Functional interdependence between septin and actin cytoskeleton
© Schmidt and Nichols; licensee BioMed Central Ltd. 2004
Received: 01 September 2004
Accepted: 12 November 2004
Published: 12 November 2004
Septin2 is a member of a highly conserved GTPase family found in fungi and animals. Septins have been implicated in a diversity of cellular processes including cytokinesis, formation of diffusion barriers and vesicle trafficking. Septin2 partially co-localises with actin bundles in mammalian interphase cells and Septin2-filamentmorphology depends upon an intact actin cytoskeleton. How this interaction is regulated is not known. Moreover, evidence that Septin2 is remodelled or redistributed in response to other changes in actin organisation is lacking.
Septin2 filaments are associated with actin fibres, but Septin2 is not associated with actin at the leading edge of moving cells or in ruffles where actin is highly dynamic. Rather, Septin2 is spatially segregated from these active areas and forms O- and C-shaped structures, similar to those previously observed after latrunculin treatment. FRAP experiments showed that all assemblies formed by Septin2 are highly dynamic with a constant exchange of Septin2 in and out of these structures, and that this property is independent of actin. A combination of RNAi experiments and expression of truncated forms of Septin2 showed that Septin2 plays a significant role in stabilising or maintaining actin bundles.
We show that Septin2 can form dynamic structures with differing morphologies in living cells, and that these morphologies are dependent on the functional state of the actin cytoskeleton. Our data provide a link between the different morphological states of Septin2 and functions of Septin2 in actin-dynamics, and are consistent with the model proposed by Kinoshita and colleagues, that Septin2 filaments play a role in stabilisation of actin stress fibres thus preventing actin turnover.
Septins are a conserved family of GTPases implicated in various cellular processes. Septin-requiring processes include cytokinesis, polarity establishment, cell cycle checkpoints and formation of a diffusion barrier in yeast , as well as cytokinesis, vesicle trafficking and exocytosis in mammalian cells [2–4]. In humans, 12 septin genes have been found so far, many of which also undergo alternative splicing generating dozens of polypeptides . Septins can be isolated from cytosol as hetero-polymeric complexes, which have the ability to polymerize and assemble into higher-order structures in vitro [6, 7]. How the polymerisation is regulated and how such higher order assemblies contribute to septin function in vivo is far from clear.
Septin2 (formerly known as Nedd5) is the best-characterised member of the septin family so far. It is ubiquitously expressed and belongs to the acidic subgroup of the septin family consisting of a short N-terminus, a conserved GTPase domain and a coiled coil structure at the C-terminus . Septin2 forms a complex together with Septin6 and Septin7 in vitro  and also co-localises with these septins in vivo . Kinoshita and colleagues showed that Septin2 is required for cytokinesis . Microinjection of an anti-Septin2 antibody interfered with cell division resulting in bi-nucleated cells. How Septin2 functions during cytokinesis is unclear. However, its localisation to the contractile ring and midbody structure in the cleavage furrow during late stages of mitosis is consistent with a functional role of Septin2 in limiting diffusion of membrane proteins across the cleavage furrow [12, 13].
In interphase cells Septin2 co-localises with actin bundles, and disruption of the actin cytoskeleton by latrunculin or cytochalasin perturbs Septin2 distribution, inducing curved or circular Septin2-containing assemblies [9, 14]. Reduction in Septin2 expression level in cells results in attenuation of actin fibres, implying a functional inter-relationship between actin and Septin2. An in vitro bundling assay showed that the interaction between actin bundles and recombinant Septin2/Septin6/Septin7 can be mediated by the bundling protein anillin . Although these in vitro results suggest a mechanism to account for the recruitment of Septin2 to the actin contractile ring during cytokinesis, it is unlikely that anillin has the same function in interphase cells. Anillin is sequestered in the nucleus in interphase cells  and the functional significance of the co-localisation between actin and Septin2 in this phase of the cell cycle is still elusive. More generally, although artificial perturbation of actin cytoskeleton can cause re-arrangement of Septin2 in cells it is not clear that this phenomenon is ever replicated during normal cell function.
Here we characterise the mutual inter-dependence of the actin cytoskeleton and Septin2 using a range of in vivo approaches. Both knock down of Septin2 expression and a specific Septin2 truncation mutant resulted in loss of visible actin fibres or bundles. Expression of dominant-negative mutants of the actin-regulating Rho-GTPases RhoA, Rac1 and CDC42 caused re-organisation of both cortical actin and Septin2. Significantly, in ruffling and migrating cells we observed wholesale redistribution of Septin2 into ring-like structures with morphology and dynamics highly similar to those previously observed upon depolymerisation of actin filaments with latrunculin. We propose that Septin2 is required for actin bundling, and that global re-organisation of the actin cytoskeleton in migrating or ruffling cells triggers concomitant re-organisation of Septin2 into a distinct functional state.
Septin2 and actin define interdependent systems
Septin2 and actin have distinct distributions in moving and ruffling cells
The filaments and rings formed by Septin2 in vivo are highly dynamic
Next we looked at the dynamics of the Septin2 positive ring-like structures formed in the cell body of ruffling cells. Time-lapse movies of these cells revealed a very active behaviour of Septin2 and a relationship between Septin2 filaments and rings. For example, as shown in Fig. 6C starting with an S-shaped filament half of a ring was formed. After 110 s an O-shaped structure with a diameter of 1.45 μm was seen which then further condensed into a smaller ring (Fig 6C, 190 s). The diameter of this ring was 0.7 μm and the morphology was comparable to rings observed after latrunculin treatment (Fig. 1C) and perturbation of actin/Septin2 organisation by Rac1-mutant (Fig. 4A). Instead of remaining in this structure the ring opened again and formed two half rings which were 1.6 μm apart from each other (Fig. 6C, 250 s). The two halves then came together again, this time forming a C-shaped structure (Fig. 6C, 440 s). Many examples of this cycle of opening and closing were observed and sometimes a disappearing and appearing of a ring-like structure was also detected. FRAP experiments of these ring structures in ruffling cells (grown on lysine-coated coverslips) confirmed that constant exchange of YFP-Septin2 into and out of these structures occurs. Halftimes for recovery (ranging from 10 s to 73.8 s) and amount of recovery (45%-86%), however, were very variable (Fig. 6D,6E. Additional files 1 and 2). Sometimes the recovered structure was distinct to the initially bleached structure in terms of position and morphology (see also Additional File 2).
Next we investigated whether Septin2 rings induced by latrunculin have equivalent properties to those seen in ruffling or migrating cells. Septin2 rings formed upon latrunculin treatment also recovered after photobleaching, with comparable dynamics (half-time for recovery was 8.5 s ± 4.5, total amount of recovery 58.3% ± 3.6; Fig. 6D,6E, movie3). Careful morphological comparison of ring structures in ruffling cells and in latrunculin treated cells again revealed considerable similarity. Rings formed upon latrunculin treatment had a slightly more uniform outer diameter (0.74 ± 0.14 μm) but were similar in size to ring-like structures in ruffling cells (0.5 μm-1.4 μm; Fig. 6F). In summary, Septin2 can participate in large-scale macromolecular assemblies with differing morphologies in response to differing physiological situations and remodelling of the actin cytoskeleton. In all instances these assemblies are highly dynamic with constant exchange of Septin2 in and out.
Previous experiments have shown that Septin2 partially co-localises with actin fibres, that actin can affect Septin2 polymerisation in vitro, that depletion of Septin2 perturbs the morphology of actin bundles, and that depolymerisation of actin fibres can cause changes in Septin2 morphology. In this study we provide additional direct evidence for the in vivo significance of actin and Septin2 interaction. Thus we show for the first time that Septin2 expression is required for maintenance of normal actin protein levels, that over-expression of a truncated version of Septin2 causes loss of visible actin bundling without perturbing the distribution of endogenous Septin2, and that the circular and ring-shaped Septin2 structures induced by actin de-polymerisation are also found in physiological situations where the actin cytoskeleton is radically remodeled.
Partial co-localisation between Septin2 and the tubulin network has also been reported . Our immunofluorescence data, however, argue, that the interaction between Septin2 and microtubules is different from the Septin2-actin interplay and is not crucial for microtubule integrity. Although Septin2 distribution is slightly affected upon nocodazole treatment, this effect is less severe and not comparable with the disruption of Septin2 organisation upon latrunculin treatment (Fig. 1). Moreover, neither knock-down of Septin2 expression upon siRNA nor overexpression of truncated Septin2 constructs affected microtubule organisation (Fig. 2, data not shown). Since in regions of the cell where Septin2 co-localises with tubulin we always find actin as well, we do not think that there is a direct interaction between Septin2 and tubulin, as has been shown for other septins [16, 23]. The distinct distribution of Septin9, which is associated with tubulin and is clearly affected upon nocodazole treatment, and Septin2 in dividing cells also suggests that the co-localisation seen between Septin2 and tubulin is not functionally significant .
Septin2 and actin distributions, however, are clearly highly interdependent. Overexpression of GDP-locked form of RhoA, Cdc42 and Rac1 highlights this interplay (Fig. 4). At this point we do not know how these GTPases act in this context, whether it is via modulating actin dynamics and/or controlling Septin2 dynamics. It is known that the Cdc42 effectors Borg1, 2 and 3 can bind to Septins in vitro and in vivo [7, 10]. Endogenous Septin6 and Septin7, which form a complex with Septin2, can be immunoprecipitated by an anti-Borg3 antibody and expression of Borg interferes with normal septin distribution. Full-length myc-Borg3 induces the formation of long and thick septin fibres and Cdc42 negatively regulates this effect by inhibiting the binding of Borg3 to septins. These data suggest that the formation of thick Septin2 filaments and actin filaments we see upon expression of the dominant-negative Cdc42 mutant is controlled by a regulatory mechanism, which modulates Septin2 function directly and not only via regulating actin organisation.
In our experiments comparing truncated versions of Septin2 in cells we could show that the GTPase domain of Septin2 is sufficient to prevent the loss of actin bundles induced by expression of the polybasic region alone. The exact role of septin GTPase activity is still unclear . Using recombinant septins, Sheffield and colleagues  demonstrated that although pre-assembled Septin6/Septin7/Septin2-filaments show only a slow GTPase activity, GTP-hydrolysis occurs during formation of heterodimers, a process before the assembling of filamentous complexes. Microinjection of non-hydrolyzable GTP (GTPγS) in cells disrupted fibrous distribution of Septin2 suggesting that the fibrous distribution of Septin2 requires GTP-hydrolysis . In contrast, there is no evidence that GTPase-activity is necessary for assembly of septin filaments in curved bundles and ring-like structures in vitro . So far we do not know how Septin2 interacts with actin bundling proteins and how it gets recruited to actin filaments in interphase cells. We do not know whether the reduction in actin expression levels induced by septin2 RNAi is a cause or an effect of the observed loss of actin bundles. Identification of binding partners for Septin2 will be necessary to elucidate the mechanisms underlying the property of Septin2 to stabilise actin bundles.
It has been shown in vitro that Septin2 is capable of forming ring-like structures and spirals in an actin-independent fashion . Here, we provide in vivo characterisation of this intrinsic property of Septin2. Moving cells and ruffling cells are the first cell systems described so far where the actin-independent distribution of Septin2 in O- and C-shaped rings has been studied in a physiological context, without interfering with cell viability and function. These model systems allow us to draw two firm conclusions: 1. That Septin2 is not associated with actin in regions where highly dynamic actin is not organised in fibres and is being constantly remodeled, and indeed Septin2 is actually efficiently excluded from these regions (e.g. at the leading edge of moving cells and in ruffles): 2. That Septin2 when not associated with actin forms rings and ring-like structures instead of filaments (Figs 5, 6). This is in agreement with in vitro data obtained from recombinant septin complexes showing their tendency to self-assemble into rings and spirals . In contrast, however, to these in vitro structures, which are highly stable, the in vivo assemblies are highly dynamic (movies 1–3). Bleaching experiments clearly showed the constant exchange of Septin2 in and out of the assemblies as well as in and out of actin-dependent Septin2 filaments. The function of actin-independent Septin2 assemblies, however, is still elusive. Since they seem to be freely localised in the cytosol and are not associated with membrane structures (unpublished observations), they might represent storage containers of Septin2. The dynamic behaviour of Septin2 explains how cells can adjust Septin2 function to different needs. The identification of further proteins involved in this process (e.g. GAPs, GEFs, bundling proteins) is necessary for molecular characterisation of the interplay between Septin2 and actin.
Our data provide a link between the different morphological states of Septin2 and functions of Septin2 in actin-dynamics, and confirm the physiological relevance of the model proposed by Kinoshita and colleagues , that Septin2 filaments play a role in stabilisation of actin stress fibres thus preventing actin turnover.
Antibodies and constructs
Anti-Septin2 polyclonal antibody was a gift from M. Kinoshita. Anti-human vinculin monoclonal antibody (clone hVIN-1), anti-c-myc monoclonal antibody (clone 9E10) and anti-α-Tubulin monoclonal antibody (clone DM1a) were all from Sigma. Anti-beta actin monoclonal antibody (AC-15) for immunoblotting was from abcam, BODIPY®FL phallacidin and Alexa Fluor ® 568 phalloidin for immunofluoresence were obtained from Molecular Probes. Anti-Endoplasmic Reticulum Protein 72 (Anti-Erp72) was from Calbiochem. Myc-constructs of GDP-locked RhoA, Rac1 and CDC42 and the anti-rac monoclonal antibody were kindly provided by H. Mellor. pEGFP-actin was obtained from Clontech. Secondary antibodies used were as follows: Donkey Anti-Rabbit IgG Cy™3 conjugated (Jackson Immuno Research Lab.), Donkey Anti-Mouse IgG Cy™5 conjugated (Jackson Immuno Research Lab.) and Alexa Fluor ® 488 goat anti-mouse IgG1 (Molecular Probes) for immunofluorescence. IRDye 800 donkey anti-rabbit IgG (Rockland) and Alexa Fluor ® 680 goat anti-mouse IgG (Molecular Probes) for immunoblotting.
Cloning of YFP-Septin2-constructs
To generate full length and truncated YFP-Septin2-constructs, PCRs of Septin2 image clone (Clone Id: 548005, from HGMP, Hinxton/UK) were performed using the following primers: 5'-GCGCTCGAGTGTCTAAGCAACAGCCAAC (sense) and 5'-ATCCCGG GTTACACGTGGTGCCCGAGAGC (antisense) for full length YFP-Septin2-PB/G/CC (nucleotides 1–1081); 5'-GCGCTCGAGTGTCTAAGCAACAGCCAAC (sense) and 5'-CGCCCCGGGTTAGCCTCTCTTGAGTCTCTC (antisense) for YFP-Septin2-PB/G (nucleotides 1–922); 5'-GCGCTCGAGTGTCTAA GCAACAGCCAAC (sense) and 5'-CGCCCCGGGTTACACCATCAGTGTGAACTC (antisense) for YFP-Septin2-PB (nucleotides 1–127); 5'-GCGCTCGAGAGTTCACACTGATGGTGGT (sense) and 5'-ATCCCGGGTTACACGTGGTGCCCGAGAGC (antisense) for YFP-Septin2-G/CC (nucleotides 113–1081). The fragments were cloned in pEYFP-C1 digested with Xho1 and Xma1. To generate untagged full length Septin2 5'-GCGCTCGAGATGTCTAAGCAACA GCCAAC (sense) and 5'-ATCCCGG GTTACACGTGGTGCCCGAGAGC (antisense) were used in the PCR reaction. The fragment was cloned in SNAG4M cut with Xho1 and Xma1. All clones were confirmed by DNA sequencing.
Cell culture, drug treatment, plasmid transfections, immunofluorescence
NRK cells were grown using standard techniques in DMEM, 10% FCS, 1% Penicillin at 10%CO2. To induce the formation of ruffles in NRK cells coverslips were coated with 0.01% poly-D-lysine for 5 min, washed with PBS and air dried before cells were seeded. A wound assay was used to investigate distribution of Septin2 in moving cells. Briefly, NRK cells were grown until confluency and a wound was scratched in the monolayer using a tip. Two hours later cells were fixed and processed for immunofluorescence. To disrupt the actin cytoskeleton or microtubules cells were incubated for 30 min in DMEM containing 150 nM Latrunculin B (Molecular Probes) or 10 μM nocodazole (Sigma), respectively. Plasmid transfections were carried out with Fugene6 (Roche).
For immunofluorescence cells were fixed with 2% Formaldehyd in either PBS or microtubule-stabilisation buffer (0.1 M Pipes, pH 6.9, 2 mM EGTA, 2 mM MgCl2, 4% PEG 8000) for 20 min, permeabilised with 0.2% saponin/10%FCS in either PBS or microtubule-stabilisation buffer for 10 min and subsequently incubated with primary and secondary antibodies.
RNA interference (RNAi) with small interfering RNA (siRNA) and immunoblotting
Two siRNAs with the following sequences: 5'-AAAGGACATGAATAAAGACCA (sense), and 5'-AAGTGAATATTGTGCCTGTCA (sense) were chosen to target nucleotides 939–957 and 521–539 of Septin2, respectively, and were supplied by Dharmacon Research Inc. siRNAs were annealed to make siRNA duplexes according to the manufacture's protocol and transfections were carried out using Oligofectamine (Invitrogen). To check for specificity of knock down we used siRNA duplexes targeting caveolin 1  and rhomboid (gift from A. McQuibban). 48 hrs after transfection cells were processed for immunofluorescence or immunoblotting. For immunoblotting cells were washed with PBS, scratched of in sample buffer (2% SDS, 80 mM Tris/HCl pH 6.8, 10% Glycerin, 0.01% bromphenolblue, 5% β-Mercaptoethanol) and sonicated. The samples were boiled at 80°C for 3 min, centrifuged and the supernatant was loaded onto the gel followed by Western blotting. Immunoreactive bands were detected by the Infrared Imaging System Odyssey (Li-Cor Biosciences).
Microscopy and photobleaching
All images and movies were obtained using BioRad Radiance and Zeiss LSM510 confocal microscopes equipped with standard filter sets and laser lines for the detection of YFP, Cy2, Alexa Fluor 488, BODIPY, Cy3 and Cy5. Live cell microscopy was carried out at 35°C in imaging medium (DMEM without phenol red, 10%FCS, 50 mM HEPES pH 7.2). Photobleaching experiments were performed with the confocal zoom set to 3 and the confocal pinhole set to 2–4 Airy units. Bleaching of actin or Septin2 filaments was carried out with a 40X 1.3 NA objective lens. A box of interest was bleached using 35 scans with the 488 and 514 laser line at full laser power. For photobleaching of ring structures a 63X 1.4 objective lens was used and a ring of interest was bleached using 25 scans with the 514 laser line at full laser power. Pre- and post-bleach images were monitored at low laser intensity. Fluorescence recovery in the bleached region and the overall fluorescence in the whole cell during the time series were quantified using the Zeiss LSM software. After subtracting the background (= mean fluorescence intensity in the bleached region after bleach) the ratio between mean fluorescence intensity of the bleached region and the mean fluorescence of the whole cell was expressed as a percentage of the pre-bleach ratio of these values. These normalised data were fitted to a single exponential curve using the PRISM software (GraphPad Software Inc., San Diego) to derive amount of recovery and characteristic diffusion time tD, which indicates the time at which half of the fluorescence has recovered.
List of abbreviations used
green fluorescent protein
yellow fluorescent protein
small interference RNA
endoplasmic reticulum protein 72
fluorescence recovery after photobleaching
region of interest
GTPase activating protein
GTP exchange factor.
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