Cytoskeletal Density Research Articles

Label-free monitoring of spatiotemporal changes in the stem cell cytoskeletons in time-lapse phase-contrast microscopy.

Investigation of the dynamic structural changes in the actin cytoskeleton during cell migration provides crucial information about the physiological conditions of a stem cell during in-vitro culture. Here we proposed a quantitative analytical model associated with texture extraction with cell tracking techniques for in situ monitoring of the cytoskeletal density change of stem cells in phase-contrast microscopy without fluorescence staining. The reliability of the model in quantifying the texture density with different orientation was first validated using a series of simulated textural images. The capability of the method to reflect the spatiotemporal regulation of the cytoskeletal structure of a living stem cell was further proved by applying it to a set of 72 h phase-contrast microscopic video of the growth dynamics of mesenchymal stem cells in vitro culture.

Constitutive Model of Erythrocyte Membranes with Distributions of Spectrin Orientations and Lengths

We present an analytical hyperelastic constitutive model of the red blood cell (erythrocyte) membrane based on recently improved characterizations of density and microscopic structure of its spectrin network from proteomics and cryo-electron tomography. The model includes distributions of both orientations and natural lengths of spectrin and updated copy numbers of proteins. By applying finite deformation to the spectrin network, we obtain the total free energy and stresses in terms of invariants of shear and area deformation. We generalize an expression of the initial shear modulus, which is independent of the number of molecular orientations within the network and also derive a simplified version of the model. We apply the model and its simplified version to analyze micropipette aspiration computationally and analytically and explore the effect of local cytoskeletal density change. We also explore the discrepancies among shear modulus values measured using different experimental techniques reported in the literature. We find that the model exhibits hardening behavior and can explain many of these discrepancies. Moreover, we find that the distribution of natural lengths plays a crucial role in the hardening behavior when the correct copy numbers of proteins are used. The initial shear modulus values we obtain using our current model (5.9–15.6 pN/μm) are close to the early estimates (6–9 pN/μm). This new, to our knowledge, constitutive model establishes a direct connection between the molecular structure of spectrin networks and constitutive laws and also defines a new picture of a much denser spectrin network than assumed in prior studies.

Tracking the 3D Rotational Dynamics in Nanoscopic Biological Systems.

The rotation of an object cannot be fully tracked without understanding a set of three angles, namely, roll, pitch, and yaw. Tracking these angles as a three-degrees-of-freedom (3-DoF) rotation is a fundamental measurement, facilitating, for example, attitude control of a ship, image stabilization to reduce camera shake, and self-driving cars. Until now, however, there has been no method to track 3-DoF rotation to measure nanometer-scale dynamics in biomolecules and live cells. Here we show that 3-DoF rotation of biomolecules can be visualized via nitrogen-vacancy centers in a fluorescent nanodiamond using a tomographic vector magnetometry technique. We demonstrate application of the method to three different types of biological systems. First, we tracked the rotation of a single molecule of the motor protein F1-ATPase by attaching a nanodiamond to the γ-subunit. We visualized the 3-step rotation of the motor in 3D space and, moreover, a delay of ATP binding or ADP release step in the catalytic reaction. Second, we attached a nanodiamond to a membrane protein in live cells to report on cellular membrane dynamics, showing that 3D rotational motion of the membrane protein correlates with intracellular cytoskeletal density. Last, we used the method to track nonrandom motions in the intestine of Caenorhabditis elegans. Collectively, our findings show that the method can record nanoscale 3-DoF rotation in vitro, in cells, and even in vivo. 3-DoF rotation tracking introduces a new perspective on microscopic biological samples, revealing in greater detail the functional mechanisms due to nanoscale dynamics in molecules and cells.

Equilibrium Structure and Mechanics of the Cellular Glycocalyx

Cells can employ distinct modes of cell migration, ranging from mesenchymal-like or amoeboid-like single cell motility to collective migration. Each mode is characterized by a unique balance of cell-matrix and cell-cell adhesion. Carbohydrates called glycans are concentrated on the cell surface in a complex structure called the glycocalyx. While the glycocalyx is known to impede cell adhesion by acting as a steric barrier, the mechanical response of the glycocalyx and its molecular constituents to force is largely unknown. Therefore, we have developed a physical model of the glycocalyx to understand and predict its deformation and reorganization under compressive loads, such as those that would be experienced by cells in confined three-dimensional environments. Simulations of our model predict that the glycocalyx can sustain substantial pressures without undergoing significant deformations, but beyond a critical pressure it experiences a pressure-sensitive response, followed by increased resistance at high strains. This suggests that cells could switch between weakly adherent and highly adherent states depending on the degree of confinement and the compression of the glycocalyx. Additionally, the simulations predict the formation of load-bearing clusters of glycoproteins in favorable regions away from the cytoskeletal membrane attachments. We elucidate the effects of the biophysical properties of the glycocalyx and the cell membrane on the mechanical behavior. Increasing the stiffness and the density of glycoproteins and the membrane bending modulus and the cytoskeletal density or decreasing the glycoprotein length increases the collective stiffness of the glycocalyx and the pressure required to generate significant deformations. Together, our results demonstrate how bulk material properties of the glycocalyx emerge from the physical properties of its molecular constituents and how these constituents rearrange under load. The understanding developed here has broad relevance in cell migration, adhesion, communication, and other receptor-mediated processes in the glycocalyx.

Carbon Ion-Irradiated Hepatoma Cells Exhibit Coupling Interplay between Apoptotic Signaling and Morphological and Mechanical Remodeling.

A apoptotic model was established based on the results of five hepatocellular carcinoma cell (HCC) lines irradiated with carbon ions to investigate the coupling interplay between apoptotic signaling and morphological and mechanical cellular remodeling. The expression levels of key apoptotic proteins and the changes in morphological characteristics and mechanical properties were systematically examined in the irradiated HCC lines. We observed that caspase-3 was activated and that the Bax/Bcl-2 ratio was significantly increased over time. Cellular morphology and mechanics analyses indicated monotonic decreases in spatial sizes, an increase in surface roughness, a considerable reduction in stiffness, and disassembly of the cytoskeletal architecture. A theoretical model of apoptosis revealed that mechanical changes in cells induce the characteristic cellular budding of apoptotic bodies. Statistical analysis indicated that the projected area, stiffness, and cytoskeletal density of the irradiated cells were positively correlated, whereas stiffness and caspase-3 expression were negatively correlated, suggesting a tight coupling interplay between the cellular structures, mechanical properties, and apoptotic protein levels. These results help to clarify a novel arbitration mechanism of cellular demise induced by carbon ions. This biomechanics strategy for evaluating apoptosis contributes to our understanding of cancer-killing mechanisms in the context of carbon ion radiotherapy.

Cytopede: A Three-Dimensional Tool for Modeling Cell Motility on a Flat Surface

When cultured on flat surfaces, fibroblasts and many other cells spread to form thin lamellar sheets. Motion then occurs by extension of the sheet at the leading edge and retraction at the trailing edge. Comprehensive quantitative models of these phenomena have so far been lacking and to address this need, we have designed a three-dimensional code called Cytopede specialized for the simulation of the mechanical and signaling behavior of plated cells. Under assumptions by which the cytosol and the cytoskeleton are treated from a continuum mechanical perspective, Cytopede uses the finite element method to solve mass and momentum equations for each phase, and thus determine the time evolution of cellular models. We present the physical concepts that underlie Cytopede together with the algorithms used for their implementation. We then validate the approach by a computation of the spread of a viscous sessile droplet. Finally, to exemplify how Cytopede enables the testing of ideas about cell mechanics, we simulate a simple fibroblast model. We show how Cytopede allows computation, not only of basic characteristics of shape and velocity, but also of maps of cell thickness, cytoskeletal density, cytoskeletal flow, and substratum tractions that are readily compared with experimental data.

Integrative Experimental and Theoretical Approach Exposes Fundamental Mechanisms of J774 Macrophage Phagocytosis

Macrophage cell lines like murine J774 cells are ideal model systems to study phagocytosis. Our unique methodology combines single-cell/single-target experiments with advanced computer simulations to elucidate the fundamental mechanisms of J774 macrophage phagocytosis. As a first step, the baseline mechanical properties of the murine macrophages were established. Micropipette-aspiration experiments were used to characterize the cortical tension and cytoplasmic viscosity of the J774 cells. Next, we used a dual-micropipette manipulation system to quantify the time courses of a number of key parameters during Fcγ-target phagocytosis. A passive cell is selected and picked up with a micropipette by partial aspiration. Another micropipette is used to bring an opsonized target bead (5-30 um diameter) into soft contact with the cell, which usually results in immediate adhesion. The target is released and images of the ensuing phagocytosis are recorded directly to a computer hard disk. Their analysis provides the time course of cell morphology, bead position, and cortical tension. Intriguingly, the macrophages maintained a constant cortical tension when engulfing targets that required a surface area expansion of up to ∼250%, indicating an extremely large membrane reservoir. The tension rose when the cells increased their surface area by an amazing ∼550%. These and other experimental observations were compared side-by-side with finite-element models of macrophage phagocytosis. This comparison allows us to assess the viability of different mechanistic models used for phagocytosis. Optimal models include a repulsion between the cytoskeleton and the free membrane (which drives protrusion), and an attraction between cytoskeleton and surface membrane newly adherent to the target (which results in thin pseudopods). Confocal inspection of the engulfment of 20 um beads by GFP-actin transfected macrophages revealed excellent agreement between the cytoskeletal density predicted by the optimal model and the observed actin distribution.

Neurofilament high molecular weight–green fluorescent protein fusion is normally expressed in neurons and transported in axons: A neuronal marker to investigate the biology of neurofilaments

The carboxy-terminal side arm of the neurofilament high subunit consists of a highly phosphorylated domain and a negatively charged region. Multiple evidences suggested that these domains are essential for the axonal phosphorylation and transport of neurofilaments and play a role in their abnormal accumulation following chemical intoxication or during neurodegenerative disorders such as amyotrophic lateral sclerosis. In order to investigate the consequences of altering this side arm of neurofilament high subunit we used a fusion protein (neurofilament high subunit–green fluorescent protein) between the mouse neurofilament high subunit missing a major part of the C-terminal domain and the reporter green fluorescent protein. In cell culture and in transgenic mice this fusion protein co-assembles and co-distributes with the endogenous intermediate filament network. Conditions known to disturb the cytoskeleton were also found to alter the distribution of the fusion protein in cell cultures. In transgenic mice the expression of the transgene evaluated by its fluorescent properties was found to be restricted to neurons, where the neurofilament high subunit–green fluorescent protein fusion protein is axonally transported. Biochemical approaches showed that the fusion protein is phosphorylated and co-purified with neurofilaments. Despite the presence of such an neurofilament high subunit–green fluorescent protein fusion protein, the axonal cytoskeletal density and the axonal caliber were not altered. Together these data show that removal of this portion of neurofilament high subunit does not affect the capacity of neurofilament high subunit to assemble and to be transported into axons, suggesting that this sequence is involved in another function. Moreover, the fluorescent properties of this fusion protein represent a useful marker.

Review and proposed action of alpha‐fetoprotein growth inhibitory peptides as estrogen and cytoskeleton‐associated factors

The (H) human growth-promoting factor, alpha-fetoprotein (AFP), has been reported to possess a growth inhibitory motif as an occult epitope in the compactly folded circulating form of the protein. Intermediate unfolded forms of the human HAFP molecule induced by stress, shock, and high ligand concentrations have revealed the presence of an encrypted growth-suppressive segment on the third domain of HAFP. A purified linear synthetic 34-mer segment termed the "growth inhibitory peptide" (GIP) exhibits various oligomeric forms with complex aggregation behaviors, in which dominant trimeric forms were found to be suppressive in assays of estrogen-induced growth. While several amino acid analogs of the cysteines of the GIP retained inhibitory activity, heavy metal binding and pre-incubation of the peptides with a variety of cations and hormone ligands were found to influence the outcomes of growth bioassays. Smaller segments of the original 34-mer were each found to display growth activities of their own, with the middle segment (P149b) also showing hydrophobic dye-binding properties. Studies of amino acid sequence identity further revealed that the GIP sequences displayed identity/similarity matches to both cytoplasmic and nucleus-cytoskeleton-associated proteins, and experimental evidence served to support these findings. That is, the peptide was capable of modulating tubulin polymerization, cell shape, and cell-surface aggregation phenomena reminiscent of a microtubule-associated protein. Immunofluorescence studies further pinpointed the localization of the GIP to cytoplasmic regions of high cytoskeletal density in the cell. Because of the involvement of the GIP in experimental models of the estrogen receptor/cytoskeleton, a mechanism of action is forwarded in which the linear GIP is proposed to be a G-coupled receptor binding ligand that is translocated across the plasma membrane via receptor-mediated endocytosis. Thus, it was predicted that the linear GIP and possibly its peptidic segments serve as decoy ligands to cell-surface receptors in order to gain access to the cytoplasmic compartment of the cell.

Membrane Tether Formation from Blebbing Cells

Membrane tension has been proposed to be important in regulating cell functions such as endocytosis and cell motility. The apparent membrane tension has been calculated from tether forces measured with laser tweezers. Both membrane-cytoskeleton adhesion and membrane tension contribute to the tether force. Separation of the plasma membrane from the cytoskeleton occurs in membrane blebs, which could remove the membrane-cytoskeleton adhesion term. In renal epithelial cells, tether forces are significantly lower on blebs than on membranes that are supported by cytoskeleton. Furthermore, the tether forces are equal on apical and basolateral blebs. In contrast, tether forces from membranes supported by the cytoskeleton are greater in apical than in basolateral regions, which is consistent with the greater apparent cytoskeletal density in the apical region. We suggest that the tether force on blebs primarily contains only the membrane tension term and that the membrane tension may be uniform over the cell surface. Additional support for this hypothesis comes from observations of melanoma cells that spontaneously bleb. In melanoma cells, tether forces on blebs are proportional to the radius of the bleb, and as large blebs form, there are spikes in the tether force in other cell regions. We suggest that an internal osmotic pressure inflates the blebs, and the pressure calculated from the Law of Laplace is similar to independent measurements of intracellular pressures. When the membrane tension term is subtracted from the apparent membrane tension over the cytoskeleton, the membrane-cytoskeleton adhesion term can be estimated. In both cell systems, membrane-cytoskeleton adhesion was the major factor in generating the tether force.

The effects of nerve growth factor and dibutyryl cyclic AMP on cytoskeletal densities in cultured sensory ganglia

The effects of nerve growth factor (NGF) and dibutyryl cyclic AMP (DBC) on the density of cytoskeletal structures in cultured dorsal root ganglia were examined using morphometric techniques. After 24 hr in culture, NGF-treated neurites were longer than either DBC-treated or control neurites. At 48 hr, neurites produced in response to NGF and DBC were of equivalent length, while controls were considerably shorter. Comparison of electron micrographs of neuritic profiles revealed some differences of area and cytoskeletal density between treatment groups. Morphometric analysis was used to determine these differences under several growth conditions, at various rates of elongation and at different neurite lengths. As shown by analysis of variance, both NGF-treated and control neurites tapered in diameter at 48 hr in vitro, while DBC-induced neurites increased in area. An increase in cytoskeletal density for all treatment groups indicated that density was not always correlated with changes in area. An increased density of microtubules as compared to neurofilaments was seen at 24 hr, with equal densities of both cytoskeletal elements present after 48 hr in vitro. Comparisons between individual groups of data indicated that NGF-treated neurites relied primarily on microtubular density at 24 hr in vitro, when NGF induced longer, faster growing neurites. At 48 hr, there was an increase in neurofilaments proximal to the explant in the presence of DBC, implying that DBC may cause increased synthesis and/or transport of these structures. A comparison of microtubule to neurofilament ratios indicated that at 24 hr, there was always a greater density of microtubules. However, after 48 hr, neurofilament density increased such that there were equivalent densities of both cytoskeletal elements, possibly due to the overall increase in length observed in each treatment group. These data imply that 1) neurites with different rates of elongation may exhibit differences in cytoskeletal density; 2) neurites of equivalent lengths may be of differing stabilities; 3) NGF and DBC produce neurites with different cytoskeletal densities, implying divergent mechanisms of neurite induction; 4) the presence or absence of NGF may be partially responsible for variations in cytoskeletal densities observed between peripheral and central processes of DRG during development.

Development of ultrastructural specializations during the formation of acetylcholine receptor aggregates on cultured myotubes

The ultrastructure of cultured rat myotubes was examined at stages in the initial assembly of acetylcholine receptor (AChR) aggregates in order to elucidate the role of cell-surface specializations in aggregate formation. Within 4-6 hr, embryonic brain extract (EBX) induces the formation of sites of AChR density elevated 5-9 X above that of surrounding regions, and the appearance of these aggregates is preceded by the formation of clouds of punctate microaggregates (Olek et al., 1983). A video image-intensification system was used to monitor this redistribution of fluorescently labeled AChR, and sites of aggregation were mapped on identified myotubes. After processing the cultures for electron microscopy, thin sections were taken through identified aggregate sites at various stages in assembly. Specializations, including a basal lamina, mound-shaped plasma membrane contours with occasional deep infoldings, and a subjacent dense cytoskeletal specialization, which tended to exclude other cytoplasmic organelles, were associated with newly formed aggregates found 4-6 hr after adding EBX to the cultures. Analysis of random thin sections through EBX-treated and untreated myotubes showed that the extent of specializations of the basal lamina and cytoplasm was approximately threefold greater in cells exposed to EBX for 4 hr, suggesting a concurrent, and possibly interdependent, organization of such specializations with AChR aggregate assembly. Examination of sections through clouds of microaggregates, which formed within 90 min, revealed mound-shaped plasma membrane contours and underlying cytoplasm depleted of organelles but relatively little basal lamina and submembrane cytoskeletal density. These results suggest that the initial stage of AChR aggregate assembly involves relatively subtle changes in the structure of the cell cortex and that the evolution of microaggregates to aggregates may require the formation of additional cytoskeletal and extracellular matrix structures.