FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2170027513> ?p ?o ?g. }

• W2170027513 endingPage "1826" @default.
• W2170027513 startingPage "1816" @default.
• W2170027513 abstract "Article6 April 2006free access Regulation of actin dynamics by annexin 2 Matthew J Hayes Matthew J Hayes Search for more papers by this author Dongmin Shao Dongmin Shao Search for more papers by this author Maryse Bailly Maryse Bailly Search for more papers by this author Stephen E Moss Corresponding Author Stephen E Moss Division of Cell Biology, Institute of Ophthalmology, University College London, London, UK Search for more papers by this author Matthew J Hayes Matthew J Hayes Search for more papers by this author Dongmin Shao Dongmin Shao Search for more papers by this author Maryse Bailly Maryse Bailly Search for more papers by this author Stephen E Moss Corresponding Author Stephen E Moss Division of Cell Biology, Institute of Ophthalmology, University College London, London, UK Search for more papers by this author Author Information Matthew J Hayes‡, Dongmin Shao‡, Maryse Bailly and Stephen E Moss 1 1Division of Cell Biology, Institute of Ophthalmology, University College London, London, UK ‡These authors contributed equally to this work *Corresponding author. Division of Cell Biology, Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, UK. Tel.: +44 207 608 6973; Fax: +44 207 608 4034; E-mail: [email protected] The EMBO Journal (2006)25:1816-1826https://doi.org/10.1038/sj.emboj.7601078 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Annexin 2 is a ubiquitous Ca2+-binding protein that is essential for actin-dependent vesicle transport. Here, we show that in spontaneously motile cells annexin 2 is concentrated in dynamic actin-rich protrusions, and that depletion of annexin 2 using siRNA leads to the accumulation of stress fibres and loss of protrusive and retractile activity. Cells co-expressing annexin 2-CFP and actin-YFP exhibit Ca2+-dependent fluorescense resonance energy transfer throughout the cytoplasm and in membrane ruffles and protrusions, suggesting that annexin 2 may directly interact with actin. This notion was supported by biochemical studies, in which we show that annexin 2 reduces the polymerisation rate of actin monomers in a dose-dependent manner. By measuring actin polymerisation rates in the presence of barbed-end and pointed-end cappers, we further demonstrate that annexin 2 specifically inhibits filament elongation at the barbed ends. These results show that annexin 2 has an essential role in maintaining the plasticity of the dynamic membrane-associated actin cytoskeleton, and that its activity in this context may be at least partly explained through direct interactions with polymerised and monomeric actin. Introduction Annexin 2 is one of a family of conserved Ca2+-binding proteins, members of which are expressed in virtually all eukaryotic cell types (Gerke et al, 2005). The unifying biochemical property of annexins, namely their ability to bind to negatively charged phospholipids in the presence of Ca2+, suggests that annexin function is likely to involve membrane dynamics. For annexin 2 there is evidence to support such a role, both in the secretory pathway (Drust and Creutz, 1988; Ali et al, 1989) and endocytic pathway, where it has been shown to associate with early endosomes (Emans et al, 1993; Harder et al, 1997; Jost et al, 1997). It has also been found on phagosomes (Diakonova et al, 1997) and actin-propelled pinosomes (Merrifield et al, 2001) via an interaction with phosphatidylinositol 4,5,bisphosphate (Hayes et al, 2004a, 2004b). Disruption of annexin 2 function, either by expression of a dominant-negative mutant or by RNA interference, leads to perturbations in endocytic and secretory pathways (Harder and Gerke, 1993; Knop et al, 2004). In most cells, annexin 2 is present both as a cytosolic monomer and as a heterotetrameric complex comprising two molecules of annexin 2 and two of S100A10 (Johnsson et al, 1988). The heterotetramer locates constitutively to the membrane-cytoskeleton where, given recent observations identifying interactions between S100A10 and various cell surface receptors and ion channels (Nilius et al, 1996; Okuse et al, 2002; van der Graaf et al, 2003), it may be involved in the organisation of receptor cytoplasmic domains with the subplasma membrane cytoskeleton. On the other hand, monomeric annexin 2 does not require S100A10 to bind to endosomes (Konig and Gerke, 2000), and is more likely to be involved in dynamic interactions with lipid microdomains (Babiychuk and Draeger, 2000). Annexin 2 has also been shown to be associated with and necessary for the formation of actin-rich tight junctions (Lee et al, 2004), and has been proposed to regulate cell–cell contacts through formation of complexes with Rac1/p21-associated kinase and cadherin (Hansen et al, 2002). The close association of annexin 2 with both the actin cytoskeleton and dynamic cellular membranes led us to test the possibility that annexin 2 may have a more direct influence on actin polymerisation than has previously been assumed. Here, we demonstrate that annexin 2 is concentrated in dynamic actin-rich protrusions, and that in cells depleted of annexin 2 ruffling and protrusive activity are virtually abolished. We further show that annexin 2 binds to G-actin and interferes with the barbed-end growth of actin filaments. Annexin 2 may thus provide a direct structural link between the growing ends of actin filaments and the plasma membrane. Results Annexin 2 is localised to regions of cells undergoing actin remodelling To examine the association of annexin 2 with actin-rich structures, we transfected MIO Müller cells with annexin 2-GFP and then imaged the living cells using confocal microscopy. MIO is a nontransformed cell line (Limb et al, 2002) that exhibits intrinsic membrane ruffling. We observed marked enrichment of annexin 2-GFP at membrane ruffles, but not in adjacent stationary areas at the cell periphery. The boxed area of the cell in Figure 1A illustrates this point, the time-lapse sequence showing that the loss of annexin 2-GFP is associated with the disappearance of the ruffle. Co-expression of CFP alone (a cell volume control) with annexin 2-YFP revealed enrichment of the latter but not the former in ruffles (Figure 1B). This is confirmed quantitatively by analysis of line scans through such ruffles, and shows that the increased fluorescence intensity of annexin 2 at these regions is not due to increased membrane thickness. In experiments using MTLn3 cells fixed at a series of time points following plating, we also observed co-localisation of annexin 2 and F-actin at the ruffles of spreading cells, which diminished once spreading had finished and the F-actin reorganised into stress fibres (Supplementary Figure 1). These observations suggest that annexin 2 predominantly co-localises with actin at regions of active cytoskeletal remodelling. Figure 1.Annexin 2 is concentrated in membrane ruffles in MIO cells. (A) Müller cells were transiently transfected with annexin 2-GFP prior to time-lapse confocal and phase image acquisition. Arrowheads point to the enrichment of annexin 2-GFP in dynamic membrane ruffles. The timed series elaborating the boxed area in (A) shows that loss of annexin 2-GFP is coincident with the collapse of these structures and cessation of ruffling. (B) Müller cells were transiently co-transfected with pECFP-C1 and annexin 2-YFP. The following day the cells were fixed with 3.7% paraformaldehyde and prepared for confocal microscopy. Thin section scans were obtained (approximately 0.2 μm in thickness near the base of the cells) using a Leica AOBS system. Images were quantified using the ‘linescan’ function of Metamorph. Annexin 2 but not CFP is enriched in the extending ruffles. Download figure Download PowerPoint To determine if annexin 2 has a functional role in membrane ruffling, we examined the effects on the actin cytoskeleton of siRNA-mediated depletion of annexin 2. Western blots of extracts prepared from cells cultured for 4 days in the presence or absence of 60 pmol/ml siRNA for annexin 2 revealed marked depletion of annexin 2 but no change in the levels of annexin 1, α-tubulin or actin (Figure 2A). Immunofluorescence microscopy showed that the treated cell population contained some cells that were unaffected, and a majority in which annexin 2 was virtually undetectable (Figure 2B). The most striking phenotype of the cells depleted of annexin 2 was a massive accumulation of stress fibres concomitant with the loss of cortical actin and the disappearance of actin-rich ruffles. This reorganisation of actin appeared in a manner temporally coincident with the loss of annexin 2, when examined in cells maintained in siRNA over a period of 6 days (Supplementary Figure 2). Annexin 2-depleted cells also became remarkably flattened (Figures 2B and C), although no difference was observed in total cellular volume when the cells were examined by FACS (data not shown). Quantitative analysis (described in detail in Figure 3) allowed us to establish that annexin 2-depleted cells have a mean footprint approximately one order of magnitude greater than control cells. This striking difference in size enabled us to differentiate control and annexin 2-depleted cells within a mixed population of living cells, with close to 100% confidence. Knockdown of annexin 2 in ARPE19 retinal epithelial cells and Rat1 fibroblasts also resulted in a stress-fibre-dominated, flattened cellular phenotype (data not shown). Co-staining with paxillin showed that the abundant actin stress fibres in the annexin 2-depleted cells are tethered to focal adhesions, suggesting that they are physiologically normal (Figure 2C). Thus, the absence of annexin 2 is associated with the loss of dynamic actin-based structures, and the proliferation, thickening and reinforcement of more stable actin filament structures. Figure 2.Loss of annexin 2 leads to reconfiguration of the actin cytoskeleton. (A) Cells were treated with siRNA for annexin 2 and extracts were analysed by Western blotting. Following 4 days of exposure to siRNA, annexin 2 levels were depressed, whereas α-tubulin, actin and annexin 1 were unaffected. The histogram shows protein levels, as judged by densitometric scanning, in siRNA-treated cells relative to those in control cells (n=3). (B) The panels show two MIO cells cultured in the presence of annexin 2 siRNA, one of which is depleted of annexin 2 (yellow arrowhead) and the other unaffected (pink arrowhead). TRITC-phalloidin staining reveals reorganisation of F-actin into prominent stress fibres in the cell lacking annexin 2. (C) MIO cells were treated with siRNA for annexin 2 and triple stained for annexin 2, F-actin and paxillin. The lower of the two cells shows the accumulation of actin stress fibres in the absence of annexin 2 (as in (B)), with paxillin positive structures at fibre ends consistent with the formation of focal adhesions (yellow arrowheads). Annexin 2 depleted cells also lacked the actin-rich ruffles observed in control cells (pink arrowhead). (D) MIO cells were depleted of annexin 2 for 3 days as in (B), then transfected with either an hd-anx2-GFP vector or GFP alone. Cells were fixed 24 h after transfection and stained with phalloidin and antisera to annexin 2. The panels show a field of cells depleted of annexin 2, in which the cell marked by the pink arrowhead expresses hd-anx2-GFP and has a normal phenotype, whereas the neighbouring cells (yellow arrowheads) display the knockdown phenotype. (E) The relationship between cellular F-actin phenotype and annexin 2 expression was quantified by counting cells in multiple fields of cells such as those represented in (D). The columns labelled ‘anx2+’ and ‘anx2−’ show the percentages of cells displaying normal and knockdown phenotypes following exposure to annexin 2 siRNA. The columns labelled ‘hd-anx2’ and ‘control’ show comparable data from siRNA-treated cells subsequently transfected with hd-anx2-GFP and GFP, respectively. (F) MIO cells were depleted of annexin 2 as in (B), and then immunostained for S100A10 and F-actin. Nuclei were counter-stained with DAPI. Cells lacking annexin 2, characterised by their F-actin phenotype, also exhibited a reduction in staining for S100A10. (G) MIO cells were depleted of S100A10 using specific siRNA oligonucleotides, then immunostained for S100A10, annexin 2 and F-actin. Cells lacking S100A10 exhibited normal levels of annexin 2 and had an unchanged F-actin phenotype. Download figure Download PowerPoint Figure 3.Annexin 2 is required for changes in cell shape. (A, B) MIO cells were cultured in the presence of siRNA for annexin 2 prior to video timelapse recording of spontaneous shape changes. See Supplementary data, Movies 1 and 2. Scale bar in A=10 μm. Scale bar in B=30 μm. (C, D) Wild type and annexin 2 knockdown cells were readily distinguished on the basis of size, since loss of annexin 2 led to an increase in cell footprint of approximately one order of magnitude (see Figure 2). The graphs show that cells exhibited only modest changes in footprint during the experiment. (E, F) The Shapefactor of large, annexin 2 depleted cells remained fairly constant over a 20-min period, but that of control cells oscillated as the cells extended and retracted actin-rich ruffles. The variances of cellular footprint and Shapefactor for knockdown and control cells were compared by the F-test and are 2.06E-07 and 8.84E-46, respectively. The graphs show data collected from 17 individual cells where each coloured trace represents one cell. (G) The cell footprint of annexin 2-depleted (scale bar=30 μm) and control (scale bar=10 μm) MIO cells was circumscribed from frames of time-lapse videos of live cells. Frames 6 min apart were superimposed and the proportion of the footprint of the cell that had extended (green) or retracted (red) between frames was calculated using Metamorph. These regions represent dynamic plasma membrane ruffles driven by rapid underlying actin polymerisation and depolymerisation. (H) Data are expressed as percentage extension/retraction of total cell footprint. Percentage retraction and extension were calculated by measuring the decrease or increase in cell footprint at 12 min when compared against the cell footprint at 6 min. Each dot on the scattergram represents a single cell. Wild type (wt) cells both extended (ext) and retracted (ret) a greater proportion of their cell footprint than did annexin 2 knockdown (kd) cells in any 6 min period. Download figure Download PowerPoint In order to test if the anomalous F-actin phenotype was a direct consequence of annexin 2 depletion rather than a more remote downstream effect, we performed experiments aimed at phenotypic reversal using a ‘hardened’ annexin 2-GFP expression construct (hd-anx2-GFP), in which three silent point mutations were introduced into the annexin 2 cDNA at the siRNA target site. Cells were cultured in the presence of annexin 2 siRNA for 2 days and then transfected either with hd-anx2-GFP or GFP before fixation and immunofluorescence microscopy (Figure 2D). Quantitative analysis revealed that >95% of cells that immunostained positively with antisera to annexin 2 exhibited a normal phenotype (n=83, Figures 2D (pink arrowhead) and 2E), whereas >90% of cells negative for annexin 2 displayed the knockdown phenotype (n=219, Figures 2D (yellow arrowheads) and 2E). In cells expressing hd-anx2-GFP, all of which also stained positively with antisera to annexin 2, >90% exhibited a normal phenotype (n=35, Figure 2E). In cultures transfected with GFP alone, approximately 85% of cells that were GFP positive but annexin 2 negative displayed the knockdown phenotype (n=22, Figure 2E). Time-lapse imaging of such cells expressing hd-anx2-GFP confirmed that reversal of the morphological phenotype was accompanied by restoration of membrane ruffling and protrusive activity (Supplementary Figure 3). These results confirm that the siRNA-resistant annexin 2-GFP, but not GFP, is able to reverse the F-actin phenotype observed in annexin 2-depleted cells. Since part of the cellular pool of annexin 2 associates in vivo with S100A10, we also examined the consequences of annexin 2 depletion on S100A10 expression, and of S100A10 depletion on annexin 2 expression and the F-actin phenotype. Whereas cells depleted of annexin 2 showed a reduction in the level of expression of S100A10 (Figure 2F), which is consistent with previous studies demonstrating co-regulation of expression of annexin 2 and S100A10 (Puisieux et al, 1996), depletion of S100A10 using siRNA altered neither annexin 2 expression nor the F-actin phenotype (Figure 2G). These results show that maintenance of a normal actin phenotype requires annexin 2 but not S100A10. Annexin 2 knockdown alters cell shape and protrusive activity To examine the effects of loss of annexin 2 in living cells, spontaneous protrusive activity was monitored in control and annexin 2-depleted cells over an approximately 20-min period (Figures 3A and B; see also Supplementary data, movies 1 and 2). Quantitative analysis of stills from these movies revealed that the overall size of the cell footprint changed little throughout the experiment, regardless of whether or not cells were depleted of annexin 2 (Figures 3C and D). Since annexin 2-depleted cells also appeared to undergo fewer shape changes, we analysed the same cohort of cells using the Shapefactor algorithm in Metamorph. This measures the deviation of a given form from a circle (which has unit value on the scale). The more elongated or irregular the form, the lower the Shapefactor value. Wild-type (wt) cells tended to have lower Shapefactor values than annexin 2-depleted cells (Figures 3E and F), indicating that loss of annexin 2 leads to fewer protrusions and invaginations, and hence a more rounded profile. Although both cell populations exhibited changes in Shapefactor during the experiment, these were significantly less marked in annexin 2-depleted cells than in wt cells, as judged by F-test analysis (wt=2.06E-07 and knockdown=8.84E-46). We also analysed protrusion dynamics by quantifying lamellipod extension and retraction (Bailly et al, 1998) in control and annexin 2-depleted cells (Figures 3G and H). The results show that when measured relative to cell area, extension and retraction of lamellipodia are significantly greater in control than annexin 2-knockdown cells. Collectively, these data show that loss of annexin 2 leads to diminished plasticity of the actin cytoskeleton and a reduction in dynamic actin-based structures such as lamellipodia and ruffles. Annexin 2 binds G-actin and inhibits actin polymerisation Although annexin 2 has been shown to bundle preformed actin filaments in the presence of millimolar calcium in vitro (Glenney, 1987), our observation that annexin 2 is preferentially associated with actin at sites of cytoskeletal remodelling prompted us to examined whether annexin 2 has a more direct role in the regulation of actin dynamics. To determine whether annexin 2 physically interacts with actin in vivo, we first examined fluorescence resonance energy transfer (FRET) between annexin 2-CFP and YFP-actin. We performed acceptor photobleaching experiments in which the CFP image was recorded before and after bleaching of YFP in selected regions of interest (Figures 4A and B). In control experiments utilising YFP-actin with CFP no FRET was observed, whereas FRET at an approximate average efficiency of >40% was observed in cells co-expressing YFP-actin and annexin 2-CFP. Although FRET was evident between annexin 2 and actin at ruffles, it was also apparent throughout the cell, indicating that annexin 2 and actin may exist in close physical proximity both in the cytosol and at the plasma membrane. To test the generality of this observation we performed identical FRET experiments in two other cell lines, A431 squamous carcinoma cells and B16F10 melanoma cells, and obtained similar results with comparable FRET efficiency values (Supplementary Figure 4). In a further set of experiments we examined whether or not the strength of the FRET signal between annexin 2-CFP and YFP-actin might be influenced by the concentration of intracellular Ca2+ ([Ca2+]i). A431 cells preloaded with BAPTA-AM to reduce [Ca2+]i exhibited a significant reduction in FRET efficiency when compared to control cells, whereas elevation of [Ca2+]i stimulated by the addition of ionomycin had the opposite effect (Figure 4C), suggesting that the interaction between annexin 2 and actin is Ca2+-dependent. Figure 4.FRET analysis of annexin 2 and actin. (A) MIO cells were co-transfected with YFP-actin and CFP, then fixed and analysed by confocal microscopy after 24 h. CFP and YFP emission signals were captured before and after 50% photobleaching YFP. FRET is indicated as the relative increase in CFP emission following YFP photobleaching, where the scale bar represents high FRET efficiency with bright colours (red). In this control experiment, there was no measurable FRET between YFP-actin and CFP. (B) MIO cells were transfected with YFP-actin and annexin 2-CFP and subjected to image analysis as described in (A). A significant increase in CFP fluorescence was observed following YFP photobleaching equivalent to an average FRET efficiency of ∼40%. Similar experiments in A431 and B16F10 cells also yielded FRET measurements of up to 40% (see Supplementary data). These data indicate that FRET occurs between annexin 2-CFP and YFP-actin, corresponding to a physical proximity of the two fluorophores of <8 nm. (C) FRET experiments were performed in cells immediately following either chelation of intracellular Ca2+ [Ca2+]i using BAPTA-AM (top three panels) or elevation of [Ca2+]i using ionomycin (bottom three panels). The histogram shows that the average FRET intensity is increased when Cai2+ is raised and diminished when Cai2+ is reduced (n=20 for each sample). Download figure Download PowerPoint The possibility of a physical interaction between annexin 2 and actin suggested that annexin 2 might directly influence actin polymerisation/depolymerisation. To test this, we investigated the effect of annexin 2 on the rate of spontaneous actin polymerisation in vitro. In kinetic measurements of fluorescence intensity in an assay employing pyrene-labelled ATP-actin monomers, recombinant annexin 2 inhibited the rate of polymerisation of Mg2+-ATP G-actin monomers in the presence of 50 μM Ca2+ in a dose-dependent manner. The inhibitory effect of 5 μM annexin 2 was completely abolished in the presence of 1 mM EGTA (Figures 5A and B). In contrast, annexin 5 had no effect in this assay (not shown), suggesting that unlike other common activities of these proteins, inhibition of actin polymerisation is not a generic annexin property. On close examination we noticed an increase in the lag-time associated with the formation of actin nuclei at the commencement of polymerisation in the presence of annexin 2 (Figure 5A inset), a significant annexin 2-dependent reduction in the rate of actin polymerisation, and (not shown) a minor reduction in the final concentration of F-actin filaments attained as polymerisation plateaued. This is consistent with annexin 2 having the capacity to bind both monomeric G-actin (suppressing filament nucleation by monomer sequestration, and sequestering a pool of G-actin from incorporation into filaments at the end of the experiment) and to interfere with filament elongation. In agreement with these observations, a quantitative morphometric analysis of actin filaments prepared in the absence and presence of equimolar annexin 2 (Figures 5C–E) showed that annexin 2 shifts the profile of actin filament length towards shorter filaments. Annexin 2 (2 μM) also Ca2+-dependently increased the apparent critical concentration for actin polymerisation measured at steady state from 0.13 to 0.45 μM, providing further evidence that annexin 2 can bind and sequester actin monomers. The Kd of annexin 2 for actin monomers estimated from the results is around 0.7 μM, a value similar to that of other monomer-sequestering proteins such as profilin (Figures 5F and G). Figure 5.Annexin 2 is a G-actin binding protein. (A, B) Effect of annexin 2 on spontaneous actin polymerisation in the presence of (A) 50 μM Ca2+ or (B) 1 mM EGTA. The inset shows a increased lag phase in presence of annexin 2 (blue) compared to the control without annexin 2 (red). (C–E) F-actin filaments (10% rhodamine-labelled Mg2+-ATP) assembled in the presence of annexin 2 are shorter. The histogram (E) represents the distribution of filament lengths observed after G–Mg2+–ATP actin was allowed to polymerise for 2 h (>200 filaments were measured, the histogram shows one experiment from three independent assays, representative images of which are shown in C, D). Filament length was calculated using Metamorph. (F, G) Effects of annexin 2 on actin polymerisation at steady state. Inclusion of annexin 2 in the polymerisation mix shifts the critical concentration plot of actin assembly in a calcium-dependent manner. Annexin 2 (2 μM) was incubated with increasing concentrations of actin in the presence of 50 μM Ca2+ (F) or 1 mM EGTA (G). Download figure Download PowerPoint Kinetic measurements were performed to evaluate the effects of annexin 2 on the assembly and disassembly of actin at the barbed and pointed-ends of actin filaments. Annexin 2 dramatically inhibited the polymerisation of actin filaments in a dose-dependent manner when actin assembly was initiated by spectrin seeds, that is, when the pointed-ends were blocked (Figure 6A). Conversely, a range of concentrations of annexin 2 up to 5 μM had little effect on actin assembly when the barbed ends were blocked with gelsolin (Figure 6B). In this case, we observed only a slight reduction in the final concentration of F-actin that polymerised as the reaction plateaued. Thus, annexin 2 may be able to sequester a small pool of G-actin but does not appear to be able to block polymerisation at the pointed-end of the filament. Representation of the effect of annexin 2 as a function of elongation rate reveals that the concentration of annexin 2 required for half-maximal inhibition of barbed end polymerisation is approximately 125 nM (Figure 6C). Furthermore, annexin 2 also decreased the depolymerisation rate of preformed actin filaments in a dose-dependent manner (Figure 6D). This is consistent with annexin 2 capping the fast depolymerising barbed ends of the filaments, allowing dissociation only from the slowly depolymerising pointed ends. Figure 6.Annexin 2 is a barbed end capping protein. (A) Spectrin seeds were preincubated with increasing concentrations of annexin 2. The rate of polymerisation of 0.45 μM actin in the presence of these seeds is inversely proportional to the concentration of annexin 2. This is consistent with inhibition of polymerisation at the barbed-end when the pointed-end of the growing filament is blocked by spectrin. (B) In the reciprocal experiment in which annexin 2 was preincubated with gelsolin seeds, which blocks the barbed-end, there was little effect of annexin 2 on the rate of polymerisation of 2 μM actin. This shows that annexin 2 does not have the capacity to block polymerisation at the slowly elongating pointed end of the growing filament. The slight reduction in the ‘equilibrium’ concentration of F-actin could be due to G-actin sequestration by annexin 2. (C) Annexin 2 participates in filament growth at the barbed-end and not the pointed-end. Annexin 2 shows concentration dependent inhibition of F-actin filament elongation when the pointed-ends of the filaments are blocked with spectrin seeds (red squares). There is no additional effect on polymerisation when the barbed ends are blocked with gelsolin (blue circles). (D) Kinetics of depolymerisation of 100 nM actin filaments in the presence of increasing concentration of annexin 2. Download figure Download PowerPoint Annexin 2 requires free barbed ends for targeting to ruffles To determine whether annexin 2 requires free barbed-ends to localise to membrane ruffles in vivo, we used the approach of Bear et al (2002), who reported that low concentrations of cytochalasin D (CD) can be used to effectively block the barbed ends of actin filaments without significant disruption of the filamentous actin cytoskeleton. We found that in control cells Wave-1 (which is not localised to ruffles by a direct interaction with actin barbed-ends) was insensitive to concentrations of CD <100 nM, and that it co-localised with annexin 2 to actin-rich membrane ruffles (Figure 7A). In contrast, annexin 2 was completely displaced from membrane ruffles following the addition of 40 nM CD to culture medium, whereas the actin cytoskeleton and Wave-1 were unaffected. In control experiments, a range of concentrations of Latrunculin B (LatB) up to 100 nM did not diminish the co-localisation of Wave-1, annexin 2 and actin at membrane ruffles. Interestingly, the lowest concentration of CD (40 nM) effective in displacing annexin 2 from membrane ruffles was also sufficient to partially reverse the F-actin phenotype in siRNA-treated cells lacking annexin 2 (Figure 7B). The flattened cells became more elongated together with redistribution of F-actin from stress fibres to the cortex. This pharmacomimetic reversal of phenotype was however only partial and there was little evidence of an increase in cellular ruffling. In similar experiments, LatB had no effect on the actin phenotype, suggesting that the knockdown phenotype was" @default.
• W2170027513 created "2016-06-24" @default.
• W2170027513 creator A5007032618 @default.
• W2170027513 creator A5028997721 @default.
• W2170027513 creator A5068986246 @default.
• W2170027513 creator A5086005970 @default.
• W2170027513 date "2006-04-06" @default.
• W2170027513 modified "2023-10-15" @default.
• W2170027513 title "Regulation of actin dynamics by annexin 2" @default.
• W2170027513 cites W1538692246 @default.
• W2170027513 cites W1604221810 @default.
• W2170027513 cites W1630348734 @default.
• W2170027513 cites W1772998384 @default.
• W2170027513 cites W1922523492 @default.
• W2170027513 cites W1969926473 @default.
• W2170027513 cites W1970849988 @default.
• W2170027513 cites W1978007602 @default.
• W2170027513 cites W1980917046 @default.
• W2170027513 cites W1984234802 @default.
• W2170027513 cites W1990300178 @default.
• W2170027513 cites W2000331018 @default.
• W2170027513 cites W2002842138 @default.
• W2170027513 cites W2004744930 @default.
• W2170027513 cites W2007861835 @default.
• W2170027513 cites W2008123634 @default.
• W2170027513 cites W2009024748 @default.
• W2170027513 cites W2014906153 @default.
• W2170027513 cites W2046445536 @default.
• W2170027513 cites W2053431375 @default.
• W2170027513 cites W2055248999 @default.
• W2170027513 cites W2057344850 @default.
• W2170027513 cites W2062176870 @default.
• W2170027513 cites W2070532752 @default.
• W2170027513 cites W2074321236 @default.
• W2170027513 cites W2077150786 @default.
• W2170027513 cites W2079313214 @default.
• W2170027513 cites W2079944376 @default.
• W2170027513 cites W2083682484 @default.
• W2170027513 cites W2093288486 @default.
• W2170027513 cites W2100601378 @default.
• W2170027513 cites W2110726109 @default.
• W2170027513 cites W2115666450 @default.
• W2170027513 cites W2122037409 @default.
• W2170027513 cites W2126496496 @default.
• W2170027513 cites W2126893470 @default.
• W2170027513 cites W2128535856 @default.
• W2170027513 cites W2131194303 @default.
• W2170027513 cites W2148489676 @default.
• W2170027513 cites W2153243435 @default.
• W2170027513 cites W2160123605 @default.
• W2170027513 cites W2173338776 @default.
• W2170027513 cites W2183064036 @default.
• W2170027513 cites W32161370 @default.
• W2170027513 doi "https://doi.org/10.1038/sj.emboj.7601078" @default.
• W2170027513 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1456940" @default.
• W2170027513 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16601677" @default.
• W2170027513 hasPublicationYear "2006" @default.
• W2170027513 type Work @default.
• W2170027513 sameAs 2170027513 @default.
• W2170027513 citedByCount "178" @default.
• W2170027513 countsByYear W21700275132012 @default.
• W2170027513 countsByYear W21700275132013 @default.
• W2170027513 countsByYear W21700275132014 @default.
• W2170027513 countsByYear W21700275132015 @default.
• W2170027513 countsByYear W21700275132016 @default.
• W2170027513 countsByYear W21700275132017 @default.
• W2170027513 countsByYear W21700275132018 @default.
• W2170027513 countsByYear W21700275132019 @default.
• W2170027513 countsByYear W21700275132020 @default.
• W2170027513 countsByYear W21700275132021 @default.
• W2170027513 countsByYear W21700275132022 @default.
• W2170027513 countsByYear W21700275132023 @default.
• W2170027513 crossrefType "journal-article" @default.
• W2170027513 hasAuthorship W2170027513A5007032618 @default.
• W2170027513 hasAuthorship W2170027513A5028997721 @default.
• W2170027513 hasAuthorship W2170027513A5068986246 @default.
• W2170027513 hasAuthorship W2170027513A5086005970 @default.
• W2170027513 hasBestOaLocation W21700275131 @default.
• W2170027513 hasConcept C121332964 @default.
• W2170027513 hasConcept C125705527 @default.
• W2170027513 hasConcept C145912823 @default.
• W2170027513 hasConcept C1491633281 @default.
• W2170027513 hasConcept C24890656 @default.
• W2170027513 hasConcept C30345302 @default.
• W2170027513 hasConcept C54355233 @default.
• W2170027513 hasConcept C70721500 @default.
• W2170027513 hasConcept C86803240 @default.
• W2170027513 hasConcept C88634738 @default.
• W2170027513 hasConcept C95444343 @default.
• W2170027513 hasConceptScore W2170027513C121332964 @default.
• W2170027513 hasConceptScore W2170027513C125705527 @default.
• W2170027513 hasConceptScore W2170027513C145912823 @default.
• W2170027513 hasConceptScore W2170027513C1491633281 @default.
• W2170027513 hasConceptScore W2170027513C24890656 @default.
• W2170027513 hasConceptScore W2170027513C30345302 @default.
• W2170027513 hasConceptScore W2170027513C54355233 @default.
• W2170027513 hasConceptScore W2170027513C70721500 @default.
• W2170027513 hasConceptScore W2170027513C86803240 @default.

• next