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.     

Cell contraction caused by microtubule disruption is accompanied by shape changes and an increased elasticity measured by scanning acoustic microscopy

The state of crosslinking of microfilaments and the state of myosin-driven contraction are the main determinants of the mechanical properties of the cell cortex underneath the membrane, which is significant for the mechanism of shaping cells. Therefore, any change in the contractile state of the actomyosin network would alter the mechanical properties and finally result in shape changes. The relationship of microtubules to the mechanical properties of cells is still obscure. The main problem arises because disruption of microtubules enhances acto-myosin-driven contraction. This reaction and its impact on cell shape and elasticity have been investigated in single XTH-2 cells. Microtubule disruption was induced by colcemid, a polymerization inhibitor. The reaction was biphasic: a change in cell shape from a fried egg shape to a convex surface topography was accompanied by an increase in elastic stiffness of the cytoplasm, measured as longitudinal sound velocity revealed by scanning acoustic microscope. Elasticity increases in the cell periphery and reaches its peak after 30 min. Subsequently while the cytoplasm retracts from the periphery, longitudinal sound velocity (elasticity) decreases. Simultaneously, a two- to threefold increase of F-actin and alignment of stress fibers from the cell center to cell-cell junctions in dense cultures are induced, supposedly a consequence of the increased tension.     

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.     

Actin organization during the cell cycle in meristematic plant cells. Actin is present in the cytokinetic phragmoplast

The distribution and organisation of F-actin during the cell cycle of meristematic root-tip cells of Allium was investigated using a rhodamine-labelled phalloidin to stain F-actin in isolated cell preparations. Such preparations could, in addition, be stained for tubulin by immunofluorescence, enabling a comparison between F-actin and microtubule distributions in the same cell. In interphase, an extensive array of actin-filament bundles was present in the cytoplasm of elongating cells, the bundles generally following the long axis of the cell and passing in close proximity to the nucleus. In contrast, the interphase microtubule array occupied the cortex of the cell and was oriented at right angles to the actin bundles. In smaller, isodiametric cells, microfilament arrays were present but less well developed. During cell division, phalloidin-specific staining was seen in the cytokinetic phragmoplast, and co-distributed with microtubules at all stages of cell plate formation; however, neither the pre-prophase band nor the mitotic spindle were stained with phalloidin. Co-distribution of F-actin and microtubules only occurs, therefore, at cytokinesis. The relationship between microfilaments and microtubules is discussed, together with the possible role of actin in the phragmoplast.     

Scanning microfluorometric measurement of immunofluorescently labelled microtubules in cultured cells. Dependence of microtubule content on cell density

A method for evaluation of microtubule content in cultured cells has been developed. The method is based on scanning microfluorometric measurement of immunofluorescently labelled microtubules. The method has been applied to the comparison of microtubule content in epithelial XTH-2 cells grown in culture at various cell densities. The results have shown that the microtubule content in the cells is not dependent on their proliferative state rather than it depends on cellular contacts.     

Scanning microfluorometric measurement of TRITC-phalloidin labelled F-actin. Dependence of F-actin content on density of normal and transformed cells

A method is described for the determination of cellular F-actin content using fluorometry of TRITC-phalloidin at very high dilution. In this case no saturation of all binding sites available is reached, however the staining is highly specific and the specificity is not affected by the preparative procedure as may be the case at high concentrations of TRITC-phalloidin. The method is based on calculation of fluorescence intensity at equilibrium conditions from measurements at two different numbers of exchange of the staining solution by using Lineweaver-Burk plotting. The relative content of F-actin has been determined for three established cell lines, an amphibian cell line XTH-2 of endothelial origin, 3T3 cells and SV 40 transformed 3T3 cells. In the two non transformed lines F-actin decreases with increasing cell density starting with the onset of confluency of the culture. SV 40 transformed 3T3 cells generally contain less F-actin and do not show any significant point of change. The decrease in F-actin with increasing cell density is accompanied by a disappearance of stress fibres. SV 40 3T3 cells generally are devoid of stress fibres. The observations are discussed considering a possible involvement of F-actin in growth control.     

Relationship of F-actin distribution to development of polar shape in human polymorphonuclear neutrophils

Polymerization of actin has been associated with development of polar shape in human neutrophils (PMN). To examine the relation of filamentous actin (F-actin) distribution to shape change in PMN, we developed a method using computerized video image analysis and fluorescence microscopy to quantify distribution of F-actin in single cells. PMN were labeled with fluorescent probe NBD-phallicidin to measure filamentous actin and Texas red to assess cell thickness. We show that Texas red fluorescence is a reasonable measure of cell thickness and that correction of the NBD-phallicidin image for cell thickness using the Texas red image permits assessment of focal F-actin content. Parameters were derived that quantify total F-actin content, movement of F-actin away from the center of the cell, asymmetry of F-actin distribution, and change from round to polar shape. The sequence of change in F-actin distribution and its relation to development of polar shape in PMN was determined using these parameters. After stimulation with chemotactic peptide at 25 degrees C, F-actin polymerized first at the rim of the PMN. This was followed by development of asymmetry of F-actin distribution and change to polar shape. The dominant pseudopod developed first in the region of lower F-actin concentration followed later by polymerization of actin in the end of the developed pseudopod. Asymmetric F-actin distribution was detected in round PMN before development of polar shape. Based upon these data, asymmetric distribution of F-actin is coincident with and probably precedes development of polar shape in PMN stimulated in suspension by chemotactic peptide.     

The protein kinase kin1 is required for cellular symmetry in fission yeast

The fission yeast Schizosaccharomyces pombe is a highly polarized unicellular eukaryote with two opposite growing poles in which F-actin cytoskeleton is focused. The KIN1/PAR-1/MARK protein family is composed of conserved eukaryotic serine/threonine kinases which are involved in cell polarity, microtubule stability or cell cycle regulation. Here, we investigate the function of the fission yeast KIN1/PAR-1/MARK member, kin1p. Using a deletion allele (kin1Delta), we show that kin1 mutation promotes a delay in septation. Kin1p regulates the structure of the new cell end after cytokinesis by modulating cell wall remodeling. Abnormal shaped interphase kin1Delta cells misplace F-actin patches and the premitotic nucleus. Thus, mitotic kin1Delta cells misposition the F-actin ring assembly site that is dependent on the position of the interphase nucleus. The resulting asymmetric cell division produces daughter cells with distinct shapes. Overexpressed kin1p accumulates asymmetrically at the cell cortex and affects cell shape, F-actin organization and microtubules. Our results suggest that correct dosage of kin1p at the cortex is required for spatial organization of the fission yeast cell.     

Measuring elastic properties of cells by evaluation of scanning acoustic microscopy V(Z) values using simplex algorithm

In this paper a new technique is proposed to determine the acoustic properties as well as the thickness (and volume) of biological cells. Variations of thickness, density, acoustic wave velocity, stiffness, and attenuation coefficient of a living or dead cell are obtained by scanning the cell by an acoustic microscope. The distance between the cell and the microscope lens is varied and several voltage curves are thus obtained. These curves are then inverted by simplex optimization technique to obtain the cell parameters. The spatial resolution of the method is limited to the resolution of the scanning acoustic microscope. It allows to take advantage of the full range of frequencies and amplification of the microscope. Characteristic distributions of stiffness are exemplified with an endothelial cell in culture. The main part of the thin, lamellar cytoplasm has high stiffness, which drops close to the lamella/cell body transition region and only slightly increases again through the central part of the cell. Acoustic attenuation seems to be related to two factors, cytoplasm accumulation (in the lamellar parts) and scattering in the central part rich in organelles.     

The role of actin, actomyosin and microtubules in defining cell shape during the differentiation of Naegleria amebae into flagellates

Differentiation of Naegleria amebae into flagellates was used to examine the interaction between actin, actomyosin and microtubules in defining cell shape. Amebae, which lack microtubules except during mitosis, differentiate into flagellates with a fixed shape and a complex microtubule cytoskeleton in 120 min. Based on earlier models of ameboid motility it has been suggested that actomyosin is quiescent in flagellates. This hypothesis was tested by following changes in the cytoskeleton using three-dimensional reconstructions prepared by confocal microscopy of individual cells stained with antibodies against actin and tubulin as well as with phalloidin and DNase I. F-actin as defined by phalloidin staining was concentrated in expanding pseudopods. Most phalloidin staining was lost as cells rounded up before the onset of flagellum formation. Actin staining with a Naegleria-specific antibody that recognizes both F- and G-actin was confined to the cell cortex of both amebae and flagellates. DNase I demonstrated G-actin throughout all stages. Most of the actin in the cortex was not bound by phalloidin yet was resistant to detergent extraction suggesting that it was polymerized. The microtubule cytoskeleton of flagellates was intimately associated with this actin cortex. Treatment of flagellates with cytochalasin D produced a rapid loss of flagellate shape and the appearance of phalloidin staining while latrunculin A stabilized the flagellate shape. These results suggest that tension produced by an actomyosin network is required to maintain the flagellate shape. The rapid loss of the flagellate shape induced by drugs, which specifically block myosin light chain kinase, supports this hypothesis.     

Scanning microfluorometric measurement of immunofluorescently labelled microtubules in cultured cells

Summary A method for evaluation of microtubule content in cultured cells has been developed. The method is based on scanning microfluorometric measurement of immunofluorescently labelled microtubules. The method has been applied to the comparison of microtubule content in epithelial XTH-2 cells grown in culture at various cell densities. The results have shown that the microtubule content in the cells is not dependent on their proliferative state rather than it depends on cellular contacts.     

Multiple actin binding domains of Ena/VASP proteins determine actin network stiffening

Vasodilator-stimulated phosphoprotein (Ena/VASP) is an actin binding protein, important for actin dynamics in motile cells and developing organisms. Though VASP’s main activity is the promotion of barbed end growth, it has an F-actin binding site and can form tetramers, and so could additionally play a role in actin crosslinking and bundling in the cell. To test this activity, we performed rheology of reconstituted actin networks in the presence of wild-type VASP or mutants lacking the ability to tetramerize or to bind G-actin and/or F-actin. We show that increasing amounts of wild-type VASP increase network stiffness up to a certain point, beyond which stiffness actually decreases with increasing VASP concentration. The maximum stiffness is 10-fold higher than for pure actin networks. Confocal microscopy shows that VASP forms clustered actin filament bundles, explaining the reduction in network elasticity at high VASP concentration. Removal of the tetramerization site results in significantly reduced bundling and bundle clustering, indicating that VASP’s flexible tetrameric structure causes clustering. Removing either the F-actin or the G-actin binding site diminishes VASP’s effect on elasticity, but does not eliminate it. Mutating the F-actin and G-actin binding site together, or mutating the F-actin binding site and saturating the G-actin binding site with monomeric actin, eliminates VASP’s ability to increase network stiffness. We propose that, in the cell, VASP crosslinking confers only moderate increases in linear network elasticity, and unlike other crosslinkers, VASP’s network stiffening activity may be tuned by the local concentration of monomeric actin.     

Scanning microfluorometric measurement of TRITC-phalloidin labelled F-actin

Summary A method is described for the determination of cellular F-actin content using fluorometry of TRITC-phalloidin at very high dilution. In this case no saturation of all binding sites available is reached, however the staining is highly specific and the specificity is not affected by the preparative procedure as may be the case at high concentrations of TRITC-phalloidin. The method is based on calculation of fluorescence intensity at equilibrium conditions from measurements at two different numbers of exchange of the staining solution by using Lineweaver-Burk plotting.The relative content of F-actin has been determined for three established cell lines, an amphibian cell line XTH-2 of endothelial origin, 3T3 cells and SV 40 transformed 3T3 cells. In the two non transformed lines F-actin decreases with increasing cell density starting with the onset of confluency of the culture. SV 40 transformed 3T3 cells generally contain less F-actin and do not show any significant point of change. The decrease in F-actin with increasing cell density is accompanied by a disappearance of stress fibres. SV 40 3T3 cells generally are devoid of stress fibres. The observations are discussed considerung a possible involvement of F-actin in growth control.     

Microtubule dynamics differentially regulates Rho and Rac activity and triggers Rho-independent stress fiber formation in macrophage polykaryons

Multinucleated giant cells (MNGC) derived from avian peripheral blood monocytes present a dense microtubular network emanating from peripherally located centrosomes. We were interested to study how microtubule and F-actin cytoskeletons cooperate in MNGC to maintain cell shape. Microtubule depolymerization by nocodazole triggered the reorganization of the F-actin cytoskeleton in MNGC that is normally organized into podosomes, cortical actin filaments and membrane ruffles. After nocodazole treatment, F-actin was redistributed into unusual transverse fibers associated with focal adhesion plaques. When microtubules were allowed to repolymerize after nocodazole removal, F-actin appeared transiently, together with the small GTPase Rac, in large membrane ruffles. Using affinity precipitation assays, we show that microtubule depolymerization leads to activation of Rho and inhibition of Rac, whereas microtubule repolymerization induces Rac activation and Rho inhibition. Thus, the level of microtubule polymerization inversely regulates Rho and Rac activity in MNGC. Moreover, using C3 exoenzyme, a known inhibitor of Rho, we demonstrate that both the F-actin fiber formation in response to microtubule depolymerization and the formation of membrane ruffles after microtubule repolymerization occur in C3-treated MNGC, indicating that Rho is not required for these events.     

Three-dimensional localization and redistribution of F-actin in higher plant mitosis and cell plate formation

The distribution of F-actin cables in dividing endosperm cells of a higher plant, Haemanthus, was visualized with the immunogold-silver-enhanced method and compared with the arrangement of immunogold-stained microtubules in the same cells. The three-dimensional distribution of F-actin cables and microtubules during mitosis and cell plate formation was analyzed using ultrathin optical sectioning of whole mounts in polarized light video microscopy. F-actin cables form a loose irregular network in the interphase cytoplasm. Much of this network remains outside of the spindle during mitosis. A few F-actin cables were detected within the spindle. Their pronounced rearrangement during mitosis appears to be related to the presence and growth of microtubule arrays. During prometaphase, actin cables located on the spindle surface and those present within the spindle tend to arrange parallel to the long axis of the spindle. Cables outside the spindle do not reorient, except those at the polar region, where they appear to be compressed by the elongating spindle. Beginning with mid-anaphase, shorter actin cables oriented in various directions accumulate at the equator. Some of them are incorporated into the phragmoplast and cell plate and are gradually fragmented as the cell plate is formed and ages. Actin cables adjacent to microtubule arrays often show a regular punctate staining pattern. Such a pattern is seldom observed in the peripheral cytoplasm, which contains few microtubules. The rearrangement of F-actin cables mimicks the behavior of spindle inclusions, such as starch grains, mitochondria, etc., implying that F-actin is redistributed passively by microtubule growth or microtubule-related transport. Thus F-actin or actomyosin-based motility does not appear to be directly involved in mitosis and cytokinesis in higher plants.     

Cortical actin nanodynamics determines nitric oxide release in vascular endothelium

The release of the main vasodilator nitric oxide (NO) by the endothelial NO synthase (eNOS) is a hallmark of endothelial function. We aim at elucidating the underlying mechanism how eNOS activity depends on cortical stiffness (К(cortex)) of living endothelial cells. It is hypothesized that cortical actin dynamics determines К(cortex) and directly influences eNOS activity. By combined atomic force microscopy and fluorescence imaging we generated mechanical and optical sections of single living cells. This approach allows the discrimination between К(cortex) and bulk cell stiffness (К(bulk)) and, additionally, the simultaneous analysis of submembranous actin web dynamics. We show that К(cortex) softens when cortical F-actin depolymerizes and that this shift from a gel-like stiff cortex to a soft G-actin rich layer, triggers the stiffness-sensitive eNOS activity. The results implicate that stiffness changes in the ～100 nm phase of the submembranous actin web, without affecting К(bulk), regulate NO release and thus determines endothelial function.     

The myosin ATPase inhibitor 2,3-butanedione-2-monoxime disorganizes microtubules as well as F-actin in Saccharomyces cerevisiae

Interactions between microtubules and filamentous actin (F-actin) are essential to many cellular processes, but their mechanisms are poorly understood. We investigated possible roles of the myosin family of proteins in the interactions between filamentous actin (F-actin) and microtubules of budding yeast Saccharomyces cerevisiae with the general myosin ATPase inhibitor 2,3-butanedione-2-monoxime (BDM). The growth of S. cerevisiae was completely inhibited by BDM at 20 mmol/L and the effect of BDM on cell growth was reversible. In more than 80% of BDM-treated budding yeast cells, the polarized distribution of F-actin was lost and fewer F-actin dots were observed. When cells were synchronized in G₁ with α-factor and released in the presence of BDM, cell number did not increase and cells were mainly arrested in G₁ DNA content without any bud, suggesting that myosin activity is required for new bud formation and the start of a new cell cycle. More than 10% of the BDM-treated cells also revealed defects in nuclear migration to the bud neck as well as in nuclear shape. Consistent with these defects, the orientation of mitotic spindles was random in the 57% of cells treated with 20 mmol/L BDM and immunostained with anti-tubulin antibody. Furthermore, microtubule structures were completely disorganized in most of the cells incubated in 50 mmol/L BDM, while similar amounts of tubulin proteins were present in both BDM-treated and untreated cells. These results show that the general myosin inhibitor BDM disorganizes microtubule structures as well as F-actin, and suggest that BDM-sensitive myosin activities are necessary for the interaction of F-actin and microtubules to coordinate polarized bud growth and the shape and migration of the nucleus in S. cerevisiae. This revised version was published online in July 2006 with corrections to the Cover Date.     

The nucleocytoplasmic microfilament network in protoplasts from cultured soybean cells is a plastic entity that pervades the cytoplasm except the central vacuole

The microfilament network of cultured Glycine max cells (SB-1 line), and protoplasts was visualized with rhodamine-phalloidin under conditions that lysed the protoplast and changed the cell shape. The whole cell had the typical microfilament distribution of a "cage" around the nucleus, from which the large subcortical cables and transvacuolar strands radiated towards the cortex until it reached the cortical microfilament network. Upon cell wall removal, the network conserved its compartmentalization. Thus, the redistribution of the shape where the vacuole becomes a central entity, made the cytoplasm displace peripherally, but the network distribution was conserved. When protoplasts were lysed in a low osmotic medium, the vacuoles were gradually released intact. Under these conditions, the F-actin staining remained within the ghost of the cell, but none was detected in either emerging or almost completely released vacuoles. Most of the released F-actin was found in debris from the cell lysate in the form of microfilaments. When the ghosts were constrained in a coverslip with an air bubble, the shape of the ghost changed accordingly, but the microfilament network distribution remained constant. These results provide further evidence that the vacuole of plant cells does not have detectable associated F-actin. In addition, we demonstrate that the actin microfilament network is a moldable entity that can change its shape but keeps its distribution under constant conditions, in these cultured cells.     

Cytoskeletal changes in actin and microtubules underlie the developing surface mechanical properties of sensory and supporting cells in the mouse cochlea

Correct patterning of the inner ear sensory epithelium is essential for the conversion of sound waves into auditory stimuli. Although much is known about the impact of the developing cytoskeleton on cellular growth and cell shape, considerably less is known about the role of cytoskeletal structures on cell surface mechanical properties. In this study, atomic force microscopy (AFM) was combined with fluorescence imaging to show that developing inner ear hair cells and supporting cells have different cell surface mechanical properties with different developmental time courses. We also explored the cytoskeletal organization of developing sensory and non-sensory cells, and used pharmacological modulation of cytoskeletal elements to show that the developmental increase of hair cell stiffness is a direct result of actin filaments, whereas the development of supporting cell surface mechanical properties depends on the extent of microtubule acetylation. Finally, this study found that the fibroblast growth factor signaling pathway is necessary for the developmental time course of cell surface mechanical properties, in part owing to the effects on microtubule structure.     

Colocalization of cortical microtubules and F-actin in<Emphasis Type="Italic">Dipodascus magnusii</Emphasis> using confocal laser scanning microscopy

Distribution of microtubules and F-actin in aerobically growing cells of Dipodascus magnusii, belonging to the class Saccharomycetes was analyzed using immunofluorescence microscopy and labeling with rhodamine-tagged phalloidin. A conspicuous system of permanent cytoplasmic microtubules was observed in association with multiple nuclei. In elongating cells, helices of cytoplasmic microtubules appeared at the cell cortex. In cells approaching cytokinesis transversely oriented microtubules were revealed at incipient division sites. Confocal laser scanning microscopy showed a continuity of these transverse microtubules with the remaining microtubule network. The actin system of D. magnusii consisted of patches and filaments. Patches were found to accumulate at the tips of growing cells. Bands of fine actin filaments were usually observed before F-actin rings were established. A close cortical association of microtubules with the F-actin ring was documented on individual optical sections of labeled cells. Cells with developing septa showed medial F-actin discs associated at both sides with microtubules. Colocalization of cytoplasmic microtubules with actin filaments at the cortex of dividing cells supports a role of both cytoskeletal components in controlling cell wall growth and septum formation in D. magnusii.