Acoustic microscopy of cultured cells

The scanning acoustic microscope (SAM) allows one to measure mechanical parameters of living cells with high lateral resolution. By analyzing single acoustic images’ sound attenuation and sound velocity, the latter corresponding to stiffness (elasticity) of the cortical cytoplasm can be determined. In this study, measurements of stiffness distribution in XTH-2 cells were compared with the organization of F-actin and microtubules. Single XTH-2 cells exhibit relatively high stiffness at the free margins; toward the cell center this value decreases and reaches a sudden minimum where the slope of the surface topography enlargens at the margin of the dome-shaped cell center. The steepness of the increase in slope is linearly related to the decrease in sound velocity at this site. Thus, a significant determinant of cell shape is paralleled by an alteration of stiffness. In the most central parts, no interferences could be distinguished, therefore, this region had to be excluded from the calculations. Stiffness distribution roughly coincided with the distribution of F-actin, but no correlation to microtubule arrangement was found. Following the treatment of XTH-2 cells with ionomycin in the presence of calcium (in the culture medium), the cell cortex first contracted as indicated by shape changes and by a marked increase in stiffness (deduced from sound velocity). This contraction phase was followed by a phase of microtubule and F-actin disassembly. Concomittantly, sound velocity decreased considerably, indicating the loss of elasticity in the cell cortex. No structural equivalent to sound attenuation has been identified.     

Acoustic microscopy of cultured cells. Distribution of forces and cytoskeletal elements.

The scanning acoustic microscope (SAM) allows one to measure mechanical parameters of living cells with high lateral resolution. By analyzing single acoustic images' sound attenuation and sound velocity, the latter corresponding to stiffness (elasticity) of the cortical cytoplasm can be determined. In this study, measurements of stiffness distribution in XTH-2 cells were compared with the organization of F-actin and microtubules. Single XTH-2 cells exhibit relatively high stiffness at the free margins; toward the cell center this value decreases and reaches a sudden minimum where the slope of the surface topography enlargens at the margin of the dome-shaped cell center. The steepness of the increase in slope is linearly related to the decrease in sound velocity at this site. Thus, a significant determinant of cell shape is paralleled by an alteration of stiffness. In the most central parts, no interferences could be distinguished, therefore, this region had to be excluded from the calculations. Stiffness distribution roughly coincided with the distribution of F-actin, but no correlation to microtubule arrangement was found. Following the treatment of XTH-2 cells with ionomycin in the presence of calcium (in the culture medium), the cell cortex first contracted as indicated by shape changes and by a marked increase in stiffness (deduced from sound velocity). This contraction phase was followed by a phase of microtubule and F-actin disassembly. Concomittantly, sound velocity decreased considerably, indicating the loss of elasticity in the cell cortex. No structural equivalent to sound attenuation has been identified.     

The microtubule system in endothelial barrier dysfunction: disassembly of peripheral microtubules and microtubules reorganization in internal cytoplasm

Endothelial cell barrier dysfunction is often associated with dramatic cytoskeletal reorganization, activation of actomyosin contraction and finally gap formation. At present time the role of microtubules in endothelial cell barrier regulation is not fully understood, however a number of observations allow to assume that microtubules reaction is the extremely important part in development of endothelial dysfunction. These observations have been forced us to examine the role of microtubule system reorganization in endothelial cell barrier regulation. In quiescent endothelial cells microtubule density is the highest in the centrosome region and insignificant near the cell margin. The analysis of microtubules distribution after specific antibodies staining using the method of measurement of their fluorescence intensity has shown that in control endothelial cells the reduction of fluorescence intensity from the cell center to its periphery is described by the equation of an exponential regression. The hormone agent, thrombin (25 nM), causes rapid increase of endothelial cell barrier permeability accompanied by fast decrease in quantity of peripheral microtubules and reorganization of microtubule system in internal cytoplasm of endothelial cells (the decrease of fluorescence intensity is described by the equation of linear regress already through 10 min after the beginning of the treatment). Both effects are reversible -- through 60 min after the beginning of the treatment the microtubule network does not differ from normal one, so the microtubule system is capable to adapt for influence of a natural regulator thrombin. The microtubules reaction develops more quickly, than reorganization of the actin filaments system, which responsible for the subsequent changes in the cell shape during barrier dysfunction. Apparently, the microtubules are the first part in a circuit of the reactions leading to the pulmonary endothelial cell barrier compromise.     

Type-I IFN signaling is required for the induction of antigen-specific CD8(+) T cell responses by adenovirus vector vaccine in the gut-mucosa

Microtubules in living cells are very important component for various cellular functions as well as to maintain the cell shape. Mechanical properties of microtubules play a vital role in their functions and structure. To understand the mechanical properties of microtubules in living cells, we developed an orthotropic-Pasternak model and investigated the vibrational behavior when microtubules are embedded in surrounding elastic medium. We considered microtubules as orthotropic elastic shell and its surrounding elastic matrix as Pasternak foundation. We found that due to mechanical coupling of microtubules with elastic medium, the flexural vibration is increased with the stiffening of elastic medium. We noticed that foundation modulus (H) and shear modulus (G) have more effect on radial vibrational mode as compared to longitudinal vibrational mode and torsional vibrational mode.     

Can elastic stresses in gels cause the elongation and division of a cell?

Some metabolites present in the cell may affect its colloidal structure. If the cytoplasm is in the state of a gel, elastic stresses may appear as a result of non-uniform concentration of some metabolites which affect mechanical properties of gels. This paper investigates the question whether mechanical stresses, thus produced, can result in such a deformation of the cell which may lead to a division. The result of this study indicates that this is not possible. Elastic stresses, produced by non-uniformities of concentration of metabolites which affect the colloidal structures of the cytoplasm, may produce only a general dilation or contraction of the system, without change in shape.     

Development of a differentiated microtubule structure: formation of the chicken erythrocyte marginal band in vivo

The microtubules of mature nucleated erythrocytes are organized into a marginal band that is confined to a single plane at the periphery and that contains essentially the same number of microtubule profiles in each individual cell. Developing erythrocytes can be isolated in homogeneous and synchronously developing populations from chicken embryos. For these reasons, these cells offer a particularly accessible system for study of the pathway leading to a specific microtubule structure in a normal, terminally differentiated animal cell. Along this developmental course, striking changes occur in the properties of the microtubules. Between the postmitotic cell and the formation of the band, a novel arrangement is found: bundles of laterally associated microtubules in each cell, coursing through the cytoplasm but not confined to the periphery. The microtubule organizing centers evident at early stages disappear by the time the band forms. The microtubules in early cells are readily depolymerized by drugs, but that drug sensitivity is lost in the mature cells. The microtubule arrangement of mature cells is faithfully recapitulated after reversible depolymerization, while that of the immature cells is not. Finally, as the band forms, the microtubules and microfilaments increasingly become coaligned. In sum, the microtubules of immature cells have many properties in common with those of cultured cells, but during maturation those properties change. The results suggest that lateral interactions become increasingly important in stabilizing and organizing the microtubules. The properties of marginal band microtubules, and comparable properties of axonal microtubules, may reflect differences between the requirements for cytoskeletal structures of cycling cells and terminally differentiated cells.     

Cyclic amp and cell morphology in cultured fibroblasts. Effects on cell shape, microfilament and microtubule distribution, and orientation to substratum

The change in shape of 3T3 and L929 cells due to Bt2cAMP treatment is accompanied by altered intracellular distribution of microfilaments and microtubules. Bt2cAMP added to cells in low density culture causes (a) microfilaments to accumulate in bundles near the plasma membrane, mainly at the cell periphery, and (b) microtubules to accumulate beneath these microfilament bundles. In narrow cell processes that form characteristically in Bt2cAMP-treated L cells, microtubules accumulate in parallel arrays near the center of these processes. A new simple method for evaluating the relative distance of the cell from its underlying substratum is desribed. In normal medium, 3T3 cells attach to their substratum near the nucleus and at the tips of cell processes, bridging irregularities in the plastic surface. With Bt2cAMP treatment, attachment occurs at the cell edge and at many isolated points under the cytoplasm, and the cells conform more closely to irregularities of the underlying substratum. A model of the mechanism by which cAMP modulates cell shape is presented.     

Gradient of rigidity in the lamellipodia of migrating cells revealed by atomic force microscopy

Changes in mechanical properties of the cytoplasm have been implicated in cell motility, but there is little information about these properties in specific regions of the cell at specific stages of the cell migration process. Fish epidermal keratocytes with their stable shape and steady motion represent an ideal system to elucidate temporal and spatial dynamics of the mechanical state of the cytoplasm. As the shape of the cell does not change during motion and actin network in the lamellipodia is nearly stationary with respect to the substrate, the spatial changes in the direction from the front to the rear of the cell reflect temporal changes in the actin network after its assembly at the leading edge. We have utilized atomic force microscopy to determine the rigidity of fish keratocyte lamellipodia as a function of time/distance from the leading edge. Although vertical thickness remained nearly constant throughout the lamellipodia, the rigidity exhibited a gradual but significant decrease from the front to the rear of the lamellipodia. The rigidity profile resembled closely the actin density profile, suggesting that the dynamics of rigidity are due to actin depolymerization. The decrease of rigidity may play a role in facilitating the contraction of the actin-myosin network at the lamellipodium/cell body transition zone.     

Motor-driven marginal band coiling promotes cell shape change during platelet activation

Platelets float in the blood as discoid particles. Their shape is maintained by microtubules organized in a ring structure, the so-called marginal band (MB), in the periphery of resting platelets. Platelets are activated after vessel injury and undergo a major shape change known as disc to sphere transition. It has been suggested that actomyosin tension induces the contraction of the MB to a smaller ring. In this paper, we show that antagonistic microtubule motors keep the MB in its resting state. During platelet activation, dynein slides microtubules apart, leading to MB extension rather than contraction. The MB then starts to coil, thereby inducing the spherical shape of activating platelets. Newly polymerizing microtubules within the coiled MB will then take a new path to form the smaller microtubule ring, in concerted action with actomyosin tension. These results present a new view of the platelet activation mechanism and reveal principal mechanistic features underlying cellular shape changes.     

How Listeria exploits host cell actin to form its own cytoskeleton. II. Nucleation, actin filament polarity, filament assembly, and evidence for a pointed end capper

Processes such as cell locomotion and morphogenesis depend on both the generation of force by cytoskeletal elements and the response of the cell to the resulting mechanical loads. Many widely accepted theoretical models of processes involving cell shape change are based on untested hypotheses about the interaction of these two components of cell shape change. I have quantified the mechanical responses of cytoplasm to various chemical environments and mechanical loading regimes to understand better the mechanisms of cell shape change and to address the validity of these models. Measurements of cell mechanical properties were made with strands of cytoplasm submerged in media containing detergent to permeabilize the plasma membrane, thus allowing control over intracellular milieu. Experiments were performed with equipment that generated sinusoidally varying length changes of isolated strands of cytoplasm from Physarum polycephalum. Results indicate that stiffness, elasticity, and viscosity of cytoplasm all increase with increasing concentration of Ca2+, Mg2+, and ATP, and decrease with increasing magnitude and rate of deformation. These results specifically challenge assumptions underlying mathematical models of morphogenetic events such as epithelial folding and cell division, and further suggest that gelation may depend on both actin cross-linking and actin polymerization.     

Dissecting regional variations in stress fiber mechanics in living cells with laser nanosurgery

The ability of a cell to distribute contractile stresses across the extracellular matrix in a spatially heterogeneous fashion underlies many cellular behaviors, including motility and tissue assembly. Here we investigate the biophysical basis of this phenomenon by using femtosecond laser nanosurgery to measure the viscoelastic recoil and cell-shape contributions of contractile stress fibers (SFs) located in specific compartments of living cells. Upon photodisruption and recoil, myosin light chain kinase-dependent SFs located along the cell periphery display much lower effective elasticities and higher plateau retraction distances than Rho-associated kinase-dependent SFs located in the cell center, with severing of peripheral fibers uniquely triggering a dramatic contraction of the entire cell within minutes of fiber irradiation. Image correlation spectroscopy reveals that when one population of SFs is pharmacologically dissipated, actin density flows toward the other population. Furthermore, dissipation of peripheral fibers reduces the elasticity and increases the plateau retraction distance of central fibers, and severing central fibers under these conditions triggers cellular contraction. Together, these findings show that SFs regulated by different myosin activators exhibit different mechanical properties and cell shape contributions. They also suggest that some fibers can absorb components and assume mechanical roles of other fibers to stabilize cell shape.     

Microtubule system in endothelial barrier dysfunction: Disassembly of peripheral microtubules and microtubule reorganization in internal cytoplasm

Endothelial cell barrier dysfunction is associated with dramatic cytoskeletal reorganization, the activation of actomyosin contraction, and, finally, gap formation. Although the role of microtubules in the regulation of endothelial cell barrier function is not fully understood, a number of observations allow for the assumption that the reaction of the microtubule is an extremely important part in the development of endothelial dysfunction. These observations have forced us to examine the role of microtubule reorganization in the regulation of the endothelial cell barrier function. In quiescent endothelial cells, microtubule density is the highest in the centrosome region; however, microtubules are also present near the cell margin. The analysis of microtubule distribution after specific antibody staining using the method of measurement of their fluorescence intensity showed that, in control endothelial cells, the reduction of fluorescence intensity from the cell center to its periphery is described by the equation of exponential regression. The edemagenic agent, thrombin (25 nM), caused the rapid increase of endothelial cell barrier permeability accompanied by a fast decrease in quantity of the peripheral microtubules and reorganization of the microtubule system in the internal cytoplasm of endothelial cells (the decrease of fluorescence intensity is described by the equation of linear regress within as little as 5 min after the beginning of treatment). Both effects are reversible; within 60 min after the beginning of treatment, the microtubule network does not differ from the standard one. Thus, the microtubule system is capable of adapting to the influence of a natural regulator, thrombin. The reorganization of microtubules develops more quickly than the reorganization of the actin filaments system responsible for the subsequent changes of the cell shape during barrier dysfunction. Apparently, the microtubules are the first part in the circuit of the reactions leading to the pulmonary endothelial cell barrier compromise.     

Microtubule-dependent control of cell shape and pseudopodial activity is inhibited by the antibody to kinesin motor domain

One of the major functions of cytoplasmic microtubules is their involvement in maintenance of asymmetric cell shape. Microtubules were considered to perform this function working as rigid structural elements. At the same time, microtubules play a critical role in intracellular organelle transport, and this fact raises the possibility that the involvement of microtubules in maintenance of cell shape may be mediated by directed transport of certain cellular components to a limited area of the cell surface (e.g., to the leading edge) rather than by their functioning as a mechanical support. To test this hypothesis we microinjected cultured human fibroblasts with the antibody (called HD antibody) raised against kinesin motor domain highly conserved among the different members of kinesin superfamily. As was shown before this antibody inhibits kinesin-dependent microtubule gliding in vitro and interferes with a number of microtubule-dependent transport processes in living cells. Preimmune IgG fraction was used for control experiments. Injections of fibroblasts with HD antibody but not with preimmune IgG significantly reduced their asymmetry, resulting in loss of long processes and elongated cell shape. In addition, antibody injection suppressed pseudopodial activity at the leading edge of fibroblasts moving into an experimentally made wound. Analysis of membrane organelle distribution showed that kinesin antibody induced clustering of mitochondria in perinuclear region and their withdrawal from peripheral parts of the cytoplasm. HD antibody does not affect either density or distribution of cytoplasmic microtubules. The results of our experiments show that many changes of phenotype induced in cells by microtubule-depolymerizing agents can be mimicked by the inhibition of motor proteins, and therefore microtubule functions in maintaining of the cell shape and polarity are mediated by motor proteins rather than by being provided by rigidity of tubulin polymer itself.     

A microtubule-based, dynein-dependent force induces local cell protrusions: Implications for neurite initiation

A key event in neurite initiation is the accumulation of microtubule bundles at the neuron periphery. We hypothesized that such bundled microtubules may generate a force at the plasma membrane that facilitates neurite initiation. To test this idea we observed the behavior of microtubule bundles that were induced by the microtubule-associated protein MAP2c. Endogenous MAP2c contributes to neurite initiation in primary neurons, and exogeneous MAP2c is sufficient to induce neurites in Neuro-2a cells. We performed nocodazol washout experiments in primary neurons, Neuro-2a cells and COS-7 cells to investigate the underlying mechanism. During nocodazol washout, small microtubule bundles formed rapidly in the cytoplasm and immediately began to move toward the cell periphery in a unidirectional manner. In neurons and Neuro-2a cells, neurite-like processes extended within minutes and concurrently accumulated bundles of repolymerized microtubules. Speckle microscopy in COS-7 cells indicated that bundle movement was due to transport, not treadmilling. At the periphery bundles remained under a unidirectional force and induced local cell protrusions that were further enhanced by suppression of Rho kinase activity. Surprisingly, this bundle motility was independent of classical actin- or microtubule-based tracks. It was, however, reversed by function-blocking antibodies against dynein. Suppression of dynein expression in primary neurons by RNA interference severely inhibited the generation of new neurites, but not the elongation of existing neurites formed prior to dynein knockdown. Together, these cell biological data suggest that neuronal microtubule-associated proteins induce microtubule bundles that are pushed outward by dynein and locally override inward contraction to initiate neurite-like cell protrusions. A similar force-generating mechanism might participate in spontaneous initiation of neurites in developing neurons. Electronic Supplementry Materials: Supplementary Materials are available in the online version of this article at     

Tensional Homeostasis in Single Fibroblasts

Adherent cells generate forces through acto-myosin contraction to move, change shape, and sense the mechanical properties of their environment. They are thought to maintain defined levels of tension with their surroundings despite mechanical perturbations that could change tension, a concept known as tensional homeostasis. Misregulation of tensional homeostasis has been proposed to drive disorganization of tissues and promote progression of diseases such as cancer. However, whether tensional homeostasis operates at the single cell level is unclear. Here, we directly test the ability of single fibroblast cells to regulate tension when subjected to mechanical displacements in the absence of changes to spread area or substrate elasticity. We use a feedback-controlled atomic force microscope to measure and modulate forces and displacements of individual contracting cells as they spread on a fibronectin-patterned atomic-force microscope cantilever and coverslip. We find that the cells reach a steady-state contraction force and height that is insensitive to stiffness changes as they fill the micropatterned areas. Rather than maintaining a constant tension, the fibroblasts altered their contraction force in response to mechanical displacement in a strain-rate-dependent manner, leading to a new and stable steady-state force and height. This response is influenced by overexpression of the actin crosslinker α-actinin, and rheology measurements reveal that changes in cell elasticity are also strain- rate-dependent. Our finding of tensional buffering, rather than homeostasis, allows cells to transition between different tensional states depending on how they are displaced, permitting distinct responses to slow deformations during tissue growth and rapid deformations associated with injury.     

Tensional Homeostasis in Single Fibroblasts

Adherent cells generate forces through acto-myosin contraction to move, change shape, and sense the mechanical properties of their environment. They are thought to maintain defined levels of tension with their surroundings despite mechanical perturbations that could change tension, a concept known as tensional homeostasis. Misregulation of tensional homeostasis has been proposed to drive disorganization of tissues and promote progression of diseases such as cancer. However, whether tensional homeostasis operates at the single cell level is unclear. Here, we directly test the ability of single fibroblast cells to regulate tension when subjected to mechanical displacements in the absence of changes to spread area or substrate elasticity. We use a feedback-controlled atomic force microscope to measure and modulate forces and displacements of individual contracting cells as they spread on a fibronectin-patterned atomic-force microscope cantilever and coverslip. We find that the cells reach a steady-state contraction force and height that is insensitive to stiffness changes as they fill the micropatterned areas. Rather than maintaining a constant tension, the fibroblasts altered their contraction force in response to mechanical displacement in a strain-rate-dependent manner, leading to a new and stable steady-state force and height. This response is influenced by overexpression of the actin crosslinker α-actinin, and rheology measurements reveal that changes in cell elasticity are also strain- rate-dependent. Our finding of tensional buffering, rather than homeostasis, allows cells to transition between different tensional states depending on how they are displaced, permitting distinct responses to slow deformations during tissue growth and rapid deformations associated with injury.     

Mechanical properties of cytoskeletal polymers

The mechanical properties of cytoplasm are dominated by microfilaments, microtubules, and intermediate filaments, collectively termed the cytoskeleton. This review discusses how the physical properties of these biopolymer systems are related to their molecular structures and interactions, and how remodelling of these biopolymers in vivo affects cell shape and motility.     

Elasticity Maps of Living Neurons Measured by Combined Fluorescence and Atomic Force Microscopy

Detailed knowledge of mechanical parameters such as cell elasticity, stiffness of the growth substrate, or traction stresses generated during axonal extensions is essential for understanding the mechanisms that control neuronal growth. Here, we combine atomic force microscopy-based force spectroscopy with fluorescence microscopy to produce systematic, high-resolution elasticity maps for three different types of live neuronal cells: cortical (embryonic rat), embryonic chick dorsal root ganglion, and P-19 (mouse embryonic carcinoma stem cells) neurons. We measure how the stiffness of neurons changes both during neurite outgrowth and upon disruption of microtubules of the cell. We find reversible local stiffening of the cell during growth, and show that the increase in local elastic modulus is primarily due to the formation of microtubules. We also report that cortical and P-19 neurons have similar elasticity maps, with elastic moduli in the range 0.1–2 kPa, with typical average values of 0.4 kPa (P-19) and 0.2 kPa (cortical). In contrast, dorsal root ganglion neurons are stiffer than P-19 and cortical cells, yielding elastic moduli in the range 0.1–8 kPa, with typical average values of 0.9 kPa. Finally, we report no measurable influence of substrate protein coating on cell body elasticity for the three types of neurons.     

Subcellular tension fields and mechanical resistance of the lamella front related to the direction of locomotion

Keratocytes derived from the epidermis of aquatic vertebrates are now widely used for investigation of the mechanism of cell locomotion. One of the main topics under discussion is the question of driving force development and concomitantly subcellular force distribution. Do cells move by actin polymerization-driven extension of the lamella, or is the lamella edge extended at regions of weakness by a flow of cytoplasm generated by hydrostatic pressure? Thus, elasticity changes were followed and the stiffness of the leading front of the lamella was manipulated by local application of phalloidin and cytochalasin D (CD). In scanning acoustic microscopy (SAM), elasticity is revealed from the propagation velocity of longitudinal sound waves (1 GHz). The lateral resolution of SAM is in the micrometer range. Using this method, subcellular tension fields with different stiffnesses (elasticity) can be determined. A typical pattern of subcellular stiffness distribution is related to the direction of migration. Cells forced to change their direction of movement by exposure to DC electric fields of varying polarity alter their pattern of subcellular stiffness in relationship to the new direction. The cells spread into the direction of low stiffness and retract at zones of high stiffness. The pattern of subcellular stiffness distribution reveals force distribution in migrating cells; i.e., if a cell moves exactly in a direction perpendicular to its long axis, then the contractile forces are largest along the long axis and decrease toward the short axis. Locomotion in any angle oblique to this axis requires an asymmetric stiffness distribution. Inhibition of actomyosin contractions by La³⁺ (2 mM), which inhibits Ca²⁺ influx, reduces cytoplasmic stiffness accompanied by an immediate cessation of locomotion and a change of cell shape. Local release of CD in front of a progressing lamella activates a cell to follow the CD gradient: The lamella thickens locally and is extended toward the tip of the microcapillary. Release of phalloidin stops extension of the lamella, and the cell turns away from the releasing microcapillary. The response to CD is assumed to be the result of local weakening of the cytoplasm due to severing of the actin fibrils. Phalloidin is supposed to stabilize the leading front by inhibition of F-actin depolymerization. These observations are in favor of the assumption that migration is due to an extension of the cell into the direction of minimum stiffness, and they are consistent with the hypothesis that local release of hydrostatic pressure provides the driving force for the flux of cytoplasm.     

Radial-organized microtubules provide cell shape support and more effective intercellular transport then free microtubules

Microtubules take part in various cell processes, including cell polarization, migration, intercellular transport, and some others. Therefore, the spatial organization of microtubules is crucial for normal cell behavior. Fibroblasts have radial microtubule arrays that consist of microtubules that run from the centrosome. Two components compose this microtubule array, i.e., (1) minus ends attached to the centrosome microtubules with their plus ends radiating to the cell periphery and (2) free microtubules with ends not attached to the centrosome. Distinctions in the dynamic properties, intercellular organization, and structure of centrosome-attached and free microtubules allow us to assume that their cellular functions are also different. To study centrosome-attached and free microtubules functions, we used cytoplasts, i.e., nucleus-lacking cellular fragments that, under certain conditions, also lose their centrosomes. In these cytoplasts, there are only free microtubules. The shape, general morphology, and size of cytoplasts that retain their centrosomes differ only slightly from whole cells. Cytoplasts who have lost their centrosomes have an extremely thin network of microtubules located in their central region; furthermore, they lose the shape that is typical for fibroblast and become rough lamellae with protrusions. The internal architecture of the cytoplasm and organoid arrangement are also broken. Saltatory movements in cytoplasts with centrosomes are similar to those in whole cells; in cytoplasts without centrosomes, saltatory movements occur with velocities that are twofold less and by shorter distances. Saltatory movements of granules in centrosome-lacking cytoplasts took place basically in the central region of cytoplast and were less ordered than in whole cells and in cytoplasts with centrosomes. We believe that radial organized microtubules ensure the effective transport and dynamical interaction of microtubule plus ends with cellular cortical structures, which is sufficient to support the common fibroblast-like shape, whereas the disorganized free microtubules are not able to maintain the external fibroblast shape and its intercellular organization.