In the current study, we hypothesized that the changes in binding affinity associated with phosphorylation of S159 may also render the podocyte vulnerable to mechanical stress and lead to eventual proteinuria and FSGS in vivo. Using CRISPR/Cas9 technology, we developed a new phosphomimetic mouse model Actn4 S160D (analogous to S159D mutation in humans). We first isolated primary podocytes from wild‐type (WT) and homozygous Actn4S160D/S160D mice. Similar to podocytes carrying disease‐causing mutant Actn4, homozygous Actn4S160D/S160D podocytes demonstrated more aligned actin filaments and increased number of focal adhesions (quantified using vinculin staining) in comparison with WT podocytes. We subjected both WT and homozygous Actn4S160D/S160D mice to subtotal nephrectomy to induce glomerular hyperfiltration and increase glomerular mechanical stress upon podocytes. We found that in comparison with WT, homozygous Actn4S160D/S/160D mice demonstrated significantly increased albuminuria at 2, 3, and 7 weeks after nephrectomy. Additionally, kidney sections were used for PAS staining and examined by a renal pathologist to assess for glomerulosclerosis. Whereas only 1% of glomeruli were found to be sclerosed in WT, 12% were sclerosed in homozygous Actn4S160D/S/160D mice (p<0.001). Our findings confirm that increased phosphorylation at S159 is pathologic, in showing that a phosphomimetic mouse model develops albuminuria and FSGS. Moreover, we show that phosphomimetic podocytes demonstrate changes in actin alignment and focal adhesion, markers of podocyte dysfunction that are shared by disease‐causing mutant ACTN4 podocytes. Altogether, these findings add to the building evidence suggesting that abnormal phosphorylation at S159 leads to changes on a protein, cellular, and whole animal level that are similar to mutant ACTN4‐mediated disease. This evidence provides new insights into the importance of a post‐translational modification affecting the podocyte cytoskeleton.Support or Funding InformationK01DK114329 and R37DK059588This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ANGUSTIFOLIA Regulates Actin Filament Alignment for Nuclear Positioning in Leaves.

During dark adaptation, plant nuclei move centripetally toward the midplane of the leaf blade; thus, the nuclei on both the adaxial and abaxial sides become positioned at the inner periclinal walls of cells. This centripetal nuclear positioning implies that a characteristic cell polarity exists within a leaf, but little is known about the mechanism underlying this process. Here, we show that ANGUSTIFOLIA (AN) and ACTIN7 regulate centripetal nuclear positioning in Arabidopsis (Arabidopsis thaliana) leaves. Two mutants defective in the positioning of nuclei in the dark were isolated and designated as unusual nuclear positioning1 (unp1) and unp2 In the dark, nuclei of unp1 were positioned at the anticlinal walls of adaxial and abaxial mesophyll cells and abaxial pavement cells, whereas the nuclei of unp2 were positioned at the anticlinal walls of mesophyll and pavement cells on both the adaxial and abaxial sides. unp1 was caused by a dominant-negative mutation in ACTIN7, and unp2 resulted from a recessive mutation in AN Actin filaments in unp1 were fragmented and reduced in number, which led to pleiotropic defects in nuclear morphology, cytoplasmic streaming, and plant growth. The mutation in AN caused aberrant positioning of nuclei-associated actin filaments at the anticlinal walls. AN was detected in the cytosol, where it interacted physically with plant-specific dual-specificity tyrosine phosphorylation-regulated kinases (DYRKPs) and itself. The DYRK inhibitor (1Z)-1-(3-ethyl-5-hydroxy-2(3H)-benzothiazolylidene)-2-propanone significantly inhibited dark-induced nuclear positioning. Collectively, these results suggest that the AN-DYRKP complex regulates the alignment of actin filaments during centripetal nuclear positioning in leaf cells.

Quantification of alignment of vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) are essential components that keep the tonus of the arterial network, which is the channel used to conduct the blood from the heart to the peripheral areas of the body. It is known that mechanical and architectural changes in VSMCs may lead to functional modifications in the cardiovascular system; therefore, the quantitative characterization of these changes can help to elucidate questions that remain unclear in pathological situations, such as hypertension, vasospasm, vascular hypertrophy, and atherosclerosis. In this work, we have developed a new framework of image processing using the Sobel operator, associated with statistical analysis, to determine the degree of local alignment of actin filaments, which we found to be directly related with the distensibility of the arterial wall. We have also compared these results with the rigidity of the cytoskeleton of VSMCs. The results suggest that the alignment degree increases from peripheral arteries, such as carotid and femoral, to central arteries, as well coronary and thoracic aorta, which can indicate that the level of local alignment of the actin fibers in VSMCs is related with the mechanical behavior of the arterial wall. © 2018 International Society for Advancement of Cytometry.

Actin assembly mediated by a nucleation promoting factor WASH is involved in MTOC-TMA formation during Xenopus oocyte maturation.

During Xenopus oocyte maturation, the microtubule organizing center and transient microtubule array (MTOC-TMA) forms at the vegetal side of the nucleus and we have previously demonstrated that this formation requires actin reorganization. However, the molecular mechanism that coordinates actin filaments with microtubules (MTs) during oocyte maturation is unknown. Here we show that the actin nucleation promoting factor WASH is involved in MTOC-TMA assembly. WASH depletion disrupted the alignment of actin filaments and MT at the base of MTOC-TMA. In vitro cosedimentation assays revealed that the Xenopus WASH VCA domain, which comprises the actin and Arp2/3 complex binding region, directly bound to MT but that the WHD2 domain, which had been previously reported to be an MT-binding domain of mammalian WASH, did not. Injection of the WHD2-deleted mutant into oocytes caused a drastic accumulation of actin filaments in the cytoplasm and malformation of MTOC-TMA, suggesting that the WHD2 domain negatively regulates the VCA domain activity during oocyte maturation. Our results suggest that WASH complex-dependent actin nucleation is involved in the assembly of MTOC-TMA.

Bending, Pushing, and Ratcheting It Up: Memories of a Modeling Effort

In 1993, I was a PhD student at the University of British Columbia, doing research with Leah Edelstein-Keshet, the eminent mathematical biologist. We were very interested in the general problem of alignment of elongated objects in biology—for example, alignment of fish swimming in groups—but really, we were thinking more about angular order in the dynamic cytoskeleton. Electron micrographs of actin filaments (1) in the “comet tail” of actin propelling Listeria pathogens inside host cells fascinated us, and we started to develop a rather abstract mathematical model for the actin filament alignment.

Electrospun nanoyarn seeded with myoblasts induced from placental stem cells for the application of stress urinary incontinence sling: An in vitro study

ObjectiveTo fabricate a novel electrospun nanoyarn using dynamic liquid electrospinning technique. The nanoyarn will be seeded with myoblasts differentiated from placental stem cells (PSCs) to evaluate the feasiblity of the cell-scaffold construct as a tissue engineering sling to treat stress urinary incontinence. Material and methodsPSCs were induced to myoblasts with 5-azacytidine and horse serum. Myoblasts differentiation was confirmed by immunofluorescence and western blot. Western blot was also used to assess the change of extracellular matrix (ECM) expression. A dynamic liquid electrospinning system was used to fabricate a novel nanoyarn scaffold for myoblast seeding. Cell morphology and proliferation on nanoyarn and nanofiber scaffold were compared with scanning electron microscopy (SEM) and MTS assay respectively. Filament actin development was tested with Rhodamine-labeled phalloidin stainning; cell infiltration into scaffolds was observed with H&E stainning. ECM expression was evaluated by a collagen assay, immunofluorescence imaging and real-time PCR. ResultsMyoblasts showed increased expression of α-SMA, desmin, and collagen type 1, 3 when compared to PSCs. The nanoyarn possessed higher porosity, larger pore size, and aligned fibers/yarns as compared to nanofiber scaffold. Cell proliferation was significantly improved on nanoyarn scaffold. Cells could infiltrate deeply in the nanoyarn scaffold after 7 days in culture, however, they could only proliferate on the surface of the nanofiber scaffold. The myoblast-nanoyarn constructs seemed to be more like a muscle tissue. The myoblasts spreading on the nanoyarn scaffold were visible with aligned actin filaments in the horizontal view, whereas myoblasts spreading on the nanofiber scaffold were visible with unaligned actin filaments. Nanoyarn myoblasts exhibited higher production and density of type 1, 3 collagen and elastin. ConclusionsPSCs could be induced into myoblast and expressed elevated myogenic markers and ECM. PSCs are potential cell source for a tissue engineered sling. The novel electrospun nanoyarn scaffold showed potential for use as a sling for treatment of stress urinary incontinence. In vitro studies demonstrated that the nanoyarn scaffold could improve cell proliferation, muscular tissue development, and ECM expression compared to random nanofiber scaffolds. The combination of myoblasts and nanoyarn scaffold could be a promising tissue engineered sling for future in vivo studies.

A Shearing-Stretching Device That Can Apply Physiological Fluid Shear Stress and Cyclic Stretch Concurrently to Endothelial Cells.

Endothelial cell (EC) morphology and functions can be highly impacted by the mechanical stresses that the cells experience in vivo. In most areas in the vasculature, ECs are continuously exposed to unsteady blood flow-induced shear stress and vasodilation-contraction-induced tensile stress/strain simultaneously. Investigations on how ECs respond to combined shear stress and tensile strain will help us to better understand how an altered mechanical environment affects EC mechanotransduction, dysfunction, and associated cardiovascular disease development. In the present study, a programmable shearing and stretching device that can apply dynamic fluid shear stress and cyclic tensile strain simultaneously to cultured ECs was developed. Flow and stress/strain conditions in the device were simulated using a fluid structure interaction (FSI) model. To characterize the performance of this device and the effect of combined shear stress-tensile strain on EC morphology, human coronary artery ECs (HCAECs) were exposed to concurrent shear stress and cyclic tensile strain in the device. Changes in EC morphology were evaluated through cell elongation, cell alignment, and cell junctional actin accumulation. Results obtained from the numerical simulation indicated that in the "in-plane" area of the device, both fluid shear stress and biaxial tensile strain were uniform. Results obtained from the in vitro experiments demonstrated that shear stress, alone or combined with cyclic tensile strain, induced significant cell elongation. While biaxial tensile strain alone did not induce any appreciable change in EC elongation. Fluid shear stress and cyclic tensile strain had different effects on EC actin filament alignment and accumulation. By combining various fluid shear stress and cyclic tensile strain conditions, this device can provide a physiologically relevant mechanical environment to study EC responses to physiological and pathological mechanical stimulation.

Biomechanistic Study of Smooth Muscle Cell Sheet during Circumferential Alignment in Circular Micropatterns.

Engineered functional blood vessels play an important role in tissue regeneration and wound healing, but they remain far from clinical practices because of the complex three-layer capillary histology. In general, the reconstruction of tunica media from vascular smooth muscle cells (SMCs) that mediate vasoconstriction and vasodilation of circulatory system is critical for engineering functional blood vessels. To date, the mechanotransduction during circumferential alignment of SMC layer in tunica media remains to be elucidated. In this study, concentric microwalls fabricated on a polydimethylsiloxane (PDMS) substrate were developed for cell traction force microscopy (CTFM) of SMC layer. First, the cell shape, density, and alignment orientation of SMCs during circumferential cell sheet formation were measured during 7-day cell culture. Second, a collective CTFM assay was carried out to measure the traction forces exerted by the confluent cell layer at various stages of circumferential alignment. It was found that the mechanotransduction of SMC layer grown between the circular microwalls was significantly altered compared to the SMC layer grown on a flat substrate. The results indicated the presence of an optimal region for formation of circumferentially aligned SMC layer verified by both quantitative analysis and biomechanical study. Moreover, the distributions of traction forces and von Mises stresses during cell alignment were affected by the cellular mechanotransduction from the extracellular physical constraints and cell-cell interactions. The immunofluorescence staining and real time quantitative polymerase chain reaction (RT-qPCR) analyses of actin filaments further revealed the relation between the monolayer-generated traction forces and the direction of aligned actin filaments.

Characterization of Biomechanical Properties of Primary Endothelial Cells Exposed to Shear Stress

Mechanotransduction, the process where cells convert biophysical signals into chemical signals, directs the behavior and function of endothelial cells (ECs) because of their unique position on the luminal surface of blood vessels. ECs are in direct contact with blood and can sense changes in shear stress caused by alterations in flow rate. To investigate the biophysical implications of this response we recapitulated physiological levels of shear stress in vitro using two systems, imaged and measured the consequent cytoskeletal changes with immunofluorescence, and quantified cortical stiffness distributions. We developed a sampling method for Atomic Force Microscopy (AFM) stiffness measurements by which force-indentation data is collected in a uniform grid (force map) over multiple cells within a monolayer. In this way, the sampling bias resulting from the selection of force-indentation measurements was reduced. Hertzian contact modeling for AFM was used to specifically isolate the cortical stiffness of the cell using a pyramidal indenter and small indentation depths (100 nm). Nonparametric statistics were used to compare the distributions of moduli from a given experiment, as the data distribution was not Gaussian. Actin filament alignment was quantified using Hough Transforms on immunofluorescence images and increased when cells were exposed to shear stress. The medians of stiffness distributions within sheared EC monolayers increased in magnitude when compared to static monolayers, yielding a method-dependent increase in cortical stiffness of up to 60%. This demonstrates that physiological levels of shear stress dramatically affect ECs morphology in a way that cannot be simulated under static conditions. We concluded that the biophysical milieu of a cell's physiological microenvironment must be recapitulated in order to represent characteristic cellular mechanical properties in vitro.

Multiscale, hierarchically patterned topography for directing human neural stem cells into functional neurons.

Various biophysical and biochemical factors are important for determining the fate of neural stem cells (NSCs). Among biophysical signals, topographical stimulation by micro/nanopatterns has been applied to control NSC differentiation. In this study, we developed a hierarchically patterned substrate (HPS) platform that can synergistically enhance the differentiation of human NSCs (hNSCs) by simultaneously providing microscale and nanoscale spatial controls to facilitate the alignment of the cytoskeleton and the formation of focal adhesions. The multiscale HPS was fabricated by combining microgroove patterns (groove size: 1.5 μm), prepared by a conventional photolithographic process, and nanopore patterns (pore diameter: 10 nm), prepared from cylinder-forming block copolymer thin films. The hNSCs grown on the HPS exhibited not only a highly aligned, elongated morphology, but also a greatly enhanced differentiation into neuronal and astrocyte lineages, compared to hNSCs on a flat substrate (FS) or single-type patterned substrates [microgroove patterned substrate (MPS) and nanopore patterned substrate (NPS)]. Interestingly, the application of the HPS directed hNSC differentiation toward neurons rather than astrocytes. The expression of focal adhesion proteins in hNSCs was also significantly increased on the HPS compared to the FS, MPS, and NPS, likely a result of the presence of more focal contact points provided by nanopore structures. Inhibition of both β1 integrin-mediated binding and the intracellular Rho-associated protein kinase pathway of hNSCs eliminated the beneficial effects of the HPS on focal adhesion formation and actin filament alignment, which subsequently reduced hNSC differentiation. More importantly, hNSCs on the HPS differentiated into functional neurons exhibiting sodium currents and action potentials. The multiscale, hierarchically patterned topography would be useful for the design of functional biomaterial scaffolds to potentiate NSC therapeutic efficacy.

Accurate polarity control and parallel alignment of actin filaments for myosin-powered transport systems

An actin filament is a micrometer-long biological filament, which serves as a track of transport systems in cells. The filament exhibits polarity while myosin transports a cargo along it unidirectionally. Here, we established a method to align many actin filaments parallel with each other on a substrate with uniform (>99%) polarity as assessed by myosin movements. This actin array is an ideal candidate for the construction of a nano-scale unidirectional transport system powered by myosin.

Rotational model for actin filament alignment by myosin

Dynamics of the actomyosin cytoskeleton regulate cellular processes such as secretion, cell division, cell motility, and shape change. Actomyosin dynamics are themselves regulated by proteins that control actin filament polymerization and depolymerization, and myosin motor contractility. Previous theoretical work has focused on translational movement of actin filaments but has not considered the role of filament rotation. Since filament rotational movements are likely sources of forces that direct cell shape change and movement we explicitly model the dynamics of actin filament rotation as myosin II motors traverse filament pairs, drawing them into alignment. Using Monte Carlo simulations we find an optimal motor velocity for alignment of actin filaments. In addition, when we introduce polymerization and depolymerization of actin filaments, we find that alignment is reduced and the filament arrays exist in a stable, asynchronous state. Further analysis with continuum models allows us to investigate factors contributing to the stability of filament arrays and their ability to generate force. Interestingly, we find that two different morphologies of F-actin arrays generate the same amount of force. We also identify a phase transition to alignment which occurs when either polymerization rates are reduced or motor velocities are optimized. We have extended our analysis to include a maximum allowed stretch of the myosin motors, and a non-uniform length for filaments leading to little change in the qualitative results. Through the integration of simulations and continuum analysis, we are able to approach the problem of understanding rotational alignment of actin filaments by myosin II motors.

Electric Field Induces Alignment of Actin Filaments

Electric Field Induces Alignment of Actin Filaments