Stress fibers stabilize the position of intranuclear DNA through mechanical connection with the nucleus in vascular smooth muscle cells

Actin stress fibers (SFs) running across the top surface of the nucleus in vascular smooth muscle cells were dissected using laser nano-dissection technique to release its pretension, and the dynamic behavior of SFs, nucleus, and intranuclear DNA were investigated. SFs shortened across the top surface of the nuclei after their dissection. The nuclei moved in the direction of SF retraction, and showed marked local deformation, indicating that SFs firmly connected to the nuclear surface. Intranuclear DNA located near and around the dissected SFs disappeared and their distribution changed markedly. These findings suggest that SFs stabilize the position of intranuclear chromatin through mechanical connection with the nucleus. The tension of SFs may be transmitted mechanically to the nucleus inducing conformational changes of intranuclear chromatin.     

Measurements of strain on single stress fibers in living endothelial cells induced by fluid shear stress

Fluid shear stress (FSS) acting on the apical surface of endothelial cells (ECs) can be sensed by mechano-sensors in adhesive protein complexes found in focal adhesions and intercellular junctions. This sensing occurs via force transmission through cytoskeletal networks. This study quantitatively evaluated the force transmitted through cytoskeletons to the mechano-sensors by measuring the FSS-induced strain on SFs using live-cell imaging for actin stress fibers (SFs). FSS-induced bending of SFs caused the SFs to align perpendicular to the direction of the flow. In addition, the displacement vectors of the SFs were detected using image correlation and the FSS-induced axial strain of the SFs was calculated. The results indicated that FSS-induced strain on SFs spanned the range 0.01-0.1% at FSSs ranging from 2 to 10 Pa. Together with the tensile property of SFs reported in a previous study, the force exerted on SFs was estimated to range from several to several tens of pN.     

Movement of stress fibers away from focal adhesions identifies focal adhesions as sites of stress fiber assembly in stationary cells

Force generated in contractile actin filament bundles (stress fibers-SFs) is transmitted to the extracellular matrix (ECM) via linker proteins and transmembrane integrins at focal adhesions (FAs). Though it has long been known that actin is rapidly exchanged in FAs, the connection between SFs and FAs has not been studied in detail. We introduced fiduciary marks on SFs by expressing GFP-palladin or GFP-alpha-actinin-1, which are both FA and dense body proteins, and by pattern bleaching of GFP-actin. Following fiduciary marks on SFs over time by time-lapse fluorescence microscopy, we detected assembly of SFs at FAs in stationary cells resulting in movement of SFs away from FAs with a velocity of 0.2-0.4 microm/min. Visualization of FAs in GFP-palladin/DsRed-paxillin double transfected cells showed that SF elongation was not accompanied by a change in FA length. SF elongation at FAs depended on actin polymerization and force as demonstrated by inhibitors of actin polymerization (cytochalasin D, jasplakinolide) and inhibitors of myosin-dependent contraction (blebbistatin, Y-27632), respectively. Our finding of SF assembly at FAs has important implications for SF formation, force transmission, and tension distribution within the actin cytoskeletal network of stationary cells.     

Intracellular stress transmission through actin stress fiber network in adherent vascular cells

Intracellular stress transmission through subcellular structural components has been proposed to affect activation of localized mechano-sensing sites such as focal adhesions in adherent cells. Previous studies reported that physiological extracellular forces produced heterogeneous spatial distributions of cytoplasmic strain. However, mechanical signaling pathway involved in intracellular force transmission through basal actin stress fibers (SFs), a mechano-responsive cytoskeletal structure, remains elusive. In the present study, we investigated force balance within the basal SFs of cultured smooth muscle cells and endothelial cells by (i) removing the cell membrane and cytoplasmic constituents except for materials physically attaching to the substrate (i.e., SF-focal adhesion complexities) or (ii) dislodging either mechanically or chemically the cell processes of the cells expressing fluorescent proteins-labeled actin and focal adhesions in order, to examine stress-release-induced deformation of the basal SFs. The result showed that a removal of mechanical restrictions for SFs resulted in a decrease in the length of the remaining SFs, which means SFs bear tension. In addition, a release of the preexisting tension in a single SF was transmitted to another SF physically linked to the former, but not transmitted to the other ones physically independent of the former, suggesting that the prestress is balanced in tensed SF networks. These results support a hypothesis regarding cell structural architecture that physiological extracellular forces can produce in the basal SF network a directional intracellular stress or strain distribution. Therefore, consideration of the coexistence of the directional stretching strain along the axial direction of SFs and the heterogeneous strain in the other cytoplasmic region will be essential for understanding intracellular stress transmission in the adherent cells.     

Stress fibers of the aortic smooth muscle cells in tissues do not align with the principal strain direction during intraluminal pressurization

Stress fibers (SFs) in cells transmit external forces to cell nuclei, altering the DNA structure, gene expression, and cell activity. To determine whether SFs are involved in mechanosignal transduction upon intraluminal pressure, this study investigated the SF direction in smooth muscle cells (SMCs) in aortic tissue and strain in the SF direction. Aortic tissues were fixed under physiological pressure of 120 mmHg. First, we observed fluorescently labeled SFs using two-photon microscopy. It was revealed that SFs in the same smooth muscle layers were aligned in almost the same direction, and the absolute value of the alignment angle from the circumferential direction was 16.8° ± 5.2° (n = 96, mean ± SD). Second, we quantified the strain field in the aortic tissue in reference to photo-bleached markers. It was found in the radial-circumferential plane that the largest strain direction was − 21.3° ± 11.1°, and the zero normal strain direction was 28.1° ± 10.2°. Thus, the SFs in aortic SMCs were not in line with neither the largest strain direction nor the zero strain direction, although their orientation was relatively close to the zero strain direction. These results suggest that SFs in aortic SMCs undergo stretch, but not maximal and transmit the force to nuclei under intraluminal pressure.

     

Finite-element analysis of the adhesion-cytoskeleton-nucleus mechanotransduction pathway during endothelial cell rounding: axisymmetric model

Endothelial cells possess a mechanical network connecting adhesions on the basal surface, the cytoskeleton, and the nucleus. Transmission of force at adhesions via this pathway can deform the nucleus, ultimately resulting in an alteration of gene expression and other cellular changes (mechanotransduction). Previously, we measured cell adhesion area and apparent nuclear stretch during endothelial cell rounding. Here, we reconstruct the stress map of the nucleus from the observed strains using finite-element modeling. To simulate the disruption of adhesions, we prescribe displacement boundary conditions at the basal surface of the axisymmetric model cell. We consider different scenarios of the cytoskeletal arrangement, and represent the cytoskeleton as either discrete fibers or as an effective homogeneous layer When the nucleus is in the initial (spread) state, cytoskeletal tension holds the nucleus in an elongated, ellipsoidal configuration. Loss of cytoskeletal tension during cell rounding is represented by reactive forces acting on the nucleus in the model. In our simulations of cell rounding, we found that, for both representations of the cytoskeleton, the loss of cytoskeletal tension contributed more to the observed nuclear deformation than passive properties. Since the simulations make no assumption about the heterogeneity of the nucleus, the stress components both within and on the surface of the nucleus were calculated. The nuclear stress map showed that the nucleus experiences stress on the order of magnitude that can be significant for the function of DNA molecules and chromatin fibers. This study of endothelial cell mechanobiology suggests the possibility that mechanotransduction could result, in part, from nuclear deformation, and may be relevant to angiogenesis, wound healing, and endothelial barrier dysfunction.     

Relationship between Apical Membrane Elasticity and Stress Fiber Organization in Fibroblasts Analyzed by Fluorescence and Atomic Force Microscopy

To investigate the relationship between cellular microelasticity and the structural features of cytoskeletons (CSKs), a microindentation test for apical cell membranes and observation of the spatio-distribution of actin CSKs of fibroblasts were performed by fluorescence and atomic force microscopy (FM/AFM). The indentation depths of apical cell membranes were measured from AFM force–indentation (f–i) curves under equal final loads and mapped two-dimensionally to show the relative distribution of local microelasticity on cell membranes. Intracellular spatial distribution of actin CSKs was visualized fluorescently by high Z-resolution cross-sectional observation of a cell on which indentation mapping analysis had been performed in advance. Structural features of stress fibers (SFs) were observed as three typical patterns of dense SF, sparse SF and sparser SF cell groups, which were quantitated using the degree of orientation in apical SFs (ASFs) that had been defined using two-dimensional Fourier analysis. In indentation depth maps, the upper nuclear region was markedly softer than the pseudopodium region. The mean indentation depth of the upper nuclear region decreased with increased SF density in whole cells and the degree of orientation of ASF, although the pseudopodium region did not exhibit such a trend. The apical membrane of adhered cells was found to tend to stiffen with the increase in both density and degree of orientation of SFs.     

A novel method for measuring tension generated in stress fibers by applying external forces

The distribution of contractile forces generated in cytoskeletal stress fibers (SFs) contributes to cellular dynamic functions such as migration and mechanotransduction. Here we describe a novel (to our knowledge) method for measuring local tensions in SFs based on the following procedure: 1), known forces of different magnitudes are applied to an SF in the direction perpendicular to its longitudinal axis; 2), force balance equations are used to calculate the resulting tensions in the SF from changes in the SF angle; and 3), the relationship between tension and applied force thus established is extrapolated to an applied force of zero to determine the preexisting tension in the SF. In this study, we measured tensions in SFs by attaching magnetic particles to them and applying known forces with an electromagnetic needle. Fluorescence microscopy was used to capture images of SFs fluorescently labeled with myosin II antibodies, and analysis of these images allowed the tension in the SFs to be measured. The average tension measured in this study was comparable to previous reports, which indicates that this method may become a powerful tool for elucidating the mechanisms by which cytoskeletal tensions affect cellular functions.     

Chromatin Arrangement and Axis Formation in the Spermiogenesis of a Pulmonate Snail

Nuclear change in relation to axis formation and condensation during spermiogenesis was investigated in the snail, Physa acuta. In the early spermatid, characteristic thick layers (termed apical and basal plates) are formed on two sides of a nuclear envelope. Soon after the formation of these plates, a developing acrosome and a flagellum attach externally to the center of the apical and basal plates, respectively. However, most (presumably all) of the chromatin filaments become attached all over the inner surface of the apical and basal plates. This means that the plates themselves are actually the specialized forms of the nuclear envelope to which chromatin filaments become connected; by means of these plates, the chromatin filaments become arranged in parallel to the antero-posterior axis as the nucleus elongates. This suggests that the formation of these two thick layers on opposing surfaces of the nucleus primarily determines the antero-posterior axis of the spermatid and the direction of the arrangement of chromatin.
The flattening of the nucleus prior to elongation is caused mainly by the enlargement of the basal plate. Subsequent nuclear shaping and condensation are discussed in relation to the change in the surface structures of the nucleus and the organization of the microtubules.     

Modelling the compartmentalization of splicing factors

Splicing factor (SF) compartments, also known as speckles, are heterogeneously distributed compartments within the nucleus of eukaryotic cells that are enriched in pre-mRNA SFs. We derive a fourth-order aggregation-diffusion model that describes a possible mechanism underlying the organization of SFs into speckles. The model incorporates two hypotheses, namely (1) that self-organization of dephosphorylated SFs, modulated by a phosphorylation-dephosphorylation cycle, is responsible for the formation and disappearance of speckles, and (2) that an underlying nuclear structure plays a major role in the organization of SFs. A linear stability analysis about homogeneous steady-state solutions of the model reveals how the self-interaction among dephosphorylated SFs can result in the onset of spatial patterns. A detailed bifurcation analysis of the model describes how phosphorylation and dephosphorylation modulate the onset of the compartmentalization of SFs.     

Fine structure of the spermatozoa of Chiton marginatus (mollusca: Amphineura), with special reference to nucleus maturation

The spermatozoon of Chiton marginatus is a long uniflagellate cell displaying structural features of “modified sperm.” The nucleus presents a conical shape with a long apical cylindrical extension. The chromatin is homogeneously dense. Scattered inside the condensed nucleus, a few nuclear lacunae are visible. The acrosomal complex is lacking. Some mitochondria are located in a laterofrontal structure side by side with the nucleus. The typical midpiece is absent. The cytoplasm forms a thin layer around the nucleus and the mitochondria. The proximal centriole is in a basal nuclear indent. The distal centriole serves to form the axoneme tail with the usual microtubular pattern. During nuclear maturation, the early spermatid nucleus is spherical and contains fine granular chromatin patches. The nuclear envelope shows a deposit of dense material at the base of the nucleus, forming a semicircular invagination occupied by a flocculent mass. In middle spermatid stage, the chromatin gets organized in filaments, coiled as a hank, attached over the inner surface of the basal thickening of the nuclear envelope. The nucleus starts to elongate anteroposteriorly. At the pointed apical portion of the spermatid, a group of microtubules is observed seeming to impose external pressure to the nucleus giving rise to the long apical nuclear point. The mitochondria have a basal position. Late spermatids have an elongated conical nucleus. The chromatin filaments are further condensed, and lacunae appear inside the nucleus. Some mitochondria migrate to a lateral position.     

Characterization of the nuclear deformation caused by changes in endothelial cell shape

We investigated the mechanotransduction pathway in endothelial cells between their nucleus and adhesions to the extracellular matrix. First, we measured nuclear deformations in response to alterations of cell shape as cells detach from a flat surface. We found that the nuclear deformation appeared to be in direct and immediate response to alterations of the cell adhesion area. The nucleus was then treated as a neo-Hookean compressible material, and we estimated the stress associated with the cytoskeleton and acting on the nucleus during cell rounding. With the obtained stress field, we estimated the magnitude of the forces deforming the nucleus. Considering the initial and final components of this adhesion-cytoskeleton-nucleus force transmission pathway, we found our estimate for the internal forces acting on the nucleus to be on the same order of magnitude as previously measured traction forces, suggesting a direct mechanical link between adhesions and the nucleus.     

Orientation of apical and basal actin stress fibers in isolated and subconfluent endothelial cells as an early response to cyclic stretching

We investigated the response of apical and basal actin stress fibers (SFs) and its dependency on cell confluency for endothelial cells subjected to cyclic stretching. Porcine aortic endothelial cells from the 2nd and 5th passages were transferred to a fibronectin-coated silicone chamber with 5000-8000 cells/cm2 (isolated condition), positioning the cells apart, or with 25,000-27,000 cells/cm2 (subconfluent condition), allowing cell-to-cell contact. The substrate was stretched cyclically by 0.5 Hz for 2 h with a peak strain on the substrate that was 15% in the stretch direction and -4% in the transverse direction. The actin filaments (AFs) were stained with rhodamine phalloidin and their orientations were examined under a confocal laser scanning microscope. In the basal region, SFs formed in all of the cells under both the isolated and subconfluent conditions. We observed an average of 5 and 9 SFs per cell under the isolated and subconfluent conditions, respectively, in the fluorescent images of the apical region. We also observed cells that were bush-like without apical AFs or apical SFs. On average, the SFs in the subconfluent cells oriented in the direction of minimal strain, while the SFs in the isolated cells oriented in the direction of a 2% compressive strain. These results suggest that such differential response may be due to differences in the transmission of mechanical stretching to the central and apical regions of the cell through the SFs. We also speculate that cell-to-cell contact might change the strength, orientation, and anchorage of apical AFs and play a critical role in mechanical signal transduction.     

Apical movement during interkinetic nuclear migration is a two-step process

Neural progenitor cells in the pseudostratified neuroepithelium in vertebrates undergo interkinetic nuclear migration, which results in mitotic cells localized to the apical surface. Interphase nuclei are distributed throughout the rest of the epithelium. How mitosis is coordinated with nuclear movement is unknown, and the mechanism by which the nucleus migrates apically is controversial. Using time-lapse confocal microscopy, we show that nuclei migrate apically in G2 phase via microtubules. However, late in G2, centrosomes leave the apical surface after cilia are disassembled, and mitosis initiates away from the apical surface. The mitotic cell then rounds up to the apical surface, which is an actin-dependent process. This behavior is observed in both chicken neural-tube-slice preparations and in mouse cortical slices, and therefore is likely to be a general feature of interkinetic nuclear migration. We propose a new model for interkinetic nuclear migration in which actin and microtubules are used to position the mitotic cell at the apical surface.     

Fine structure of the germinating cells during the spermiogenesis of Arion rufus (Mollusca,Pulmonate Gastropoda)

Changes in spermatozoan ultrastructure have been studied during spermiogenesis of the slug Arion rufus (Gastropoda, Pulmonata, Stylommatophora). The ovotestis was investigated during the male stage, definite by the presence of spermatozoa. Some peculiar characteristics are shown by early spermatids: Around the nucleus, the nuclear envelope presents two thick layers located on opposite sides, the apical and basal plates, that will determine the antero-posterior axis of the spermatid. The chromatin, first dispersed throughout the nucleoplasm gives later on thick filaments which become attached over the inner surface of these plates. The chromatin filaments are then arranged parallel to the antero-posterior axis as the nucleus elongates. The position of the plates determines the antero-posterior axis of the spermatid. In the mature spermatozoa, the chromatin is more condensed and the nucleus presents an helical organization. The acrosome and flagellum are respectively attached externally to the center of the apical and basal plates. The acrosome consists of a membrane-bound vesicle and forms a column of homogeneous material. In the middle piece, the mitochondria have been transformed into a mitochondrial derivate by the way of a complicated metamorphosis. The axoneme is surrounded by three mitochondrial helices but only one of them contains glycogene granules. © 1996 Wiley-Liss, Inc.     

Mechanical properties of aponeurosis and tendon of the cat soleus muscle during whole-muscle isometric contractions

Recent studies have suggested that the mechanical properties of aponeurosis are not similar to the properties of external tendon. In the present study, the lengths of aponeurosis, tendon, and muscle fascicles were recorded individually, using piezoelectric crystals attached to the surface of each structure during isometric contractions in the cat soleus muscle. We used a surgical microscope to observe the surface of the aponeurosis, which revealed a confounding effect on measures of aponeurosis length due to sliding of a thin layer of epimysium over the proximal aponeurosis. After correcting for this artifact, the stiffness computed for aponeurosis was similar to tendon, with both increasing from around 8 F₀/L_c (F₀ is maximum isometric force and L_c is tissue length) at 0.1 F₀ to 30 F₀/L_c at forces greater than 0.4 F₀. At low force levels only (0.1 F₀), aponeurotic stiffness increased somewhat as fascicle length increased. There was a gradient in the thickness of the aponeurosis along its length: its thickness was minimal at the proximal end and maximal at the distal end, where it converged to form the external tendon. This gradient in thickness appeared to match the gradient in tension transmitted along this structure. We conclude that the specific mechanical properties of aponeurosis are similar to those of tendon. © 1995 Wiley-Liss, Inc.     

Atomic force and total internal reflection fluorescence microscopy for the study of force transmission in endothelial cells

This paper describes the combined use of atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRFM) to examine the transmission of force from the apical cell membrane to the basal cell membrane. A Bioscope AFM was mounted on an inverted microscope, the stage of which was configured for TIRFM imaging of fluorescently labeled human umbilical vein endothelial cells (HUVECs). Variable-angle TIRFM experiments were conducted to calibrate the coupling angle with the depth of penetration of the evanescent wave. A measure of cellular mechanical properties was obtained by collecting a set of force curves over the entire apical cell surface. A linear regression fit of the force-indentation curves to an elastic model yields an elastic modulus of 7.22 +/- 0. 46 kPa over the nucleus, 2.97 +/- 0.79 kPa over the cell body in proximity to the nucleus, and 1.27 +/- 0.36 kPa on the cell body near the edge. Stress transmission was investigated by imaging the response of the basal surface to localized force application over the apical surface. The focal contacts changed in position and contact area when forces of 0.3-0.5 nN were applied. There was a significant increase in focal contact area when the force was removed (p < 0.01) from the nucleus as compared to the contact area before force application. There was no significant change in focal contact coverage area before and after force application over the edge. The results suggest that cells transfer localized stress from the apical to the basal surface globally, resulting in rearrangement of contacts on the basal surface.     

Reinforcing the LINC complex connection to actin filaments: the role of FHOD1 in TAN line formation and nuclear movement

Positioning the nucleus is critical for many cellular processes including cell division, migration and differentiation. The linker of nucleoskeleton and cytoskeleton (LINC) complex spans the inner and outer nuclear membranes and has emerged as a major factor in connecting the nucleus to the cytoskeleton for movement and positioning. Recently, we discovered that the diaphanous formin family member FHOD1 interacts with the LINC complex component nesprin-2 giant (nesprin-2G) and that this interaction plays essential roles in the formation of transmembrane actin-dependent nuclear (TAN) lines and nuclear movement during cell polarization in fibroblasts. We found that FHOD1 strengthens the connection between nesprin-2G and rearward moving dorsal actin cables by providing a second site of interaction between nesprin-2G and the actin cable. These results indicate that the LINC complex connection to the actin cytoskeleton can be enhanced by cytoplasmic factors and suggest a new model for TAN line formation. We discuss how the nesprin-2G-FHOD1 interaction may be regulated and its possible functional significance for development and disease.     

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.