BACKGROUND
cytokinesis is essential to cell growth and has to be tightly regulated to insure equal distribution of the cytoplasm content and newly replicated chromosomes within two daughter cells at the end of mitosis.
defective chromosome segregation or daughter cell separation can alter chromosome number, which is commonly found in tumour cells and potentially involved in cancer development  <cit> .
for these reasons it is essential to understand the signalling pathways that coordinate mitotic exit and cytokinesis.
in metazoans, the central spindle forms during anaphase between the two sets of chromosomes.
simultaneously, the cleavage furrow composed by actin and myosin ii filaments is established at the cell equator under the control of peripheral microtubules   <cit> .
thus, constriction of the actomyosin ring can only start once the two sets of chromosomes are segregated, insuring genome integrity.
the two daughter cells stay connected by a cytoplasmic bridge with the midbody in the centre for several hours until the bridge is finally severed during abscission  <cit> .
the small gtpase rhoa has been shown to play a major role in cleavage furrow establishment.
indeed, the gtpase exchange factor  ect <dig> is specifically recruited at the cell equator under the control of the centralspindlin complex  also required for central spindle formation  <cit> .
once activated, rhoa induces, through its effector rock, the phosphorylation of myosin regulatory light chain , which is necessary for contractile ring formation and ingression  <cit> .
in mammals, actin filaments present in the contractile ring derive from preexisting f-actin flux and de novo actin polymerisation  <cit> .
in s. pombe, cleavage furrow establishment and activity depend on de novo actin polymerisation driven by the two main actin nucleation factors: the arp2/ <dig> complex and a formin   <cit> .
arp2/ <dig> complex is activated by myo <dig> and wsp <dig> at the equator and is involved in the maturation and/or the maintenance of the cleavage furrow  <cit> .
in mammals the mdia formins, orthologs of cdc <dig> and rhoa effectors, are required for cytokinesis  <cit> .
in agreement with a conserved mechanism for cleavage furrow formation and activity, the arp2/ <dig> complex and its activator wasp/n-wasp, ortholog of wsp <dig>  were found to potentially play a role during cytokinesis  <cit> .
in this study the functional role of the arp2/ <dig> complex during cytokinesis in mammalian cells was evaluated.
we demonstrated that arp2/ <dig> complex is recruited late to the cleavage furrow.
consequences of the inhibition of wasp/n-wasp activity towards the arp2/ <dig> complex during cytokinesis were then studied using the newly identified inhibitor: wiskostatin  <cit> .
we show that wiskostatin strongly inhibits cytokinesis inducing cell binucleation without affecting the cell cycle.
however, these results are unlikely related to the arp2/ <dig> complex function since the wiskostatin induced phenotype could not be validated using n-wasp and/or arp2/ <dig> rna interference approach.
this novel effect of wiskostatin on cytokinesis is also not related to another known unexpected effect of this compound i.e.
atp depletion  <cit> .
this study that was originally designed to study n-wasp/arp2/ <dig> complex pathway during cytokinesis, unexpectedly highlighted a novel effect of wiskostatin.
for this reason, wiskostatin will have to be used with caution in future experiments.
RESULTS
localisation of arp2/ <dig> complex during mitosis
in mammalian cells, the localisation of endogenous arp2/ <dig> complex during mitosis is currently unknown.
in order to study the function of the n-wasp/arp2/ <dig> complex pathway during cytokinesis, we thus analyzed the subcellular localisation of arp <dig> and p <dig>  subunits of the arp2/ <dig> complex by indirect immunofluorescence.
in interphase hela cells using monoclonal anti-arp <dig> antibody, the arp2/ <dig> complex was found to be diffuse and associated with vesicle-like structures in the cytoplasm but also accumulated in cortical f-actin-rich structures, which are likely lamellipodia .
to better visualize cytoskeleton bound arp2/ <dig> complex in mitotic cells, the cytosolic unbound fraction was eliminated by permeabilising the cells prior to fixation .
under these conditions, arp <dig> presented a diffuse vesicular-like localisation and was not particularly enriched into the nascent cleavage furrow during anaphase .
however, during late telophase, once the cleavage furrow is fully contracted, arp <dig> was strongly enriched in the close vicinity of the contractile ring, as visualized by the f-actin staining .
furthermore, the vesicular-like localisation of arp <dig> was organised around the minus-end and along microtubules .
in cytokinesis, when the cytoplasmic bridge between the two daughter cells extended, arp <dig> colocalised with f-actin in the cortex facing the cytoplasmic bridge  and within vesicle-like structures .
furthermore, the arp2/ <dig> complex also localised within the cytoplasmic bridge but not in the midbody .
this subcellular localisation of arp2/ <dig> complex was further confirmed by using a polyclonal antibody directed against p <dig> subunit .
thus, the localization of arp2/ <dig> complex is in agreement with a potential functional role during cytokinesis.
wiskostatin inhibits cytokinesis but not mitosis
the mammalian ortholog of wsp <dig>  wasp/n-wasp, is regulated by autoinhibition essentially relieved by binding of activated small gtpase cdc <dig> and/or phosphatidyl inositol  <dig> -biphosphate   <cit> .
gtp loaded cdc <dig> binds to the gbd  of wasp/n-wasp, which is preceded by a basic region where ptdins <cit> p <dig> interacts  <cit> .
wiskostatin is a recently identified wasp/n-wasp chemical inhibitor designed to bind to the gbd  <cit> .
wiskostatin not only prevents cdc <dig> binding, but also stabilises the autoinhibited state of this actin nucleation promoting factor  <cit> .
this led us to use wiskostatin to investigate the role of the arp2/ <dig> complex activated by wasp/n-wasp during cytokinesis.
hela cells stably expressing gfp-tagged histone h2b  were synchronised in prometaphase with nocodazole and were released for various times after shake-off in presence of vehicle  or increasing concentration of wiskostatin .
cell dna content was determined by flow cytometry at  <dig>   <dig> and  <dig> minutes after nocodazole release.
in control conditions, at t =  <dig>  cells had a 4n content confirming mitosis synchronisation mediated by nocodazole.
<dig> minutes later half of the cells had exit mitosis and displayed a 2n content.
finally,  <dig> min after release, control cells were mostly in g <dig> phase  .
in contrast, under wiskostatin treatment, the number of tetraploid cells  was abnormally elevated at  <dig> min after release suggesting a mitotic delay both at  <dig> and  <dig> μm doses .
<dig> minutes after nocodazole release under  <dig> μm wiskostatin, half of the cells had a 2n content while under  <dig> μm wiskostatin treatment all cells were tetraploid indicating that they did not proceed through mitosis .
thus wiskostatin inhibits mitotic progression in a dose dependent manner.
the increased number of tetraploid cells under wiskostatin could either result from an inhibition of mitosis or cytokinesis.
to answer this question, we first analysed the status of cyclin b <dig>  which is degraded at the metaphase/anaphase transition.
mitotic hela cells were released in presence of vehicle alone or wiskostatin , and cyclin b <dig> levels were determined by immunoblot analysis .
no difference in the kinetics of cyclin b <dig> degradation was observed between control and wiskostatin treated cells.
this result was further confirmed by studying another mitotic marker, the phosphorylation of histone h <dig> on serine  <dig>  which correlates with chromatin condensation  <cit> .
as expected, histone h <dig> phosphorylation was not affected by wiskostatin treatment .
equal sample loading was confirmed by cofilin immunoblot analysis .
these results clearly demonstrate that wiskostatin does not inhibit mitotic progression.
taken altogether, our results indicate an effect of wiskostatin on cytokinesis.
cytokinesis failure is generally associated with cell binucleation.
to investigate this hypothesis, nocodazole synchronised hela gfp h2b cells were grown in presence of vehicle alone or wiskostatin , fixed after  <dig> minutes and binucleated cells were quantified.
in control condition, around 7% of cells were found to be binucleated .
in contrast, 85% of wiskostatin treated cells contained two nuclei  .
to rule out any cooperative effects of nocodazole and wiskostatin, asynchronous hela gfp-h2b cells were grown in presence of  <dig> μm wiskostatin for  <dig> hours and binucleated cells were counted as previously described.
under these conditions more than 26% of cells were found to be binucleated  compared to  <dig> %  of cells treated with vehicle alone .
similar results were obtained with nih3t <dig> and xl <dig> cells .
this result clearly demonstrates that wiskostatin affects mitotic exit.
in order to analyse wiskostatin induced cytokinesis defects in more details, time-lapse microscopy was used.
in vehicle treated hela gfp-h2b cells cytokinesis started around  <dig> minutes after release from nocodazole block .
after complete cleavage furrow contraction, daughter cells were linked to each other by a cytoplasmic bridge, which extended over time with the midbody in its centre .
finally, after several hours, abscission occurred.
under wiskostatin treatment, cytokinesis started with the same kinetics as control cells  but prior and during cleavage furrow contraction blebs were visible at cell poles .
finally, once the cleavage furrow fully contracted, the cytoplasmic bridge did not extend, the contractile ring relaxed and daughter cells finally fused-back together .
we then studied the organisation of the cytoskeleton in more details.
mitotic hela cells stably expressing gfp-tagged β-tubulin were fixed two hours after nocodazole release in presence of vehicle or  <dig> μm wiskostatin, and were stained for f-actin and dna.
during cytokinesis, the microtubule bundles of the central spindle, which are not associated with kinetochores, are compacted by the contraction of the cleavage furrow.
in control cells, these compacted bundles were clearly visible between the two daughter cells .
similarly in wiskostatin treated cells, compacted microtubule bundles were also present between newly assembled nuclei but cells were binucleated .
cleavage furrow relaxation induced by wiskostatin was not associated with obvious f-actin reorganisation and some cleavage furrow remnant could be observed surrounding the central spindle as evidenced by f-actin staining .
relocalisation of ect <dig> from the central spindle in anaphase to the midbody during cytokinesis was also not altered by wiskostatin treatment  confirming that wiskostatin did not affect cleavage furrow formation and initial contraction.
we next studied the localisation of arp <dig> during cytokinesis under wiskostatin treatment.
as previously showed, arp <dig> was enriched within the cleavage furrow once fully contracted in vehicle treated cells at the end of telophase .
under wiskostatin treatment, arp <dig> localisation within the contractile ring was reduced .
in addition, wiskostatin induced the clustering of arp <dig> within vesicle-like structures throughout the cell .
altogether, our results showed that wiskostatin inhibited late cell cycle events required for abscission to occur.
wiskostatin induced cleavage furrow regression was associated with a reduced localisation of arp2/ <dig> complex within the contractile ring.
wiskostatin effects onto cytokinesis are not reproduced by alternative inhibition of the n-wasp/arp2/ <dig> pathway
in order to study whether the mislocalisation of arp <dig>  induced by wiskostatin, was responsible for defective cytokinesis, we used alternative approaches to inhibit n-wasp and arp2/ <dig> complex activities.
we first knocked-down n-wasp and arp <dig> protein levels using an sirna approach.
n-wasp and arp <dig> expressions were strongly reduced in cells treated for  <dig> hours with specific sirna  as demonstrated by immunoblot analysis .
as previously shown, arp <dig> knockdown is associated with down-regulation of other arp2/ <dig> subunits  <cit>  as revealed by the strong inhibition of p <dig> expression in arp <dig> null cells .
the dna content of sirna treated cells was then evaluated by flow cytometry in order to study the consequences of n-wasp and/or arp <dig> depletion on cytokinesis.
no major differences between control cells and cells transfected with n-wasp and/or arp <dig> sirnas were observed .
in contrast, tetraploid and also octaploid cells accumulated when transfected with sirna inhibiting ect <dig> protein expression , a major effector of cytokinesis .
the phenotypes resulting from sirna transfection were also evaluated by indirect immunofluorescence analysis.
cellular morphology, f-actin organisation and arp <dig> staining were not affected in control and n-wasp knockdown cells .
in contrast, arp <dig> staining was greatly reduced in cells transfected with arp <dig> sirna for  <dig> hours.
the morphology of these cells was altered as they appeared rounder and smaller, a phenotype likely due to the disorganization of the f-actin network .
during cytokinesis, arp <dig> recruitment to the contractile ring was only reduced in arp <dig> null cells but not in control and n-wasp null cells .
we obtained similar results using other sirnas targeting n-wasp and arp <dig> sequences .
our data suggest that arp2/ <dig> complex localisation within the cleavage furrow does not rely on n-wasp and is not mandatory for cytokinesis to occur.
in order to investigate possible subtle consequences of n-wasp and/or arp <dig> losses on mitosis/cytokinesis, sirna treated cells were studied by time-lapse microscopy.
the time-lapse experiment began  <dig> hours after sirna transfection when protein expression was significantly reduced  and cells were imaged every  <dig> minutes for  <dig> hours.
no differences in terms of frequency and kinetics of mitosis and cytokinesis were observed between control cells , n-wasp null-cells , arp <dig> null-cells , n-wasp and arp <dig> null-cells .
in contrast, ect <dig> knockdown strongly interfered with cell division inducing binucleation and tetranucleation over time .
surprisingly, these results suggest that the loss of n-wasp and/or arp2/ <dig> complex, are not associated with cytokinesis failure as we were expecting based on the effects of wiskostatin.
in order to confirm these unexpected results, various n-wasp mutants known to interfere with arp2/ <dig> complex activity were overexpressed and their effects on cytokinesis evaluated.
wasp family proteins are characterized by the presence, at the carboxy-terminus, of three independent domains: the verprolin homology  , central  and acidic  regions.
these domains form the so-called vca module, which is necessary and sufficient to activate arp2/3-dependent actin polymerisation in vitro.
the v and a regions bind actin monomers and arp2/ <dig> respectively, whilst the c region binds arp2/ <dig> and induces crucial changes in the tertiary and quaternary structures of the arp2/ <dig> complex, thereby regulating the ability of the arp2/ <dig> complex to induce actin polymerisation  <cit> .
deletion of the a region within the sequence of wasp family proteins compromises binding to the arp2/ <dig> complex.
this mutation acts as a dominant negative protein.
on the other hand, overexpression of vca and ca modules from wasp family proteins strongly induces mislocalised arp2/ <dig> complex activation and unproductive arp2/ <dig> binding respectively  <cit> .
these constructs titrate endogenous arp2/ <dig> complex and inhibit f-actin processes depending onto arp2/ <dig> activity  <cit> .
vector expressing gfp, gfp-tagged n-wasp, n-wasp Δa , vca or ca modules from n-wasp were transfected in nih3t <dig> fibroblasts.
three days after transfection cells were fixed and binucleated transfected cells counted.
results from a representative experiment are presented in table  <dig>  as shown, no major differences were observed between gfp, gfp-tagged n-wasp, n-wasp Δa and ca overexpressing cells, although, overexpression of gfp-tagged vca slightly, but consistently, increased cell binucleation .
nih3t <dig> cells were transfected with indicated constructs.
three days after transfection cells were harvested and a fifth seeded onto glass coverslips.
two hours after seeding cells were fixed.
nuclei number of transfected cells was determined after f-actin and dna staining respectively with tritc-coupled phalloidin and dapi.
total cell number and the number of binucleated cells from a representative experiment are presented as well as the deduced percentage of binucleated cells.
altogether, our data demonstrated that the inhibition of n-wasp/arp2/ <dig> pathway is not sufficient to mimic wiskostatin effects on cytokinesis.
therefore, it is likely that wiskostatin affect the activity of additional targets involved in cytokinesis.
wiskostatin effect onto cytokinesis is independent of atp depletion
taken altogether, our results suggest that wiskostatin effects on cytokinesis progression cannot solely be explained by the inhibition of n-wasp.
during the course of our study, wiskostatin was shown to induce cellular atp depletion  <cit> .
we thus evaluated whether atp depletion may interfere with cytokinesis progression.
mitotic hela cells released in presence of vehicle or wiskostatin  from a nocodazole block were harvested at various time points and lysed for  <dig> minutes at 100°c in distilled water.
atp levels were then determined from clarified lysates using a bioluminescent luciferase assay.
in vehicle  or wiskostatin  treated cells no major variation of atp levels was observed during mitosis/cytokinesis .
in contrast, atp levels strongly decreased in cells released in presence of energy poisons such as sodium azide and 2-deoxy-d-glucose .
in order to confirm that inhibition of cytokinesis induced by wiskostatin was independent of atp depletion, dna content of hela cells synchronised and treated as mentioned was determined by flow cytometry.
whereas vehicle or atp depleted treated cells were mainly diploids after  <dig> minutes from nocodazole release, wiskostatin treated cells were massively tetraploids and likely binucleated .
interestingly, we observed a delay in exit to mitosis in atp depleted cells , which was correlated with a delay of cyclin b <dig> degradation as demonstrated by immunoblot analysis .
as previously demonstrated, there was no effect of wiskostatin on cyclin b <dig> degradation.
time-lapse microscopy confirmed that atp depletion delayed mitotic exit whereas wiskostatin did not .
our results showed that atp depletion does not block mitosis/cytokinesis but simply delays it.
furthermore, we established that inhibition of cytokinesis by wiskostatin is independent of atp depletion suggesting additional targets for wiskostatin.
discussion
partitioning of the two daughter cells following cytokinesis is crucial to allow faithful segregation of chromosomes.
disrupting the final stages of cytokinesis can lead to multinucleated cells and genome instability.
the involvement of the actin cytoskeleton during cytokinesis is now well established.
the cleavage furrow is formed during anaphase by actin and myosin filaments and its complete ingression allows daughter cell separation at the end of mitosis.
however, little data regarding actin dynamics during cytokinesis is available.
a recent study showed that actin filaments present within the cleavage furrow of mammalian cells come from the flux of preexisting f-actin and de novo in situ actin polymerisation  <cit> .
however the functional importance of de novo actin polymerisation or the identity of the nucleation factor involved remain unanswered questions.
in s. pombe, the two main actin nucleation factors, the arp2/ <dig> complex and a formin, are required for cleavage furrow establishment and activity  <cit> .
in mammals, the involvement of formins during cytokinesis is now well established suggesting a conserved mechanism between yeast and mammals for cleavage furrow formation  <cit> .
however, in metazoan the involvement of wasp/n-wasp and the arp2/ <dig> complex during cytokinesis is still a matter of debate.
very little information regarding the function of wasp/n-wasp during cell growth in general and cytokinesis in particular are available.
in nematodes, knockdown of wsp- <dig> protein, the wasp/n-wasp ortholog, leads to cytokinesis defects  <cit> .
n-wasp knockout in mouse is embryonic lethal  and primary fibroblasts isolated from knockout embryos are unable to grow without being transformed suggesting an involvement of n-wasp in cell growth  <cit> .
data regarding the involvement of the arp2/ <dig> complex during cytokinesis in metazoans are more controversial.
in nematode, the requirement of the arp2/ <dig> complex is unclear since two studies using rna interference lead to conflicting results.
two groups described that knocking-down most of the arp2/ <dig> complex subunits lead to embryonic lethality, however only the group of skop et al.
showed that these depletions were associated with early or late cytokinesis defects  <cit> .
in drosophila, the picture is more readable since arp <dig> knockdown in s <dig> cells led to cell binucleation  <cit> .
furthermore, the arp2/ <dig> complex is required for the assembly of two drosophila embryonic structures highly related to the cleavage furrow, the pseudocleavage furrow in syncytial embryos and the ring canal  <cit> .
finally, in mammals, p <dig>  subunit of the arp2/ <dig> complex is an essential gene, as knockdown results in a block in cell growth phenotype  <cit> .
however, a potential relation with a cytokinesis defect was not reported.
in summary, the data available in the literature on the functional importance of the arp2/ <dig> complex for cell growth are clear, however because of the pleïotropic functions of the actin cytoskeleton, specific requirements of arp2/ <dig> complex activity for efficient cytokinesis are still questionable.
in this study, we showed that endogenous arp <dig>  despite being localised to the cell cortex, did not accumulate in the nascent contractile ring.
however, once the cleavage furrow was completely contracted during cytokinesis, arp2/ <dig> complex colocalised with f-actin within this structure.
arp2/ <dig> complex was also found within the cytoplasmic bridge linking daughter cells at the end of mitosis in agreement with the biochemical association of arp2/ <dig> subunits with the central spindle  <cit> .
the absence of early specific localisation of arp <dig> within the cleavage furrow suggests that the arp2/ <dig> complex is not involved in its establishment.
this is in agreement with the proposed role of the arp2/ <dig> complex in fission yeast where it is recruited late to/around the contractile ring and seems to be involved in cleavage furrow maintenance by allowing new plasma membrane addition  <cit> .
new membrane addition relies on vesicle recruitment and is essential for cleavage furrow ingression and to seal up daughter cells at time of abscission  <cit> .
the arp2/ <dig> complex is involved in endocytosis, vesicular trafficking but also in the polarised delivery of certain proteins to the plasma membrane  <cit> .
all these processes are known or supposed to be important during cytokinesis  <cit> .
we showed that arp2/ <dig> complex was enriched in vesicle-like structure localised on microtubules facing the cytoplasmic bridge between daughter cells.
we found that inhibition of n-wasp activity towards the arp2/ <dig> complex using wiskostatin strongly impaired cytokinesis without affecting mitosis in a dose dependent manner.
wiskostatin inhibited final stages of cytokinesis, which correlated with a decrease of the arp2/ <dig> complex from the contractile ring and the clustering of the arp2/ <dig> complex within vesicle-like structures.
the late inhibition of cytokinesis by wiskostatin suggests that the arp2/ <dig> complex is required at the end of cytokinesis and/or for abscission in agreement with its late recruitment within the cleavage furrow.
however, we were unable to confirm these results by knocking-down n-wasp and/or arp <dig> protein expression by rna interference.
moreover, n-wasp depletion showed that arp <dig> localisation within the cleavage furrow is not dependent upon n-wasp.
different possibilities can explain the discrepancy between the results we obtained using rna interference versus wiskostatin.
first, we cannot exclude that remaining n-wasp and/or arp <dig> proteins, in sirna treated cells, are sufficient to trigger cytokinesis bearing in mind that complete depletion of these proteins is associated with cell growth defects and embryonic lethality  <cit> .
second, wiskostatin could have additional targets related or not to n-wasp.
wiskostatin binds purified n-wasp in vitro but nothing is known about binding to native n-wasp.
native n-wasp is predominantly associated with wip , a protein involved in n-wasp inhibition but also required for n-wasp activation  <cit> .
wip can regulate f-actin organisation independently of its effects on n-wasp  <cit> .
wiskostatin could prevent the release or the binding of wip or other unknown proteins.
this could have major consequences for the progression of cytokinesis if such proteins play a role during this process.
furthermore, this could explain the discrepancy between wiskostatin and n-wasp knockdown results.
finally, wiskostatin could inhibit additional targets unrelated to n-wasp.
indeed, wiskostatin was recently shown to deplete cellular atp in an irreversible manner perturbing membrane transport  <cit> .
atp in general and membrane trafficking in particular, as mentioned above, play crucial roles during mitosis and cytokinesis.
however, we showed that wiskostatin, at the highest concentration used in this study , did not affect cellular atp levels during mitosis/cytokinesis.
thus, inhibition of cytokinesis induced by wiskostatin is likely independent to its atp depleting activity.
the existence of additional targets of wiskostatin is supported by the results we obtained using n-wasp mutants.
the expression of a dominant negative mutant of n-wasp towards the arp2/ <dig> complex  did not block cytokinesis suggesting again that n-wasp inhibition is not sufficient.
in contrast, overexpression of n-wasp vca domain slightly increased nuclei number.
the vca module alters arp2/ <dig> activity by titrating and delocalising it.
vca overexpression could either block the activation of the arp2/ <dig> complex by another factor than n-wasp involved in cytokinesis.
on the other hand, the increase of f-actin generated by over-activation of arp2/ <dig> complex triggered by vca overexpression could affect cytokinesis.
this hypothesis is supported by a recent study showing that expression of a constitutively active mutant of wasp towards the arp2/ <dig> complex increases cell binucleation in a same extend as vca  <cit> .
CONCLUSIONS
wiskostatin has been designed, as numerous inhibitors, to specifically inhibit n-wasp activity towards the arp2/ <dig> complex.
our studies reveal the caveat of using such an in vitro approach to identify new compounds.
indeed, in vivo test may not parallel the in vitro activity and should be included in the compound characterisation.
we show that wiskostatin inhibits cytokinesis and it is likely that n-wasp is not its sole target during this process.
our study is the second report of a non-expected effect of wiskostatin, which suggests that this compound will have to be used in future with caution.
however, it will be of interest to characterise wiskostatin targets involved in cytokinesis.
the identification of these proteins could be achieved using a proteomic approach based upon mass spectrometry using immobilised wiskostatin over mitotic cell extracts.

 cytokinesis is essential to cell growth and has to be tightly regulated to insure equal distribution of the cytoplasm content and newly replicated chromosomes within two daughter cells at the end of mitosis. defective chromosome segregation or daughter cell separation can alter chromosome number, which is commonly found in tumour cells and potentially involved in cancer development. for these reasons it is essential to understand the signalling pathways that coordinate mitotic exit and cytokinesis. in metazoans, the central spindle forms during anaphase between the two sets of chromosomes. simultaneously, the cleavage furrow composed by actin and myosin ii filaments is established at the cell equator under the control of peripheral microtubules. thus, constriction of the actomyosin ring can only start once the two sets of chromosomes are segregated, insuring genome integrity. the two daughter cells stay connected by a cytoplasmic bridge with the midbody in the centre for several hours until the bridge is finally severed during abscission. the small gtpase rhoa has been shown to play a major role in cleavage furrow establishment. indeed, the gtpase exchange factor ect is specifically recruited at the cell equator under the control of the centralspindlin complex also required for central spindle formation. once activated, rhoa induces, through its effector rock, the phosphorylation of myosin regulatory light chain, which is necessary for contractile ring formation and ingression. in mammals, actin filaments present in the contractile ring derive from preexisting f-actin flux and de novo actin polymerisation. in s. pombe, cleavage furrow establishment and activity depend on de novo actin polymerisation driven by the two main actin nucleation factors: the arp2/ complex and a formin. arp2/ complex is activated by myo and wsp at the equator and is involved in the maturation and/or the maintenance of the cleavage furrow. in mammals the mdia formins, orthologs of cdc and rhoa effectors, are required for cytokinesis. in agreement with a conserved mechanism for cleavage furrow formation and activity, the arp2/ complex and its activator wasp/n-wasp, ortholog of wsp were found to potentially play a role during cytokinesis. in this study the functional role of the arp2/ complex during cytokinesis in mammalian cells was evaluated. we demonstrated that arp2/ complex is recruited late to the cleavage furrow. consequences of the inhibition of wasp/n-wasp activity towards the arp2/ complex during cytokinesis were then studied using the newly identified inhibitor: wiskostatin. we show that wiskostatin strongly inhibits cytokinesis inducing cell binucleation without affecting the cell cycle. however, these results are unlikely related to the arp2/ complex function since the wiskostatin induced phenotype could not be validated using n-wasp and/or arp2/ rna interference approach. this novel effect of wiskostatin on cytokinesis is also not related to another known unexpected effect of this compound i.e. atp depletion. this study that was originally designed to study n-wasp/arp2/ complex pathway during cytokinesis, unexpectedly highlighted a novel effect of wiskostatin. for this reason, wiskostatin will have to be used with caution in future experiments.  localisation of arp2/ complex during mitosis in mammalian cells, the localisation of endogenous arp2/ complex during mitosis is currently unknown. in order to study the function of the n-wasp/arp2/ complex pathway during cytokinesis, we thus analyzed the subcellular localisation of arp and p subunits of the arp2/ complex by indirect immunofluorescence. in interphase hela cells using monoclonal anti-arp antibody, the arp2/ complex was found to be diffuse and associated with vesicle-like structures in the cytoplasm but also accumulated in cortical f-actin-rich structures, which are likely lamellipodia. to better visualize cytoskeleton bound arp2/ complex in mitotic cells, the cytosolic unbound fraction was eliminated by permeabilising the cells prior to fixation. under these conditions, arp presented a diffuse vesicular-like localisation and was not particularly enriched into the nascent cleavage furrow during anaphase. however, during late telophase, once the cleavage furrow is fully contracted, arp was strongly enriched in the close vicinity of the contractile ring, as visualized by the f-actin staining. furthermore, the vesicular-like localisation of arp was organised around the minus-end and along microtubules. in cytokinesis, when the cytoplasmic bridge between the two daughter cells extended, arp colocalised with f-actin in the cortex facing the cytoplasmic bridge and within vesicle-like structures. furthermore, the arp2/ complex also localised within the cytoplasmic bridge but not in the midbody. this subcellular localisation of arp2/ complex was further confirmed by using a polyclonal antibody directed against p subunit. thus, the localization of arp2/ complex is in agreement with a potential functional role during cytokinesis. wiskostatin inhibits cytokinesis but not mitosis the mammalian ortholog of wsp wasp/n-wasp, is regulated by autoinhibition essentially relieved by binding of activated small gtpase cdc and/or phosphatidyl inositol -biphosphate. gtp loaded cdc binds to the gbd of wasp/n-wasp, which is preceded by a basic region where ptdins p interacts. wiskostatin is a recently identified wasp/n-wasp chemical inhibitor designed to bind to the gbd. wiskostatin not only prevents cdc binding, but also stabilises the autoinhibited state of this actin nucleation promoting factor. this led us to use wiskostatin to investigate the role of the arp2/ complex activated by wasp/n-wasp during cytokinesis. hela cells stably expressing gfp-tagged histone h2b were synchronised in prometaphase with nocodazole and were released for various times after shake-off in presence of vehicle or increasing concentration of wiskostatin. cell dna content was determined by flow cytometry at and minutes after nocodazole release. in control conditions, at t = cells had a 4n content confirming mitosis synchronisation mediated by nocodazole. minutes later half of the cells had exit mitosis and displayed a 2n content. finally, min after release, control cells were mostly in g phase. in contrast, under wiskostatin treatment, the number of tetraploid cells was abnormally elevated at min after release suggesting a mitotic delay both at and μm doses. minutes after nocodazole release under μm wiskostatin, half of the cells had a 2n content while under μm wiskostatin treatment all cells were tetraploid indicating that they did not proceed through mitosis. thus wiskostatin inhibits mitotic progression in a dose dependent manner. the increased number of tetraploid cells under wiskostatin could either result from an inhibition of mitosis or cytokinesis. to answer this question, we first analysed the status of cyclin b which is degraded at the metaphase/anaphase transition. mitotic hela cells were released in presence of vehicle alone or wiskostatin, and cyclin b levels were determined by immunoblot analysis. no difference in the kinetics of cyclin b degradation was observed between control and wiskostatin treated cells. this result was further confirmed by studying another mitotic marker, the phosphorylation of histone h on serine which correlates with chromatin condensation. as expected, histone h phosphorylation was not affected by wiskostatin treatment. equal sample loading was confirmed by cofilin immunoblot analysis. these results clearly demonstrate that wiskostatin does not inhibit mitotic progression. taken altogether, our results indicate an effect of wiskostatin on cytokinesis. cytokinesis failure is generally associated with cell binucleation. to investigate this hypothesis, nocodazole synchronised hela gfp h2b cells were grown in presence of vehicle alone or wiskostatin, fixed after minutes and binucleated cells were quantified. in control condition, around 7% of cells were found to be binucleated. in contrast, 85% of wiskostatin treated cells contained two nuclei. to rule out any cooperative effects of nocodazole and wiskostatin, asynchronous hela gfp-h2b cells were grown in presence of μm wiskostatin for hours and binucleated cells were counted as previously described. under these conditions more than 26% of cells were found to be binucleated compared to % of cells treated with vehicle alone. similar results were obtained with nih3t and xl cells. this result clearly demonstrates that wiskostatin affects mitotic exit. in order to analyse wiskostatin induced cytokinesis defects in more details, time-lapse microscopy was used. in vehicle treated hela gfp-h2b cells cytokinesis started around minutes after release from nocodazole block. after complete cleavage furrow contraction, daughter cells were linked to each other by a cytoplasmic bridge, which extended over time with the midbody in its centre. finally, after several hours, abscission occurred. under wiskostatin treatment, cytokinesis started with the same kinetics as control cells but prior and during cleavage furrow contraction blebs were visible at cell poles. finally, once the cleavage furrow fully contracted, the cytoplasmic bridge did not extend, the contractile ring relaxed and daughter cells finally fused-back together. we then studied the organisation of the cytoskeleton in more details. mitotic hela cells stably expressing gfp-tagged β-tubulin were fixed two hours after nocodazole release in presence of vehicle or μm wiskostatin, and were stained for f-actin and dna. during cytokinesis, the microtubule bundles of the central spindle, which are not associated with kinetochores, are compacted by the contraction of the cleavage furrow. in control cells, these compacted bundles were clearly visible between the two daughter cells. similarly in wiskostatin treated cells, compacted microtubule bundles were also present between newly assembled nuclei but cells were binucleated. cleavage furrow relaxation induced by wiskostatin was not associated with obvious f-actin reorganisation and some cleavage furrow remnant could be observed surrounding the central spindle as evidenced by f-actin staining. relocalisation of ect from the central spindle in anaphase to the midbody during cytokinesis was also not altered by wiskostatin treatment confirming that wiskostatin did not affect cleavage furrow formation and initial contraction. we next studied the localisation of arp during cytokinesis under wiskostatin treatment. as previously showed, arp was enriched within the cleavage furrow once fully contracted in vehicle treated cells at the end of telophase. under wiskostatin treatment, arp localisation within the contractile ring was reduced. in addition, wiskostatin induced the clustering of arp within vesicle-like structures throughout the cell. altogether, our results showed that wiskostatin inhibited late cell cycle events required for abscission to occur. wiskostatin induced cleavage furrow regression was associated with a reduced localisation of arp2/ complex within the contractile ring. wiskostatin effects onto cytokinesis are not reproduced by alternative inhibition of the n-wasp/arp2/ pathway in order to study whether the mislocalisation of arp induced by wiskostatin, was responsible for defective cytokinesis, we used alternative approaches to inhibit n-wasp and arp2/ complex activities. we first knocked-down n-wasp and arp protein levels using an sirna approach. n-wasp and arp expressions were strongly reduced in cells treated for hours with specific sirna as demonstrated by immunoblot analysis. as previously shown, arp knockdown is associated with down-regulation of other arp2/ subunits as revealed by the strong inhibition of p expression in arp null cells. the dna content of sirna treated cells was then evaluated by flow cytometry in order to study the consequences of n-wasp and/or arp depletion on cytokinesis. no major differences between control cells and cells transfected with n-wasp and/or arp sirnas were observed. in contrast, tetraploid and also octaploid cells accumulated when transfected with sirna inhibiting ect protein expression, a major effector of cytokinesis. the phenotypes resulting from sirna transfection were also evaluated by indirect immunofluorescence analysis. cellular morphology, f-actin organisation and arp staining were not affected in control and n-wasp knockdown cells. in contrast, arp staining was greatly reduced in cells transfected with arp sirna for hours. the morphology of these cells was altered as they appeared rounder and smaller, a phenotype likely due to the disorganization of the f-actin network. during cytokinesis, arp recruitment to the contractile ring was only reduced in arp null cells but not in control and n-wasp null cells. we obtained similar results using other sirnas targeting n-wasp and arp sequences. our data suggest that arp2/ complex localisation within the cleavage furrow does not rely on n-wasp and is not mandatory for cytokinesis to occur. in order to investigate possible subtle consequences of n-wasp and/or arp losses on mitosis/cytokinesis, sirna treated cells were studied by time-lapse microscopy. the time-lapse experiment began hours after sirna transfection when protein expression was significantly reduced and cells were imaged every minutes for hours. no differences in terms of frequency and kinetics of mitosis and cytokinesis were observed between control cells, n-wasp null-cells, arp null-cells, n-wasp and arp null-cells. in contrast, ect knockdown strongly interfered with cell division inducing binucleation and tetranucleation over time. surprisingly, these results suggest that the loss of n-wasp and/or arp2/ complex, are not associated with cytokinesis failure as we were expecting based on the effects of wiskostatin. in order to confirm these unexpected results, various n-wasp mutants known to interfere with arp2/ complex activity were overexpressed and their effects on cytokinesis evaluated. wasp family proteins are characterized by the presence, at the carboxy-terminus, of three independent domains: the verprolin homology, central and acidic regions. these domains form the so-called vca module, which is necessary and sufficient to activate arp2/3-dependent actin polymerisation in vitro. the v and a regions bind actin monomers and arp2/ respectively, whilst the c region binds arp2/ and induces crucial changes in the tertiary and quaternary structures of the arp2/ complex, thereby regulating the ability of the arp2/ complex to induce actin polymerisation. deletion of the a region within the sequence of wasp family proteins compromises binding to the arp2/ complex. this mutation acts as a dominant negative protein. on the other hand, overexpression of vca and ca modules from wasp family proteins strongly induces mislocalised arp2/ complex activation and unproductive arp2/ binding respectively. these constructs titrate endogenous arp2/ complex and inhibit f-actin processes depending onto arp2/ activity. vector expressing gfp, gfp-tagged n-wasp, n-wasp Δa, vca or ca modules from n-wasp were transfected in nih3t fibroblasts. three days after transfection cells were fixed and binucleated transfected cells counted. results from a representative experiment are presented in table as shown, no major differences were observed between gfp, gfp-tagged n-wasp, n-wasp Δa and ca overexpressing cells, although, overexpression of gfp-tagged vca slightly, but consistently, increased cell binucleation. nih3t cells were transfected with indicated constructs. three days after transfection cells were harvested and a fifth seeded onto glass coverslips. two hours after seeding cells were fixed. nuclei number of transfected cells was determined after f-actin and dna staining respectively with tritc-coupled phalloidin and dapi. total cell number and the number of binucleated cells from a representative experiment are presented as well as the deduced percentage of binucleated cells. altogether, our data demonstrated that the inhibition of n-wasp/arp2/ pathway is not sufficient to mimic wiskostatin effects on cytokinesis. therefore, it is likely that wiskostatin affect the activity of additional targets involved in cytokinesis. wiskostatin effect onto cytokinesis is independent of atp depletion taken altogether, our results suggest that wiskostatin effects on cytokinesis progression cannot solely be explained by the inhibition of n-wasp. during the course of our study, wiskostatin was shown to induce cellular atp depletion. we thus evaluated whether atp depletion may interfere with cytokinesis progression. mitotic hela cells released in presence of vehicle or wiskostatin from a nocodazole block were harvested at various time points and lysed for minutes at 100°c in distilled water. atp levels were then determined from clarified lysates using a bioluminescent luciferase assay. in vehicle or wiskostatin treated cells no major variation of atp levels was observed during mitosis/cytokinesis. in contrast, atp levels strongly decreased in cells released in presence of energy poisons such as sodium azide and 2-deoxy-d-glucose. in order to confirm that inhibition of cytokinesis induced by wiskostatin was independent of atp depletion, dna content of hela cells synchronised and treated as mentioned was determined by flow cytometry. whereas vehicle or atp depleted treated cells were mainly diploids after minutes from nocodazole release, wiskostatin treated cells were massively tetraploids and likely binucleated. interestingly, we observed a delay in exit to mitosis in atp depleted cells, which was correlated with a delay of cyclin b degradation as demonstrated by immunoblot analysis. as previously demonstrated, there was no effect of wiskostatin on cyclin b degradation. time-lapse microscopy confirmed that atp depletion delayed mitotic exit whereas wiskostatin did not. our results showed that atp depletion does not block mitosis/cytokinesis but simply delays it. furthermore, we established that inhibition of cytokinesis by wiskostatin is independent of atp depletion suggesting additional targets for wiskostatin. discussion partitioning of the two daughter cells following cytokinesis is crucial to allow faithful segregation of chromosomes. disrupting the final stages of cytokinesis can lead to multinucleated cells and genome instability. the involvement of the actin cytoskeleton during cytokinesis is now well established. the cleavage furrow is formed during anaphase by actin and myosin filaments and its complete ingression allows daughter cell separation at the end of mitosis. however, little data regarding actin dynamics during cytokinesis is available. a recent study showed that actin filaments present within the cleavage furrow of mammalian cells come from the flux of preexisting f-actin and de novo in situ actin polymerisation. however the functional importance of de novo actin polymerisation or the identity of the nucleation factor involved remain unanswered questions. in s. pombe, the two main actin nucleation factors, the arp2/ complex and a formin, are required for cleavage furrow establishment and activity. in mammals, the involvement of formins during cytokinesis is now well established suggesting a conserved mechanism between yeast and mammals for cleavage furrow formation. however, in metazoan the involvement of wasp/n-wasp and the arp2/ complex during cytokinesis is still a matter of debate. very little information regarding the function of wasp/n-wasp during cell growth in general and cytokinesis in particular are available. in nematodes, knockdown of wsp- protein, the wasp/n-wasp ortholog, leads to cytokinesis defects. n-wasp knockout in mouse is embryonic lethal and primary fibroblasts isolated from knockout embryos are unable to grow without being transformed suggesting an involvement of n-wasp in cell growth. data regarding the involvement of the arp2/ complex during cytokinesis in metazoans are more controversial. in nematode, the requirement of the arp2/ complex is unclear since two studies using rna interference lead to conflicting results. two groups described that knocking-down most of the arp2/ complex subunits lead to embryonic lethality, however only the group of skop et al. showed that these depletions were associated with early or late cytokinesis defects. in drosophila, the picture is more readable since arp knockdown in s cells led to cell binucleation. furthermore, the arp2/ complex is required for the assembly of two drosophila embryonic structures highly related to the cleavage furrow, the pseudocleavage furrow in syncytial embryos and the ring canal. finally, in mammals, p subunit of the arp2/ complex is an essential gene, as knockdown results in a block in cell growth phenotype. however, a potential relation with a cytokinesis defect was not reported. in summary, the data available in the literature on the functional importance of the arp2/ complex for cell growth are clear, however because of the pleïotropic functions of the actin cytoskeleton, specific requirements of arp2/ complex activity for efficient cytokinesis are still questionable. in this study, we showed that endogenous arp despite being localised to the cell cortex, did not accumulate in the nascent contractile ring. however, once the cleavage furrow was completely contracted during cytokinesis, arp2/ complex colocalised with f-actin within this structure. arp2/ complex was also found within the cytoplasmic bridge linking daughter cells at the end of mitosis in agreement with the biochemical association of arp2/ subunits with the central spindle. the absence of early specific localisation of arp within the cleavage furrow suggests that the arp2/ complex is not involved in its establishment. this is in agreement with the proposed role of the arp2/ complex in fission yeast where it is recruited late to/around the contractile ring and seems to be involved in cleavage furrow maintenance by allowing new plasma membrane addition. new membrane addition relies on vesicle recruitment and is essential for cleavage furrow ingression and to seal up daughter cells at time of abscission. the arp2/ complex is involved in endocytosis, vesicular trafficking but also in the polarised delivery of certain proteins to the plasma membrane. all these processes are known or supposed to be important during cytokinesis. we showed that arp2/ complex was enriched in vesicle-like structure localised on microtubules facing the cytoplasmic bridge between daughter cells. we found that inhibition of n-wasp activity towards the arp2/ complex using wiskostatin strongly impaired cytokinesis without affecting mitosis in a dose dependent manner. wiskostatin inhibited final stages of cytokinesis, which correlated with a decrease of the arp2/ complex from the contractile ring and the clustering of the arp2/ complex within vesicle-like structures. the late inhibition of cytokinesis by wiskostatin suggests that the arp2/ complex is required at the end of cytokinesis and/or for abscission in agreement with its late recruitment within the cleavage furrow. however, we were unable to confirm these results by knocking-down n-wasp and/or arp protein expression by rna interference. moreover, n-wasp depletion showed that arp localisation within the cleavage furrow is not dependent upon n-wasp. different possibilities can explain the discrepancy between the results we obtained using rna interference versus wiskostatin. first, we cannot exclude that remaining n-wasp and/or arp proteins, in sirna treated cells, are sufficient to trigger cytokinesis bearing in mind that complete depletion of these proteins is associated with cell growth defects and embryonic lethality. second, wiskostatin could have additional targets related or not to n-wasp. wiskostatin binds purified n-wasp in vitro but nothing is known about binding to native n-wasp. native n-wasp is predominantly associated with wip, a protein involved in n-wasp inhibition but also required for n-wasp activation. wip can regulate f-actin organisation independently of its effects on n-wasp. wiskostatin could prevent the release or the binding of wip or other unknown proteins. this could have major consequences for the progression of cytokinesis if such proteins play a role during this process. furthermore, this could explain the discrepancy between wiskostatin and n-wasp knockdown results. finally, wiskostatin could inhibit additional targets unrelated to n-wasp. indeed, wiskostatin was recently shown to deplete cellular atp in an irreversible manner perturbing membrane transport. atp in general and membrane trafficking in particular, as mentioned above, play crucial roles during mitosis and cytokinesis. however, we showed that wiskostatin, at the highest concentration used in this study, did not affect cellular atp levels during mitosis/cytokinesis. thus, inhibition of cytokinesis induced by wiskostatin is likely independent to its atp depleting activity. the existence of additional targets of wiskostatin is supported by the results we obtained using n-wasp mutants. the expression of a dominant negative mutant of n-wasp towards the arp2/ complex did not block cytokinesis suggesting again that n-wasp inhibition is not sufficient. in contrast, overexpression of n-wasp vca domain slightly increased nuclei number. the vca module alters arp2/ activity by titrating and delocalising it. vca overexpression could either block the activation of the arp2/ complex by another factor than n-wasp involved in cytokinesis. on the other hand, the increase of f-actin generated by over-activation of arp2/ complex triggered by vca overexpression could affect cytokinesis. this hypothesis is supported by a recent study showing that expression of a constitutively active mutant of wasp towards the arp2/ complex increases cell binucleation in a same extend as vca.  wiskostatin has been designed, as numerous inhibitors, to specifically inhibit n-wasp activity towards the arp2/ complex. our studies reveal the caveat of using such an in vitro approach to identify new compounds. indeed, in vivo test may not parallel the in vitro activity and should be included in the compound characterisation. we show that wiskostatin inhibits cytokinesis and it is likely that n-wasp is not its sole target during this process. our study is the second report of a non-expected effect of wiskostatin, which suggests that this compound will have to be used in future with caution. however, it will be of interest to characterise wiskostatin targets involved in cytokinesis. the identification of these proteins could be achieved using a proteomic approach based upon mass spectrometry using immobilised wiskostatin over mitotic cell extracts. 
we conclude that the wiskostatin induced defective cytokinesis does not occur through the inhibition of the n-wasp/arp2/ pathway. wiskostatin is likely to either directly target other proteins required for cytokinesis progression or alternately wiskostatin bound to n-wasp could affect the activity of other factors involved in cytokinesis. we found that the arp2/ complex is recruited late to the cleavage furrow suggesting a potential involvement of arp2/ complex during this process. cytokinesis is the final step of cell division taking place at the end of mitosis during which the cytoplasmic content and replicated chromosomes of a cell are equally partitioned between the two daughter cells. furthermore, wiskostatin a potent inhibitor of n-wasp activity towards the arp2/ complex blocked cytokinesis without affecting mitosis. the mechanisms of contractile ring formation are not fully understood but involve recruitment of preexisting actin filaments and de novo actin polymerisation. nonetheless, this inhibition could not be reproduced using alternative approaches targeting the n-wasp/arp2/ complex pathway.
 cytokinesis is the final step of cell division taking place at the end of mitosis during which the cytoplasmic content and replicated chromosomes of a cell are equally partitioned between the two daughter cells. this process is achieved by the formation and the ingression of an actomyosin contractile ring under the control of equatorial microtubules. the mechanisms of contractile ring formation are not fully understood but involve recruitment of preexisting actin filaments and de novo actin polymerisation.  in this study, we evaluated the role of the actin nucleation factor, arp2/ complex, during cytokinesis. we found that the arp2/ complex is recruited late to the cleavage furrow suggesting a potential involvement of arp2/ complex during this process. furthermore, wiskostatin a potent inhibitor of n-wasp activity towards the arp2/ complex blocked cytokinesis without affecting mitosis. nonetheless, this inhibition could not be reproduced using alternative approaches targeting the n-wasp/arp2/ complex pathway.  we conclude that the wiskostatin induced defective cytokinesis does not occur through the inhibition of the n-wasp/arp2/ pathway. wiskostatin is likely to either directly target other proteins required for cytokinesis progression or alternately wiskostatin bound to n-wasp could affect the activity of other factors involved in cytokinesis. 