RGD-containing peptides and cyclic AMP have antagonistic roles in the morphology of Mucor rouxii

Summary. The tripeptide Arg-Gly-Asp (RGD) (1 mM) as well as the polymer ProNectin F (20 nM) added to culture medium of the fungus Mucor rouxii (defined medium) produced a delay in the switch from isodiametric growth to tip growth; at the time of germination the mother cell had a 4.6 times larger volume with 3.6 times more germ tubes per cell than control germinating sporangiospores. Disruption of the actin network with 2 µg of cytochalasin A per ml blocked the switch to tip growth; the effect was analogous to the one of 150 µM dibutyryl–cyclic AMP (cAMP), which we previously described to promote isodiametric growth via protein kinase A. 150 µM dibutyryl-cAMP antagonises partially the effect of 1 mM RGD; the cells still emit several germ tubes per mother cell but their number is smaller and the volume of the cell at germ tube emission is larger than with RGD alone. At higher concentrations the dibutyryl-cAMP overrides completely the effect of RGD. Our results suggest that M. rouxii has an RGD recognition system and demonstrate that RGD-containing peptides have a profound effect on the isotropic stage of growth and on the establishment of cell polarity and that cAMP analogues can override this effect.Correspondence and reprints: Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Pabell'n 2, Ciudad Universitaria, Universidad de Buenos Aires, 1428 Buenos Aires, ArgentinaReceived January 29, 2003; accepted March 10, 2003; published online September 23, 2003     

Dibutyryl cAMP inhibits expression of transformation-related properties in Kirsten murine sarcoma virus transformed Balb/c-3T3 cells despite continued presence of p21v-Ki-ras

We studied the effects of simultaneous treatment with 0.1 mM N6, O2'-dibutyryl cAMP (dbcAMP) and 1 mM theophylline on several transformation-specific properties and on levels of the Kirsten murine sarcoma virus (Ki-MSV) transforming gene product p21v-Ki-ras, in a Ki-MSV-transformed mouse cell line (Balb/c-3T3, clone A31; KA31). The rate of logarithmic growth, cell motility, and final saturation density were reduced in dbcAMP-treated KA31 cultures. Capabilities for anchorage-independent growth were reduced in treated cells, to levels similar to those observed for the untransformed parental A31 cell line. Treatment with dbcAMP had no observable effect on the binding of 125I-labeled epidermal growth factor and did not alter fluorescence staining patterns for actin microfilaments and fibronectin which, although characteristic of normal cells, were also present in KA31 cells. Changes induced by dbcAMP were readily reversible, except for loss of anchorage-independent growth. However, this property was also reversible, provided removal of dbcAMP occurred 48 h prior to inoculation into soft agar medium. Immunoprecipitation with a monoclonal antibody directed against the protein p21v-Ki-ras (Y13-259) revealed the continued presence of this protein in dbcAMP-treated KA31 cells. We, therefore, conclude that cAMP mediates the inhibition of growth-related transformation-specific properties either by acting at steps subsequent to the expression of p21v-Ki-ras or on a pathway independent of p21ras function.     

Changes of the actin filament system in the green algaMicrasterias denticulata induced by different cytoskeleton inhibitors

Summary Two different techniques have been adapted forMicrasterias denticulata to depict the actin cytoskeleton of both untreated and inhibitor-treated developing cells: the quickstaining method, where the cells are fixed in a mixture of glutaraldehyde and formaldehyde followed by staining with phalloidin without embedding, and the methacrylate method, where the cells are also fixed by aldehydes and where the embedding medium is removed prior to incubation with an actin antibody. Both methods produce sufficient preservation and visualization of actin microfilaments (MFs) and confirm earlier observations on the presence of a cortical actin MF network in both the growing and the nongrowing semicell as well as of a basketlike MF arrangement around the migrating nucleus. The results show that a network of actin MFs is essential for the proper development of the young lobes ofM. denticulata. Early developmental stages expanding uniformly at the beginning of growth lack any netlike actin MF arrangement. The actin cytoskeleton in developing cells treated with the actin-targeting agents cytochalasin D and latrunculin B is markedly influenced. Cytochalasin D, which produces the most pronounced effects, causes a breakdown of the network of actin MFs, resulting in bright actin clusters as well as in short and abnormally thick actin fragments particularly in cortical cell regions. In latrunculin B-treated cells remnants of the former actin MF network are still visible, yet most of the actin cytoskeleton appears collapsed and is reduced to short filament pieces. The disturbance of the actin MF system visualized in the present study correlates with the severe morphological and ultrastructural changes occurring in desmid cells as a consequence of both drugs. The dinitroanilin herbicide oryzalin, known to deploymerize cytoplasmic microtubules, causes also an impairment of the actin cytoskeleton inM. denticulata though not sufficient to influence normal cell growth and differentiation.Abbreviations CB cytochalasin B - CD cytochalasin D - DMSO dimethyl sulfoxide - FA formaldehyde - GA glutaraldehyde - LAT-A latrunculin A - LAT-B latrunculin B - MFs microfilaments - MT microtubule Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

Role of actin and Myo2p in polarized secretion and growth of Saccharomyces cerevisiae

We examined the role of the actin cytoskeleton in secretion in Saccharomyces cerevisiae with the use of several quantitative assays, including time-lapse video microscopy of cell surface growth in individual living cells. In latrunculin, which depolymerizes filamentous actin, cell surface growth was completely depolarized but still occurred, albeit at a reduced level. Thus, filamentous actin is necessary for polarized secretion but not for secretion per se. Consistent with this conclusion, latrunculin caused vesicles to accumulate at random positions throughout the cell. Cortical actin patches cluster at locations that correlate with sites of polarized secretion. However, we found that actin patch polarization is not necessary for polarized secretion because a mutant, bee1Delta(las17Delta), which completely lacks actin patch polarization, displayed polarized growth. In contrast, a mutant lacking actin cables, tpm1-2 tpm2Delta, had a severe defect in polarized growth. The yeast class V myosin Myo2p is hypothesized to mediate polarized secretion. A mutation in the motor domain of Myo2p, myo2-66, caused growth to be depolarized but with only a partial decrease in the level of overall growth. This effect is similar to that of latrunculin, suggesting that Myo2p interacts with filamentous actin. However, inhibition of Myo2p function by expression of its tail domain completely abolished growth.     

Actin as a cytoskeletal basis for cell architecture and a protein essential for ecdysis in Prorocentrum minimum (Dinophyceae,Prorocentrales)

The specific cell architecture of prorocentroid dinoflagellates is reflected in the internal cell structure, particularly, in cytoskeleton organization. Cytoskeleton arrangement in a Prorocentrum minimum cell was investigated using fluorescent labeling approaches, electron‐microscopy and immunocytochemical methods. The absence of cortical microtubules was confirmed. Phalloidin – tetramethylrhodamine isothiocyanate conjugate staining demonstrated that F‐actin forms a dense layer in the cortical region of the cell; besides, it was detected in the ‘archoplasmic sphere’ adjacent to the nucleus. In some cells the rest of the cytoplasm and the nucleus were also slightly stained. In dividing cells, F‐actin was mainly distributed in the cortical region and in the cleavage furrow. Fluorescent deoxyribonuclease I staining demonstrated more evenly distributed cytoplasmic non‐polymerized actin; the basis of the nuclear actin pool is monomeric actin. It concentrates in the nucleoplasm and forms a meshwork around chromosomes. The significant amount of G‐actin is apparently localized in the P. minimum nucleolus. Assumed involvement of F‐actin in the process of stress‐induced ecdysis – cell cover shedding – was examined. A sharp decrease in the level of ecdysis was observed after treatment with actin‐depolymerizing agent latrunculin B. The fluorescent staining of treated cells demonstrated disturbance of the actin cytoskeleton and disappearance of the cortical F‐actin layer. Our results support the recent data on the actin involvement in fundamental nuclear processes: cytoplasmic F‐actin appears to participate in cell shape determination, cell cover rearrangement and development. Actin may play a substitute role in the absence of cortical microtubules, representing the cytoskeletal basis of P. minimum cell architecture.     

Investigation of cell adhesion to structured surfaces using total internal reflection fluorescence and confocal laser scanning microscopy

Adhesion of adherent cells on structured surfaces is influenced by the surface pattern given. Here, we designed a structured gold relief surface based on cell adhesion patterns we had previously observed. We analysed the geometric parameters and the overall distribution of focal adhesion kinase in focal adhesions on unstructured glass surfaces using optical microscopy. The basic structural elements obtained from this analysis were arranged in regular clusters that resembled the shape of a polarised migratory cell. In time-lapse studies we observed that the cells adhere preferentially to the gold pads and adopt the shape of the clusters. Staining of the actin cytoskeleton revealed that the actin filaments are aligned to the gold pads of the elementary structure.     

Detection of dehydrin-like proteins in embryos and endosperm of mature Euterpe edulis seeds

Summary. Euterpe edulis Martius, a tropical palm species characterized as highly recalcitrant, accumulated dehydrin proteins in both the endosperm and the embryo of the mature seed, as detected by Western blot analysis and immunogold electron microscopy. Three major bands at molecular masses of approximately 16, 18, and 24 kDa were identified in both samples analysed. Immunogold electron microscopy studies detected the presence of dehydrins in the embryo and endosperm. In both cases, dehydrins were immunolocalized in cytoplasm and chromatin. No labelling associated with either membranes or organelles was detected. It is known that dehydrins are produced as part of the developmental program of orthodox seeds and are also present in some recalcitrant seeds of temperate regions. The constitutive presence of dehydrins in embryos of extremely recalcitrant species of tropical origin has not been previously reported. Correspondence and reprints: Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, C1428EHA, Ciudad Autónoma de Buenos Aires, Argentina.     

Segregation of GM1 and GM3 clusters in the cell membrane depends on the intact actin cytoskeleton

Gangliosides have been implicated in exerting multiple physiological functions, and it is important to understand how their distribution is regulated in the cell membrane. By using freeze-fracture immunolabeling electron microscopy, we showed that GM1 and GM3 make independent clusters that are significantly reduced by cholesterol depletion. In the present study, we examined the effects of actin depolymerization/polymerization and Src-family kinase inhibition on the GM1 and GM3 clusters. Both GM1 and GM3 clustering was reduced when the actin cytoskeleton was perturbed by latrunculin A or jasplakinolide, but the decrease was less significant than that induced by cholesterol depletion. On the other hand, inhibition of Src-family kinases decreased GM3 clustering more drastically than did cholesterol depletion, whereas its effect on GM1 clustering was less significant. GM1 and GM3 were segregated from each other in unperturbed cells, but co-clustering increased significantly after actin depolymerization. Our results indicate that the GM1 and GM3 clusters in the cell membrane are regulated in different ways and that segregation of the two gangliosides depends on the intact actin cytoskeleton.     

Pollen tube growth: coping with mechanical obstacles involves the cytoskeleton

Cellular growth and movement require both the control of direction and the physical capacity to generate forces. In animal cells directional control and growth forces are generated by the polymerization of and traction between the elements of the cytoskeleton. Whether actual forces generated by the cytoskeleton play a role in plant cell growth is largely unknown as the interplay between turgor and cell wall is considered to be the predominant structural feature in plant cell morphogenesis. We investigated the mechano-structural role of the cytoskeleton in the invasive growth of pollen tubes. These cells elongate rapidly by tip growth and have the ability to penetrate the stigmatic and stylar tissues in order to drill their way to the ovule. We used agents interfering with cytoskeletal functioning, latrunculin B and oryzalin, in combination with mechanical in vitro assays. While microtubule degradation had no significant effect on the pollen tubes’ capacity to invade a mechanical obstacle, latrunculin B decreased the pollen tubes’ ability to elongate in stiffened growth medium and to penetrate an obstacle. On the other hand, the ability to maintain a certain growth direction in vitro was affected by the degradation of microtubules but not actin filaments. To find out whether both cytoskeletal elements share functions or interact we used both drugs in combination resulting in a dramatic synergistic response. Fluorescent labeling revealed that the integrity of the microtubule cytoskeleton depends on the presence of actin filaments. In contrast, actin filaments seemed independent of the configuration of microtubules.     

Actin assembly plays a variable, but not obligatory role in receptor-mediated endocytosis in mammalian cells

Three cell-permeant compounds, cytochalasin D, latrunculin A and jasplakinolide, which perturb intracellular actin dynamics by distinct mechanisms, were used to probe the role of filamentous actin and actin assembly in clathrin-mediated endocytosis in mammalian cells. These compounds had variable effects on receptor-mediated endocytosis of transferrin that depended on both the cell line and the experimental protocol employed. Endocytosis in A431 cells assayed in suspension was inhibited by latrunculin A and jasplakinolide, but resistant to cytochalasin D, whereas neither compound inhibited endocytosis in adherent A431 cells. In contrast, endocytosis in adherent CHO cells was more sensitive to disruption of the actin cytoskeleton than endocytosis in CHO cells grown or assayed in suspension. Endocytosis in other cell types, including nonadherent K562 human erythroleukemic cells or adherent Cos-7 cells was unaffected by disruption of the actin cytoskeleton. While it remains possible that actin filaments can play an accessory role in receptor-mediated endocytosis, these discordant results indicate that actin assembly does not play an obligatory role in endocytic coated vesicle formation in cultured mammalian cells.     

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

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

Localised depletion of polymerised actin at the front of Walker carcinosarcoma cells increases the speed of locomotion

Spontaneously migrating Walker carcinosarcoma cells usually form lamellipodia at the front. Combined treatment with 10(-5)M colchicine and 10(-7)M latrunculin A produces large defects in the cortical F-actin layer at the leading front and suppresses lamellipodia. However, the cortical actin layer at the rear is intact and shows myosin IIA accumulation. These cells, showing no or little detectable cortical F-actin at the front and no morphologically recognisable protrusions, migrate faster than control cells with lamellipodia and an intact cortical actin layer. This documents that the cortical actin layer or actin-powered force generation at the front is redundant for locomotion. Colchicine and latrunculin A have synergistic effects in compromising the cortical layer at the front and in increasing the speed of locomotion, but antagonistic effects on the relative amount of F-actin per cell. Colchicine but not latrunculin A, can increase the proportion of polarised and locomoting cells under appropriate conditions. Locomotion and polarity of cells treated with latrunculin A and colchicine is inhibited at latrunculin A concentrations >10(-7)M, by the myosin inhibitor BDM or the ROCK inhibitor Y-27632. Colchicine and Y-27632 have antagonistic effects on polarity and the speed of locomoting cells. The data show that locomotion of metazoan cells, which normally form lamellipodia, can be driven by actomyosin contraction behind the front (cell body, uropod). They are best compatible with a cortical contraction/frontal expansion model, but they are not compatible with models implying that actin polymerisation or actomyosin contraction at the front drive locomotion of the cells studied.     

PKB phosphorylation and survivin expression are cooperatively regulated by disruption of microfilament cytoskeleton

Changes in cell shape can lead to detachment and cell death, and the disruption in the actin cytoskeletal network, as one marker of cell shape changes, can itself induce apoptosis. In this study, the effects of cytochalasin B on the apoptosis-related proteins, protein kinase B and survivin were investigated. Apoptosis induced by disruption of microfilaments with cytochalasin B was found, although it happened at a low level, to simultaneously occur with G2/M arrest in 50% of the cytochalasin B-treated cells. During apoptosis, PKB phosphorylation and survivin expression was decreased by cytochalasin B, and the decline in survivin expression were preceded by PKB dephosphorylation, which implicated that survivin may be a target of PKB protein. The G2/M arrest of cytochalasin B-treated cells may be the direct function of cytochalasin B to microfilaments or the subsequent inhibition of survivin expression, or both. These results suggest that PKB/survivin signaling pathway may be responsible for the apoptosis induced by the disruption of actin cytoskeleton.     

Role of the actin cytoskeleton in T cell absorption and internalization of ligands from APC

A feature of T-APC interaction is that, via either TCR or CD28, T cells can absorb molecules from APC on to the cell surface and then internalize these molecules. Here, using both normal and TCR-transgenic T cells, we investigated the mechanism of T cell absorption of molecules from APC and the role of the cytoskeleton. The results show that although activated T cells could absorb APC molecules in the form of cell fragments, uptake of molecules by resting T cells required direct T-APC interaction. Based on studies with latrunculin B, surface absorption of molecules by resting T cells was crucially dependent upon the actin cytoskeleton for both CD28- and TCR-mediated absorption. Significantly, however, TCR-mediated absorption became strongly resistant to latrunculin B when the concentration of MHC-bound peptide on APC was raised to a high level, implying that the actin cytoskeleton is only important for absorption when the density of receptor/ligand interaction is low. By contrast, in all situations tested, the actin cytoskeleton played a decisive role in controlling T cell internalization of ligands from the cell surface.     

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.     

The Mitotic Exit Network and Cdc14 phosphatase initiate cytokinesis by counteracting CDK phosphorylations and blocking polarised growth

Polarisation of the actin cytoskeleton must cease during cytokinesis, to support efficient assembly and contraction of the actomyosin ring at the site of cell division, but the underlying mechanisms are still understood poorly in most species. In budding yeast, the Mitotic Exit Network (MEN) releases Cdc14 phosphatase from the nucleolus during anaphase, leading to the inactivation of mitotic forms of cyclin-dependent kinase (CDK) and the onset of septation, before G1-CDK can be reactivated and drive re-polarisation of the actin cytoskeleton to a new bud. Here, we show that premature inactivation of mitotic CDK, before release of Cdc14, allows G1-CDK to divert the actin cytoskeleton away from the actomyosin ring to a new site of polarised growth, thereby delaying progression through cytokinesis. Our data indicate that cells normally avoid this problem via the MEN-dependent release of Cdc14, which counteracts all classes of CDK-mediated phosphorylations during cytokinesis and blocks polarised growth. The dephosphorylation of CDK targets is therefore central to the mechanism by which the MEN and Cdc14 initiate cytokinesis and block polarised growth during late mitosis.     

Tobacco WLIM1 is a novel F-actin binding protein involved in actin cytoskeleton remodeling

We used confocal microscopy and in vitro analyses to show that Nicotiana tabacum WLIM1, a LIM domain protein related to animal Cys-rich proteins, is a novel actin binding protein in plants. Green fluorescent protein (GFP)-tagged WLIM1 protein accumulated in the nucleus and cytoplasm of tobacco BY2 cells. It associated predominantly with actin cytoskeleton, as demonstrated by colabeling and treatment with actin-depolymerizing latrunculin B. High-speed cosedimentation assays revealed the ability of WLIM1 to bind directly to actin filaments with high affinity. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed a highly dynamic in vivo interaction of WLIM1-GFP with actin filaments. Expression of WLIM1-GFP in BY2 cells significantly delayed depolymerization of the actin cytoskeleton induced by latrunculin B treatment. WLIM1 also stabilized actin filaments in vitro. Importantly, expression of WLIM1-GFP in Nicotiana benthamiana leaves induces significant changes in actin cytoskeleton organization, specifically, fewer and thicker actin bundles than in control cells, suggesting that WLIM1 functions as an actin bundling protein. This hypothesis was confirmed by low-speed cosedimentation assays and direct observation of F-actin bundles that formed in vitro in the presence of WLIM1. Taken together, these data identify WLIM1 as a novel actin binding protein that increases actin cytoskeleton stability by promoting bundling of actin filaments.     

Maxi-K channels localize to caveolae in human myometrium: a role for an actin-channel-caveolin complex in the regulation of myometrial smooth muscle K+ current

Multiple cell-signaling pathways converge to modulate large-conductance, voltage- and Ca²⁺-sensitive K⁺ channel (maxi-K channel) activity and buffer cell excitability in human myometrial smooth muscle cells (hMSMCs). Recent evidence indicates that maxi-K channel proteins can target to membrane microdomains; however, their association with other proteins within these macromolecular complexes has not been elucidated. Biochemical isolation of detergent-resistant membrane fractions from human myometrium demonstrates the presence of maxi-K channels in lipid raft microdomains, which cofractionate with caveolins. In both nonpregnant and late-pregnant myometrium, maxi-K channels associate and colocalize with caveolar scaffolding proteins caveolin-1 and caveolin-2, but not caveolin-3. Disruption of cultured hMSMC caveolar complexes by cholesterol depletion with cyclodextrin increases an iberiotoxin-sensitive K⁺ current. Coimmunoprecipitations have indicated that the maxi-K channel also is associated with both - and -actin. Immunocytochemical analysis indicates colocalization of maxi-K channels, actin, and caveolin-1 in primary cultures of hMSMCs. Further experiments using immunoelectron microscopy have shown the proximity of both actin and the maxi-K channel within the same cell surface caveolar structures. Functionally, disruption of the actin cytoskeleton in cultured hMSMCs by cytochalasin D and latrunculin A greatly increased the open-state probability of the channel, while stabilization of actin cytoskeleton with jasplakinolide abolished the effect of latrunculin A. These data indicate that the actin cytoskeleton is involved as part of a caveolar complex in the regulation of myometrial maxi-K channel function. potassium channel; membrane microdomain     

Stimulus-induced selective association of actin-associated proteins (alpha-actinin) and protein kinase C isoforms with the cytoskeleton of human neutrophils.

We report a selective, differential stimulus-dependent enrichment of the actin-associated protein alpha-actinin and of isoforms of the signaling enzyme protein kinase C (PKC) in the neutrophil cytoskeleton. Chemotactic peptide, activators of PKC, and cell adhesion all induce a significant increase in the amount of cytoskeletal alpha-actinin and actin. Increased association of PKCbetaI and betaII with the cytoskeletal fraction of stimulated cells was also observed, with phorbol ester being more effective than chemotactic peptide. A fraction of phosphatase 2A was constitutively associated with the cytoskeleton independent of cell activation. None of the stimuli promoted association of vinculin or myosin II with the cytoskeleton. Phosphatase inhibitors okadaic acid and calyculin A prevented increases in cytoskeletal actin, alpha-actinin, and PKCbetaII induced by phorbol ester, suggesting the requirement for phosphatase activity in these events. Increases in cytoskeletal alpha-actinin and PKCbetaII showed differing sensitivity to agents that prevent actin polymerization (cytochalasin D, latrunculin A). Latrunculin A (1 microM) completely blocked PMA-induced increases in cytoskeletal alpha-actinin but reduced cytoskeletal recruitment of PKCbetaII only by 16%. Higher concentrations of latrunculin A (4 microM), which almost abolished the cytoskeletal actin pool, reduced cytoskeletal PKCbetaII by 43%. In conclusion, a selective enrichment of cytoskeletal and signaling proteins in the cytoskeleton of human neutrophils is induced by specific stimuli.     

Unstable F-actin specifies the area and microtubule direction of cell expansion in Arabidopsis root hairs

Plant cells expand by exocytosis of wall material contained in Golgi-derived vesicles. We examined the role of local instability of the actin cytoskeleton in specifying the exocytosis site in Arabidopsis root hairs. During root hair growth, a specific actin cytoskeleton configuration is present in the cell's subapex, which consists of fine bundles of actin filaments that become more and more fine toward the apex, where they may be absent. Pulse application of low concentrations of the actin-depolymerizing drugs cytochalasin D and latrunculin A broadened growing root hair tips (i.e., they increased the area of cell expansion). Interestingly, recovery from cytochalasin D led to new growth in the original growth direction, whereas in the presence of oryzalin, a microtubule-depolymerizing drug, this direction was altered. Oryzalin alone, at the same concentration, had no influence on root hair elongation. These results represent an important step toward understanding the spatial and directional regulation of root hair growth.