Actin and septin ultrastructures at the budding yeast cell cortex

Budding yeast has been a powerful model organism for studies of the roles of actin in endocytosis and septins in cell division and in signaling. However, the depth of mechanistic understanding that can be obtained from such studies has been severely hindered by a lack of ultrastructural information about how actin and septins are organized at the cell cortex. To address this problem, we developed rapid-freeze and deep-etch techniques to image the yeast cell cortex in spheroplasted cells at high resolution. The cortical actin cytoskeleton assembles into conical or mound-like structures composed of short, cross-linked filaments. The Arp2/3 complex localizes near the apex of these structures, suggesting that actin patch assembly may be initiated from the apex. Mutants in cortical actin patch components with defined defects in endocytosis disrupted different stages of cortical actin patch assembly. Based on these results, we propose a model for actin function during endocytosis. In addition to actin structures, we found that septin-containing filaments assemble into two kinds of higher order structures at the cell cortex: rings and ordered gauzes. These images provide the first high-resolution views of septin organization in cells.     

Apparent stiffness of vimentin intermediate filaments in living cells and its relation with other cytoskeletal polymers

The cytoskeleton is a complex network of interconnected biopolymers intimately involved in the generation and transmission of forces. Several mechanical properties of microtubules and actin filaments have been extensively explored in cells. In contrast, intermediate filaments (IFs) received comparatively less attention despite their central role in defining cell shape, motility and adhesion during physiological processes as well as in tumor progression. Here, we explored relevant biophysical properties of vimentin IFs in living cells combining confocal microscopy and a filament tracking routine that allows localizing filaments with ~20 nm precision. A Fourier-based analysis showed that IFs curvatures followed a thermal-like behavior characterized by an apparent persistence length (lp*) similar to that measured in aqueous solution. Additionally, we determined that certain perturbations of the cytoskeleton affect lp* and the lateral mobility of IFs as assessed in cells in which either the microtubule dynamic instability was reduced or actin filaments were partially depolymerized. Our results provide relevant clues on how vimentin IFs mechanically couple with microtubules and actin filaments in cells and support a role of this network in the response to mechanical stress.     

Analysis of turnover dynamics of the submembranous actin cortex

The cell cortex is a thin network of actin, myosin motors, and associated proteins that underlies the plasma membrane in most eukaryotic cells. It enables cells to resist extracellular stresses, perform mechanical work, and change shape. Cortical structural and mechanical properties depend strongly on the relative turnover rates of its constituents, but quantitative data on these rates remain elusive. Using photobleaching experiments, we analyzed the dynamics of three classes of proteins within the cortex of living cells: a scaffold protein (actin), a cross-linker (α-actinin), and a motor (myosin). We found that two filament subpopulations with very different turnover rates composed the actin cortex: one with fast turnover dynamics and polymerization resulting from addition of monomers to free barbed ends, and one with slow turnover dynamics with polymerization resulting from formin-mediated filament growth. Our data suggest that filaments in the second subpopulation are on average longer than those in the first and that cofilin-mediated severing of formin-capped filaments contributes to replenishing the filament subpopulation with free barbed ends. Furthermore, α-actinin and myosin minifilaments turned over significantly faster than F-actin. Surprisingly, only one-fourth of α-actinin dimers were bound to two actin filaments. Taken together, our results provide a quantitative characterization of essential mechanisms under­lying actin cortex homeostasis.     

Stretching actin filaments within cells enhances their affinity for the myosin II motor domain

To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli.     

Arabidopsis actin depolymerizing factor4 modulates the stochastic dynamic behavior of actin filaments in the cortical array of epidermal cells

Actin filament arrays are constantly remodeled as the needs of cells change as well as during responses to biotic and abiotic stimuli. Previous studies demonstrate that many single actin filaments in the cortical array of living Arabidopsis thaliana epidermal cells undergo stochastic dynamics, a combination of rapid growth balanced by disassembly from prolific severing activity. Filament turnover and dynamics are well understood from in vitro biochemical analyses and simple reconstituted systems. However, the identification in living cells of the molecular players involved in controlling actin dynamics awaits the use of model systems, especially ones where the power of genetics can be combined with imaging of individual actin filaments at high spatial and temporal resolution. Here, we test the hypothesis that actin depolymerizing factor (ADF)/cofilin contributes to stochastic filament severing and facilitates actin turnover. A knockout mutant for Arabidopsis ADF4 has longer hypocotyls and epidermal cells when compared with wild-type seedlings. This correlates with a change in actin filament architecture; cytoskeletal arrays in adf4 cells are significantly more bundled and less dense than in wild-type cells. Several parameters of single actin filament turnover are also altered. Notably, adf4 mutant cells have a 2.5-fold reduced severing frequency as well as significantly increased actin filament lengths and lifetimes. Thus, we provide evidence that ADF4 contributes to the stochastic dynamic turnover of actin filaments in plant cells.     

Filamentous actin organization in the unfertilized sea urchin egg cortex

We have investigated the organization of filamentous actin in the cortex of unfertilized eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus variegatus. Rhodamine phalloidin and anti-actin immunofluorescent staining of isolated cortices reveal a punctate pattern of fluorescent sources. Comparison of this pattern with SEM images of microvillar morphology and distribution indicates that filamentous actin in the cortex is predominantly localized in the microvilli. Thin-section TEM and quick-freeze deep-etch ultrastructure of isolated cortices demonstrates that this microvillar-associated actin is in a novel organizational state composed of very short filaments arranged in a tight network and that these filament networks form mounds that extend beyond the plane of the plasma membrane. Actin filaments within the networks do not exhibit free ends and make end-on attachments with the membrane only within the region of the evaginating microvilli. Myosin S-1 dissociable crosslinks, 2-3 nm in diameter, are observed between network filaments and between network filaments and the membrane. A second population of long, individual actin filaments is observed in close lateral association with the plasma membrane and frequently complexes with the microvillar actin networks. The filamentous actin of the unfertilized egg cortex may participate in establishing the mechanical properties of the egg surface and may function in nucleating the assembly of cortical actin following fertilization.     

Lateral Motion and Bending of Microtubules Studied with a New Single-Filament Tracking Routine in Living Cells

The cytoskeleton is involved in numerous cellular processes such as migration, division, and contraction and provides the tracks for transport driven by molecular motors. Therefore, it is very important to quantify the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization. It has been demonstrated that relevant mechanical properties of microtubules can be extracted from the analysis of their motion and shape fluctuations. However, tracking individual filaments in living cells is extremely complex due, for example, to the high and heterogeneous background. We introduce a believed new tracking algorithm that allows recovering the coordinates of fluorescent microtubules with ∼9 nm precision in in vitro conditions. To illustrate potential applications of this algorithm, we studied the curvature distributions of fluorescent microtubules in living cells. By performing a Fourier analysis of the microtubule shapes, we found that the curvatures followed a thermal-like distribution as previously reported with an effective persistence length of ∼20 μm, a value significantly smaller than that measured in vitro. We also verified that the microtubule-associated protein XTP or the depolymerization of the actin network do not affect this value; however, the disruption of intermediate filaments decreased the persistence length. Also, we recovered trajectories of microtubule segments in actin or intermediate filament-depleted cells, and observed a significant increase of their motion with respect to untreated cells showing that these filaments contribute to the overall organization of the microtubule network. Moreover, the analysis of trajectories of microtubule segments in untreated cells showed that these filaments presented a slower but more directional motion in the cortex with respect to the perinuclear region, and suggests that the tracking routine would allow mapping the microtubule dynamical organization in cells.     

High frequency force generation in outer hair cells from the mammalian ear

Mammalian outer hair cells generate mechanical forces at acoustic frequencies and can thus amplify the sound stimulus within the inner ear. The mechanism of force generation depends upon the plasma membrane potential but not upon either calcium or ATP. Forces are generated in the lateral cortex along the full length of the cell. The cortex includes a two-dimensional cytoskeletal lattice composed of circumferential filaments 6-7 nm thick that are cross-linked by filaments 3-4 nm thick and 40-60 nm long. The two filament types may, respectively, be actin and some form of spectrin. The lattice reinforces the cylindrical shape of the cell and permits limited changes in length. Beneath it lie the lateral cisternae, a regular system of multi-layered membranes. Force-generation may depend upon voltage-dependent shape changes in proteins that lie either in the plasma membrane or in the cytoskeletal lattice.     

Distinct pathways for the early recruitment of myosin II and actin to the cytokinetic furrow

Equatorial organization of myosin II and actin has been recognized as a universal event in cytokinesis of animal cells. Current models for the formation of equatorial cortex favor either directional cortical transport toward the equator or localized de novo assembly. However, this process has never been analyzed directly in dividing mammalian cells at a high resolution. Here we applied total internal reflection fluorescence microscope (TIRF-M), coupled with spatial temporal image correlation spectroscopy (STICS) and a new analytical approach termed temporal differential microscopy (TDM), to image the dynamics of myosin II and actin during the assembly of equatorial cortex. Our results indicated distinct and at least partially independent mechanisms for the early equatorial recruitment of myosin and actin filaments. Cortical myosin showed no detectable directional flow during early cytokinesis. In addition to equatorial assembly, we showed that localized inhibition of disassembly contributed to the formation of the equatorial myosin band. In contrast to myosin, actin filaments underwent a striking flux toward the equator. Myosin motor activity was required for the actin flux, but not for actin concentration in the furrow, suggesting that there was a flux-independent, de novo mechanism for actin recruitment along the equator. Our results indicate that cytokinesis involves signals that regulate both assembly and disassembly activities and argue against mechanisms that are coupled to global cortical movements.     

Mechanical Detection of a Long-Range Actin Network Emanating from a Biomimetic Cortex

Actin is ubiquitous globular protein that polymerizes into filaments and forms networks that participate in the force generation of eukaryotic cells. Such forces are used for cell motility, cytokinesis, and tissue remodeling. Among those actin networks, we focus on the actin cortex, a dense branched network beneath the plasma membrane that is of particular importance for the mechanical properties of the cell. Here we reproduce the cellular cortex by activating actin filament growth on a solid surface. We unveil the existence of a sparse actin network that emanates from the surface and extends over a distance that is at least 10 times larger than the cortex itself. We call this sparse actin network the “actin cloud” and characterize its mechanical properties with optical tweezers. We show, both experimentally and theoretically, that the actin cloud is mechanically relevant and that it should be taken into account because it can sustain forces as high as several picoNewtons (pN). In particular, it is known that in plant cells, actin networks similar to the actin cloud have a role in positioning the nucleus; in large oocytes, they play a role in driving chromosome movement. Recent evidence shows that such networks even prevent granule condensation in large cells.     

Lateral Motion and Bending of Microtubules Studied with a New Single-Filament Tracking Routine in Living Cells

The cytoskeleton is involved in numerous cellular processes such as migration, division, and contraction and provides the tracks for transport driven by molecular motors. Therefore, it is very important to quantify the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization. It has been demonstrated that relevant mechanical properties of microtubules can be extracted from the analysis of their motion and shape fluctuations. However, tracking individual filaments in living cells is extremely complex due, for example, to the high and heterogeneous background. We introduce a believed new tracking algorithm that allows recovering the coordinates of fluorescent microtubules with ∼9 nm precision in in vitro conditions. To illustrate potential applications of this algorithm, we studied the curvature distributions of fluorescent microtubules in living cells. By performing a Fourier analysis of the microtubule shapes, we found that the curvatures followed a thermal-like distribution as previously reported with an effective persistence length of ∼20 μm, a value significantly smaller than that measured in vitro. We also verified that the microtubule-associated protein XTP or the depolymerization of the actin network do not affect this value; however, the disruption of intermediate filaments decreased the persistence length. Also, we recovered trajectories of microtubule segments in actin or intermediate filament-depleted cells, and observed a significant increase of their motion with respect to untreated cells showing that these filaments contribute to the overall organization of the microtubule network. Moreover, the analysis of trajectories of microtubule segments in untreated cells showed that these filaments presented a slower but more directional motion in the cortex with respect to the perinuclear region, and suggests that the tracking routine would allow mapping the microtubule dynamical organization in cells.     

Mechanical Detection of a Long-Range Actin Network Emanating from a Biomimetic Cortex

Actin is ubiquitous globular protein that polymerizes into filaments and forms networks that participate in the force generation of eukaryotic cells. Such forces are used for cell motility, cytokinesis, and tissue remodeling. Among those actin networks, we focus on the actin cortex, a dense branched network beneath the plasma membrane that is of particular importance for the mechanical properties of the cell. Here we reproduce the cellular cortex by activating actin filament growth on a solid surface. We unveil the existence of a sparse actin network that emanates from the surface and extends over a distance that is at least 10 times larger than the cortex itself. We call this sparse actin network the “actin cloud” and characterize its mechanical properties with optical tweezers. We show, both experimentally and theoretically, that the actin cloud is mechanically relevant and that it should be taken into account because it can sustain forces as high as several picoNewtons (pN). In particular, it is known that in plant cells, actin networks similar to the actin cloud have a role in positioning the nucleus; in large oocytes, they play a role in driving chromosome movement. Recent evidence shows that such networks even prevent granule condensation in large cells.     

Jasplakinolide reversibly disrupts actin filaments in suspension-cultured tobacco BY-2 cells

Summary. Jasplakinolide is potentially a useful pharmacological tool for the study of actin organization and dynamics in living cells, since it induces actin polymerization in vitro and, unlike phalloidin, is membrane permeative. In the present work, the effect of jasplakinolide on the actin cytoskeleton of living suspension-cultured Nicotiana tabacum ‘Bright Yellow 2’ cells was investigated. Actin filaments in the living cells were disrupted by jasplakinolide. The effect of jasplakionlide on the actin cytoskeleton was concentration and time dependent. When cells were treated with a moderate concentration (150 nM) of jasplakinolide, cortical actin filaments were disrupted preferentially, whereas actin aggregated at the perinuclear region. With concentrations higher than 400 nM and exposure times longer than 30 min, actin filaments in the cell disappeared completely. The effect of jasplakinolide on the actin cytoskeleton was reversible even at high concentration. Actin bundles appeared first in the perinuclear region within 5 min, and the cortical actin array was reestablished in 15 min, suggesting that actin filaments might be organized at this region. Received July 31, 2001 Accepted December 14, 2001     

Distribution of myosin and relationship to actin organization in cortical and subcortical areas of antibody-labelled, quick-frozen, deep-etched fibroblast cytoskeletons

In this study I describe the ultrastructural distribution of myosin in cortical and subcortical areas of antibody-labelled, quick-frozen fibroblasts. In many cells myosin was present in small variably spaced and sized (0.23-0.39 micron long), nonaligned patches, while in other cells much larger periodically spaced patches of more uniform length (0.27 micron) were found. In all regions of the cytoskeleton myosin was found, primarily on linear bundles of actin filaments running parallel to the cell's long axis. Myosin was absent from single actin filaments, actin filaments perpendicular to actin bundles aligned with the cell's long axis, and actin filaments, such as geodome vertices and parts of the cortex, which had a complex interwoven appearance. These data indicate that in motile non-muscle cells myosin exerts force only in a unidirectional manner. Recognisable myosin filaments were never observed even in cells incubated either in N-ethylmaleimide or sodium azide. The presence of myosin in, and almost to the very edge of, the cortex suggests that the cellular control of actomyosin based movement is direct and over short-range distances. Large numbers of small cross-linking filaments were found in association with cortical and subcortical actin. Their relationship to myosin and overall actin geometry is discussed.     

Micromechanical architecture of the endothelial cell cortex

Mechanical properties of living cells are important for cell shape, motility, and cellular responses to biochemical and biophysical signals. Although these properties are predominantly determined by the cytoskeleton, relatively little is known about the mechanical organization of cells at a subcellular level. We have studied the cell cortex of bovine pulmonary artery endothelial cells (BPAECs) using atomic force microscopy (AFM) and confocal fluorescence microscopy (CFM). We show that the contrast in AFM imaging of these cells derives in large part from differences in local mechanical properties, and AFM images of BPAEC reveal the local micromechanical architecture of their apical cortex at approximately 125 nm resolution. Mechanically the cortex in these cells is organized as a polygonal mesh at two length scales: a coarse mesh with mesh element areas approximately 0.5-10 microm2, and a finer mesh with areas <0.5 microm2. These meshes appear to be intertwined, which may have interesting implications for the mechanical properties of the cell. Correlated AFM-CFM experiments and pharmacological treatments reveal that actin and vimentin are components of the coarse mesh, but microtubules are not mechanical components of the BPAEC apical cortex.     

A Prestressed Cable Network Model of the Adherent Cell Cytoskeleton

A prestressed cable network is used to model the deformability of the adherent cell actin cytoskeleton. The overall and microstructural model geometries and cable mechanical properties were assigned values based on observations from living cells and mechanical measurements on isolated actin filaments, respectively. The models were deformed to mimic cell poking (CP), magnetic twisting cytometry (MTC) and magnetic bead microrheometry (MBM) measurements on living adherent cells. The models qualitatively and quantitatively captured the fibroblast cell response to the deformation imposed by CP while exhibiting only some qualitative features of the cell response to MTC and MBM. The model for CP revealed that the tensed peripheral actin filaments provide the key resistance to indentation. The actin filament tension that provides mechanical integrity to the network was estimated at ~158 pN, and the nonlinear mechanical response during CP originates from filament kinematics. The MTC and MBM simulations revealed that the model is incomplete, however, these simulations show cable tension as a key determinant of the model response.     

Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton

By combining astigmatism imaging with a dual-objective scheme, we improved the image resolution of stochastic optical reconstruction microscopy (STORM) and obtained <10-nm lateral resolution and <20-nm axial resolution when imaging biological specimens. Using this approach, we resolved individual actin filaments in cells and revealed three-dimensional ultrastructure of the actin cytoskeleton. We observed two vertically separated layers of actin networks with distinct structural organizations in sheet-like cell protrusions.     

Mechanism of the formation of contractile ring in dividing cultured animal cells. II. Cortical movement of microinjected actin filaments

The contractile ring in dividing animal cells is formed primarily through the reorganization of existing actin filaments (Cao, L.-G., and Y.-L. Wang. 1990. J. Cell Biol. 110:1089-1096), but it is not clear whether the process involves a random recruitment of diffusible actin filaments from the cytoplasm, or a directional movement of cortically associated filaments toward the equator. We have studied this question by observing the distribution of actin filaments that have been labeled with fluorescent phalloidin and microinjected into dividing normal rat kidney (NRK) cells. The labeled filaments are present primarily in the cytoplasm during prometaphase and early metaphase, but become associated extensively with the cell cortex 10-15 min before the onset of anaphase. This process is manifested both as an increase in cortical fluorescence intensity and as movements of discrete aggregates of actin filaments toward the cortex. The concentration of actin fluorescence in the equatorial region, accompanied by a decrease of fluorescence in polar regions, is detected 2-3 min after the onset of anaphase. By directly tracing the distribution of aggregates of labeled actin filaments, we are able to detect, during anaphase and telophase, movements of cortical actin filaments toward the equator at an average rate of 1.0 micron/min. Our results, combined with previous observations, suggest that the organization of actin filaments during cytokinesis probably involves an association of cytoplasmic filaments with the cortex, a movement of cortical filaments toward the cleavage furrow, and a dissociation of filaments from the equatorial cortex.     

A mechanochemical model of actin filaments

In eukaryotic cells, actin filaments are involved in important processes such as motility, division, cell shape regulation, contractility, and mechanosensation. Actin filaments are polymerized chains of monomers, which themselves undergo a range of chemical events such as ATP hydrolysis, polymerization, and depolymerization. When forces are applied to F-actin, in addition to filament mechanical deformations, the applied force must also influence chemical events in the filament. We develop an intermediate-scale model of actin filaments that combines actin chemistry with filament-level deformations. The model is able to compute mechanical responses of F-actin during bending and stretching. The model also describes the interplay between ATP hydrolysis and filament deformations, including possible force-induced chemical state changes of actin monomers in the filament. The model can also be used to model the action of several actin-associated proteins, and for large-scale simulation of F-actin networks. All together, our model shows that mechanics and chemistry must be considered together to understand cytoskeletal dynamics in living cells.     

AFM functional imaging on vascular endothelial cells

Vascular endothelial (VE)-cadherin is predominantly responsible for the mechanical linkage between endothelial cells, where VE-cadherin molecules are clustered and linked through their cytoplasmic domain to the actin-based cytoskeleton. Clustering and linkage of VE-cadherin to actin filaments is a dynamic process and changes according to the functional state of the cells. Here nano-mapping of VE-cadherin was performed using simultaneous topography and recognition imaging (TREC) technique onto microvascular endothelial cells from mouse myocardium (MyEnd). The recognition maps revealed prominent 'dark' spots (domains or clusters) with the sizes from 10 to 250 nm. These spots arose from a decrease of oscillation amplitude during specific binding between VE-cadherin cis-dimers. They were assigned to characteristic structures of the topography images. After treatment with nocodazole so as to depolymerize microtubules, VE-cadherin domains with a typical ellipsoidal form were still found to be collocalized with cytoskeletal filaments supporting the hypothesis that VE-cadherin is linked to actin filaments. Compared to other conventional techniques such as immunochemistry or single molecule optical microscopy, TREC represents an alternative method to quickly obtain the local distribution of receptors on cell surface with an unprecedented lateral resolution of several nanometers.