Orientation Of Actin Filaments Research Articles

Mechanoresponsive Alignment of Molecular Self-Assembled Negatively Charged Nanofibrils.

Inspired by the mechanoresponsive orientation of actin filaments in cell, we introduce a design paradigm of synthetic molecular self-assembling fibrils that respond to external mechanical force by transforming from a macroscopically disorder state to a highly ordered uniaxial aligned state. The incorporation of aromatic-containing amino acids and negatively charged amino acids lead to self-assembly motifs that transform into uniform nanofibrils in acidic solution. Adjusting the pH level of aqueous solution introduces optimal negative charge to the surface of self-assembling nanofibrils inducing long-range electrostatic repulsion forming a nematic phase. Upon external mechanical force, nanofibrils align in the force direction. Via evaporation casting in capillary confinement, the solvated synthetic self-assembling nanofibrils transform into scalable lamellar domains. Adjusting capillary geometry and drying procedure offers further parameters for tuning the mesoscale alignment of nanofibrils generating a variety of interference colors. The design paradigm of mechanoresponsive alignment of self-assembled nanofibrils as an addition of nanofabrication techniques is potentially employable for realizing biomimetic optical structures.

Localization of actin filaments and cortical microtubules in wood-forming tissues of conifers

ABSTRACT The aim of the present study was to investigate the orientation and localization of actin filaments and cortical microtubules in wood-forming tissues in conifers to understand wood formation. Small blocks were collected from the main stems of Abies firma, Pinus densiflora, and Taxus cuspidata during active seasons of the cambium. Bundles of actin filaments were oriented axially or longitudinally relative to the cell axis in fusiform and ray cambial cells. In differentiating tracheids, actin filaments were oriented longitudinally relative to the cell axis during primary and secondary wall formation. In contrast, the orientation of well-ordered cortical microtubules in tracheids changed from transverse to longitudinal during secondary wall formation. There was no clear relationship between the orientation of actin filaments and cortical microtubules in cambial cells and cambial derivatives. Aggregates of actin filaments and a circular band of cortical microtubules were localized around bordered pits and cross-field pits in differentiating tracheids. In addition, rope-like bands of actin filaments were observed during the formation of helical thickenings at the final stage of formation of secondary walls in tracheids. Actin filaments might not play a major role in changes in the orientation of cortical microtubules in wood-forming tissues. However, since actin filaments were co-localized with cortical microtubules during the formation of bordered pits, cross-field pits and helical thickenings at the final stage of formation of the secondary wall in tracheids, it seems plausible that actin filaments might be closely related to the localization of cortical microtubules during the development of these modifications of wood structure.

Aluminum toxicity-induced pollen tube growth inhibition in apple (Malus domestica) is mediated by interrupting calcium dynamics and modification of cell wall components

Aluminum (Al) is known the most abundant metal in the Earth’s crust which is a major problem limiting crop production and yield throughout the world. Al could inhibit pollen tube elongation, however the mechanism is largely unknown. The aim of this study was to investigate the influence of Al toxicity on Malus domestica pollen tube growth and possible cytological mechanism. The results showed that Al inhibited pollen germination and tube growth in a concentration dependent manner. An non-invasive ion-selective electrode technique together with fluorescent labeling indicated that Al toxicity decreased pollen tube apex calcium influx and disrupted [Ca2+]c gradient. Al toxicity also altered actin filament orientation. Results from immuno-localization and fluorescence labeling indicated that Al toxicity affected the accumulation and distribution of callose, acid pectins, esterified pectins and arabinogalactan proteins in pollen tubes, which were supported by Fourier-transform infrared analysis. In a word, Al induced a series of the cellular toxic changes concerning calcium influx, [Ca2+]c gradient, actin and cell wall components. Based on the information provided in this article, it is concluded that Al toxicity inhibits apical growth of pollen tubes by changing calcium influx and [Ca2+]c and cell wall components.

Guiding cellular activity with polarized light.

Actin, cytoskeleton protein forming microfilaments, play a crucial role in cellular motility. Here we show that exposure to very low levels of polarized light guide their orientation in-vivo within the live cell. Using a simple model to describe the role of actin-filament orientation in directional cellular motion, we demonstrate that the actin polymerization/depolymerization mechanism develops primarily along this direction and, under certain conditions, can lead to guidance of the cell movement. Our results also show a dose dependent increase in actin activity in direct correspondence to the level of laser irradiance. We found that total expression of Tau protein, which stabilize microtubules, was decreased by the irradiance, indicating that exposure to the light may change the activity of kinase, leading to increased cell activity.

In vivo dynamics of the cortical actin network revealed by fast-scanning atomic force microscopy

Together with lamellipodia and stress fibers, a dynamic network of actin filaments in the cell cortex plays a major role in the maintenance of cell morphology and motility. In contrast to lamellipodia, which have been well studied in various motile cells, the dynamics of actin filaments in the cell cortex have not yet been clarified due to a lack of proper imaging techniques. Here, we utilized high-speed atomic force microscopy for live-cell imaging and analyzed cortical actin dynamics in living cells. We successfully measured the polymerization rate and the frequency of filament synthesis in living COS-7 cells, and examined the associated effects of various inhibitors and actin-binding proteins. Actin filaments are synthesized beneath the plasma membrane and eventually descend into the cytoplasm. The inhibitors, cytochalasin B inhibited the polymerization, while jasplakinolide, inhibited the turnover of actin filaments as well as descension of the newly synthesized filaments, suggesting that actin polymerization near the membrane drives turnover of the cortical actin meshwork. We also determined how actin turnover is maintained and regulated by the free G-actin pool and G-actin binding proteins such as profilin and thymosin β4, and found that only a small amount of free G-actin was present in the cortex. Finally, we analyzed several different cell types, and found that the mesh size and the orientation of actin filaments were highly divergent, indicating the involvement of various actin-binding proteins in the maintenance and regulation of cortical actin architecture in each cell type.

Trajectories of Gliding Filaments on Optically Patterned Myosin Surfaces

Interfaces are ubiquitous in biology. For a sub-cellular component moving inside a cell any change in its local environment across an interface whether chemical concentration, density, or other physical variables can produce novel dynamics. Recent advances in bioengineering allow us to control motor proteins’ velocities when prompted by an optical trigger. Using a photoactive LOV-domain in a myosin-XI construct and an optical diaphragm we can spatially pattern light to create interfaces across which speed of a gliding actin filament can differ as much as by a factor of two. We observe that when a gliding actin filament crosses an interface that has a discontinuous velocity jump, it buckles and changes its angle of orientation due to the velocity mismatch. We tracked the position and orientation of actin filaments using an in-house MATLAB tracking algorithm and measured that, for small angles of incidence, the angle of emergence increases linearly. If we increase the angle of incidence further we observe that the angle of emergence saturates. For actin filaments approaching the interface near-tangentially we observe total internal reflection as they fail to cross over the boundary. We have modeled our system using Cytosim software package and find excellent agreement with experimental data. These results have given insight into the dynamics of actin-myosin mechanics and have resulted in a better understanding of how to control direction changes in filaments and create dynamically programmable gliding trajectories.

Vinculin forms a Catch Bond with F-Actin that Depends on Actin Filament Orientation

Vinculin forms a Catch Bond with F-Actin that Depends on Actin Filament Orientation

Coronin 1C-free primary mouse fibroblasts exhibit robust rearrangements in the orientation of actin filaments, microtubules and intermediate filaments

Coronin 1C is an established modulator of actin cytoskeleton dynamics. It has been shown to be involved in protrusion formation, cell migration and invasion. Here, we report the generation of primary fibroblasts from coronin 1C knock-out mice in order to investigate the impact of the loss of coronin 1C on cellular structural organisation. We demonstrate that the lack of coronin 1C not only affects the actin system, but also the microtubule and the vimentin intermediate filament networks. In particular, we show that the knock-out cells exhibit a reduced proliferation rate, impaired cell migration and protrusion formation as well as an aberrant subcellular localisation and function of mitochondria. Moreover, we demonstrate that coronin 1C specifically interacts with the non-α-helical amino-terminal domain (“head”) of vimentin. Our data suggest that coronin 1C acts as a cytoskeletal integrator of actin filaments, microtubules and intermediate filaments.

Hyaluronan-positive plasma membrane protrusions exist on mesothelial cells in vivo.

Previous observations of our research group showed that HAS2 and HAS3 overexpression in cultured cells induces the formation of long and numerous microvillus-like cell protrusions, which are present also in cultured cell types with naturally high hyaluronan secretion and the cell protrusions resemble those found in mesothelial cells. The aim of this study was to investigate whether these hyaluronan secreting, actin-dependent protrusions exist also in vivo. It was found that rat mesothelium in vivo is positive for hyaluronan and Has1-3. Also microvilli in rat mesothelium and live primary cultures of mesothelial cells were found to be hyaluronan positive, and the cells expressed all Has isoforms. Furthermore, ultrastructure of the cell protrusions in rat mesothelium was similar to that induced by overexpression of HAS2 and HAS3, and the number and orientation of actin filaments supporting the cell protrusions was identical. The results of this study show that HA-positive protrusions exist in vivo and support the idea that hyaluronan secretion from plasma membrane protrusions is a general process. This mechanism is potentially crucial for the normal function and maintenance of tissues and body fluids and may be utilized in many therapeutic applications.

The non-processive rice kinesin-14 OsKCH1 transports actin filaments along microtubules with two distinct velocities.

Microtubules and actin filaments function coordinately in many cellular processes(1-3). Although much of this coordination is mediated by proteins that statically bridge the two cytoskeletal networks(4-6), kinesin-14 motors with an actin binding calponin homology domain (KCHs) have been discovered as putatively dynamic crosslinkers in plants(7,8). OsKCH1, a KCH from rice, interacts with both microtubules and actin filaments in vivo and in vitro(9). However, it has remained unclear whether this interaction is dynamic or if actin binding reduces or even abolishes the motor's motility on microtubules(10,11). Here, we directly show in vitro that OsKCH1 is a non-processive, minus-end-directed motor that transports actin filaments along microtubules. Interestingly, we observe two distinct transport velocities dependent on the relative orientation of the actin filaments with respect to the microtubules. In addition, torsional compliance measurements on individual molecules reveal low flexibility in OsKCH1. We suggest that the orientation-dependent transport velocities emerge from OsKCH1's low torsional compliance combined with an inherently oriented binding to the actin filament. Together, our results imply a central role of OsKCH1 in the polar orientation of actin filaments along microtubules, and thus a contribution to the organization of the cytoskeletal architecture.

Homocysteine Disrupts Outgrowth of Microvascular Endothelium by an iNOS‐Dependent Mechanism

To test the hypothesis that Hcy impairs angiogenic outgrowth through an iNOS-dependent mechanism. Adult C57Bl/6 mouse choroid explants were used in angiogenic outgrowth assays. Mouse microvascular endothelial cells were studied in culture during scrape-induced migration and dispersed cell locomotion experiments. Activity of iNOS was manipulated with pharmacology (1400 W), siRNA, and by use of choroid explants from iNOS knockout mice (iNOS(-/-)). Hcy (20 μM) significantly decreased the area of endothelial outgrowth without altering the number of cells in the choroid explant angiogenic assay, resulting in more densely packed outgrowth. Hcy prevented the outward orientation of actin filaments and decreased the number of actin projections along the leading edge of outgrowth. Hcy also slowed outgrowth from the edge of a scraped endothelial monolayer and in cultures of dispersed cells, Hcy impaired cell locomotion without affecting proliferation. Inhibition of iNOS activity rescued the effect of Hcy on area of explant outgrowth, cell density, number of projections, cell locomotion, and rate of outgrowth following scraping. Hcy impairs microvascular endothelial outgrowth, but not proliferation, by disrupting cell locomotion through an iNOS-dependent mechanism.

Specific Sorting and Post-Golgi Trafficking of Dendritic Potassium Channels in Living Neurons

Proper membrane localization of ion channels is essential for the function of neuronal cells. Particularly, the computational ability of dendrites depends on the localization of different ion channels in specific subcompartments. However, the molecular mechanisms that control ion channel localization in distinct dendritic subcompartments are largely unknown. Here, we developed a quantitative live cell imaging method to analyze protein sorting and post-Golgi vesicular trafficking. We focused on two dendritic voltage-gated potassium channels that exhibit distinct localizations: Kv2.1 in proximal dendrites and Kv4.2 in distal dendrites. Our results show that Kv2.1 and Kv4.2 channels are sorted into two distinct populations of vesicles at the Golgi apparatus. The targeting of Kv2.1 and Kv4.2 vesicles occurred by distinct mechanisms as evidenced by their requirement for specific peptide motifs, cytoskeletal elements, and motor proteins. By live cell and super-resolution imaging, we identified a novel trafficking machinery important for the localization of Kv2.1 channels. Particularly, we identified non-muscle myosin II as an important factor in Kv2.1 trafficking. These findings reveal that the sorting of ion channels at the Golgi apparatus and their subsequent trafficking by unique molecular mechanisms are crucial for their specific localizations within dendrites.

Orientation of actin filaments in teleost retinal pigment epithelial cells, and the effect of the lectin, Concanavalin A, on melanosome motility.

Retinal pigment epithelial cells of teleosts contain numerous melanosomes (pigment granules) that exhibit light-dependent motility. In light, melanosomes disperse out of the retinal pigment epithelium (RPE) cell body (CB) into long apical projections that interdigitate with rod photoreceptors, thus shielding the photoreceptors from bleaching. In darkness, melanosomes aggregate through the apical projections back into the CB. Previous research has demonstrated that melanosome motility in the RPE CB requires microtubules, but in the RPE apical projections, actin filaments are necessary and sufficient for motility. We used myosin S1 labeling and platinum replica shadowing of dissociated RPE cells to determine actin filament polarity in apical projections. Actin filament bundles within RPE apical projections are uniformly oriented with barbed ends toward the distal tips. Treatment of RPE cells with the tetravalent lectin, Concanavalin A, which has been shown to suppress cortical actin flow by crosslinking of cell-surface proteins, inhibited melanosome aggregation and stimulated ectopic filopodia formation but did not block melanosome dispersion. The polarity orientation of F-actin in apical projections suggests that a barbed-end directed myosin motor could effect dispersion of melanosomes from the CB into apical projections. Inhibition of aggregation, but not dispersion, by ConA confirms that different actin-dependent mechanisms control these two processes and suggests that melanosome aggregation is sensitive to treatments previously shown to disrupt actin cortical flow.

Big Cells Cleave as Fast as Small Ones: the Physics of Cytokinesis

Big Cells Cleave as Fast as Small Ones: the Physics of Cytokinesis

Directed Actin Assembly and Contractility

The organization of actin filaments into higher-ordered structures governs eukaryotic cell shape and movement. Global actin network size and architecture is maintained in a dynamic steady-state through regulated assembly and disassembly. We have developed a micropatterning method that enables the spatial control of actin nucleation sites for in vitro assays (Reymann, Nat Mat, 2010). These actin templates were used to evaluate the response of oriented actin structures to myosin-induced contractility. We determine that myosins selectively contract and disassemble anti-parallel actin structures while parallel actin bundles remain unaffected. In addition, the local distribution of nucleation sites and the resulting orientation of actin filaments regulate the scalability of the contraction process. This “orientation selection” mechanism for selective contraction and disassembly reveals how the dynamics of the cellular actin cytoskeleton is spatially controlled by actomyosin contractility. Further application of the micropatterning method will be presented in particular recent data on the reconstitution of a lamellipodium-type of actin organization and the fabrication of three-dimensional electrical connections by means of directed actin self-organization.

Homocysteine disrupts actin organization and vascular outgrowth by an iNOS‐dependent mechanism during angiogenesis

ObjectiveHyperhomocysteinemia (HHcy) impairs angiogenesis. The goal of this study was to test the hypothesis that HHcy impairs angiogenic growth through an iNOS dependent mechanism.MethodsWe used an ex‐vivo adult mouse choroid tissue model of VEGF‐stimulated angiogenesis. Mouse microvascular endothelial cells were studied in culture in parallel experiments.Results20μM homocysteine (Hcy) treatments significantly decreased the area of angiogenic outgrowth (8.9mm2 ± 0.31 vs 4.1mm2 ± 0.58; p<0.05). Hcy 1) canted the orientation of actin filaments (orientation in relation to explant center: 38.6ܰ±13.0ܰ vs 95.4ܰ± 6.5ܰ; p<0.05), 2) decreased the occurrence of cellular extensions beyond the leading edge of outgrowth (7 ± 0.89 vs 2.2 ±0.52; p<0.05), and 3) reduced the motility of dispersed cells (19.8μm/hr ±1.4 vs 13.1μm/hr ±1.7; p<0.05). Inhibition of iNOS with 1400W (10μM) rescued the effect of Hcy on area of outgrowth, occurrence of cellular extensions, and cell motility.ConclusionsHcy decreases angiogenic outgrowth by inhibiting motility and disrupting actin filament organization in part via an iNOS‐dependent mechanism. This work was supported by NIH grant #HL106548(SEB).

Networks of Polarized Actin Filaments in the Axon Initial Segment Provide a Mechanism for Sorting Axonal and Dendritic Proteins

Trafficking of proteins specifically to the axonal or somatodendritic membrane allows neurons to establish and maintain polarized compartments with distinct morphology and function. Diverse evidence suggests that an actin-dependent vesicle filter within the axon initial segment (AIS) plays a critical role in polarized trafficking; however, no distinctive actin-based structures capable of comprising such a filter have been found within the AIS. Here, using correlative light and scanning electron microscopy, we visualized networks of actin filaments several microns wide within the AIS of cortical neurons in culture. Individual filaments within these patches are predominantly oriented with their plus ends facing toward the cell body, consistent with models of filter selectivity. Vesicles carrying dendritic proteins are much more likely to stop in regions occupied by the actin patches than in other regions, indicating that the patches likely prevent movement of dendritic proteins to the axon and thereby act as a vesicle filter.

Single Amino Acid Substitutions in the Chemotactic Sequence of Urokinase Receptor Modulate Cell Migration and Invasion

The receptor for urokinase-type plasminogen activator (uPAR) plays an important role in controlling cell migration. uPAR binds urokinase and vitronectin extracellular ligands, and signals in complex with transmembrane receptors such as Formyl-peptide Receptors (FPR)s and integrins. Previous work from this laboratory has shown that synthetic peptides, corresponding to the uPAR88–92 chemotactic sequence, when carrying the S90P or S90E substitutions, up- or down-regulate cell migration, respectively. To gain mechanistic insights into these opposite cell responses, the functional consequences of S90P and S90E mutations in full-length uPAR were evaluated. First, (HEK)-293 embryonic kidney cells expressing uPARS90P exhibit enhanced FPR activation, increased random and directional cell migration, long-lasting Akt phosphorylation, and increased adhesion to vitronectin, as well as uPAR/vitronectin receptor association. In contrast, the S90E substitution prevents agonist-triggered FPR activation and internalization, decreases binding and adhesion to vitronectin, and inhibits uPAR/vitronectin receptor association. Also, 293/uPARS90P cells appear quite elongated and their cytoskeleton well organized, whereas 293/uPARS90E cells assume a large flattened morphology, with random orientation of actin filaments. Interestingly, when HT1080 cells co-express wild type uPAR with uPAR S90E, the latter behaves as a dominant-negative, impairing uPAR-mediated signaling and reducing cell wound repair as well as lung metastasis in nude mice. In contrast, signaling, wound repair and in vivo lung metastasis of HT1080 cells bearing wild type uPAR are enhanced when they co-express uPARS90P. In conclusion, our findings indicate that Ser90 is a critical residue for uPAR signaling and that the S90P and S90E exert opposite effects on uPAR activities. These findings may be accommodated in a molecular model, in which uPARS90E and uPARS90P are forced into inactive and active forms, respectively, suggesting important implications for the development of novel drugs targeting uPAR function.

The Geometry of Branching Actin Networks from Capping Branching and Filament Growth

Branching actin network growth is the primary engine driving cell motility. At the front of a motile cell, inside the lamellipodium, is a dense mesh of actin filaments pushing the membrane forward. The structure of the mesh is believed to be largely regulated by three processes: branching of new filaments off of existing ones, capping of filament tips stopping filament growth, and filament growth. Filaments inside the lamellipodium have been observed to organize into a strict orientation pattern where filaments are angled approximately −35/35 degrees from the direction normal to the membrane. It has been previously hypothesized that the three processes above are sufficient to generate the unique orientation pattern.We derive and analyze an integro-differential PDE for the angular density of branching actin network by incorporating the three constituent processes. Our analysis implies that there exist multiple equilibrium angular distributions, which strongly suggests additional process that regulates actin filament orientation.

Modelling planar polarity of epithelia: the role of signal relay in collective cell polarization

Collective cell polarization is an important characteristic of tissues. Epithelia commonly display cellular structures that are polarized within the plane of the tissue. Establishment of this planar cell polarity requires mechanisms that locally align polarized structures between neighbouring cells, as well as cues that provide global information about alignment relative to an axis of a tissue. In the Drosophila ovary, the cadherin Fat2 is required to orient actin filaments located at the basal side of follicle cells perpendicular to the long axis of the egg chamber. The mechanisms directing this orientation of actin filaments, however, remain unknown. Here we show, using genetic mosaic analysis, that fat2 is not essential for the local alignment of actin filaments between neighbouring cells. Moreover, we provide evidence that Fat2 is involved in the propagation of a cue specifying the orientation of actin filaments relative to the tissue axis. Monte Carlo simulations of actin filament orientation resemble the results of the genetic mosaic analysis, if it is assumed that a polarity signal can propagate from a signal source only through a connected chain of wild-type cells. Our results suggest that Fat2 is required for propagating global polarity information within the follicle epithelium through direct cell-cell contact. Our computational model might be more generally applicable to study collective cell polarization in tissues.