Chondrocyte Deformation Induces Mitochondrial Distortion and Heterogeneous Intracellular Strain Fields

Chondrocyte mechanotransduction is poorly understood but may involve cell deformation and associated distortion of intracellular structures and organelles. This study quantifies the intracellular displacement and strain fields associated with chondrocyte deformation and in particular the distortion of the mitochondria network, which may have a role in mechanotransduction. Isolated articular chondrocytes were compressed in agarose constructs and simultaneously visualised using confocal microscopy. An optimised digital image correlation technique was developed to calculate the local intracellular displacement and strain fields using confocal images of fluorescently labelled mitochondria. The mitochondria formed a dynamic fibrous network or reticulum, which co-localised with microtubules and vimentin intermediate filaments. Cell deformation induced distortion of the mitochondria, which collapsed in the axis of compression with a resulting loss of volume. Compression generated heterogeneous intracellular strain fields indicating mechanical heterogeneity within the cytoplasm. The study provides evidence supporting the potential involvement of mitochondrial deformation in chondrocyte mechanotransduction, possibly involving strain-mediated release of reactive oxygen species. Furthermore the heterogeneous strain fields, which appear to be influenced by intracellular structure and organisation, may generate significant heterogeneity in mechanotransduction behaviour for cells subjected to identical levels of deformation.     

Mechanical compression and hydrostatic pressure induce reversible changes in actin cytoskeletal organisation in chondrocytes in agarose

In numerous cell types, the cytoskeleton has been widely implicated in mechanotransduction pathways involving stretch-activated ion channels, integrins and deformation of intracellular organelles. Studies have also demonstrated that the cytoskeleton can undergo remodelling in response to mechanical stimuli such as tensile strain or fluid flow. In articular chondrocytes, the mechanotransduction pathways are complex, inter-related and as yet, poorly understood. Furthermore, little is known of how the chondrocyte cytoskeleton responds to physiological mechanical loading. This study utilises the well-characterised chondrocyte-agarose model and an established confocal image-analysis technique to demonstrate that both static and cyclic, compressive strain and hydrostatic pressure all induce remodelling of actin microfilaments. This remodelling was characterised by a change from a uniform to a more punctate distribution of cortical actin around the cell periphery. For some loading regimes, this remodelling was reversed over a subsequent 1h unloaded period. This reversible remodelling of actin cytoskeleton may therefore represent a mechanism through which the chondrocyte alters its mechanical properties and mechanosensitivity in response to physiological mechanical loading.     

Compression-induced changes in the shape and volume of the chondrocyte nucleus

Changes in cell shape and volume are believed to play a role in the process of mechanical signal transduction by chondrocytes in articular cartilage. One proposed pathway through which chondrocyte deformation may be transduced to an intracellular signal is through cytoskeletally mediated deformation of intracellular organelles, and more specifically, of the cell nucleus. In this study, confocal scanning laser microscopy was used to perform in situ three-dimensional morphometric analyses of the nuclei of viable condrocytes during controlled compression of articular cartilage explants from the canine patellofemoral groove. Unconfined compression of the tissue to a 15% surface-to-surface strain resulted in a significant decrease of chondrocyte height and volume by 14.7 ± 6.4 and 11.4 ± 8.4%, respectively, and of nuclear height and volume by 8.8 ± 6.2% and 9.8 ± 8.8%, respectively. Disruption of the actin cytoskeleton using cytochalasin D altered the relationship between matrix deformation and changes in nuclear height and shape, but not volume. The morphology and deformation behavior of the chondrocytes were not affected by cytochalasin treatment. These results suggest that the actin cytoskeleton plays an important role in the link between compression of the extracellular matrix and deformation of the chondrocyte nuclei and imply that chondrocytes and their nuclei undergo significant changes in shape and volume in vivo.     

Confocal analysis of cytoskeletal organisation within isolated chondrocyte sub-populations cultured in agarose

This study reports the cytoskeletal organisation within chondrocytes, isolated from the superficial and deep zones of articular cartilage and seeded into agarose constructs. At day 0, marked organisation of actin microfilaments was not observed in cells from both zones. Partial or clearly organised microtubules and vimentin intermediate filaments cytoskeletal components were present, however, in a proportion of cells. Staining for microtubules and vimentin intermediate filaments was less marked after 1 day in culture however than on initial seeding. For all three cytoskeletal components there was a dramatic increase in organisation between days 3 and 14 and, in general, organisation was greater within deep zone cells. Clear organisation for actin microfilaments was characterised by a cortical network and punctate staining around the periphery of the cell, while microtubules and vimentin intermediate filaments formed an extensive fibrous network. Cytoskeletal organisation within chondrocytes in agarose appears, therefore, to be broadly similar to that described in situ. Variations in the organisation of actin microfilaments between chondrocytes cultured in agarose and in monolayer are consistent with a role in phenotypic modulation. Vimentin intermediate filaments and microtubules form a link between the plasma membrane and the nucleus and may play a role in the mechanotransduction process.     

Cytoskeletal arrays in the cells of soybean root nodules: The role of actin microfilaments in the organisation of symbiosomes

Summary Within the infected cells of root nodules there is evidence of stratification and organisation of symbiosomes and other organelles. This organisation is likely to be important for the efficient exchange of nutrients and metabolites during functioning of the nodules. Using immunocytochemical labelling and confocal microscopy we have determined the organisation of cytoskeletal elements, micro tubules and actin microfilaments in soybean nodule cells, with a view to assessing their possible role in organelle distribution. Most microtubule arrays occurred in the cell cortex where they formed disorganised arrays in both uninfected and infected cells from mature nodules. In infected cells from developing nodules, parallel arrays of microtubules, transverse to the long axis of the cell, were observed. In incipient nodules, before release of rhizobia into the plant cells, the cells also had an array of microtubules which radiated from the nucleus into the cytoplasm. Three actin arrays were identified in the infected cells of mature nodules: an aster-like array which emanated from the surface of the nucleus, a cortical array which had an arrangement similar to that of the cortical microtubules, and, throughout the cytoplasm, an array of fine filaments which had a honeycomb arrangement consistent with a distribution between adjacent symbiosomes. Uninfected cells from mature nodules had only a random cortical array of actin filaments. In incipient nodules, the density of actin microfilaments associated with the nucleus and radiating through the cytoplasm was much less than that seen in mature infected cells. The cortical array of actin also differed, being composed of swirling configurations of filaments. After invasion of nodule cells by the rhizobia, the number of actin filaments emanating from the nucleus increased markedly and formed a network through the cytoplasm. Conversely, the cytoplasmic array in uninfected cells of developing nodules was identical to that in the cells of incipient nodules. The cytoplasmic network in infected cells of developing nodules is likely to be the precursor of the honeycomb array seen in mature nodule cells. We propose that this actin array plays a role in the spatial organisation of symbiosomes and that the microtubules are involved in the localisation of mitochondria and plastids at the cell periphery in the infected cells of root nodules.     

Mechanical properties of cancer cytoskeleton depend on actin filaments to microtubules content: Investigating different grades of colon cancer cell lines

Biomechanical properties of cancer cells have been proposed as promising biomarkers to investigate cancer progression. Cytoskeletal reorganization alters these characteristics in different grades of cancer cells. In the present study based on the micropipette aspiration method, whole body evaluation for two different colon cancer cells was performed to determine viscoelastic parameters of the cells. A finite element model was developed for verification of experiments and predicting some behaviors of cells. Western blot analysis and fluorescence intensity for actin microfilaments and microtubules were performed to measure cell content of the proteins. It was illustrated that the proportion of microtubules and actin microfilaments is different in grade I and grade IV colon cancer cells in a manner that microtubules attain an effectual role in progressive reorganization of cytoskeleton in transition from nonaggressive to malignant phenotypes in cancer cells. Furthermore, it was concluded that larger instantaneous Young's modulus value for high grade cells is related to the existence of extensively build-up actin networks at the cell cortex. Based on the cell mechanics results, a simple parameter is suggested for sorting different grades of colon cancer cells.     

Actin cytoskeleton and calcium-ATPase in the process of abomasal mucus secretion in cattle

Summary The distribution of actin filaments in pyloric gland cells of cattle was studied with respect to their functional significance in the process of exocrine secretion by use of rhodamine-phalloidin labelling and immunogold-electron microscopy based on the biotinstreptavidin bridge technique. Actin concentrates on the filamentous network of the luminal cell cortex. Membranes of secretory vesicles accumulating in the cell cortex are also labelled for actin. The present results support the concept of a barrier function of cortical microfilaments entrapping vesicles and linking them to the cytoskeleton. In addition, intracellular localization of calcium-ATPase activity was determined. Enzyme activity associated with the microfilamentous cortical matrix is supposed to be of cytoskeletal nature indicating participation of myosin (-like) structures in the dynamic secretion event. Deposition on the interior aspect of secretory vesicle membranes points to an ATPase transporting calcium into these organelles and enabling them to participate via storage of the cation in intracellular calcium homeostasis, thereby influencing the functional architecture of the cortical cytoskeleton.     

Global cytoskeletal control of mechanotransduction in kidney epithelial cells

Studies of mechanotransduction mediated by stress-sensitive ion channels generally focus on the site of force application to the cell. Here we show that global, cell-wide changes in cytoskeletal structure and mechanics can regulate mechanotransduction previously shown to be triggered by activation of the mechanosensitive calcium channel, polycystin-2, in the apical primary cilium of renal epithelial cells [S.M. Nauli, F.J. Alenghat, Y. Luo, E. Williams, P. Vassilev, X. Li, A.E. Elia, W. Lu, E.M. Brown, S.J. Quinn, D.E. Ingber, J. Zhou, Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. Nat. Genet. 33 (2003) 129-37]. Disrupting cytoplasmic microfilaments or microtubules in these cells eliminated fluid shear stress-induced increase of intracellular calcium. Altering the cytoskeletal force balance by inhibiting actomyosin-based tension generation (using 2,3-butanedione monoxime), interfering with microtubule polymerization (using nocodazole, cochicine, or taxol), or disrupting basal integrin-dependent extracellular matrix adhesions (using soluble GRGDSP peptide or anti-beta1 integrin antibody), also inhibited the calcium spike in response to fluid stress. These data indicate that although fluid stress-induced displacement of the primary cilium may be transduced into a calcium spike through activation of polycystin-2 and associated calcium-induced calcium release from intracellular stores, this mechanotransduction response is governed by global mechanical cues, including isometric tension (prestress) within the entire cytoskeleton and intact adhesions to extracellular matrix.     

Activation of chondrocytes calcium signalling by dynamic compression is independent of number of cycles

Mechanical loading is necessary for the development and maintenance of healthy articular cartilage through the control of extracellular matrix synthesis and catabolism. However, the underlying process of chondrocyte mechanotransduction remains unclear. This study examined the influence of cyclic compression on intracellular calcium (Ca(2+)) signalling within isolated articular chondrocytes cultured in agarose constructs. A validated experimental system was developed for applying controlled cyclic cell deformation. Cell-agarose constructs were subjected to 1Hz cyclic compression between 0 and 10% gross strain for 1, 10, 100 or 300 cycles. The cells were subsequently visualised for 300s in the unstrained state using confocal microscopy and the Ca(2+) indicator, Fluo-4 AM. Within unloaded control constructs, a sub-population of approximately 50% of chondrocytes exhibited characteristic spontaneous Ca(2+) transients each lasting approximately 40-60s. Cyclic compression, for only 1 cycle, significantly up-regulated the percentage of cells exhibiting Ca(2+) transients in the subsequent 5min period (p<0.05). Increasing the number of cycles to 10 or 100 had no additional effect. The up-regulated Ca(2+) signalling was maintained for up to 5min before returning to basal levels. By contrast, 300 cycles were followed by Ca(2+) signalling that was not significantly different from that in unloaded controls. However, this response was shown to be due to the increased time following the start of compression. In conclusion, this study indicates that chondrocyte Ca(2+) signalling is stimulated by dynamic compression, probably mediated by cyclic cell deformation. The overall response appears to be independent of the number of cycles or duration of cyclic compression. The sustained up-regulation of Ca(2+) signalling after 1, 10 or 100 cycles suggests the involvement of an autocrine-paracrine signalling mechanism. Furthermore, the reduced response following 300 cycles indicates a possible receptor desensitisation mechanism. Therefore, Ca(2+) signalling may be part of a mechanotransduction pathway through which chondrocyte populations can modulate their metabolic activity in response to changing mechanical stimuli.     

Dynamic study of cell mechanical and structural responses to rapid changes of calcium level

Cell shape control is complex since it may involve multiple cytoskeletal components and metabolic pathways. Here we present a kinetic study of the mechanical and structural responses of cells from the monocytic THP-1 line to a rapid increase of cytosolic calcium level. Cells were exposed to ionomycin in a medium of varying calcium concentration and they were probed at regular intervals for (1) cortical rigidity as determined with micropipette aspiration, and (2) content and distribution of polymerized actin, myosin or ABP-280, as determined with flow cytometry and/or confocal microscopy. An increase of free intracellular calcium level induced: (1) a biphasic deformability change with marked stiffening within a second, and significant softening a minute later; (2) a biphasic change of actin polymerization with initial decrease (within less than a second) and rapid recovery (within a few seconds); (3) a topographical redistribution of microfilaments with an oscillatory behavior of the cortical fraction, while no substantial redistribution of myosin or ABP-280 was detected. It is suggested that a regulation of cell rigidity might be achieved without any structural change by suitable modulation of the lifetime of bridges formed between microfilaments by actin binding proteins.     

Outer segment growth and periciliary vesicle turnover in developing photoreceptors of Xenopus laevis

Summary Cytoskeletal alterations in the cytoplasm of chromatolytic neurons of the dorsal root ganglia were studied in chickens after transection of the sciatic nerves. These studies were carried out using cryofixation with a nitrogencooled propane jet. By this method, the morphological complexity of the cytoskeleton in normal perikarya and cell processes can be visualized. The cytoskeleton of the dorsal root ganglion cells (DRG) is composed of an intricate network of microtubules, neurofilaments and microfilaments. The membrane-bounded cell organelles, as well as the cell nucleus and the plasmalemma, are linked to the microtubules and neurofilaments by microfilaments (or crosslinkers). As a result of the transection of the axon, chromatolysis takes place, characterized by dislocation of cell organelles, eccentric position of the nucleus and dispersion of the parallel cisternae of the rough endoplasmic reticulum throughout the cytoplasm. This characteristic phenomenon coincides with a regression of the neurocytoskeletal network. The neurofilaments and microtubules become shorter, and the microfilaments are replaced by strands of globular or granular material. The temporary regression of the microfilaments leads to a dispersion of the cell organelles. During the remodelling of the cytoskeletal structures, proliferation of the neurofilaments in the regenerating neurons may occasionally be observed. These results show that the cytoskeletal structures are responsible not only for the preservation of cell shape, but also for the maintenance of the normal distributional pattern (location and mobility) of the intracellular components.     

Cell-type-specific disruption and recovery of the cytoskeleton in Arabidopsis thaliana epidermal root cells upon heat shock stress

Summary. The cytoskeleton in plant cells plays an important role in controlling cell shape and mediating intracellular signalling. However, almost nothing is known about the reactions of cytoskeletal elements to heat stress, which represents one of the major environmental challenges for plants. Here we show that living epidermal root cells of Arabidopsis thaliana could cope with short-term heat shock stress showing disruption and subsequent recovery of microtubules and actin microfilaments in a time-dependent manner. Time-lapse imaging revealed a very dynamic behavior of both cytoskeletal elements including transient depolymerization and disassembly upon heat shock (40–41 °C) followed by full recovery at room temperature (20 °C) within 1–3 h. Reaction of microtubules, but not actin filaments, to heat shock was dependent on cell type and developmental stage. On the other hand, recovery of actin filaments, but not microtubules, from heat shock stress was dependent on the same parameters. The relevance of this adaptive cytoskeletal behavior to intracellular signalling is discussed. Correspondence and reprints: Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, 53115 Bonn, Federal Republic of Germany.     

Cytoskeletal dynamics in living plant cells

Microtubules and microfilaments have been imaged in living plant cells and their dynamic changes recorded during division, growth and development. Carboxyfluorescein labeled brain tubulin has been injected into cells that are maintained in an active state in a culture chamber on the microscope stage. Subsequent imaging with the confocal microscope reveals microtubules in the preprophase band, the mitotic apparatus, the phragmoplast, and the cortical array. The structural changes of these microtubules have been observed during transitional stages. In addition, their dynamic features are demonstrated by depolymerization in elevated calcium, low temperature, and in the drug oryzalin, and by repolymerization when returned to normal conditions. Examination of living Tradescantia stamen hair cells, which have been injected with fluorescent phalloidin to label the actin microfilaments, reveals hitherto undisclosed aspects of the preparation of the division site and dynamics of the phragmoplast cytoskeleton. During prophase microfilaments occur throughout the cell cortex, with those in the region of the preprophase band becoming transversely aligned. At nuclear envelope breakdown, these specifically disassemble, leaving a circumferential zone from which microfilaments remain absent throughout division. During cytokinesis microfilaments arise within the phragmoplast, oriented parallel to the microtubules, but excluded from the zone where the MTs overlap and where cell plate vesicles aggregate. The phragmoplast microfilaments, in a manner similar to microtubules, shorten in length, expand in girth, and eventually disassemble when the cell plate is complete.     

Association of actin filaments with axonal microtubule tracts

Axoplasmic organelles move on actin as well as microtubules in vitro and axons contain a large amount of actin, but little is known about the organization and distribution of actin filaments within the axon. Here we undertake to define the relationship of the microtubule bundles typically found in axons to actin filaments by applying three microscopic techniques: laser-scanning confocal microscopy of immuno-labeled squid axoplasm; electronmicroscopy of conventionally prepared thin sections; and electronmicroscopy of touch preparations-a thin layer of axoplasm transferred to a specimen grid and negatively stained. Light microscopy shows that longitudinal actin filaments are abundant and usually coincide with longitudinal microtubule bundles. Electron microscopy shows that microfilaments are interwoven with the longitudinal bundles of microtubules. These bundles maintain their integrity when neurofilaments are extracted. Some, though not all microfilaments decorate with the S1 fragment of myosin, and some also act as nucleation sites for polymerization of exogenous actin, and hence are definitively identified as actin filaments. These actin filaments range in minimum length from 0.5 to 1.5 µm with some at least as long as 3.5 µm. We conclude that the microtubule-based tracks for fast organelle transport also include actin filaments. These actin filaments are sufficiently long and abundant to be ancillary or supportive of fast transport along microtubules within bundles, or to extend transport outside of the bundle. These actin filaments could also be essential for maintaining the structural integrity of the microtubule bundles.     

Understanding Sensory Nerve Mechanotransduction through Localized Elastomeric Matrix Control

Background

While neural systems are known to respond to chemical and electrical stimulation, the effect of mechanics on these highly sensitive cells is still not well understood. The ability to examine the effects of mechanics on these cells is limited by existing approaches, although their overall response is intimately tied to cell-matrix interactions. Here, we offer a novel method, which we used to investigate stretch-activated mechanotransduction on nerve terminals of sensory neurons through an elastomeric interface.

Methodology/Principal Findings

To apply mechanical force on neurites, we cultured dorsal root ganglion neurons on an elastic substrate, polydimethylsiloxane (PDMS), coated with extracellular matrices (ECM). We then implemented a controlled indentation scheme using a glass pipette to mechanically stimulate individual neurites that were adjacent to the pipette. We used whole-cell patch clamping to record the stretch-activated action potentials on the soma of the single neurites to determine the mechanotransduction-based response. When we imposed specific mechanical force through the ECM, we noted a significant neuronal action potential response. Furthermore, because the mechanotransduction cascade is known to be directly affected by the cytoskeleton, we investigated the cell structure and its effects. When we disrupted microtubules and actin filaments with nocodozale or cytochalasin-D, respectively, the mechanically induced action potential was abrogated. In contrast, when using blockers of channels such as TRP, ASIC, and stretch-activated channels while mechanically stimulating the cells, we observed almost no change in action potential signalling when compared with mechanical activation of unmodified cells.

Conclusions/Significance

These results suggest that sensory nerve terminals have a specific mechanosensitive response that is related to cell architecture.     

Calcium, microfilaments and morphogenesis

Morphogenesis, the generation of tissue form, is important not only in the embryogenesis of a new individual, but also because a change in morphogenesis may be involved in the establishment of differences between individuals during evolution. Morphogenetic movements are effected in part by coordinated changes in the shapes of individual cells and over the past decade the cellular organelles responsible for cell shape have been identified as microfilaments and microtubules. In non-embryonic systems the contraction of microfilaments is controlled by the level of intracellular free calcium, and so calcium is implicated as an intermediate control mechanism in morphogenisis. Through techniques which perturb the calcium balance of cells, or which measure calcium ion concentration directly, evidence is accumulating that calcium is involved in morphogenetic movements such as gastrulation and neurulation, and related phenomena such as wound healing. Thus fundamental questions about the control of morphogenesis in embryogenesis and evolution may now be couched in more precise terms of the control of intracellular calcium ion balance.     

Dynamic reorganization of microtubules and microfilaments in flax cells during the resistance response to flax rust infection

The cytoskeleton in plant cells is a dynamic structure that can rapidly respond to extracellular stimuli. Alteration of the organization of microtubules and actin microfilaments was examined in mesophyll cells of flax, Linum usitatissimum L., during attempted infection by the flax rust fungus, Melampsora lini (Ehrenb.) Lev. Flax leaves that had been inoculated with either a compatible (yielding a susceptible reaction) or an incompatible (yielding a resistant reaction) strain of M. lini were embedded in butyl-methylmethacrylate resin; sections of this material were immunofluorescently labelled with anti-tubulin or anti-actin and examined using confocal laser scanning microscopy. In uninfected leaves, microtubules in the mesophyll cells formed a transverse array in the cell cortex. Microfilaments radiated through the cytoplasm from the nucleus. In an incompatible interaction, microtubules and microfilaments were extensively reorganized in mesophyll cells that were in contact with fungal infection hyphae or haustorial mother cells before penetration of the cell by the infection peg. After the initiation of haustorium development, microtubules disappeared from the infected cells, and growth of the haustoria ceased. In an incompatible interaction, hypersensitive cell death occurred in more than 70% of infected cells but occurred in less than 20% of cells in compatible interactions. After the infected cell had undergone hypersensitive cell death, the cytoskeleton in neighbouring cells became focused on the walls shared with the necrotic cell. In compatible interactions, reorganization of the cytoskeleton was either not observed at all or was observed much less frequently up to 48 h after inoculation.Abbreviations FITC fluorescein isothiocyanate - WGA wheatgerm agglutinin We thank Dr. G.J. Lawrence for providing valuable discussions and materials.     

Conformation of cytoskeletal elements during the division of infected Lupinus albus L. nodule cells

Lupin nodule cells maintain their ability to divide for several cycles after being infected by endosymbiotic rhizobia. The conformation of the cytoskeletal elements of nodule cells was studied by fluorescence labelling, immunocytochemistry, and laser confocal and transmission electron microscopy. The dividing infected cells showed the normal microtubule and actin patterns of dividing plant cells. The clustered symbiosomes were tethered to the spindle-pole regions and moved to the cell poles during spindle elongation. In metaphase, anaphase, and early telophase, the symbiosomes were found at opposite cell poles where they did not interfere with the spindle filaments or phragmoplast. This symbiosome positioning was comparable with that of the organelles (which ensures organelle inheritance during plant cell mitosis). Tubulin microtubules and actin microfilaments appeared to be in contact with the symbiosomes. The possible presence of actin molecular motor myosin in nodules was analysed using a monoclonal antibody against the myosin light chain. The antigen was detected in protein extracts of nodule and root cytosol as bands of approximately 20 kDa (the size expected). In the nodules, an additional polypeptide of 65 kDa was found. Immunogold techniques revealed the antigen to be localized over thin microfilaments linked to the cell wall, as well as over the thicker microfilament bundles and surrounding the symbiosomes. The pattern of cytoskeleton rearrangement in dividing infected cells, along with the presence of myosin antigen, suggests that the positioning of symbiosomes in lupin nodule cells might depend on the same mechanisms used to partition genuine plant cell organelles during mitosis.     

Viscoelastic Cell Mechanics and Actin Remodelling Are Dependent on the Rate of Applied Pressure

Background

Living cells are subjected to external and internal mechanical stresses. The effects of these stresses on the deformation and subsequent biological response of the cells remains unclear. This study tested the hypothesis that the rate at which pressure (or stress) is applied influence the viscoelastic properties of the cell associated with differences in the dynamics of the actin cytoskeleton.

Principal Finding

Micropipette aspiration was used to determine the instantaneous and equilibrium moduli and the viscosity of isolated chondrocytes based on the standard linear solid (SLS) model and a variation of this incorporating Boltzmann superposition. Cells were visualised for 180 seconds following aspiration to 7 cmH₂O at 0.35, 0.70 and 5.48 cmH₂O/sec. Cell recovery was then examined for a further 180 seconds once the pressure had been removed. Reducing the rate of application of pressure reduced the levels of cell deformation and recovery associated with a significant increase in modulus and viscosity. Using GFP transfection and confocal microscopy, we show that chondrocyte deformation involves distortion, disassembly and subsequent reassembly of the cortical actin cytoskeleton. At faster pressure rates, cell deformation produced an increase in cell volume associated with membrane bleb formation. GFP-actin transfection inhibited the pressure rate dependent variation in cell mechanics indicating that this behaviour is regulated by GFP-sensitive actin dynamics.

Conclusion

We suggest that slower rates of aspiration pressure enable greater levels of cortical actin distortion. This is partially inhibited by GFP or faster aspiration rates leading to membrane bleb formation and an increase in cell volume. Thus the rate of application of pressure regulates the viscoelastic mechanical properties of living cells through pressure rate sensitive differences in actin dynamics. Therefore cells appear softer when aspirated at a faster rate in contrast to what is expected of a normal viscoelastic material.     

Vacuolar and cytoskeletal dynamics during elicitor-induced programmed cell death in tobacco BY-2 cells

Responses of plant cells to environmental stresses often involve morphological changes, differentiation and redistribution of various organelles and cytoskeletal network. Tobacco BY-2 cells provide excellent model system for in vivo imaging of these intracellular events. Treatment of the cell cycle-synchronized BY-2 cells with a proteinaceous oomycete elicitor, cryptogein, induces highly synchronous programmed cell death (PCD) and provide a model system to characterize vacuolar and cytoskeletal dynamics during the PCD. Sequential observation revealed dynamic reorganization of the vacuole and actin microfilaments during the execution of the PCD. We further characterized the effects cryptogein on mitotic microtubule organization in cell cycle-synchronized cells. Cryptogein treatment at S phase inhibited formation of the preprophase band, a cortical microtubule band that predicts the cell division site. Cortical microtubules kept their random orientation till their disruption that gradually occurred during the execution of the PCD twelve hours after the cryptogein treatment. Possible molecular mechanisms and physiological roles of the dynamic behavior of the organelles and cytoskeletal network in the pathogenic signal-induced PCD are discussed.Key words: actin microfilament, cell cycle, cryptogein, microtubules, nuclei, programmed cell death, tobacco BY-2 cells, vacuoles