Accessible Synthetic Probes for Staining Actin inside Platelets and Megakaryocytes by Employing Lifeact Peptide

Lifeact is a 17-residue peptide that can be employed in cell microscopy as a probe for F-actin when fused to fluorescent proteins, but therefore is not suitable for all cell types. We have conjugated fluorescently labelled Lifeact to three different cell-penetrating systems (a myristoylated carrier (myr), the pH low insertion peptide (pHLIP) and the cationic peptide TAT) as a strategy to deliver Lifeact into cells and developed new tools for actin staining with improved synthetic accessibility and low toxicity, focusing on their suitability in platelets and megakaryocytes. Using confocal microscopy, we characterised the cell distribution of the new hybrids in fixed cells, and found that both myr– and pHLIP–Lifeact conjugates provide efficient actin staining upon cleavage of Lifeact from the carriers, without affecting cell spreading. This new approach could facilitate the design of new tools for actin visualisation.


Introduction
Actin is one of the most abundant proteins in eukaryotes that can exist as filamentous actin (F-actin) formed from ATP-mediated polymerisation of monomeric actin (G-actin) and existing in highly dynamic supramolecular organisations. It is one of the major components of the cytoskeleton playing ak ey role in cell morphogenesis,d ivision and motility; it is regulated and organisedb ys everal actin-binding and signallingp roteins. [1] Methods to visualise actin dynamics without interfering with their complex activity are particularly important for cell biologists, and one major challenge is to achieve this by using live cell microscopy. [2] Current approaches to observe real-time F-actin dynamics use the incorporation of fluorescently labelledG -actin during F-actin polymerisation or employ labelled F-actin binding domains( ABDs, usually deriving from actin binding proteins) as fluorescent markers. [3] Among these,a17-residue peptide sequence, named Lifeact, from the yeast actin crosslinker Abp140h as been identified as the shortest sequence to interact with F-actin. [4] Lifeact is ideally suited as ap robe as it has low toxicity and interference with naturala ctin dynamics as well as the ability to tag al arge distribution of actin structures; therefore there is great interesti nL ifeact-based markersf or imaging actin. However, existing strategies employing Lifeact, as wella so ther ABDs or labelledG -actin, requiref usion with fluorescent proteins (GFP,R FP,e tc.) and insertioni nc ells with geneticm odificationso rb ym icroinjection techniques; [4b, 5] these procedures do not alwaysg uarantee ap roper controlo f the level of the probe in cells, thus affecting its efficiency,r eproducibility and toxicity; [6] more importantly,t hey are not suitable for all cell types or accessible for all research laboratories. Platelets are one significant example of primary cells that cannotb et ransformed (they are anucleate) or efficientlym icroinjected (due to their small size, 1-3 mmd iameter), therefore real-time actin dynamic studies are limited to platelets isolated from transgenic mice expressing GFP-actin markers. [7] Cell staining upon incubation with synthetic actin markers, such as labelledp hallotoxinso rj asplakinolide and their derivatives, are the most employed alternative tools for visualising actin structures by fluorescent microscopy.B right, high-resolutions images can be achieved thankstothe wide range of fluorophore-phallotoxinc onjugates commercially available, but these compounds are either toxic (in fact they have also been investigated as potentialc ytotoxic drugs), cell impermeable or interferew ith actin polymerisation. Consequently their use is typicallyl imited to the study of fixed cells or only for specific experiments in whichk nown effectso na ctin polymerisation can be taken into account. [8] Attempts to reduce the toxicity of known synthetic actin-bindingc ompounds by designing new derivatives have often been limited by the complexity and costs of syntheses;h owever,o ne fluorogenic and cell-permea-Lifeact is a1 7-residue peptide that can be employed in cell microscopy as ap robe for F-actin when fused to fluorescent proteins, butt herefore is not suitable for all cell types. We have conjugated fluorescently labelled Lifeact to three differentc ellpenetrating systems (a myristoylated carrier (myr), the pH low insertionp eptide( pHLIP) and the cationic peptide TAT) as a strategyt od eliver Lifeact into cells and developed new tools for actin staining with improved synthetic accessibility and low toxicity, focusingo nt heir suitabilityi np latelets and megakaryocytes. Using confocal microscopy,w ec haracterised the cell distribution of the new hybrids in fixed cells, andf ound that both myr-and pHLIP-Lifeact conjugates provide efficient actin stainingu pon cleavage of Lifeact from the carriers, without affectingc ell spreading. This new approach could facilitatet he design of new tools for actin visualisation.
ble actin marker with remarkably reducedt oxicity has recently been identified amongs everals yntheticj asplakinolide derivatives. [9] This is ap romising tool, although its potential needs to be characterised further as it shows different behaviours in different cell lines.
We aimed to develop alternative synthetic actin markers with improved synthetic accessibility and reduced toxicity; these being the main limits of the few existing compounds. We focuso nt he markers' suitability for actin stainingi n human platelets, as developing new approaches for these cells would help to understand important events in thrombosis and haemostasis;w ea lso verify their versatility in megakaryocytes (MKs), which are responsible for platelet production.D ue to the known actin affinity/low toxicityc ombination, fluorescently labelledL ifeact should guarantee minimum interference with actin dynamics (compared to other existing actin binders), therefore it is an ideal component for an actin marker.B ecause it is not cell permeable, we explore three different carriers able to promote the delivery and release of labelled Lifeact into the cytosol of platelets and MKs. To the best of our knowledge, there is only one successful example of am embrane-permeable synthetic carrier-Lifeactc onjugate, which was designed exclusively for live imaging in plant cells, in which Lifeact was fused to the antimicrobial peptideB P100 as as pecific vector for these cells. [10] We investigate three different systems as potential vectors for Lifeact:amyristoylated (myr) carrier, [11] the pH low insertion peptide( pHLIP) [12] and the cationic cell-penetrating peptide (CPP) TAT. [13] We conjugated Lifeact to each of these carriers both throughd isulfide-based linkers and covalent bonds, and investigated the efficiency of the new hybridsa sa ctin probesa nd the cell distribution of the carriers by fluorescent confocal microscopy with actin staining as an unambiguous read out.

Results and Discussion
Design and synthesis Platelets are small cells (1-3 mm) that can be readily isolated from blood and kept in buffer for up to 6-8 h. In this study,w ei nvestigated three potentialc arriers for Lifeact,s electing relatively small ones amongt he large variety of cell-penetrating materials availablea nd known to promote fast cell uptake of their cargos (< 30-60min). [14] Also, they are hypothesised to penetrate cell membranes by different mechanisms, thus we were able to in-vestigate the compatibility of differentd elivery methods into platelets.L ipid-based carriers, such as the myr system that we employed here, are believed to be independent from membrane-recognition events and are, therefore, more versatile than peptide-based carriers. [11,15] Furthermore, both palmitoylation and myristoylation have been employed to deliver potential antiplatelet drugs,t hus proving their compatibilityw ith platelets. [16] pHLIP is a3 8-residue transmembrane peptideh elix isolated from bacteriorhodopsin Ct hat is ablet od eliver several cargos( conjugated at the Cterminus by ac leavable link) in responset op Hc hanges:a tl ow pH (between 6.5 and 7.0), pHLIP folds into an a-helixc onformation able to penetrate the cell membrane, translocate the cargo and release it upon cytosolic disulfide reduction.A ni ncreasei np H( between 7.0 and 7.5) unfolds the a-helix and releases the carrier to the extracellular environment. [17] This carrier is also compatible with platelets;wehave recently described its ability to deliver nanomaterials into thesec ells. [18] Finally,w ee mployed the TATp eptide, which is one of the mostc ommon CPPs and widely employed in drug-delivery research.
We conjugated Lifeact to each carrier through ad isulfidebond-based linker to allow cleavage and releaseo ft he probe in the reductive environment in the cytosola fter membrane penetration. We modifiedL ifeact at the C-terminal by adding aL ys residue labelled with carboxyfluorescein (FAM) so as to allow fluorescent detection of actin staining;a tt he N-terminal we added aC ys residue for disulfide bond formation with the carrier. Each of the three selected carriers was modified at the Scheme1.Synthesis of carrier-Lifeact hybrids. Scheme of conjugation between carriers (labelled with TAMRA)a nd Lifeact (labelled with FAM) by disulfide bond exchange and alist of the cleavable (by disulfidereduction) and uncleavable compounds investigated for cellimaging. www.chembiochem.org C-terminal by adding aC ys followed by aL ys residue labelled with carboxytetramethylrhodamine (TAMRA) for fluorescent detection of the carrier;t he thiol group of the Cys was protected by a2 -thiopyridyl group to allow thiol-disulfide exchangew ith the cargo Lifeact (Scheme 1). We preparedt hree cleavable compounds:M yr-S-S-Life (5), pHLIP-S-S-Life (6)a nd TAT-S-S-Life (7). To understand the role of cleavage in Lifeact delivery,w e also prepared three analogousu ncleavable systems in which carrier and Lifeact are covalentlyb ound:M yr-Life( 8), pHLIP-Life (9)a nd TAT-Life (10).
The compounds were obtained by one-step conjugationb etween commercial peptides or by following standard and reliable synthetic procedures (peptide synthesis, amino group labellinga nd disulfidee xchange);t his is one advantage compared to other actin markerso btained by complex multistep organic syntheses.
The FAM/TAMRA pair (donor and acceptor,r espectively) is suitable for FRET spectroscopy and this plays ad ual function: providing ana dditional tool to monitor cleavage and cell uptake [19] and reducing the background fluorescenceo riginating from extracellularo ru ncleavedL ifeact(FAM). Emission scans of the cleavable compounds 5, 6 and 7 in the presence of ar educinga gent confirmt hat energy transfer occurs when the disulfide bond is intact andt hat its cleavage causesad ecrease in FRET and al arge increasei nd onor emission intensity ( Figure S1).

Confocal microscopy in fixed platelets and megakaryocytes
We investigated the synthesised compounds' efficiency as actin probesb yc onfocal microscopy in fixed human platelets, aiming to achieve al evel of actin staining that is comparable with that observedf or Alexa Fluor 488 Phalloidin. Figure 1A shows phalloidin marking distinctive actin organisations in platelets, such as stress fibres, lamellipodia and filopodia.
Suspensionso fw ashed human platelets( 210 7 cells mL À1 ) in Tyrode's buffer were incubated with differentc arrier-Lifeact systems( concentrationso fi ncubation between 0.5a nd 10 mm were investigated), transferred onto cover slips for spreading on fibrinogen and fixed. Detection of FAMa nd TAMRA emissions by confocal microscopy allowed visualisation of actin stainingb yL ifeact andt he distribution of the carriers in cells, respectively.T he image in Figure 1B confirms that Lifeact(FAM) (1)a lone does not penetrate cellsw ithout an appropriate carrier,a sn oa ctin staining or any other fluorescencew as observed, although plateletss preading was unaffected (Figure 1C). Only al arge increase in both gain and laser power allowed visualisationo fw eak fluorescencet hat could not be recognised as actin filaments taining (Figure1D). Figure 1E-L shows fluorescencei mageso fp latelets treated with 5 (4 mm,E and F), 6 (4 mm,Gand H) and 7 (0.5 mm,Iand J). The emission of Lifeact(FAM) and carrier(TAMRA) are on the left and right, respectively,a nd representative enlargements are indicated. By employing both Myr-S-S-Life and pHLIP-S-S-Life ( Figure 1E and G), we observed normals preading of platelets and typical stainingo fc ommon F-actin structures (stress fibres, lamellipodia and filopodia);t his is comparable to the staininga chieved with phalloidin ( Figure 1A). In both cases, carriers(TAMRA) (Figure 1F and H) are present insidec ells, but clearly separate from Lifeact(FAM) and not involved in actin staining, thus indicating that cleavage betweenc arriers and Lifeact had occurred (additional images in Figures S2 and S3). In contrast, TAT-S-S-Life significantly affects platelet viability;e ven at relatively low concentrations (1-2 mm)o fc ompound, more than 50 %o fc ells are incorrectly spread on fibrinogen ( Figure S2 O-R). At lower concentrations (0.5-1 mm,n ecessary to observe emission), platelets treated with this probe presentareduced mean surface area compared to controlsa nd to cells incubated with the other two probes. In addition, both cleavageand actin staining are uncertain as Lifeact(FAM) ( Figure 1I)a nd TAT(TAMRA) staining ( Figures 1J and S2) appear to overlap.
The procedure employedf or sample preparation is based on preincubation of cells with the probes, thereby proving that 1) the new compounds do not need ap ermeabilisation step, which is necessary for penetration of phalloidin, and 2) cell spreading is not influencedb ypreincubation with the new systems. However,a safurther control,w ep repared slides according to the same procedure employed for platelets stainedw ith phalloidin, in which spread cells were fixed, permeabiliseda nd treated with the carrier-Lifeacts ystems; no significant improvement in actin staining was observed ( Figure S4). Ar ange of incubation times (0-45 min) was also explored;t his showed that images of plateletsp reincubated with the probe for 45 min prior spreading are brighter;h owever,s atisfactory staining was also observed when using incubation times of a few minutes ( Figure S5), thus indicating fast uptake. We decided to keep incubationt imes of 30 min for all our experiments.
In order to characterise and compare Myr-S-S-Life and pHLIP-S-S-Life, we quantified both florescence intensities and areas of relevant regions of the cells. In particular, we observed that both myr and pHLIP carriers accumulated in very distinct regions of the cells, possibly in membrane-dense regions. We selected and analysed these "carrier-dense" regions (black lines in Figure 2A)u sing as threshold ar atio between meanT AMRA intensities of carrier-dense areas and mean TAMRA intensities of the corresponding entire cell above 1.5 (for each selected cell). The chart in Figure 2B reports the percentage area of selected carrier-dense regions relative to the cell area at different concentrations of Myr-S-S-Life and pHLIP-S-S-Life. Generally, "myr-dense" areas are 1.9 times larger than "pHLIP-dense" areas at the lowest concentration of probe and 2.8 times at the highest concentration,t hus indicating that myristoylation introduces ah igherc oncentration of carriers tack in cells. Furthermore, the concentration of compound during incubation affects myr-dense areas (threefold increase overt he concentration range of 0.5 to 10 mm)m ore than pHLIP-dense areas (twofold increase over the same range). The higher accumulation of myr in platelets could be simply the consequenceo f ah igherc ell uptake of myr-Lifeact hybrid. Alternatively, the differentc ell distribution observed is consistentw ith proposed theories about the internalisation processes of these carriers. Myristoylated carriers are believed to penetrate cells based on their affinity for lipid cell membranes, and the concentrationdependenta ccumulation that we observe in plateletss uggests that the vector remainsa nchored at the platelets' membranes. This is also in agreement with studies in which myristoylation and palmitoylation were employed to enhancet he activity of anti-platelet drugs targeting receptors at the inner leaflet of transmembrane proteins. [16c, 20] As ac ontrast, we incubated platelets with pHLIP-S-S-Life at pH 6.5 to induce carrierp enetration and cargo delivery,f ollowed by washes at pH 7.4 (controls confirmed that this does not affect cell spreading), which should promote release of membrane-attached carrier back to the extracellulare nvironment. [12] This would explain why pHLIPa ccumulationi sl ower than that of myr.W ea lso observeds ignificant delocalisation between FAMa nd TAMRA emission signals in myr-dense regions, whereas the two fluorophores partially colocalise in pHLIP-dense regions (compare images in Figure 1E and F, and Ga nd H; more details are reportedi nF iguresS2, S3 and S6); this suggests that release of the cargo-Lifeact is more efficient when employing myristoylation rathert han pHLIP.T his observation is important because detection of Lifeact(FAM) bound to the carrier and not involved in actin staining might lead to misinterpretation in actin studies;h owever,l abelling the carrier allowed uncleaved Lifeact to be identified in relativelys mall regions (between 3a nd 8% of total cell area);t his did not influ-ence the mean emission intensityo fF AM ( Figure S7). Measurements of mean emissioni ntensity of Lifeact(FAM) inside cells ( Figure 2C), associated with actin staining efficiency,i ndicate that the brightnesso ft he staining improves when the concentrationso fb oth Myr-S-S-Life and pHLIP-S-S-Life are increased, but higher emission intensitiesw ith lower concentrationso f probe are achieved with Myr-S-S-Life (additional controls in Figure S7).
We treated human platelets with the uncleavable derivatives Myr-Life (8;F igure 3A and B) and pHLIP-Life (9;F igure 3C and D);n oa ctin staining was observed in either case (0.5-10 mm concentrations of compounds were explored), thusi ndicating that releaseofLifeact is essential for its binding with actin. Furthermore, 8 affects platelet spreading even at 1 mm (Figure3B). As imilar pattern of resultsw as observedw hen platelets were treated with carriers only:M yr(TAMRA) (2;F igure 3E and F) severely affected cell spreading, althought his could be ac onsequenceofcarrier insolubility when it is not bound to apeptide, although pHLIP(TAMRA) (3;F igure 3G and H) is randomly distributed in correctly spread cells. Images in Figure 3A and Br einforce the hypothesis that Myr remains anchored at the membrane in such aw ay that its covalentl ink with the actin binder affects cell viability.I nc ontrast, the transmembranef ragment pHLIP is unable to diffuse into the cytosol, as expected, but the covalentb inding with Lifeact does not interferew ith cell spreading, probablyd ue to ad ifferent motilityo fthe system in membranes. We also observed that accumulation of both 9 and 3 ( Figure 3C and G) is broad and random rather than concentrated in relativelys mall pHLIP-dense regions (as is that of the cleavable pHLIP-S-S-Life);t his suggests that the presence of ac leavable cargo influencesp enetration and/orr eleaseo f the carrier.
Uncleavable TAT-Life (10)a nd carrier TAT(TAMRA) (4)w ere also tested in platelets.L ike the analogous cleavable system, both compoundsp revented cell spreading at low concentra- www.chembiochem.org tions, and staining by TAT-Life had no clear definition (Figure S8), thus confirming the incompatibility of the polycationic TATw ith platelets. Thef ailure of ac ell-penetrating system could dependo nacombinationo ff actors( e.g.,t he cargo-carrier-fluorophore combination,t he type of linker,t he type of cell and its set of surfacer eceptors). However,o ur images show that all TAT-containing compounds affect platelets preading;t his suggestst hat TATi st he disturbingc omponent. There are af ew studies describing how polycationic-containings ystems (including polylysine and TAT) influence both platelet aggregation anda ctivation, either by adhering to the negatively chargedp latelet membranes and forming bridgesb etween adjacent cells or by interfering with specific membrane receptors. [20,21] These might be related to the behaviour that we observe,a nd this aspect should be taken into consideration when planningc argo deliveryi np latelets employing polycationic carriers.
Since 5 and 6 are promisinga ctin markers for platelets,w e tested their efficiency in megakaryocytes (MKs), which are responsible for plateletp roduction.F igure 4A and Bs hows two representative examples of MKsp retreated with 5 and 6,r espectively,s pread on fibrinogen and fixed. Actin staining was observedf or compounds (Lifact(FAM) emission images on the left), including marking of typical podosome structures (indicated in the Figure 4A). [22] As observed in platelets, myristoylated carrieri sd enselya ccumulated in cells (carrier(TAMRA) emission images in the middle), whereas the emission of pHLIP is significantly lower.D ue to the highest complexity of actin filament organisation in thesec ells, colocalisation between carriers and Lifeact is complex, and FRET microscopy is important for interpretation:t he images on the right wereo btainedb y detecting TAMRA emission caused by energy transfer upon excitation of FAMa t4 88 nm;t his occurs only if Lifeact is anchored to the carrier. FRET intensity was higher than FAMe mission intensity in cells treated with 5 ( Figure 4A), thus indicating al arge amounto fu ncleaved compound in cells. Instead, only low FRET was detected in MKs treated with 6 ( Figure 4B), thus indicatingl ow accumulation of uncleaved conjugate (see analysisinF igure 4C).
In summary,w ef ound that the known cell-penetrating system TATi sn ot as uitable carrierf or platelets, whereas both Myr-S-S-Life (5)a nd pHLIP-S-S-Life (6)a re efficient probesi n both fixed platelets and MKs, displaying F-actins taining properties that are comparable to those of phalloidin. Ac ytosolic cleavable linker between the carrier and Lifeact is essential to achieve actin staining, and incubation of the cells with the probe (up to 10-20 mm)d oes not influence their correct spreading on fibrinogen.I nt he case of myristoylation, cleavage from the cargo is also essential to avoid inhibitiono fc ell spreading. Both myr and pHLIP concentrate in carrier-dense regions in platelets;t hese are larger when myristoylation is employed (~15 %o ft he cell area at 4 mm), although uncleaved Lifeact was not observed. pHLIP-dense regions are significantly reduced compared to myr-dense regions, and al ow percentage of uncleaved Lifeact(FAM) was detected, although this did not influence total Lifeact(FAM) emission intensity,r elatedt o actin staining, in cells. pHLIP-S-S-Life is also able to deliver and releaseL ifeact in MKs, whereas Myr-S-S-Life is not suitable for these cells, due to the significant accumulationo fu ncleaved compound, which would lead to misinterpretation of signals from Lifeact(FAM). Further designso fa nalogousc ompounds for MKs should consideri nserting the cleavable bond in am ore extended linker to facilitate intracellularcleavage.

Real-timeimaging in platelets.
Achieving real-time images of F-actin,e specially in difficult and important targets such as human platelets, is ac riticalc hallenge in cell microscopy.W ep erformed live imaging experiments with 5 and 6,b ut the resultsw ere not satisfactory.A lthoughr eflection images display correctly spreading cells and compound was detected in cell areas, delocalisation between carriers and Lifeact was not observed, and actin staining com- www.chembiochem.org parable to that observed in mice platelets by Lifeact-GFP [7] was not achieved (Figures 5and S9).
To verify whether cleavage of compound occurs in the presence of live cells, we measured FRET changes in 5 in the presence of plateletsb yf luorescences cans ( Figure 6). In the control experiment in Tyrode's buffer and in the absence of platelets, upon excitation at 488 nm, the uncleaved compound presentsastable emission profile (monitoredf or 20 min) with two bands corresponding to FAM( the donor) and TAMRA (the acceptor) at 520 and5 80 nm, respectively,a saconsequence of energy transfer.A dding increasing concentrations of platelets to as olution of Myr-S-S-Life (1 mm)i nT yrode's buffer,i nduces an increasei nF AM emission and ad ecrease in TAMRA emission, with the anticorrelation that is typical for FRET decrease ( Figure 6A and B). In ap arallel experiment ( Figure 6C), we prepared separates uspensions of human platelets (2 10 7 cells mL À1 )i nT yrode's buffer with different concentrations of Myr-S-S-Life:a tl ower concentration of probe, we only ob-servedF AM emission, thusi ndicating that most of the compound in solutioni sc leaved and confirming that platelets cleave the disulfide bond. At the highest concentration, we observed both FAMa nd TAMRA emission;t his must indicate extracellular excesso fc ompound. These experiments demonstrate rapid cleavage of compound in the presence of living platelets(changes in emission were detected immediatelya fter additions)a nd inside cells, as ar eductive environmenti sn ecessary for disulfide bond rupture.
Nevertheless,d espite the images of fixed cells above and the fact that cleavageo ccurs as planned in platelets, live cell images remainu nsatisfactory.D ue to ac ombination of factors, images of actin in live transgenic mouse plateletsl abelledw ith Lifeact-GFP are not nearly as cleara si nf ixed cell samples: [7] the kinetics of the actin assembling/disassembling process, the reversible nature of the actin-Lifeact interaction, background issues particularly influential in such small cells and the physical-chemical properties of the fluorophore are all key components affecting the quality of images of F-actin in living platelets. Although we do not expect comparable quality between liveand fixed-cell images, these synthetic Lifeact vectors do not give stainingt hat is comparable to Lifeact-GFP,a nd af ew explanations are possible.
The delivery process might not be complete or adequate, and af ixation step might be necessary to promote access to the actin. However,i ft his were the case, we might expect fixation to have the same beneficial effecto nu ncleavable Myr-Life or Lifeact only,whichitd oes not. Moreover,the live-cell experiments in Figure 6c learly indicatet hat cytosolic release of Lifeact(FAM) occurs and that it is complete when suspensions of platelets( 210 7 cells mL À1 )a re treated with concentrations of hybrids < 1 mm.
Alternativelyt he Lifeact-FAM combination might not be the most appropriate marker in terms of affinityfor F-actin:the position and/ort he nature of the fluorophore and/or the linker betweent he fluorophore andL ifeact might affect the interaction with F-actin resulting in poor visualisation of dynamic events in real time. For example, Lukinavicius et al. identified one efficient actin marker for live-cell imaging among al ibrary  www.chembiochem.org of many derivatives, and modifications to the linker between the fluorophore and the actin binder were key determinants of activity. [9b] An et al. used pHLIP to deliver phalloidin into cancer cells as an antiproliferatived rug and observed that the presence of fluorophore on both carriera nd cargo strongly influenced the efficiency of the antiproliferative activity. [23] Finally, the plant-cell-specific cell-penetrating carrier-Lifeacth ybrid described by Eggenberger et al. [10] highlightst he importance of the positiono ft he fluorophore/carrier relative to the Lifeact sequence and suggestst hat involving the Cterminus of Lifeact in the conjugation might affect the binding affinity with actin.
Thus, these myr-and pHLIP-Lifeactc onjugates are suitable as scaffolds for further elaboration and optimisation that might enable live-as well as fixed-cell imaging. However, future design aimingt oe nhancet he affinity of the probe for F-actin must be undertaken with caution, as this might also affect the kinetics of actin assembly/disassembly, and the measurement of "biological" real time, which is the ultimate goal.

Conclusions
There is ag reat interesti nd eveloping new methods to visualise F-actin dynamics by cell microscopy,e specially because current methods cannot be appliedt oa ll cell types or rely on the use of synthetic markers that are toxic or difficult to modify. We have designed new systems for actin stainingw ith improveds ynthetic accessibility by employing the 17-residue se-quenceLifeact as the actin-binding component so as to ensure low interference with actin filaments and improve toxicity. We conjugated Lifeact to three different cell-penetrating carriers and investigated their ability to promotet he delivery and release of Lifeact, and consequently actin staining, in difficult targets such as human platelets and megakaryocytes. We found that the cationic carrier TATi sn ot suitablef or these cells, whereas both am yristoylated carrier and pHLIP,apH-responsive peptidic vector,p romote Lifeact insertion without affecting cell spreading. Actin staining,c omparable with that of commercially available phalloidin was observed in fluorescent images of fixed cells upon cytosolic releaseo ft he cargo-Lifeact. We highlight the importance of both carriera nd cargo labelling, possibly with ap air of fluorophores able to provide a cleavage-responsive signal such as FRET,a st ools to achieve a better understanding of our hybrids.
Our studies confirm that bringing together delivery vectors, cleavage sites and labelled Lifeact by employing fast and versatile synthetic procedures,p rovide a" plug and play" accessible strategy useful for simplifying the design of new actin markersw ith customisablep hysical, chemical and biological features. This valuable approach might facilitate the screening and identification of systems suitable for real-time images, bearing in mind that the binding affinity of potential probes with actin filaments, which is necessary for satisfactory visualisation of dynamic events, must not affect their kinetics.

Experimental Section
Experimental procedures and instruments employed are detailed in the Supporting Information. Here we briefly describe principal methods employed.
Synthesis:T he following peptides were purchased from Peptide Protein Research Ltd. (Fareham, UK): Lifeact (1), pHLIP(TAMRA) (3) and TAT(TAMRA) (4)w ere supplied with certified purity > 80 %a nd employed for the following disulfide-exchange steps without further purification;t he uncleavable systems Myr-Life (8), pHLIP-Life (9)a nd TAT-Life (10)w ere supplied with certified purity > 98 %a nd employed for cell microscopy studies without further purification. Both FAMa nd TAMRA were conjugated at the a-amino group of the C-terminal lysine residue.
Cell microscopy.S uspensions of washed platelets (300 mL, 2 10 7 cells mL À1 )w ere preincubated with different volumes of probe solutions (150 mm,0 .5-10 mm concentration of probe during incubation was the range explored) in Tyrode'sb uffer containing glucose (5 mm,p H7.4) at 37 8Cf or 30 min. Suspensions were transferred onto glass coverslips precoated with fibrinogen and allowed to spread at 37 8Cf or 45 min. For pHLIP-Lifeact systems, incubation and spreading were in Tyrode's buffer containing glucose (5 mm)a tp H6.5. Spread cells were washed with phosphate-buffered saline (PBS), fixed with 10 %f ormalin, treated with NH 4 Cl 2 (50 mm), washed again with PBS and mounted on slides for microscopy.T he same procedure was employed to stain MKs, except that cell suspensions of 510 8 cells mL À1 were employed for incubation and spreading was allowed in medium (and not buffer) for 3h prior to fixation and mounting. For control platelets stained with phalloidin, platelet suspension (2 10 7 cells mL À1 in Tyrode's buffer) was allowed to spread on glass coverslips precoated with fibrinogen for 45 min at 37 8C, fixed with formalin, permeabilised with 0.1 %T riton X-100 in PBS and stained with Alexa Fluor 488 Phalloidin (15 nm)f or 30 min at room temperature. Stained cells were washed with PBS and deionised water and mounted on slides for microscopy.
To allow proper comparison between different compounds, the same microscope parameters (PMT,e nlargement, laser power) were employed in all the experiments (unless indicated differently). Samples were imaged by using the 488 (Ar/ArKr laser), 543 and 633 nm (He/Ne laser) laser lines on aL eica DMIRE 2l aser scanning confocal microscope with 63 , 1.4 N/A oil objective. Single images were collected by selecting the best optical plane (i.e.,t he one that looked most in focus) and using averaging (3 scan accumulations) to improve S/N. Post-imaging analyses (cell selections, mean area and mean fluorescence intensity calculations) were performed by using ImageJ 1.48.