Dual-wavelength fluorescent speckle microscopy reveals coupling of microtubule and actin movements in migrating cells

Interactions between microtubules (MTs) and filamentous actin (f-actin) are involved in directed cell locomotion, but are poorly understood. To test the hypothesis that MTs and f-actin associate with one another and affect each other's organization and dynamics, we performed time-lapse dual-wavelength spinning-disk confocal fluorescent speckle microscopy (FSM) of MTs and f-actin in migrating newt lung epithelial cells. F-actin exhibited four zones of dynamic behavior: rapid retrograde flow in the lamellipodium, slow retrograde flow in the lamellum, anterograde flow in the cell body, and no movement in the convergence zone between the lamellum and cell body. Speckle analysis showed that MTs moved at the same trajectory and velocity as f-actin in the cell body and lamellum, but not in the lamellipodium or convergence zone. MTs grew along f-actin bundles, and quiescent MT ends moved in association with f-actin bundles. These results show that the movement and organization of f-actin has a profound effect on the dynamic organization of MTs in migrating cells, and suggest that MTs and f-actin bind to one another in vivo.     

FMLP诱导的嗜中性白细胞呼吸爆发与凋亡的关系研究

The relationship between apoptosis of neutrophils and the change of their intracellular free Ca2+ concentration [Ca2+]i was studied. FMLP and A23187 were used to elevate the [Ca2+]i while BAPTA was used to deplete it. Fluorescence microscope, flow cytometry and gel electrophoresis were used to study the percentage of cell apoptosis and the change of f-actin during apoptosis. The results showed that the apoptosis was obviously inhibited by fMLP and A23187, while accelerated by BAPTA. The detection of f-actin showed that the f-actin depolymerized obviously during apoptosis. The elevation of [Ca2+]i inhibit the actin depolymerization while depletion of [Ca2+]i accelerated it. This result indicated that the apoptosis of neutrophil was obviously inhibited by [Ca2+]i elevation but accelerated by [Ca2+]i depletion.     

Target cell lysis: ultrastructural and cytoskeletal alterations

The cytoskeleton, with particular emphasis on filamentous actin (f-actin), was studied in target L cells treated with the cytotoxic lymphokine, alpha-lymphotoxin (LT), or with cytotoxic T lymphocytes. The f-actin distribution was also examined in cytotoxic T cells interacting with targets. LT-treated targets retracted their processes, rounded up, and subsequently underwent lysis. These changes were accompanied by loss of stress fibers and rearrangement of f-actin, although there was no appreciable change of total cytoskeletal protein or f-actin content. Thus, the arrangement of f-actin was altered, but not its cellular concentration. Similar morphologic changes were seen at the light microscopic level in targets affected by T cells. T lymphocytes interacting with targets showed marked polarization of f-actin adjacent to the effector target interface; some f-actin rearrangement was also noted in the underlying target cell cytoplasm. Ultrastructurally, the LT-affected targets showed a decrease of f-actin organization within microvilli and underneath the plasma membrane associated with loss of villi and cytoplasmic bleb formation. Furthermore, prominent bundles of intermediate filaments were apparent and clusters of vesicles were noted which may be an expression of plasma membrane repair. These findings suggest that target cell cytoskeletal changes may lead to the morphologic changes in LT- and T cell-mediated cytotoxicity and lysis.     

Converging populations of f-actin promote breakage of associated microtubules to spatially regulate microtubule turnover in migrating cells

BACKGROUND: In migrating cells, the retrograde flow of filamentous actin (f-actin) from the leading edge toward the cell body is accompanied by the synchronous motion of microtubules (MTs, ), whose plus ends undergo net growth. Thus, MTs must depolymerize elsewhere in the cell to maintain polymer mass over time. The source and location of depolymerized MTs is unknown. Here, we test the hypothesis that MT polymer loss occurs in central cell regions and is induced by the convergence of actin retrograde and anterograde flow, which buckles and breaks associated MTs and promotes minus-end depolymerization. RESULTS: We characterized the effects of calyculin A and ML-7 on the movement of f-actin and MTs by multi-spectral fluorescence recovery after photobleaching (FRAP) and fluorescent speckle microscopy (FSM). Our studies show that these drugs affect the rate of f-actin and MT convergence and MT buckling in a central cell region we call the "convergence zone." Increases in f-actin convergence are associated with faster MT turnover and an increase in both MT breakage and minus-end depolymerization, but they have no effect on MT plus end dynamic instability. CONCLUSIONS: We propose that f-actin movement into the convergence zone plays a major role in spatially modulating MT turnover during cell migration by regulating MT breakage, and thus minus-end dynamics, in central cell regions.     

FMLP诱导的嗜中性白细胞呼吸爆发与凋亡的关系研究

本文对fMLP诱导的嗜中性白细胞胞内钙浓度变化与凋亡的关系进行了研究。用膜受体激动剂fMLP和钙离子载体A23187诱导细胞内钙浓度升高,BAPTA螯合胞质钙。运用荧光显微镜,流式细胞仪,电泳等方法对培养细胞的凋亡百分率及细胞骨架变化进行了研究。结果表明:fMLP和A23187均有效地抑制了凋亡,而BAPTA促进了凋亡。对骨架测定表明随细胞凋亡微丝解聚明显,胞内钙升高抑制骨架解聚,胞内钙降低促进其解聚。故嗜中性白细胞凋亡过程中伴随有微丝的解聚,胞内钙浓度升高时凋亡被有效抑制,胞内钙浓度降低时促进了凋亡。     

Transitions in trophectoderm cellular shape and cytoskeletal organization in the elongating pig blastocyst

Changes in cellular shape and filamentous actin (f-actin) organization within the trophectoderm of pig embryos have been studied by fluorescence microscopy during the transitions from spherical to filamentous blastocysts. Cells comprising the trophectoderm of spherical, ovoid, tubular, and filamentous blastocysts are distinctive in their shape, size, and organization of membrane-associated f-actin. Trophectodermal cells of spherical and ovoid embryos are both generally circular in shape. However, as the spherical embryo acquires an ovoid shape, uniformally distributed apical cell surface microvilli relocate to the apical intercellular margins of adjoining trophectodermal cells. Transitional modifications in cellular shape and f-actin organization are observed in tubular blastocysts when apical cell surface microvilli reappear. In elongating filamentous blastocysts, trophectodermal cells assume a spindle-shaped morphology. The f-actin associated with the apical surface is diminished whereas the associated with the basolateral membrane predominates, especially in constricted regions of the blastocyst. These observations, in conjunction with morphometric parameters of trophectodermal cells and whole blastocysts, are discussed in relation to the role of the actin cytoskeleton in processes that modify trophectodermal cell shape and function in the elongating pig blastocyst.     

Supercoiling of f-Actin filaments

In the X-ray diffraction pattern from oriented gels of actin-containing filaments sampling of layer lines indicating the development of a well-ordered pseudo-hexagonal lattice within the gels at interfilament spacings as large as 13 nm is observed. This value exceeds by 3 nm the largest estimate of an external diameter of pure f-actin. The development of layer line sampling is always accompanied by: (i) the appearance of strong forbidden meridional reflections on the 5.9- and 5.1-nm layer lines; (ii) a drastic intensification of the first (expected) 2.75-nm meridional reflection by a factor of about 4; (iii) the appearance of streaks, connecting near-meridional reflections on the 5.9-, 5.1-, and 37-nm layer lines; and (iv) a slight decrease in the number of subunits per turn of the basic f-actin helix. All these features strongly indicate that f-actin filaments are supercoiled and make regular local contacts between themselves, which may lead to periodic distortions of the mobile external domain in the actin subunits.     

Rho plays a central role in regulating local cell-matrix mechanical interactions in 3D culture

The purpose of this study was to assess quantitatively the role of the small GTPase Rho on cell morphology, f-actin organization, and cell-induced matrix remodeling in 3D culture. Human corneal fibroblasts (HTK) were infected with adenoviruses that express green fluorescent protein (GFP) or GFP-N19Rho (dominant negative Rho). One day later cells were plated inside collagen matrices and allowed to spread for 24 h. Cells were fixed and stained for f-actin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) images were acquired using confocal microscopy. Fourier transform analysis was used to assess local collagen fibril alignment, and changes in cell morphology and collagen density were measured using MetaMorph. The decrease in matrix height was used as an indicator of global matrix contraction. HTK and HTK-GFP cells induced significant global matrix contraction; this was inhibited by N19Rho. HTK and HTK-GFP fibroblasts generally had a bipolar morphology and occasional intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. In contrast, HTK-GFPN19 cells were elongated, and had a more cortical f-actin distribution. Numerous small extensions were also observed along the cell body. In addition, both local collagen fibril density and alignment were significantly reduced. Rho plays a key role in regulating both the morphology and mechanical behavior of corneal fibroblasts in 3D culture. Overall, the data suggest that Rho-kinase dependent cell contractility contributes to global and local matrix remodeling, whereas Rho dependent activation of mDia and/or other downstream effectors regulates the structure and number of cell processes.     

A CALCIUM-DEPENDENT PROTEIN KINASE FUNCTIONS IN WOUND HEALING IN VENTRICARIA VENTRICOSA (CHLOROPHYTA)

The cytoplasm around a wound made in the multinucleate unicellular green alga Ventricaria ventricosa (  J. Agardh) Olsen et West formed an aggregation-ring surrounding the wound immediately after injury. A contraction of the ring then brought about wound healing in culture medium containing Ca^{2 +} . Involvement of a calcium-dependent protein kinase (CDPK) as a regulator of wound healing was examined using an anti- Dunaliella tertiolecta CDPK antibody. A 52-kDa protein cross-reacting with the antibody was detected by Western blotting. Protein kinases of 60 kDa and 52 kDa, which were markedly activated by Ca^{2 +} , and a 40-kDa Ca^{2 +} -independent protein kinase were detected by an in-gel protein kinase assay using myelin basic protein as the substrate. A 52-kDa band with Ca^{2 +} -dependent protein kinase activity was immunoprecipitated from the cytoplasmic extract, indicating that these 52-kDa proteins are identical and possess CDPK activity. Microscopic observation showed that the contraction of the aggregation ring was suppressed by application of the anti-CDPK to the culture medium. A protein kinase inhibitor, K-252a, and the calmodulin inhibitors, calmidazolium and compound 48   /   80, which inhibit CDPK activity, also suppressed the contraction of the aggregation-ring. Immunofluorescence microscopy showed a similar distribution of 52-kDa CDPK to the distribution of f-actin, which was randomly distributed in an intact cell and formed a bundle during wound healing. Further, f-actin was not recruited after injury in the presence of the antibody to CDPK. These results suggest that the 52-kDa CDPK functions as a Ca^{2 +} receptor in wound healing and simultaneously participates in the organization and contraction of f-actin to heal the wound.     

1,2-dioctanoyl-s,n-glycerol-induced activation of protein kinase C results in striking, but reversible growth cone shape changes and an accumulation of f-actin and serine 41-phosphorylated GAP-43 in the axonal process

In spinal cord explant cultures from embryonic chicken (E7) we found that both a long-time downregulation of PKC by phorbol-12,13-dibutyrate (PDBu) and an inhibition of PKC by RO-31-8220 strongly reduce neurite outgrowth. Unlike this, in the presence of a high dose of 1,2-dioctanoyl-s,n-glycerol (diC8, 60 microM), PKCalpha,beta isoforms are not downregulated, but neurite outgrowth appeared reduced up to 37 %. A low dose of diC8 (5 microM), however, was found to stimulate neurite outgrowth up to 25 %. Using this tissue culture system as well as neuronal cell culture we then studied the effects of diC8 on the shapes and actin-based motility of distal axonal processes and growth cones as well as on the spatial distribution of f-actin and serine 41-phosphorylated GAP-43 (neuromodulin, B50). High-resolution microscopy showed that addition of 30-60 microM diC8 leads within a few minutes to a retraction of filopodia and to an increased protrusion of lamellipodia followed by the formation of club-shaped dense growing tips, axonal varicosities, and a cessation of any actin dynamics. These striking shape changes are completely reversed after replacement of the medium by drug-free medium. Presence of cytochalasins and a panel of different PKC inhibitors prevent or respectively attenuate the diC8 effects. Immuno- and phalloidin-staining confirmed that in control neurons f-actin and serine 41-phosphorylated GAP-43 are confined to and enriched in the growth cones. In parallel with diC8-induced shape changes there is an accretion of f-actin and serine 41-phosphorylated GAP-43 in the entire axonal processes and the rounded growing tips. With respect to the fundamental role of the actin dynamics in growth cone steering and neuronal pathfinding, the data supports the view that in neurons local PKC-regulated phosphorylation of GAP-43 may represent an important mechanism to transduce guiding signals into actincytoskeletal responses mediating directed axonal growth.     

Regulation of expression of microvillus membrane proteins by estrogen in baboon fetal ovarian oocytes

We previously demonstrated that the number and height of oocyte microvilli were reduced in baboon fetuses deprived of estrogen in utero and restored to normal in animals supplemented with estradiol. Phosphorylated ezrin and Na+/H+ exchange regulatory factor 1 (NHERF, now termed SLC9A3R1) link f-actin bundles to the membrane, whereas alpha-actinin cross-links f-actin to form microvilli. Therefore, we determined whether these proteins were expressed in oocytes of the fetal baboon ovary and whether expression and/or localization were altered between mid and late gestation in association with an increase in estrogen and in late gestation in animals in which estrogen was suppressed (>95%) or restored by treatment with an aromatase inhibitor with or without estradiol. Expression of alpha-actinin was low at mid gestation, increased on the surface of oocytes of primordial follicles in late gestation, and was negligible in the ovaries of estrogen-suppressed fetuses and normal in animals treated with estrogen. Ezrin (total and phosphorylated) and SLC9A3R1 expression was localized to the surface of oocytes at mid and late gestation in estrogen-replete baboons and to the cytoplasm in late gestation after estrogen suppression. These results are the first to show that the fetal baboon oocyte expressed ezrin, SLC9A3R1, and alpha-actinin, and that these proteins were localized to the oocyte surface consistent with their role in microvilli development in epithelial cells. The current study also showed that the developmental increase in oocyte expression of alpha-actinin is regulated by estrogen and correlated with the estrogen-dependent increase in oocyte microvilli demonstrated previously. Therefore, we propose that development of oocyte microvilli requires expression of alpha-actinin and that expression of alpha-actinin and localization of ezrin-phosphate and SLC9A3R1 to the oocyte membrane are regulated by estrogen.     

Diphasic transformations of F-actin. Effects of urea and MgCl2 on F-actin.

Asakura, Taniguchi and Oosawa [1]proposed that muscle actin polymer under sonic vibration is in a different state from that of the ordinary double stranded helical structure (F-actin), characterised by partially interrupted structures of F-actin, a state of "f-actin". In order to confirm different states for actin polymers [1, 2], physicochemical studies were made by measurements of viscosity, flow birefringence, electric birefringence, fluorescence, electron microscopy, quasielastic light scattering and ATP splitting. The following results were obtained. (1) F-actin polymers can undergo two processes of depolymerization upon treatment with urea and various salts as judged by measurements of flow birefringence and viscosity: one is a rapid process in a solution containing K+ or Ca2+ and urea; the other is a slow process following a brief rapid one in a solution containing Mg2+ and urea. (2) In the presence of Mg2+ and a suitable concentration of urea, F-actin (FMU-actin) appeared to exhibit different properties than ordinary F-actin; it had lower viscosity and lower flow birefringence and it had on the whole a more flexible polymer structure, also judging from experiments of quasielastic light scattering, electric birefringence. The different structure was confirmed directly be electron microscopic observation. The aromatic side chains of FMU-actin were also more mobile than those of F-actin judging from fluorescence measurements. The transformation between F-actin and FMU-actin was reversible. (3) The state of FMU-actin polymers was also characterized by ATP splitting; FMU-actin split about one mole of ATP into ADP and inorganic phosphate per mole of actin monomer at room temperature, where F-actin did not. A molar excess of Mg2+ with respect to actin monomer at room temperature, where F-actin did not. A molar excess of Mg2+ with respect to actin monomer is required for ATP splitting. F-actin in solutions containing K+ or Ca2+ and urea did not split ATP. FMU-actin activated on Mg-ATP-ase of myosin at nearly the same rate as that of F-actin. (4) We have postulated a flexible filament model (f-actin). The relationships between the structure of f-actin and its functional role for force generation during contraction are discussed.     

Probing f-actin flow by tracking shape fluctuations of radial bundles in lamellipodia of motile cells

We examined the dynamics of radial actin bundles based on time-lapse movies of polarized light images of living neuronal growth cones. Using a highly sensitive computer vision algorithm for tracking, we analyzed the small shape fluctuations of radial actin bundles that otherwise remained stationary in their positions in the growth cone lamellipodium. Using the tracking software, we selected target points on radial bundles and measured both the local bundle orientations and the lateral displacements between consecutive movie frames. We found that the local orientation and the lateral displacement of a target point are correlated. The correlation can be explained using a simple geometric relationship between the lateral travel of tilted actin bundles and the retrograde flow of f-actin structures. Once this relationship has been established, we have turned the table and used the radial bundles as probes to measure the velocity field of f-actin flow. We have generated a detailed map of the complex retrograde flow pattern throughout the lamellipodium. Such two-dimensional flow maps will give new insights into the mechanisms responsible for f-actin-mediated cell motility and growth.     

Actin depolymerization mediated loss of SNTA1 phosphorylation and Rac1 activity has implications on ROS production,cell migration and apoptosis

Alpha-1-syntrophin (SNTA1) and Rac1 are part of a signaling pathway via the dystrophin glycoprotein complex (DGC). Both SNTA1 and Rac1 proteins are over-expressed in various carcinomas. It is through the DGC signaling pathway that SNTA1 has been shown to act as a link between the extra cellular matrix, the internal cell signaling apparatus and the actin cytoskeleton. SNTA1 is involved in the modulation of the actin cytoskeleton and actin reorganization. Rac1 also controls actin cytoskeletal organization in the cell. In this study, we present the interplay between f-actin, SNTA1 and Rac1. We analyzed the effect of actin depolymerization on SNTA1 tyrosine phosphorylation and Rac1 activity using actin depolymerizing drugs, cytochalasin D and latrunculin A. Our results indicate a marked decrease in the tyrosine phosphorylation of SNTA1 upon actin depolymerization. Results suggest that actin depolymerization mediated loss of SNTA1 phosphorylation leads to loss of interaction between SNTA1 and Rac1, with a concomitant loss of Rac1 activation. The loss of SNTA1tyrosine phosphorylation and Rac1 activity by actin depolymerization results in increased apoptosis, decreased cell migration and decreased reactive oxygen species (ROS) levels in breast carcinoma cells. Collectively, our results present a possible role of f-actin in the SNTA1-Rac1 signaling pathway and implications of actin depolymerization on cell migration, ROS production and apoptosis.     

Viscoelastic properties of f-actin, microtubules, f-actin/alpha-actinin, and f-actin/hexokinase determined in microliter volumes with a novel nondestructive method

A nondestructive method to determine viscoelastic properties of gels and fluids involves an oscillating glass fiber serving as a sensor for the viscosity of the surrounding fluid. Extremely small displacements (typically 1-100 nm) are caused by the glass rod oscillating at its resonance frequency. These displacements are analyzed using a phase-sensitive acoustic microscope. Alterations of the elastic modulus of a fluid or gel change the propagation speed of a longitudinal acoustic wave. The system allows to study quantities as small as 10 microliters with temporal resolution >1 Hz. For 2-100 microM f-actin gels a final viscosity of 1.3-9.4 mPa s and a final elastic modulus of 2.229-2.254 GPa (corresponding to 1493-1501 m/s sound velocity) have been determined. For 10- to 100-microM microtubule gels (native, without stabilization by taxol), a final viscosity of 1.5-124 mPa s and a final elastic modulus of 2.288-2. 547 GPa (approximately 1513-1596 m/s) have been determined. During polymerization the sound velocity in low-concentration actin solutions increased up to +1.3 m/s (approximately 1.69 kPa) and decreased up to -7 m/s (approximately 49 kPa) at high actin concentrations. On polymerization of tubulin a concentration-dependent decrease of sound velocity was observed, too (+48 to -12 m/s approximately 2.3-0.1 MPa, for 10- to 100-microM tubulin). This decrease was interpreted by a nematic phase transition of the actin filaments and microtubules with increasing concentration. 2 mM ATP (when compared to 0.2 mM ATP) increased polymerization rate, final viscosity and elastic modulus of f-actin (17 microM). The actin-binding glycolytic enzyme hexokinase also accelerated the polymerization rate and final viscosity but elastic modulus (2.26 GPa) was less than for f-actin polymerized in presence of 0.2 mM ATP (2.28 GPa).     

Calpain-Mediated Degradation of Drebrin by Excitotoxicity In vitro and In vivo

The level of drebrin, an evolutionarily conserved f-actin-binding protein that regulates synaptic structure and function, is reduced in the brains of patients with chronic neurodegenerative diseases such as Alzheimer’s disease (AD) and Down’s syndrome (DS). It was suggested that excitotoxic neuronal death caused by overactivation of NMDA-type glutamate receptors (NMDARs) occurs in AD and DS; however, the relationship between excitotoxicity and drebrin loss is unknown. Here, we show that drebrin is a novel target of calpain-mediated proteolysis under excitotoxic conditions induced by the overactivation of NMDARs. In cultured rodent neurons, degradation of drebrin was confirmed by the detection of proteolytic fragments, as well as a reduction in the amount of full-length drebrin. Notably, the NMDA-induced degradation of drebrin in mature neurons occurred concomitantly with a loss of f-actin. Furthermore, pharmacological inhibition of f-actin loss facilitated the drebrin degradation, suggesting a functional linkage between f-actin and drebrin degradation. Biochemical analyses using purified drebrin and calpain revealed that calpain degraded drebrin directly in vitro. Furthermore, cerebral ischemia also induced the degradation of drebrin in vivo. These findings suggest that calpain-mediated degradation of drebrin is a fundamental pathology of neurodegenerative diseases mediated by excitotoxicity, regardless of whether they are acute or chronic. Drebrin regulates the synaptic clustering of NMDARs; therefore, degradation of drebrin under excitotoxic conditions may modulate NMDAR-mediated signal transductions, including pro-survival signaling. Overall, the results presented here provide novel insights into the molecular basis of cellular responses to excitotoxicity in vitro and in vivo.     

Regional differences in the apical F-actin cytoskeleton of silk gland cells of Calpodes ethlius (Stöll) (Lepidoptera : Hesperiidae)

Silk glands of the larvae of Calpodes ethlius, Stöll (Lepidoptera : Hesperiidae) were labelled with rhodaminyl-phalloin, which is specific for f-actin. Most f-actin lies below the apical cell surface in bundles oriented in rings around the lumen of the gland. By the fifth larval stadium, the density of bundles differs in the 5 regions of the gland, the greatest density occurring in the narrower anterior regions. The greater bundle density may be correlated with the increased hoop stress where the gland has the smallest diameter. F-actin from the bundles redistributes during larval moults to coat vacuoles in the cytoplasm, except in the 2 most anterior regions where it keeps its arrangement from stage to stage, even through the moult.     

Microfilaments, cell shape changes, and the formation of primary mesenchyme in sea urchin embryos.

Primary mesenchyme formation in sea urchin embryos occurs when a subset of epithelial cells of the blastula move from the epithelial layer into the blastocoel. The role of microfilaments in producing the cell shape changes that characterize this process, referred to as ingression, was investigated in this study. f-Actin was localized by confocal microscopy using labeled phalloidin. The distribution of f-actin was observed before, during, and after ingression and was correlated with cellular movements. Prior to the onset of ingression, staining became intense in the apical region of putative primary mesenchyme and disappeared following the completion of mesenchyme formation. The apical end of these cells constricted coincidentally with the appearance of the intensified staining, indicating that f-actin may be involved in this constriction. In addition, papaverine, a smooth muscle cell relaxant that interferes with microfilament-based contraction, and that was shown in this study to inhibit cytokinesis, diminished apical constriction and delayed ingression. Despite this interference with apical constriction, the basal surface of ingressing cells protruded into the blastocoel. It is suggested that apical constriction, while not necessary for ingression, does contribute to the efficient production of mesenchyme and that protrusion of the basal surface results from changes that occur independent of apical constriction.     

Regulation of Astrocyte Morphology by RhoA and Lysophosphatidic Acid

Astrocytes in the CNS undergo morphological changes and start to proliferate after breakdown of the blood–brain barrier. In culture, proliferating astrocytes have a flat, polygonal shape. When treated with cAMP-raising agents, astrocytes adopt a stellate, process-bearing morphology resembling theirin vivoappearance. Stellation is accompanied by loss of actin stress fibers and focal adhesions. Lysophosphatidic acid (LPA), a blood-borne mitogen that signals through its cognate G protein-coupled receptor, stimulates DNA synthesis in astrocytes and causes rapid reversal of cAMP-induced stellation. LPA reversal of stellation is initiated by f-actin reassembly and tyrosine phosphorylation of focal adhesion proteins such as paxillin. Botulinum C3 toxin, which inactivates the Rho GTPase, mimics cAMP-raising agents in inducing stellation, f-actin disassembly, paxillin dephosphorylation, and growth arrest. However, unlike cAMP-induced stellation, C3-induced stellation cannot be reversed by LPA. Conversely, astrocytes expressing activated RhoA fail to undergo cAMP-induced stellation. Thus, RhoA controls astrocyte morphology in that active RhoA directs LPA reversal of stellation, while inactivation of RhoA is sufficient to induce stellation.     

Quantitative assessment of local collagen matrix remodeling in 3-D culture: the role of Rho kinase

The purpose of this study was to quantitatively assess the role of Rho kinase in modulating the pattern and amount of local cell-induced collagen matrix remodeling. Human corneal fibroblasts were plated inside 100-microm thick fibrillar collagen matrices and cultured for 24 h in media with or without the Rho kinase inhibitor Y-27632. Cells were then fixed and stained with phalloidin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. Fourier transform analysis was used to assess collagen fibril alignment, and 3-D cell morphology and local collagen density were measured using MetaMorph. Culture in serum-containing media induced significant global matrix contraction, which was inhibited by blocking Rho kinase (p<0.001). Fibroblasts generally had a bipolar morphology and intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. When Rho kinase was inhibited, cells had a more cortical f-actin distribution and dendritic morphology. Both local collagen fibril density and alignment were significantly reduced (p<0.01). Overall, the data suggests that Rho kinase-dependent contractile force generation leads to co-alignment of cells and collagen fibrils along the plane of greatest resistance, and that this process contributes to global matrix contraction.