Dynamic motion of paxillin on actin filaments in living endothelial cells

Our three-dimensional (3-D) images showed that paxillin co-localized on actin filaments as fibrous structures, as well as clusters, in endothelial cells (ECs). In living ECs under flow condition, we monitored concurrently the intracellular dynamics of DsRed2-paxillin and GFP-actin by time-lapse video recording and dual-color fluorescence imaging. The results showed that the dynamic motion of paxillin as fibrous structures was associated with actin filaments, but not with microtubules. Our findings suggest that the actin network plays an important role not only in the assembly/disassembly of paxillin at focal adhesions, but also as a track for the intracellular transport of paxillin, which is involved in signaling pathway.     

Sodium channel activity in leukemia cells is directly controlled by actin polymerization

The actin cytoskeleton has been shown to be involved in the regulation of sodium-selective channels in non-excitable cells. However, the molecular mechanisms underlying the changes in channel function remain to be defined. In the present work, inside-out patch experiments were employed to elucidate the role of submembranous actin dynamics in the control of sodium channels in human myeloid leukemia K562 cells. We found that the application of cytochalasin D to the cytoplasmic surface of membrane fragments resulted in activation of non-voltage-gated sodium channels of 12 picosiemens conductance. Similar effects could be evoked by addition of the actin-severing protein gelsolin to the bath cytosol-like solution containing 1 microm [Ca(2+)](i). The sodium channel activity induced by disassembly of submembranous microfilaments with cytochalasin D or gelsolin could be abolished by intact actin added to the bath cytosol-like solution in the presence of 1 mm MgCl(2) to induce actin polymerization. In the absence of MgCl(2), addition of intact actin did not abolish the channel activity. Moreover, the sodium currents were unaffected by heat-inactivated actin or by actin whose polymerizability was strongly reduced by cleavage with specific Escherichia coli A2 protease ECP32. Thus, the inhibitory effect of actin on channel activity was observed only under conditions promoting rapid polymerization. Taken together, our data show that sodium channels are directly controlled by dynamic assembly and disassembly of submembranous F-actin.     

Dynamic actin patterns and Arp2/3 assembly at the substrate-attached surface of motile cells

BACKGROUND: In the cortical region of motile cells, the actin network rapidly reorganizes as required for movement in various directions and for cell-to-substrate adhesion. The analysis of actin network dynamics requires the combination of high-resolution imaging with a specific fluorescent probe that highlights the filamentous actin structures in live cells. RESULTS: Combining total internal reflection fluorescence (TIRF) microscopy with a method for labeling actin filaments, we analyze the dynamics of actin patterns in the highly motile cells of Dictyostelium. A rapidly restructured network of single or bundled actin filaments provides a scaffold for the assembly of differentiated actin complexes. Recruitment of the Arp2/3 complex characterizes stationary foci with a lifetime of 7-10 s and traveling waves. These structures are also formed in the absence of myosin-II. Arp2/3-actin assemblies similar to those driving the protrusion of a leading edge form freely at the inner face of the plasma membrane. CONCLUSIONS: The actin system of highly motile cells runs far from equilibrium and generates a multitude of patterns within a dynamic filamentous network. Traveling waves are the most complicated patterns based on recruitment of the Arp2/3 complex. They are governed by the propagated induction of actin polymerization. We hypothesize that the actin system autonomously generates primordia of specialized structures such as phagocytic cups or lamellipodia. These primordia would represent an activated state of the actin system and enable cells to respond within seconds to local stimuli by chemotaxis or phagocytic-cup formation.     

Cofilin overexpression affects actin cytoskeleton organization and migration of human colon adenocarcinoma cells

The dynamic reorganization of actin cytoskeleton is regulated by a large number of actin-binding proteins. Among them, the interaction of ADF/cofilin with monomeric and filamentous actin is very important, since it severs actin filaments. It also positively influences actin treadmilling. The activity of ADF/cofilin is reversibly regulated by phosphorylation and dephosphorylation at Ser-3, with the phosphorylated form (P-cofilin) being inactive. Here, we studied the effects of overexpression of cofilin and two cofilin variants in the human colon adenocarcinoma LS180 cell line. We have generated the LS180 cells expressing three different cofilin variants: WT (wild type), Ser 3 Ala (S3A) (constitutively active) or Ser 3 Asp (S3D) (constitutively inactive cofilin). The cells expressing WT cofilin were characterized by abundant cell spreading and colocalization of cofilin with the submembranous F-actin. Similar effects were observed in cells expressing S3A cofilin. In contrast, LS180 cells expressing S3D cofilin remained longitudinal in morphology and cofilin was equally distributed within the cell body. Furthermore, the migration ability of LS180 cells expressing different cofilin mutants was analyzed. In comparison to control cells, we have noticed a significant, approximately fourfold increase in the migration factor value of cells overexpressing WT type cofilin. The overexpression of S3D cofilin resulted in an almost complete inhibition of cell motility. The estimation of actin pool in the cytosol of LS180 cells expressing S3A cofilin has shown a significantly lower level of total actin in reference to control cells. The opposite effect was observed in LS180 cells overexpressing S3D cofilin. In summary, the results of our experiments indicate that phosphorylation “status” of cofilin is a factor affecting the actin cytoskeleton organization and migration abilities of colon adenocarcinoma LS180 cells.     

Autoregulation of actin synthesis requires the 3'-UTR of actin mRNA and protects cells from actin overproduction

Monomeric (G) actin was shown to be involved in inhibiting its own synthesis by an autoregulatory mechanism that includes enhanced degradation of the actin mRNA [Bershadsky et al., 1995; Lyubimova et al., 1997]. We show that the 3'-untranslated region (3'-UTR) of beta-actin mRNA, but not its 5'-untranslated region, is important for this regulation. The level of full-length beta-actin mRNA in cells was reduced when actin filaments were depolymerized by treatment with latrunculin A and elevated when actin polymerization was induced by jasplakinolide. By contrast, the level of actin mRNA lacking the 3'-UTR remained unchanged when these drugs modulated the dynamics of actin assembly in the cell. Moreover, the transfection of cells with a construct encoding the autoregulation-deficient form of beta-actin mRNA led to very high levels of actin expression compared with transfection with the control actin construct and was accompanied by characteristic changes in cell morphology and the structure of the actin cytoskeleton. These results suggest that the autoregulatory mechanism working via the 3'-UTR of actin mRNA is involved in controlling the maintenance of a defined pool of actin monomers that could be necessary for the proper organization of the microfilament system and the cytoskeleton-mediated signaling.     

Antioxidants-induced rearrangements of actin cytoskeleton in 3T3 and 3T3-SV40 fibroblasts

Effect of antioxidants on actin cytoskeleton in 3T3 fibroblasts and 3T3 fibroblasts transformed with SV40 virus (3T3-SV40 cells) was studied. Antioxidants used were as follows: N-acetyl-L-cysteine (NAC), (-)-2-oxo-4-thiazolidine-carboxylic acid (OTZ), and glutathione in the reduced form (GSH). Both NAC and OTZ are precursors of GSH in the cell, but, in contrast to NAC, OTZ reduces inside the cell forming L-cysteine. The presence of NAC (5-20 mM) in the culture medium of both cell types resulted in loosening of monolayer, fragmentation of stress fibers, and the appearance of amorphous actin structures. As 3T3-SV40 cells contain less actin stress fibers than 3T3 cells, the NAC-induced rearrangements of actin cytoskeleton were stronger in these cells than in 3T3 cells. In contrast to NAC, OTZ (10-20 mM) did not destroy monolayer and did not induce any visible disappearance of stress fibers either in 3T3 or 3T3-SV40 cells. However, in the presence of OTZ, amorphous actin-containing structures were observed in 3T3-SV40 cells. The effect of glutathione on both cell types was similar to that of NAC. The time required for GSH-induced alterations of actin cytoskeleton (about 5 h) was consistent with the increase in the intracellular level of reactive oxygen species (4 h after addition of GSH to the culture medium). Upon removal of the antioxidants from the medium, actin filament structures were reconstructed. However, within 24 h after withdrawal of NAC or GSH, only a partial reconstruction of stress fibers was observed in 3T3 cells. On the contrary, 3T3-SV40 cells demonstrated formation of well-structured actin fibers similar to normal fibroblasts. These results suggest that GSH can act as a pro-oxidant in the absence of oxidative stress.     

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.     

ARP2 and ARP3 are localized to sites of actin filament nucleation in tobacco BY-2 cells

Summary. Complete depolymerization of actin filaments (AFs) at low temperature (0 °C) is followed by the formation of transient actin structures at 25 °C in tobacco BY-2 cells (Nicotiana tabacum L.). Using antibodies against fission yeast actin-related proteins (ARP2 and ARP3), we show here that transient actin structures (dots, dotted filaments, rods) colocalize with epitopes stained by these antibodies and thus are likely to represent sites of actin filament nucleation (SANs). In contrast to the cold-induced disassembly of AFs, no transient actin structures were detectable during recovery of AFs from latrunculin B-induced depolymerization. However, the staining pattern obtained with ARP antibodies in latrunculin B-treated cells was similar to that in controls and cold-treated cells. This suggests that, in addition to the complete depolymerization of AFs, disruption of other cellular structures is needed for the formation of transient actin structures during the early phase of recovery from cold treatment. Correspondence and reprints: Department of Plant Physiology, Faculty of Science, Charles University, Viničná 5, 128 44 Prague 2, Czech Republic.     

Localization of nonerythroid spectrin and actin in mouse oocytes and preimplantation embryos

Mouse oocytes, cleavage-stage embryos, and blastocyst-stage embryos were studied to show the distribution of both an immunoanalog to nonerythroid spectrin (p 230) and F-actin. Using antibodies to nonerythroid spectrin, diffuse, positive cytoplasmic fluorescence was regularly seen in oocytes and embryo cells. The presence of nonerythroid spectrin in oocytes was confirmed by immunoblotting. Oocytes usually exhibited an inconspicuous submembranous layer of nonerythroid spectrin, which was more pronounced in the area of the polar body. Oocytes regularly exhibited a peripheral concentration of actin. Throughout the cleavage and blastocyst stages, a cortical layer of nonerythroid spectrin and actin was usually observed in embryo cells. These submembranous layers on the outer surface of the embryo were relatively thin as compared to those in areas of intercellular contact. The contact areas regularly showed distinct positive staining, including a concentration of label at the most peripheral region of each contact area. This resulted in the presence of ring-like fluorescence around each blastomere. Nonerythroid spectrin and actin showed concentration to the contact area between the oocyte and the polar body. Although the general localization patterns of nonerythroid spectrin and actin were similar, double-staining experiments revealed that slightly different planes of focus were necessary to obtain sharp definition of the fluorescence of these components in areas of intercellular contact: the ring-like concentration of nonerythroid spectrin appeared to be localized more peripherally than that of actin. The cells of preimplantation embryos show motile features that include actual cell movements and striking changes in cell shape (e.g., during compaction). The submembraneous layers of nonerythroid spectrin and actin may contribute to the regulation of the deformability and thus the shape of embryo cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlated waves of actin filaments and PIP3 in Dictyostelium cells

Chemotaxis-deficient amiB-null mutant Dictyostelium cells show two distinct movements: (1) they extend protrusions randomly without net displacements; (2) they migrate persistently and unidirectionally in a keratocyte-like manner. Here, we monitored the intracellular distribution of phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)) to gain insight into roles PIP(3) plays in those spontaneous motilities. In keratocyte-like cells, PIP(3) showed convex distribution over the basal membrane, with no anterior enrichment. In stalled cells, as well as in wild type cells, PIP(3) repeated wave-like changes, including emergence, expansion and disappearance, on the basal membrane. The waves induced lamellipodia when they approached the cell edge, and the advancing speed of the waves was comparable to the migration speed of the keratocyte-like cells. LY294002, an inhibitor of PI3 kinase, abolished PIP(3) waves in stalled cells and stopped keratocyte-like cells. These results together suggested that keratocyte-like cells are "surfing" on the PIP(3) waves by coupling steady lamellipodial protrusions to the PIP(3) waves. Simultaneous live observation of actin filaments and PIP(3) in wild type or stalled amiB(-) cells indicated that the PIP(3) waves were correlated with wave-like distributions of actin filaments. Most notably, PIP(3) waves often followed actin waves, suggesting that PIP(3) induces local depolymerization of actin filaments. Consistent with this idea, cortical accumulation of PIP(3) was often correlated with local retraction of the periphery. We propose that the waves of PIP(3) and actin filaments are loosely coupled with each other and play important roles in generating spontaneous cell polarity.     

A dynamin-cortactin-Arp2/3 complex mediates actin reorganization in growth factor-stimulated cells

The mechanisms by which mammalian cells remodel the actin cytoskeleton in response to motogenic stimuli are complex and a topic of intense study. Dynamin 2 (Dyn2) is a large GTPase that interacts directly with several actin binding proteins, including cortactin. In this study, we demonstrate that Dyn2 and cortactin function to mediate dynamic remodeling of the actin cytoskeleton in response to stimulation with the motogenic growth factor platelet-derived growth factor. On stimulation, Dyn2 and cortactin coassemble into large, circular structures on the dorsal cell surface. These "waves" promote an active reorganization of actin filaments in the anterior cytoplasm and function to disassemble actin stress fibers. Importantly, inhibition of Dyn2 and cortactin function potently blocked the formation of waves and subsequent actin reorganization. These findings demonstrate that cortactin and Dyn2 function together in a supramolecular complex that assembles in response to growth factor stimulation and mediates the remodeling of actin to facilitate lamellipodial protrusion at the leading edge of migrating cells.     

Decreased actin and tubulin synthesis in 3T3 cells after transformation by SV40 virus.

Actin and tubulin are major protein constituents of 3T3 and SV40 virus-transformed 3T3 cells. We have fractionated growing, confluent and SV403T3 cells into particulate and soluble fractions using conditions designed to sediment microtubules, actin filaments or membrane associated actin or tubulin. The ratio of particulate to soluble actin synthesized in growing or confluent 3T3 cells is 2 to 1, while the ratio is reversed in transformed cells. There is also a 60% decrease in particulate tubulin synthesis in SV403T3 cells when compared with that in normal cells. Similar results are obtained when total actin and tubulin amounts are determined. The half-lives of actin, tubulin and total protein are over 3 days in growing 3T3 and SV40 cells and decrease over two-fold in confluent 3T3 cells. The significance of these results with respect to loss of contact inhibition and development of malignancy by these cells after transformation is discussed.     

The sperm entry site during fertilization of the zebrafish egg: localization of actin.

The sperm entry site (SES) of zebrafish (Brachydanio rerio) eggs was studied before and during fertilization by fluorescence, scanning, and transmission electron microscopy. Rhodamine phalloidin (RhPh), used to detect polymerized filamentous actin, was localized to microvilli of the SES and to cytoplasm subjacent to the plasma membrane in the unfertilized egg. The distribution of RhPh staining at the SES correlated with the ultrastructural localization of a submembranous electrondense layer of cortical cytoplasm approximately 500 nm thick and containing 5- to 6-nm filaments. Actin, therefore, was organized at the SES as a tightly knit meshwork of filaments prior to fertilization. Contact between the fertilizing sperm and the filamentous actin network was observed by 15-20 sec postinsemination or just before the onset of fertilization cone formation. Growing fertilization cones of either artificially activated or inseminated eggs exhibited intense RhPh staining and substantial increase in thickness of the actin meshwork. Collectively, TEM and RhPh fluorescence images of inseminated eggs demonstrated that the submembranous actin became rearranged in fertilization cones to form a thickened meshwork around the sperm nucleus during incorporation. The results reported here suggest that activation of the egg triggers a dramatic polymerization of actin beneath the plasma membrane of the fertilization cone. Furthermore, the actin involved in sperm incorporation is sensitive to the action of cytochalasins.     

Immunocytochemical localization of actin in dendritic spines of the cerebral cortex using colloidal gold as a probe

Immunocytochemical localization of actin in rat cerebral cortex embedded in the resin LR White was performed using 5 nm colloidal gold as a probe. Antigenicity is maintained throughout the embedding procedure and the low electron opacity of LR White permits fine filamentous structures to be visualized. Control experiments included incubating the sections with normal goat serum or mouse IgG instead of the primary antibody, preadsorbing the antibody with actin from bovine muscle or liver acetone powder, and heat treating the primary antibody. Immunoreactive actin was identified primarily in dendritic spines, particularly in the postsynaptic density (PSD), the subsynaptic web, and the spine apparatus and endothelial and smooth muscle cells of blood vessels. Within dendritic spines, actin which is labeled in the PSD is in continuity with the filaments of the subsynaptic web. These filaments, in turn, are in continuity with the spine apparatus and/or the spine membranes adjacent to the PSD. The PSD may therefore function like other submembranous filamentous arrays which communicate events occurring at the membrane, in this case, the postsynaptic membrane, to the underlying cytoskeletal network, i.e., the subsynaptic web of the spine. It is also suggested that the actin present in the spine may play a role in changes in spine shape and synaptic curvature. Some actin was also seen in the presynaptic process in association with synaptic vesicles, the filamentous network that is contiguous with the synaptic vesicle membrane, and the presynaptic dense projections. Actin may be involved in dynamic processes in the presynaptic ending which include vesicle translocation.     

Differences between nucleus and cytoplasm in the degree of actin polymerization

For purposes of studying the degree of polymerization of actin in nuclei, nuclei from 35S-labeled amoebas (Amoeba proteus) were transplanted into unlabeled cells, which were immediately lysed and extracted under conditions considered to stabilize preexisting fibrous actin. The enucleated 35S-donor cells were similarly treated for analysis of cytoplasmic actin. The extraction conditions permitted separation of soluble (unpolymerized or G) actin from pelletable (polymerized or F) actin, and the radioactivity of each was determined after the actin was separated from other proteins by polyacrylamide gel electrophoresis. We found that about 2/3 of the actin within the nucleus is pelletable, whereas only about 1/3 of the cytoplasmic actin is pelletable. We speculate that polymerized actin in the nucleus is involved in the condensation of chromatin.     

Mobile actin clusters and traveling waves in cells recovering from actin depolymerization

At the leading edge of a motile cell, actin polymerizes in close apposition to the plasma membrane. Here we ask how the machinery for force generation at a leading edge is established de novo after the global depolymerization of actin. The depolymerization is accomplished by latrunculin A, and the reorganization of actin upon removal of the drug is visualized in Dictyostelium cells by total internal reflection fluorescence microscopy. The actin filament system is reorganized in three steps. First, F-actin assembles into globular complexes that move along the bottom surface of the cells at velocities up to 10 microm/min. These clusters are transient structures that eventually disassemble, fuse, or divide. In a second step, clusters merge into a contiguous zone at the cell border that spreads and gives rise to actin waves traveling on a planar membrane. Finally, normal cell shape and motility are resumed. These data show that the initiation of actin polymerization is separated in Dictyostelium from front protrusion, and that the coupling of polymerization to protrusion is a later step in the reconstitution of a leading edge.     

Desmin and actin in the identification of Ito cells and in monitoring their evolution to myofibroblasts in experimental liver fibrosis

It has been reported that myofibroblasts contain actin and that Ito cells are positive for desmin. The distribution of desmin and actin detected by immunofluorescence, of vitamin A autofluorescence and of Sudan III staining of lipid droplets has been evaluated in sequential stages of experimental liver fibrosis induced in rats by intraperitoneal injections of swine serum. In the normal rat liver Ito cells were positive for desmin and weakly positive for actin. Prior to the development of hepatic fibrosis a clearcut increase in number and desmin staining of lobular Ito cells was observed in treated rats, but the overall actin pattern was unchanged. In the fibrotic rat livers, highly cellular septa contained large numbers of strongly desmin-positive, actin-weakly positive Ito cells and strongly desmin- and actin-positive myofibroblasts. These observations indicate that both Ito cells and myofibroblasts are positive for desmin, but only myofibroblasts contain large amounts of actin. Visualization of actin and desmin using relatively simple techniques, allows the monitoring of Ito cells proliferation, the accumulation of these cells in fibrous septa and their evolution into myofibroblasts as characterized by their increased desmin and actin content; it also allows an indirect evaluation of the process of fibrogenesis.     

Heat shock induction of intranuclear actin rods in cultured mammalian cells

Incubation of cultured cells of mouse C3H-2K fibroblastic cell line and other mammalian cell lines at 42.0-43.0 degrees C for 30 min or longer caused disintegration of normal actin structures including stress fibers, and induced formation of intranuclear actin paracrystal-like structures, called actin rods. When cells exposed to the elevated temperatures were shifted back to 37 degrees C, normal actin structures were regained. Pretreatment of cells at moderately high temperatures such as 38.5 degrees C inhibited formation of the actin rods upon subsequent exposure to 42.0 degrees C. Neither microtubules nor intermediate filaments were disrupted by the heat treatment. Several heat shock proteins were found to be synthesized under the conditions where actin rods were induced. However, there is no causal relationship between two cellular events, the induction of intranuclear actin rods and the synthesis of heat shock proteins.     

Phosphatidic acid induces actin polymerization by activating protein kinases in soybean cells

Phosphatidic acid (PA) levels rise in response to wounding, stress and elicitors, suggesting that it mediates defense responses in plants. During such responses, actin filaments are altered. Since PA induces actin polymerization in animal cells we examined its effect on actin structures in suspension-cultured soybean cells. PA caused a three to four fold increase in cells containing filamentous actin. Immunoblotting with anti-actin antibody showed that actin polymerized within 30 min of treatment. The effect of PA on actin polymerization appears to be mediated by protein kinases because: 1) the effect was suppressed by staurosporin, a general protein kinase inhibitor, and by the protein kinase C-specific inhibitor, calphostin, 2) calyculin A, an inhibitor of protein phosphatase 1 and 2A, mimicked the effect of PA on actin polymerization, and 3) PA activated protein kinases in soybean cells. We suggest that a 54 kDa Ca2+-dependent protein kinase may transduce the PA signal because EGTA inhibited the 54 kDa kinase and the PA-induced actin polymerization, and similar protein kinases have been reported to co-localize with and regulate actin filaments. Our results support the role of PA as a signal mediator and identify actin as a downstream target of PA.     

Localization and dynamics of nonfilamentous actin in cultured cells [published erratum appears in J Cell Biol 1993 Nov;123(3):following 767]

Although the distribution of filamentous actin is well characterized in many cell types, the distribution of nonfilamentous actin remains poorly understood. To determine the relative distribution of filamentous and nonfilamentous actin in cultured NRK cells, we have used a number of labeling agents that differ with respect to their specificities toward the filamentous or nonfilamentous form, including monoclonal and polyclonal anti-actin antibodies, vitamin D-binding protein (DBP), and fluorescent phalloidin. Numerous punctate structures were identified that bind poorly to phalloidin but stain positively with several anti-actin antibodies. These bead structures also stain with DBP, suggesting that they are enriched in nonfilamentous actin. Similar punctate structures were observed after the microinjection of fluorescently labeled actin into living cells, allowing us to examine their dynamics in living cells. The actin-containing punctate structures were observed predominantly in the region behind lamellipodia, particularly in spreading cells induced by wounding confluent monolayers. Time-lapse recording of cells injected with fluorescent actin indicated that they form continuously near the leading edge and move centripetally toward the nucleus. Our results suggest that at least part of the unpolymerized actin molecules are localized at discrete sites, possibly as complexes with monomer sequestering proteins. These structures may represent transient storage sites of G-actin within the cell which can be transformed rapidly into actin filaments upon stimulation by specific signals.