Cytochalasin D acts as an inhibitor of the actin-cofilin interaction

Cofilin, a key regulator of actin filament dynamics, binds to G- and F-actin and promotes actin filament turnover by stimulating depolymerization and severance of actin filaments. In this study, cytochalasin D (CytoD), a widely used inhibitor of actin dynamics, was found to act as an inhibitor of the G-actin-cofilin interaction by binding to G-actin. CytoD also inhibited the binding of cofilin to F-actin and decreased the rate of both actin polymerization and depolymerization in living cells. CytoD altered cellular F-actin organization but did not induce net actin polymerization or depolymerization. These results suggest that CytoD inhibits actin filament dynamics in cells via multiple mechanisms, including the well-known barbed-end capping mechanism and as shown in this study, the inhibition of G- and F-actin binding to cofilin.     

State of actin in gastric parietal cells

Remodeling of theapical membrane-cytoskeleton has been suggested to occur when gastricparietal cells are stimulated to secrete HCl. The present experimentsassayed the relative amounts of F-actin and G-actin in gastric glandsand parietal cells, as well as the changes in the state of actin onstimulation. Glands and cells were treated with a Nonidet P-40extraction buffer for separation into detergent-soluble (supernatant)and detergent-insoluble (pellet) pools. Two actin assays were used toquantitate actin: the deoxyribonuclease I binding assay to measureG-actin and F-actin content in the two pools and a simple Western blotassay to quantitate the relative amounts of actin in the pools.Functional secretory responsiveness was assayed by aminopyrineaccumulation. About 5% of the total parietal cell protein is actin,with about 90% of the actin present as F-actin. Stimulation of acidsecretion resulted in no measurable change in the relative amounts ofG-actin and cytoskeletal F-actin. Treatment of gastric glands withcytochalasin D inhibited acid secretion and resulted in a decrease inF-actin and an increase in G-actin. No inhibition of parietal cellsecretion was observed when phalloidin was used to stabilize actinfilaments. These data are consistent with the hypothesis thatmicrofilamentous actin is essential for membrane recruitment underlyingparietal cell secretion. Although the experiments do not eliminate theimportance of rapid exchange between G- and F-actin for the secretoryprocess, the parietal cell maintains actin in a highly polymerizedstate, and no measurable changes in the steady-state ratio of G-actin to F-actin are associated with stimulation to secrete acid.

     

Evidence for a gelsolin-rich, labile F-actin pool in human polymorphonuclear leukocytes.

Filamentous (F) actin is a major cytoskeletal element in polymorphonuclear leukocytes (PMNs) and other non-muscle cells. Exposure of PMNs to agonists causes polymerization of monomeric (G) actin to F-actin and activates motile responses. In vitro, all purified F-actin is identical. However, in vivo, the presence of multiple, diverse actin regulatory and binding proteins suggests that all F-actin within cells may not be identical. Typically, F-actin in cells is measured by either NBDphallacidin binding or as cytoskeletal associated actin in Triton-extracted cells. To determine whether the two measures of F-actin in PMNs, NBDphallacidin binding and cytoskeletal associated actin, are equivalent, a qualitative and quantitative comparison of the F-actin in basal, non-adherent endotoxin-free PMNs measured by both techniques was performed. F-actin as NBDphallacidin binding and cytoskeletal associated actin was measured in cells fixed with formaldehyde prior to cell lysis and fluorescent staining (PreFix), or in cells lysed with Triton prior to fixation (PostFix). By both techniques, F-actin in PreFix cells is higher than in PostFix cells (54.25 +/- 3.77 vs. 23.5 +/- 3.7 measured as mean fluorescent channel by NBDphallacidin binding and 70.3 +/- 3.5% vs. 47.2 +/- 3.6% of total cellular actin measured as cytoskeletal associated actin). These results show that in PMNs, Triton exposure releases a labile F-actin pool from basal cells while a stable F-actin pool is resistant to Triton exposure. Further characterizations of the distinct labile and stable F-actin pools utilizing NBDphallacidin binding, ultracentrifugation, and electron microscopy demonstrate the actin released with the labile pool is lost as filament. The subcellular localization of F-actin in the two pools is documented by fluorescent microscopy, while the distribution of the actin regulatory protein gelsolin is characterized by immunoblots with anti-gelsolin. Our studies show that at least two distinct F-actin pools coexist in endotoxin-free, basal PMNs in suspension: 1) a stable F-actin pool which is a minority of total cellular F-actin, Triton insoluble, resistant to depolymerization at 4 degrees C, gelsolin-poor, and localized to submembranous areas of the cell; and 2) a labile F-actin pool which is the majority of total cellular F-actin, Triton soluble, depolymerizes at 4 degrees C, is gelsolin-rich, and distributed diffusely throughout the cell. The results suggest that the two pools may subserve unique cytoskeletal functions within PMNs, and should be carefully considered in efforts to elucidate the mechanisms which regulate actin polymerization and depolymerization in non-muscle cells.     

Levels of filamentous and globular actin in Chinese hamster ovary cells throughout the cell cycle

Synchronous Chinese hamster ovary (CHO) cells were obtained by mitotic selection and the levels of globular (G) actin, filamentous (F) actin, and cytoskeletal-associated F-actin were determined as cells progressed through the cell cycle. Total actin levels remained quite constant when expressed as a percent of the total protein. An increase in F-actin occurred upon plating the mitotic cells, but this increase was shown to be a result of attachment to the substratum, since cells which remained attached during the second mitosis failed to show these changes. No large variation in the levels of either F-actin or cytoskeletal-associated F-actin occurred throughout the cell cycle. Therefore, changes in the morphology of the CHO cells which are accompanied by a reorganization of actin-containing microfilaments during the cell cycle are not accompanied by significant changes in the size of the monomeric actin pool.     

Regulation of Actin Tension in Plant Cells by Kinases and Phosphatases

Changes in the organization and mechanical properties of the actin network within plant and animal cells are primary responses to cell signaling. These changes are suggested to be mediated through the regulation of G/F-actin equilibria, alterations in the amount and/or type of actin-binding proteins, the binding of myosin to F-actin, and the formation of myosin filaments associated with F-actin. In the present communication, the cell optical displacement assay was used to investigate the role of phosphatases and kinases in modifying the tension and organization within the actin network of soybean cells. The results from these biophysical measurements suggest that: (a) calcium-regulated kinases and phosphatases are involved in the regulation of tension, (b) calcium transients induce changes in the tension and organization of the actin network through the stimulation of proteins containing calmodulin-like domains or calcium/calmodulin-dependent regulatory proteins, (c) myosin and/or actin cross-linking proteins may be the principal regulator(s) of tension within the actin network, and (d) these actin cross-linking proteins may be the principal targets of calcium-regulated kinases and phosphatases.     

Viscoelasticity of F-actin measured with magnetic microparticles

Dispersed submicroscopic magnetic particles were used to probe viscoelasticity for cytoplasm and purified components of cytoplasm. An externally applied magnetic field exerted force on particles in cells, in filamentous actin (F-actin) solutions, or in F-actin gels formed by the addition of the actin gelation factor, actin-binding protein (ABP). The particle response to magnetic torque can be related to the viscoelastic properties of the fluids. We compared data obtained on F-actin by the magnetic particle method with data obtained on F-actin by means of a sliding plane viscoelastometer. F-actin solutions had a significant elasticity, which increased by 20-fold when gels were formed by ABP addition. Both methods gave consistent results, but the dispersed magnetic particles indicated quantitatively greater rigidity than the viscoelastometer (two and six times greater for F-actin solutions and for F-actin plus ABP gels, respectively). These differences may be due to the fact that, compared with traditional microrheometers, dispersed particle measurements are less affected by long-range heterogeneity or domain-like structure. The magnetometric method was used to examine the mechanical properties of cytoplasm within intact macrophages; the application of the same magnetometric technique to both cells and well-defined, purified protein systems is a first step toward interpreting the results obtained for living cells in molecular terms. The magnetic particle probe system is an effective nonoptical technique for determining the motile and mechanical properties of cells in vitro and in vivo.     

The quantitation of G- and F-actin in cultured cells

An improved method to quantitate the amounts of filamentous (F-actin) and monomeric (globular) actin (G-actin) in cultured cells was developed. Cells are lysed into a myosin-containing buffer and F-actin is removed by centrifugation. The pelleted F-actin is then depolymerized to G-actin in a 1 mM ATP-containing buffer for 1 h before measuring the levels of G-actin using the DNase I inhibition assay. Partitioning of G-actin in the supernatant (greater than 95%) and recovery of actin in both fractions (greater than 85%) were measured by adding [3H]actin to cultured cells. Actin in the separated fractions is stable for at least 72 h at 0 degree C. Asynchronous monolayer cultures of Chinese hamster ovary (CHO) cells contain 2.5 +/- 0.2% of the total protein as actin with 72.4 +/- 5.7% as F-actin. About 10% of this F-actin is not associated with the readily sedimented Triton-cytoskeleton. CHO cells grown in suspension contain 55.8% of the actin as F-actin; following plating about 90 min is required for these cells to flatten and for the F-actin level to reach the monolayer value of about 70%.     

Polymerization of additional actin is not required for capping of surface antigens in B-lymphocytes

CH12 is a murine B-cell lymphoma whose surface immunoglobulin (sIg) and concanavalin A (Con A) receptors patch and cap readily. Actin may be involved in CH12 patching and capping, since fodrin and F-actin collect under the cap, and cytochalasin D inhibits sIg capping. We have examined the state of the actin cytoskeleton during patching and capping. A wide range of concentrations of rabbit anti-mouse antibody (RAM) and Con A were used to patch or cap CH12 cells. G-actin was quantitated by DNase I inhibition, F-actin was quantitated by fluorescence-activated cell sorter analysis of fluorescent phalloidin staining, and actin nucleation sites were measured by pyrene actin polymerization. None of these methods detected any significant changes in actin when compared to control cells or untreated cells, leading us to conclude that increased actin polymerization is not necessary for capping to occur. The significance of these data to the membrane flow and cytoskeletal models of capping is discussed.     

Rearrangements of F-actin during Stomatogenesis Visualised by Confocal Microscopy in Fixed and Permeabilised Tradescantia Leaf Epidermis

Abstract: New details of F-actin organisation in leaf epidermal and stomatal cells were revealed by rhodamine — and fluorescein — phalloidin staining of fixed epidermal peels of Tradescantia virginiana and visualisation by confocal microscopy. Non-specialised epidermal cells contain highly organised arrays of fine cortical actin filaments aligned in transverse or oblique orientations. In interphase guard mother cells (GMCs), the arrangement of cortical F-actin changes on the periclinal and anticlinal cell walls at different times during differentiation. Initially, cortical F-actin on the periclinal surfaces is oriented transversely and F-actin is evenly distributed around the anticlinal walls. Following polarisation of the adjacent subsidiary mother cells (SMCs), actin in GMCs concentrates on the lateral anticlinal walls, but not on the transverse walls. Subsequently, F-actin on the periclinal walls reorients to radial and then longitudinal. Organisation of F-actin in SMCs appears to be influenced by the adjacent GMCs and co-ordination in F-actin arrangements in cells of the stomatal complex continues through to the formation of the guard cell pair. Our studies indicate that actin bands marking the division site in prophase cells, and detected in microinjected living material, are a particularly labile subset of F-actin. Actin bands were difficult to preserve, even when aldehyde fixation was avoided, in contrast to all interphase and mitotic F-actin.     

The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis

Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools – Utrophin, Lifeact, and F-tractin – for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling tool within the tissue and cell type of interest in order to identify the tool that represents the best compromise between acceptable labeling and minimal disruption of the phenomenon being observed. In this case, we find that F-tractin, and perhaps Utrophin, when Utrophin expression levels are optimized to label efficiently without causing actin defects, can be used to study F-actin dynamics within the Drosophila nurse cells.     

Actin depolymerization and inhibition of capping induced by pentoxifylline in human lymphocytes and neutrophils

Pentoxifylline is used clinically for the treatment of intermittent claudication. It is believed to exert its effect by altering the rheologic properties of blood. The cytoskeleton plays an important role in the maintenance of cell structure and function. In particular, alterations in the state of actin seem to play an important role in cell motility. Therefore, we examined the effect of pentoxifylline on the actin state in human polymorphonuclear leukocytes (PMN) and mononuclear cells. Pentoxifylline (10 mM final concentration) decreased F-actin content in both PMN and mononuclear cells. Pentoxifylline also inhibited concanavalin A-induced capping in PMN and mononuclear cells. Similarly, surface immunoglobulin capping in B lymphocytes was also inhibited. Pretreatment of cells with pertussis toxin did not inhibit pentoxifylline-induced decrease in F-actin, suggesting pentoxifylline does not act through pertussis toxin-sensitive G-proteins. Dibutyryl cyclic AMP failed to show any significant effect on the F-actin content in PMN. Therefore, the effect of pentoxifylline cannot be attributed to changes in cyclic AMP levels. Chemotactic peptide-induced actin polymerization was unaffected in PMN when expressed as percent changes in F-actin. The observations reported here suggest that the rheological effects of pentoxifylline might be due to its effects on the actin state in the cellular elements of the blood. Further studies on the mechanism of action of pentoxifylline on actin state in leukocytes will prove useful in delineating the physiological mechanisms regulating actin state in leukocytes.     

A mechanochemical model of actin filaments

In eukaryotic cells, actin filaments are involved in important processes such as motility, division, cell shape regulation, contractility, and mechanosensation. Actin filaments are polymerized chains of monomers, which themselves undergo a range of chemical events such as ATP hydrolysis, polymerization, and depolymerization. When forces are applied to F-actin, in addition to filament mechanical deformations, the applied force must also influence chemical events in the filament. We develop an intermediate-scale model of actin filaments that combines actin chemistry with filament-level deformations. The model is able to compute mechanical responses of F-actin during bending and stretching. The model also describes the interplay between ATP hydrolysis and filament deformations, including possible force-induced chemical state changes of actin monomers in the filament. The model can also be used to model the action of several actin-associated proteins, and for large-scale simulation of F-actin networks. All together, our model shows that mechanics and chemistry must be considered together to understand cytoskeletal dynamics in living cells.     

Actin filament organization in activated mast cells is regulated by heterotrimeric and small GTP-binding proteins

Rat peritoneal mast cells, both intact and permeabilized, have been used widely as model secretory cells. GTP-binding proteins and calcium play a major role in controlling their secretory response. Here we have examined changes in the organization of actin filaments in intact mast cells after activation by compound 48/80, and in permeabilized cells after direct activation of GTP-binding proteins by GTP-gamma-S. In both cases, a centripetal redistribution of cellular F-actin was observed: the content of F-actin was reduced in the cortical region and increased in the cell interior. The overall F-actin content was increased. Using permeabilized cells, we show that AIF4-, an activator of heterotrimeric G proteins, induces the disassembly of F-actin at the cortex, while the appearance of actin filaments in the interior of the cell is dependent on two small GTPases, rho and rac. Rho was found to be responsible for de novo actin polymerization, presumably from a membrane-bound monomeric pool, while rac was required for an entrapment of the released cortical filaments. Thus, a heterotrimeric G-protein and the small GTPases, rho and rac, participate in affecting the changes in the actin cytoskeleton observed after activation of mast cells.     

Endocytosis and the development of cell polarity in yeast require a dynamic F-actin cytoskeleton

Studies using drugs that cause the disassembly of filamentous actin (F-actin) have demonstrated the importance of an intact actin cytoskeleton for polarised secretion by yeast cells [1,2]. To address the level of dynamic turnover needed for such processes, however, drugs or mutants that confer stabilising properties on F-actin are needed. Jasplakinolide is the only readily available drug that stabilises F-actin structures both in vivo and in vitro [3-6]. Yeast strains have been generated in which two of the ABC multidrug resistance transporter genes have been deleted, rendering normally jasplakinolide-resistant yeast cells sensitive to its effects. Treatment of these cells with jasplakinolide caused rapid and dramatic effects on the actin cytoskeleton, resulting in the accumulation of single large actin structures in cells. These structures, however, still contained components that are normally associated with cortical actin patches. A dynamic actin cytoskeleton was found to be critical for the generation of cell polarity and endocytosis.     

Role of actin cortex in the subplasmalemmal transport of secretory granules in PC-12 cells

In neuroendocrine PC-12 cells, evanescent-field fluorescence microscopy was used to track motions of green fluorescent protein (GFP)-labeled actin or GFP-labeled secretory granules in a thin layer of cytoplasm where cells adhered to glass. The layer contained abundant filamentous actin (F-actin) locally condensed into stress fibers. More than 90% of the granules imaged lay within the F-actin layer. One-third of the granules did not move detectably, while two-thirds moved randomly; the average diffusion coefficient was 23 x 10(-4) microm(2)/s. A small minority (<3%) moved rapidly and in a directed fashion over distances more than a micron. Staining of F-actin suggests that such movement occurred along actin bundles. The seemingly random movement of most other granules was not due to diffusion since it was diminished by the myosin inhibitor butanedione monoxime, and blocked by chelating intracellular Mg(2+) and replacing ATP with AMP-PNP. Mobility was blocked also when F-actin was stabilized with phalloidin, and was diminished when the actin cortex was degraded with latrunculin B. We conclude that the movement of granules requires metabolic energy, and that it is mediated as well as limited by the actin cortex. Opposing actions of the actin cortex on mobility may explain why its degradation has variable effects on secretion.     

F-actin is a Nuclear and Chromosomal Component of the Meristematic Cells of Allium sativum

It is known that actin functionates in the form of F-actin. However, the presence of Factin in eukaryotic nuclei and chromosomes has not been well established. The authors labeled meristematic cells of Allium sativum L. with rabbit anti-chicken actin antibody and FITC-conjugated goat anti-rabbit IgG antibody and observed with fluorescence microscopy. Both the nuclei and chromosomes showed prominent yellow-green fluorescence, indicating the presence of actin in them. Fluorescence examination with TR1TC-conjugated phalloidin demonstrated prominent red fluorescence in the intact interphase cells, cytoplasm-free interphase nuclei, prophase and metaphase chromosomes as well as the daughter nuclei at telophase indicating the presence of F-actin; but the fluorescence was absent or very weak in the cells exposed to cytochalasin D before fixation. When double labeling of the anti-actin antibody and phalloidin was applied, the same nuclei and chromosomes were found to emanate yellow-green fluorescence representing actin at the excitation wavelength of F1TC, and red fluorescence representing F-actin at the excitation wavelength of TRITC, respectively. The FITC fluorescence and TRITC fluorescence shared the same distribution among the nuclei and chromosomes. These results indicate that F-actin is a component of the nuclei and chromosomes of the meristematic cells of A. sativum. It also suggests that F-actin may be the major existing form of actin in them.     

Polymerization of actin in RBL-2H3 cells can be triggered through either the IgE receptor or the adenosine receptor but different signaling pathways are used.

Crosslinking of the IgE receptor on rat basophilic leukemia (RBL) cells using the multivalent antigen DNP-BSA leads to a rapid and sustained increase in the filamentous actin content of the cells. Stimulation of RBL cells through the adenosine receptor also induces a very rapid polymerization of actin, which peaks in 45-60 s and is equivalent in magnitude to the F-actin response elicited through stimulation of the IgE receptor. However, in contrast to the IgE mediated response, which remains elevated for over 30 min, the F-actin increase induced by the adenosine analogue 5'-(N-ethylcarboxamido)-adenosine (NECA) is relatively transient and returns to baseline values within 5-10 min. While previous work has shown that the polymerization of actin in RBL cells stimulated through the IgE receptor is mediated by protein kinase C (PKC), protein kinase inhibitors have no effect on the F-actin response activated through the adenosine receptor. In contrast, pretreatment of the cells with pertussis toxin completely inhibits the F-actin response to NECA but has relatively little effect on the response induced through the IgE receptor. Stimulation of RBL cells through either receptor causes increased production of phosphatidylinositol mono-phosphate (PIP) and phosphatidylinositol bis-phosphate (PIP2), which correlates with the F-actin response. Production of PIP and PIP2 may be important downstream signals since these polyphosphoinositides are able to regulate the interaction of gelsolin and profilin with actin. Thus the polymerization of actin can be triggered through either the adenosine receptor or the IgE receptor, but different upstream signaling pathways are being used. The IgE mediated response requires the activation of PKC while stimulation through the adenosine receptor is PKC independent but involves a G protein.     

Green fluorescent protein fusions to Arabidopsis fimbrin 1 for spatio-temporal imaging of F-actin dynamics in roots

The visualization of green fluorescent protein (GFP) fusions with microtubule or actin filament (F-actin) binding proteins has provided new insights into the function of the cytoskeleton during plant development. For studies on actin, GFP fusions to talin have been the most generally used reporters. Although GFP-Talin has allowed in vivo F-actin imaging in a variety of plant cells, its utility in monitoring F-actin in stably transformed plants is limited particularly in developing roots where interesting actin dependent cell processes are occurring. In this study, we created a variety of GFP fusions to Arabidopsis Fimbrin 1 (AtFim1) to explore their utility for in vivo F-actin imaging in root cells and to better understand the actin binding properties of AtFim1 in living plant cells. Translational fusions of GFP to full-length AtFim1 or to some truncated variants of AtFim1 showed filamentous labeling in transient expression assays. One truncated fimbrin-GFP fusion was capable of labeling distinct filaments in stably transformed Arabidopsis roots. The filaments decorated by this construct were highly dynamic in growing root hairs and elongating root cells and were sensitive to actin disrupting drugs. Therefore, the fimbrin-GFP reporters we describe in this study provide additional tools for studying the actin cytoskeleton during root cell development. Moreover, the localization of AtFim1-GFP offers insights into the regulation of actin organization in developing roots by this class of actin cross-linking proteins.     

Comparison of cryofixation and aldehyde fixation for plant actin immunocytochemistry: aldehydes do not destroy F-actin

For walled plant cells, the immunolocalization of actin microfilaments, also known as F-actin, has proved to be much trickier than that of microtubules. These difficulties are commonly attributed to the high sensitivity of F-actin to aldehyde fixatives. Therefore, most plant studies have been accomplished using fluorescent phallotoxins in fresh tissues. Nevertheless, concerns regarding the questionable ability of phallotoxins to bind the whole complement of F-actin necessitate further optimization of actin immunofluorescence methods. We have compared two procedures: (1) formaldehyde fixation and (2) rapid freezing and freeze substitution (cryofixation), both followed by embedding in low-melting polyester wax. Actin immunofluorescence in sections of garden cress (Lepidium sativum L.) root gave similar results with both methods. The compatibility of aldehydes with actin immunodetection was further confirmed by the freeze-shattering technique that does not require embedding after aldehyde fixation. It appears that rather than aldehyde fixation, some further steps in the procedures used for actin visualization are critical for preserving F-actin. Wax embedding, combined with formaldehyde fixation, has proved to be also suitable for the detection of a wide range of other antigens.     

Real-time monitoring of actin polymerization in living cells using split luciferase

Real-time monitoring of actin polymerization in living cells is beneficial for characterizing cellular activities such as migration, proliferation, and death. We developed new bioluminescence-based probe proteins that enable the monitoring of actin polymerization in living cells. Unlike other ordinary split luciferase probes, our probes were incorporated in endogenous actin filament that enabled it to measure the actin polymerization quantitatively. The probe proteins exhibited a dose-responsive decrease in photon emission intensity in response to the filamentous (F)-actin-disrupting agent latrunculin A. This technique has a high sensitivity with a high signal-to-noise ratio and is nontoxic compared with other methods of monitoring actin polymerization in living cells. Using this technique, we succeeded in monitoring the F-actin level in living cells during apoptosis progression induced by UV irradiation continuously for 12 h. F-actin was transiently upregulated after UV irradiation. Since UV-induced cell death was enhanced by treatment with latrunculin A during the period which F-actin is increased, transient upregulation of F-actin after UV is likely a protective reaction against UV-induced cell death. Our novel technique is an effective tool for investigating actin polymerization in living cells.