Actin Alignment Research Articles

Emergence and stability of endoplasmic reticulum network streaming in plant cells

The endoplasmic reticulum (ER) network is highly complex and highly dynamic in its geometry, and undergoes extensive remodeling and bulk flow. It is known that the ER dynamics are driven by actin–myosin dependent processes. ER motion through the cytoplasm will cause forces on the cytoplasm that will induce flow. However, ER will also clearly be passively transported by the bulk cytoplasmic streaming. We take the complex ER network structure into account and propose a positive-feedback mechanism among myosin-like motors, actin alignment, ER network dynamics for the emergence of ER flow. Using this model, we demonstrate that ER streaming may be an emergent feature of this three-way interaction and that the persistent-point density may be a key driver of the emergence of ER streaming.

Single-cell mechanical assay unveils viscoelastic similarities in normal and neoplastic brain cells

Understanding cancer cell mechanics allows for the identification of novel disease mechanisms, diagnostic biomarkers, and targeted therapies. In this study, we utilized our previously established fluid shear stress assay to investigate and compare the viscoelastic properties of normal immortalized human astrocytes and invasive human glioblastoma (GBM) cells when subjected to physiological levels of shear stress that are present in the brain microenvironment. We used a parallel-flow microfluidic shear system and a camera-coupled optical microscope to expose single cells to fluid shear stress and monitor the resulting deformation in real time, respectively. From the video-rate imaging, we fed cell deformation information from digital image correlation into a three-parameter generalized Maxwell model to quantify the nuclear and cytoplasmic viscoelastic properties of single cells. We further quantified actin cytoskeleton density and alignment in immortalized human astrocytes and GBM cells via fluorescence microscopy and image analysis techniques. Results from our study show that contrary to the behavior of many extracranial cells, normal and cancerous brain cells do not exhibit significant differences in their viscoelastic properties. Moreover, we also found that the viscoelastic properties of the nucleus and cytoplasm as well as the actin cytoskeletal densities of both brain cell types are similar. Our work suggests that malignant GBM cells exhibit unique mechanical behaviors not seen in other cancer cell types. These results warrant future studies to elucidate the distinct biophysical characteristics of the brain and reveal novel mechanical attributes of GBM and other primary brain tumors.

Abstract 194: Single-cell mechanical analysis reveals that astrocytes and glioblastoma cells have similar viscoelastic properties

Abstract Despite recent advances in treatment, the survival rate of glioblastoma (GBM) patients remains low due to the cancer’s resistance to current treatment modalities. Solid tumors like GBM are known to harbor physical abnormalities at the tissue and cell level. An understanding of GBM cell mechanics may therefore lead to the identification of novel disease mechanisms, biomarkers, and therapeutic strategies to improve treatment outcomes. In this study, we aimed to compare the mechanics of human GBM cells and their normal counterpart, immortalized human astrocytes (IHAs). To accomplish this, we used a parallel-plate flow system to expose single cells to physiological fluid shear stress and recorded the resulting deformation using a camera-coupled microscope. We then utilized digital image correlation analysis to create strain-time profiles of individual cells and fit the profiles with a three-parameter generalized Maxwell model to characterize their nuclear and cytoplasmic viscoelastic properties. We also used fluorescence microscopy and atomic force microscopy to quantify actin density and alignment and adhesion energy in both cell types, respectively. Our results showed that, contrary to extracranial cancer cells, the viscoelastic properties of GBM cells are similar to their normal counterpart. This may explain why metastasis in GBM patients is rare and conventional treatment options are unsuccessful. We also found that actin organization and localization and adhesion energy is different between GBM cells and IHAs. Thus, future research which links the genetic and microenvironmental factors that modulate these unique mechanical features may lead to the development of rational and novel clinical strategies against GBM. Citation Format: Julian Najera, Killian Onwudiwe, Luke Holen, Alice A. Burchett, Dorielis Rodriguez, Maksym Zarodniuk, Saeed Siri, Meenal Datta. Single-cell mechanical analysis reveals that astrocytes and glioblastoma cells have similar viscoelastic properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 194.

The emergence of spontaneous coordinated epithelial rotation on cylindrical curved surfaces.

Three-dimensional collective epithelial rotation around a given axis represents a coordinated cellular movement driving tissue morphogenesis and transformation. Questions regarding these behaviors and their relationship with substrate curvatures are intimately linked to spontaneous active matter processes and to vital morphogenetic and embryonic processes. Here, using interdisciplinary approaches, we study the dynamics of epithelial layers lining different cylindrical surfaces. We observe large-scale, persistent, and circumferential rotation in both concavely and convexly curved cylindrical tissues. While epithelia of inverse curvature show an orthogonal switch in actomyosin network orientation and opposite apicobasal polarities, their rotational movements emerge and vary similarly within a common curvature window. We further reveal that this persisting rotation requires stable cell-cell adhesion and Rac-1–dependent cell polarity. Using an active polar gel model, we unveil the different relationships of collective cell polarity and actin alignment with curvatures, which lead to coordinated rotational behavior despite the inverted curvature and cytoskeleton order.

Stress fiber growth and remodeling determines cellular morphomechanics under uniaxial cyclic stretch.

Stress fibers in the cytoskeleton are essential in maintaining cellular shape and influence cellular adhesion and migration. Cyclic uniaxial stretching results in cellular reorientation orthogonal to the applied stretch direction. The mechanistic cues underlying changes to cellular form and function to stretch stimuli are currently underexplored. We show stretch-induced stress fiber lengthening, their realignment, and increased cortical actin inNIH 3T3 fibroblasts stretched over varied amplitudes and durations. Higher amounts of actin and stress fiber alignment were accompanied with an increase in the effective elastic modulus of cells. Microtubules did not contribute to the measured stiffness or reorientation response but were essential to the nuclear reorientation. We used a phenomenological growth and remodeling law, based on the experimental data, to model stress fiber elongation and reorientation dynamics based on a nonlinear, orthotropic, fiber-reinforced continuum representation of the cell. The model predicts the changes observed fibroblast morphology and increased cellular stiffness under uniaxial cyclic stretch which agrees with experimental results. Such studies are important in exploring the differences underlying mechanotransduction and cellular contractility under stretch.

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.

A simple method to align cells on 3D hydrogels using 3D printed molds

Vascular smooth muscle cells align circumferentially around the vessel lumen, which allows these cells to control vascular tone by contracting and relaxing. It is essential that this circumferential alignment is recapitulated in tissue engineered blood vessels. While many methods have been reported to align cells on 2D polymeric substrates, few techniques enable cell alignment on a 3D physiologically relevant hydrogel substrate. We hypothesized that the ridges inherent to the sides of fused deposition modeling 3D printed molds could be used to topographically pattern both stiff and soft substrates and thereby align cells on flat and curved surfaces. Flat and curved molds with 150, 250, and 350 µm ridges were 3D printed and used to topographically pattern polydimethylsiloxane and gelatin-methacryloyl. The ridges transferred to both substrates with less than 10% change in ridge size. Vascular smooth muscle cells were then seeded on each substrate, and nuclear and actin alignment were quantified. Cells were highly aligned with the molded ridges to a similar extent on both the stiffer polydimethylsiloxane and the softer gelatin-methacryloyl substrates. These data confirm that fused deposition modeling 3D printed molds are a rapid, cost-effective way to topographically pattern stiff and soft substrates in varied 3D shapes. This method will enable investigators to align cells on 3D polymeric and hydrogel structures for tissue engineering and other applications.

Simulating the Architecture of the Actin Cortex during Bleb Nucleation

Blebs are round membrane protrusions involved in important cellular processes such as cell motility, cytokinesis, and apoptosis. Recent studies have focused on the importance of cells that use blebs for cell migration in 3D fibrous environments. Blebs are initiated either by a local delamination of the actin cortex from the membrane due to loss of membrane-cortex adhesion proteins or by a local disruption of the actin cortex. In either case, the cortex degrades in the region of bleb initiation and then reassembles in the bleb. Myosin contractility has been shown to be necessary for bleb formation. It has further been hypothesized that a localized increase in myosin contractility on the cortex results in a rupture of the actin network that can lead to bleb nucleation. A minimal model consisting of actin filaments, myosin molecular motors, and actin cross-linker proteins is used to quantify conditions where holes, or defects, can develop in an initially isotropic cytoskeletal network. The model is simulated using the Cytosim computational platform. Results show that increased myosin fosters actin alignment, while increasing the concentration of actin cross-linkers decreases the pore size of the cytoskeletal network. Holes in the actin network develop at relatively high concentration of both cross-linkers and myosin. Simulation results suggest that a spatially localized defect, representing cortical actin network architecture during bleb nucleation, requires localized actin depolymerization rather than localized distributions of cross-linkers and myosin.

WHAMM is essential for spindle formation and spindle actin polymerization in maturing mouse oocytes

ABSTRACT WHAMM (WAS Protein Homolog Associated with Actin, Golgi Membranes, and Microtubules) is involved in Golgi membrane association, microtubule binding, and actin nucleation as a nucleation-promoting factor, which activates the actin-related protein 2/3 complex (the Arp2/3 complex). However, the role of WHAMM in mammalian oocyte maturation is poorly understood. The presence of WHAMM mRNA and protein during all stages of mouse oocyte maturation has been verified. It is mainly co-localized with the actin cage permeating the spindle during mouse oocyte maturation. Through the knockdown of WHAMM, we confirmed that it regulates spindle formation and affects the localization of the microtubule-organizing center (MTOC) during the early stages of spindle formation. Moreover, depletion of WHAMM impaired the formation of the spindle actin and chromosome alignment, which might be the cause of chromosomal aneuploidy and abnormal, asymmetric division. Treatment with brefeldin A (BFA), an inhibitor of vesicle transport from the endoplasmic reticulum (ER) to the Golgi apparatus, induced abnormal and dispersed localization of WHAMM. Taken together, these findings show that WHAMM is an essential component of the actin cytoskeleton machinery and plays a crucial role in oocyte maturation, presumably by controlling the formation of spindles with normal length by activating the formation of the spindle actin via the Arp2/3 complex.

3D Microwell Platforms for Control of Single Cell 3D Geometry and Intracellular Organization.

Cell structure and migration is impacted by the mechanical properties and geometry of the cell adhesive environment. Most studies to date investigating the effects of 3D environments on cells have not controlled geometry at the single-cell level, making it difficult to understand the influence of 3D environmental cues on single cells. Here, we developed microwell platforms to investigate the effects of 2D vs. 3D geometries on single-cell F-actin and nuclear organization. We used microfabrication techniques to fabricate three polyacrylamide platforms: 3D microwells with a 3D adhesive environment (3D/3D), 3D microwells with 2D adhesive areas at the bottom only (3D/2D), and flat 2D gels with 2D patterned adhesive areas (2D/2D). We measured geometric swelling and Young's modulus of the platforms. We then cultured C2C12 myoblasts on each platform and evaluated the effects of the engineered microenvironments on F-actin structure and nuclear shape. We tuned the mechanical characteristics of the microfabricated platforms by manipulating the gel formulation. Crosslinker ratio strongly influenced geometric swelling whereas total polymer content primarily affected Young's modulus. When comparing cells in these platforms, we found significant effects on F-actin and nuclear structures. Our analysis showed that a 3D/3D environment was necessary to increase actin and nuclear height. A 3D/2D environment was sufficient to increase actin alignment and nuclear aspect ratio compared to a 2D/2D environment. Using our novel polyacrylamide platforms, we were able to decouple the effects of 3D confinement and adhesive environment, finding that both influenced actin and nuclear structure.

Regulation of calcium dynamics and propagation velocity by tissue microstructure in engineered strands of cardiac tissue.

Disruptions to cardiac tissue microstructure are common in diseased or injured myocardium and are known substrates for arrhythmias. However, we have a relatively coarse understanding of the relationships between myocardial tissue microstructure, propagation velocity and calcium cycling, due largely to the limitations of conventional experimental tools. To address this, we used microcontact printing to engineer strands of cardiac tissue with eight different widths, quantified several structural and functional parameters and established correlation coefficients. As strand width increased, actin alignment, nuclei density, sarcomere index and cell aspect ratio decreased with unique trends. The propagation velocity of calcium waves decreased and the rise time of calcium transients increased with increasing strand width. The decay time constant of calcium transients decreased and then slightly increased with increasing strand width. Based on correlation coefficients, actin alignment was the strongest predictor of propagation velocity and calcium transient rise time. Sarcomere index and cell aspect ratio were also strongly correlated with propagation velocity. Actin alignment, sarcomere index and cell aspect ratio were all weak predictors of the calcium transient decay time constant. We also measured the expression of several genes relevant to propagation and calcium cycling and found higher expression of the genes that encode for connexin 43 (Cx43) and a subunit of L-type calcium channels in thin strands compared to isotropic tissues. Together, these results suggest that thinner strands have higher values of propagation velocity and calcium transient rise time due to a combination of favorable tissue microstructure and enhanced expression of genes for Cx43 and L-type calcium channels. These data are important for defining how microstructural features regulate intercellular and intracellular calcium handling, which is needed to understand mechanisms of propagation in physiological situations and arrhythmogenesis in pathological situations.

Static spatial growth restriction micropatterning of endothelial colony forming cells influences their morphology and gene expression.

BackgroundEndothelialization of small diameter synthetic vascular grafts is a potential solution to the thrombosis and intimal hyperplasia that plague current devices. Endothelial colony forming cells, which are blood-derived and similar to mature endothelial cells, are a potential cell source. Anisotropic spatial growth restriction micropatterning has been previously shown to affect the morphology and function of mature endothelial cells in a manner similar to unidirectional fluid shear stress. To date, endothelial colony forming cells have not been successfully micropatterned. This study addresses the hypothesis that micropatterning of endothelial colony forming cells will induce morphological elongation, cytoskeletal alignment, and changes in immunogenic and thrombogenic–related gene expression.MethodsSpatially growth restrictive test surfaces with 25 μm-wide lanes alternating between collagen-I and a blocking polymer were created using microfluidics. Case-matched endothelial colony forming cells and control mature carotid endothelial cells were statically cultured on either micropatterned or non-patterned surfaces. Cell elongation was quantified using shape index. Using confocal microscopy, cytoskeletal alignment was visualized and density and apoptotic rate were determined. Gene expression was measured using quantitative PCR to measure KLF-2, eNOS, VCAM-1, and vWF.ResultsEndothelial colony forming cells were successfully micropatterned for up to 50 hours. Micropatterned cells displayed elongation and actin alignment. Micropatterning increased the packing densities of both cell types, but did not affect apoptotic rate, which was lower in endothelial colony forming cells. KLF-2 gene expression was increased in micropatterned relative to non-patterned endothelial colony forming cells after 50 hours. No significant differences were seen in the other genes tested.ConclusionsEndothelial colony forming cells can be durably micropatterned using spatial growth restriction. Micropatterning has a significant effect on the gross and subcellular morphologies of both cell types. Further study is required to fully understand the effect of micropatterning on endothelial colony forming cell gene expression.

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.

Emergent patterns of collective cell migration under tubular confinement

Collective epithelial behaviors are essential for the development of lumens in organs. However, conventional assays of planar systems fail to replicate cell cohorts of tubular structures that advance in concerted ways on out-of-plane curved and confined surfaces, such as ductal elongation in vivo. Here, we mimic such coordinated tissue migration by forming lumens of epithelial cell sheets inside microtubes of 1–10 cell lengths in diameter. We show that these cell tubes reproduce the physiological apical–basal polarity, and have actin alignment, cell orientation, tissue organization, and migration modes that depend on the extent of tubular confinement and/or curvature. In contrast to flat constraint, the cell sheets in a highly constricted smaller microtube demonstrate slow motion with periodic relaxation, but fast overall movement in large microtubes. Altogether, our findings provide insights into the emerging migratory modes for epithelial migration and growth under tubular confinement, which are reminiscent of the in vivo scenario.

Human aortic endothelial cells respond to shear flow in well-plate microfluidic devices.

Although chronic progressive cardiovascular diseases such as atherosclerosis are often challenging to fully model in vitro, it has been shown that certain in vitro methods can effectively evaluate some aspects of disease progression. This has been demonstrated in in vitro and in vivo studies of endothelial cells that have illustrated the effects of nitric oxide (NO) production, filamentous actin (F-actin) formation, and cell and actin angle alignment on vascular function and homeostasis. Systems utilising shear flow have been established, in order to create a physiologically relevant environment for cells that require shear flow for homeostasis. Here, we investigated the use of a well-plate microfluidic system and associated devices (0-20dyn/cm²) to demonstrate applied shear effects on primary Human Aortic Endothelial Cells (HAECs). Changes in cell and actin alignment in the direction of flow, real-time production of NO and gross cell membrane shape changes in response to physiological shear flow were observed. These commercial systems have a range of potential applications, including within the consumer and pharmaceutical industries, thereby reducing the dependency on animal testing for regulatory safety assessments.

A magnetically actuated cellular strain assessment tool for quantitative analysis of strain induced cellular reorientation and actin alignment.

Commercially available cell strain tools, such as pneumatically actuated elastomer substrates, require special culture plates, pumps, and incubator setups. In this work, we present a magnetically actuated cellular strain assessment tool (MACSAT) that can be implemented using off-the-shelf components and conventional incubators. We determine the strain field on the MACSAT elastomer substrate using numerical models and experimental measurements and show that a specific region of the elastomer substrate undergoes a quasi-uniaxial 2D stretch, and that cells confined to this region of the MACSAT elastomer substrate undergo tensile, compressive, or zero axial strain depending on their angle of orientation. Using the MACSAT to apply cyclic strain on endothelial cells, we demonstrate that actin filaments within the cells reorient away from the stretching direction, towards the directions of minimum axial strain. We show that the final actin orientation angles in strained cells are spread over a region of compressive axial strain, confirming previous findings on the existence of a varied pre-tension in the actin filaments of the cytoskeleton. We also demonstrate that strained cells exhibit distinctly different values of actin alignment coherency compared to unstrained cells and therefore propose that this parameter, i.e., the coherency of actin alignment, can be used as a new readout to determine the occurrence/extent of actin alignment in cell strain experiments. The tools and methods demonstrated in this study are simple and accessible and can be easily replicated by other researchers to study the strain response of other adherent cells.

The Effect of Gradations in Mineral Content, Matrix Alignment, and Applied Strain on Human Mesenchymal Stem Cell Morphology within Collagen Biomaterials.

The tendon-bone junction is a unique, mechanically dynamic, structurally graded anatomical zone, which transmits tensile loads between tendon and bone. Current surgical repair techniques rely on mechanical fixation and can result in high re-failure rates. A new class of collagen biomaterial that contains discrete mineralized and structurally aligned regions linked by a continuous interface to mimic the graded osteotendinous insertion has been recently described. Here the combined influence of graded biomaterial environment and increasing levels of applied strain (0%-20%) on mesenchymal stem cell (MSC) orientation and alignment have been reported. In osteotendinous scaffolds, which contain opposing gradients of mineral content and structural alignment characteristic of the native osteotendinous interface, MSC nuclear, and actin alignment is initially dictated by the local pore architecture, while applied tensile strain enhances cell alignment in the direction of strain. Comparatively, in layered scaffolds that do not contain any structural alignment cues, MSCs are randomly oriented in the unstrained condition, then become oriented in a direction perpendicular to applied strain. These findings provide an initial understanding of how scaffold architecture can provide significant, potentially competitive, feedback influencing MSC orientation under applied strain, and form the basis for future tissue engineering efforts to regenerate the osteotendinous enthesis.

MEDYAN: Mechanochemical Simulations of Contraction and Polarity Alignment in Actomyosin Networks.

Active matter systems, and in particular the cell cytoskeleton, exhibit complex mechanochemical dynamics that are still not well understood. While prior computational models of cytoskeletal dynamics have lead to many conceptual insights, an important niche still needs to be filled with a high-resolution structural modeling framework, which includes a minimally-complete set of cytoskeletal chemistries, stochastically treats reaction and diffusion processes in three spatial dimensions, accurately and efficiently describes mechanical deformations of the filamentous network under stresses generated by molecular motors, and deeply couples mechanics and chemistry at high spatial resolution. To address this need, we propose a novel reactive coarse-grained force field, as well as a publicly available software package, named the Mechanochemical Dynamics of Active Networks (MEDYAN), for simulating active network evolution and dynamics (available at www.medyan.org). This model can be used to study the non-linear, far from equilibrium processes in active matter systems, in particular, comprised of interacting semi-flexible polymers embedded in a solution with complex reaction-diffusion processes. In this work, we applied MEDYAN to investigate a contractile actomyosin network consisting of actin filaments, alpha-actinin cross-linking proteins, and non-muscle myosin IIA mini-filaments. We found that these systems undergo a switch-like transition in simulations from a random network to ordered, bundled structures when cross-linker concentration is increased above a threshold value, inducing contraction driven by myosin II mini-filaments. Our simulations also show how myosin II mini-filaments, in tandem with cross-linkers, can produce a range of actin filament polarity distributions and alignment, which is crucially dependent on the rate of actin filament turnover and the actin filament’s resulting super-diffusive behavior in the actomyosin-cross-linker system. We discuss the biological implications of these findings for the arc formation in lamellipodium-to-lamellum architectural remodeling. Lastly, our simulations produce force-dependent accumulation of myosin II, which is thought to be responsible for their mechanosensation ability, also spontaneously generating myosin II concentration gradients in the solution phase of the simulation volume.

The F-actin bundler α-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast.

The actomyosin contractile ring is a network of cross-linked actin filaments that facilitates cytokinesis in dividing cells. Contractile ring formation has been well characterized in Schizosaccharomyces pombe, in which the cross-linking protein α-actinin SpAin1 bundles the actin filament network. However, the specific biochemical properties of SpAin1 and whether they are tailored for cytokinesis are not known. Therefore we purified SpAin1 and quantified its ability to dynamically bind and bundle actin filaments in vitro using a combination of bulk sedimentation assays and direct visualization by two-color total internal reflection fluorescence microscopy. We found that, while SpAin1 bundles actin filaments of mixed polarity like other α-actinins, SpAin1 has lower bundling activity and is more dynamic than human α-actinin HsACTN4. To determine whether dynamic bundling is important for cytokinesis in fission yeast, we created the less dynamic bundling mutant SpAin1(R216E). We found that dynamic bundling is critical for cytokinesis, as cells expressing SpAin1(R216E) display disorganized ring material and delays in both ring formation and constriction. Furthermore, computer simulations of initial actin filament elongation and alignment revealed that an intermediate level of cross-linking best facilitates filament alignment. Together our results demonstrate that dynamic bundling by SpAin1 is important for proper contractile ring formation and constriction.

Cell Shapes and Actin Alignment of Mesenchymal Stem Cells on Different Elastic Matrices with the Same Surface Topography

Mesenchymal stem cells (MSCs) can differentiate into multiline age cells. Thus, many efforts have been focused on finding effective ways to selectively differentiate the pluripotent cells into defined lineagecommitted progenitor cells for their suitable use in regenerative medicine. Some studies showed that the lineage of MSCs was influenced by the surface topography where the cells were attached, while other studies showed that its lineage was decided by the rigidity of the matrix where the cells interfaced. Our study showed that both the surface topography and rigidity of the matrix simultaneously affected the shapes of MSCs through the alignment and organization of actin cytoskeletons when MSCs were grown on either soft (1 M Pa) or hard (2 ~ 5 GPa) polymers with groove patterns in various dimensions ranging from 500 nm to 50 μm. Cells were mostly aligned and elongated along all the patterns irrespective of the rigidity of the substrate. However, the degree of the elongation was affected by both the matrix rigidity and dimension of the patterns. This is further supported by the results showing that the orientation of alignment of actin cytoskeletons and their organization were affected by both the rigidity and the topography of the substrate.