A unique profilin-actin interface is important for malaria parasite motility
[['Motility of adherent cells is important for both uni- and multicellular organisms .', 'In higher eukaryotic cells , motility plays a key role in , for example embryo- and organogenesis [1] , immune responses [2] , and cancer metastasis [3] .', 'Cell motility depends on an actin-myosin motor that is linked via transmembrane proteins to the substrate [4] ( S1 Fig ) .', 'In unicellular Plasmodium parasites that cause malaria , motility is essential for the progression through their complex life cycle [5] .', 'The active substrate-dependent gliding motility of these parasites is essential for them to enter red blood cells , to pass through the epithelium of the mosquito midgut , and for transmission from the mosquito to the vertebrate host [5] .', 'While the red blood cell entering merozoites are motile for just a few seconds [6] , the midgut penetrating ookinetes can move for tens of minutes at approximately 5 μm/min [7–9]—almost as fast as neutrophils of the innate immune system [10] .', 'However , the mosquito-transmitted sporozoites are the fastest form of the parasite , moving with average speeds of 1–2 μm/s for tens of minutes [11] .', 'Sporozoites are formed in oocysts at the midgut wall and actively enter the salivary gland of the mosquito , where they can stay for weeks prior to transmission via a mosquito bite into the dermis of the vertebrate host [12] .', 'In the Plasmodium spp .', 'infecting mammals , the sporozoites first migrate rapidly within the dermis and subsequently enter either the blood or the lymph vessels [13] .', 'Those entering the blood stream adhere to the liver endothelium and actively penetrate the liver parenchyma to infect hepatocytes [14] .', 'Apicomplexan gliding motility relies on an actin-myosin motor , which resides in the narrow space delimited by the inner membrane complex ( IMC ) , a flattened post-Golgi organelle , subtending the plasma membrane [15–17] .', 'While myosin is anchored to the IMC , actin filaments appear to be anchored to plasma membrane traversing adhesins [18] .', 'These provide the link between the actin filaments and the substratum [16 , 17] and are essential for force transmission [19 , 20] .', 'Parasites can exert forces on various substrates in the range of 100 pN , which has been measured in vitro for sporozoites using traction force microscopy and optical traps [20 , 21] .', 'Efficient motility of the different stages of Plasmodium as well as related parasites involves unusually dynamic and short actin filaments [17 , 21–24] .', 'These remarkable properties are due to a structural divergence of the parasite actin from canonical actins [22–25] as well as a small set of regulatory actin-binding proteins , which often have non-canonical functions [26–28] .', 'Compared to higher eukaryotic cells , there are only a few actin-binding proteins encoded in the Plasmodium genomes [26–29] .', 'Gene deletion studies have shown that at least two of these proteins are not needed for the entry of merozoites into red blood cells , while they are important for the motility of ookinetes and/or sporozoites [30 , 31] .', 'Parasites lacking one subunit of the actin capping protein [31] have reduced ookinete and sporozoite motility and ultimately fail to enter the salivary glands .', 'The actin filament binding protein coronin [32] is only expressed in P . berghei sporozoites and coronin knockout ( coronin ( - ) ) sporozoites are not motile in vitro and are impaired in salivary gland invasion [30] .', 'Curiously , coronin ( - ) sporozoites deposited into the skin by mosquito bites migrate like wild-type sporozoites [30] .', 'Profilin , one of the core actin regulators , has been suggested to be essential in the blood stage , where transfection and selection of transgenic parasites is performed [33] .', 'Indeed , conditional down-regulation of profilin , likely leading to a defect in merozoite invasion of red blood cells , suggested that it is important for efficient blood stage growth [34] , while overexpression had no effect on the blood stage [35] .', 'Canonical profilins bind monomeric ADP-actin , catalyze ADP-to-ATP exchange , and enhance actin polymerization in conjunction with the actin nucleators of the formin family [36] .', 'Importantly , apicomplexan formins contain only rudimentary or no profilin-binding motifs , and both P . falciparum and T . gondii profilins appear to sequester actin monomers also in the presence of formins [37 , 38] .', 'Furthermore , T . gondii profilin inhibits , rather than accelerates , nucleotide exchange on actin monomers [38 , 39] .', 'Thus , it was suggested that T . gondii profilin functions mainly as an actin monomer sequestering protein , possibly limiting actin polymerization in vivo [38] .', 'Profilin may have evolved via gene fusion from two smaller proteins [40] .', 'Subsequent divergent evolution seems to have resulted in two structurally very different branches: the classical profilins in most organisms as well as the apicomplexan ones with a large sequence insertion at the border of the two small ancestral proteins [33 , 40] .', 'This insertion , a unique arm-like ß-hairpin domain seems optimally positioned to participate in actin binding [33 , 39] .', 'However , a possible functional contribution of this motif in both actin binding and parasite motility remained uncharacterized so far .', 'To probe this potential interaction and to investigate the structure-function relationships of profilin across the Plasmodium life cycle , we generated a series of transgenic parasites , featuring two mutations at the tip of the ß-hairpin .', 'We further expressed the corresponding proteins in vitro and show that the arm-like domain contributes critically to the actin-binding capacity of Plasmodium profilin .', 'Interestingly , this additional motif appears important only during the rapid motility of sporozoites , where a weakened profilin-actin interaction leads to a decrease in their capacity to generate force .', 'Strikingly , one mutant still retained continuous gliding motility , while the other one lost this ability , although for both mutants , the capacity to produce force was similarly weak .', 'Molecular dynamics simulations suggested differences in the actin-binding mode of the two mutants , which appears to be at the core of the different gliding phenotypes .', 'Taken together , our data show how sporozoites fine-tune actin dynamics to achieve gliding motility .'], ['P . berghei profilin has previously been shown by RT-PCR analysis to be expressed across the entire parasite life cycle [33] .', 'To investigate profilin protein abundance and localization across the life cycle , we endogenously tagged profilin with mCherry ( S2 Fig ) .', 'Since the profilin gene consists of 4 exons and 3 introns , we measured expression of profilin-mCherry constructs with and without introns ( Pfn-i mCh and Pfn+i mCh ) , as these could influence gene expression [41 , 42] .', 'The fluorescent parasites progressed through the life cycle in a manner largely comparable to wild-type parasites , suggesting that the mCherry fusion did not detectably impair parasite viability ( Table 1 ) .', 'Western blot analysis showed that profilin-mCherry migrated at the expected size of the fusion protein ( S3A Fig ) .', 'Imaging parasites at the ring , trophozoite , and sporozoite stages revealed uniform profilin localization in the cytosol and the nucleus , while in gametocytes and ookinetes the profilin-mCherry fluorescence was more pronounced in the nucleus ( S3B Fig ) .', 'Profilin localization is consistent with the localization of GFP-actin , which was also found in the cytosol and the nucleus [43] .', 'Addition of actin filament stabilizing ( jasplakinolide ) or depolymerizing ( cytochalasin D ) compounds did not alter the localization of profilin , as expected for an actin monomer binding protein .', 'Quantitative analysis of images from different stages showed that the profilin-mCherry expression levels varied slightly across the stages .', 'The only significant differences in expression depending on the presence or absence of introns occurred in the blood stages ( Fig 1A ) .', 'Intriguingly , the parasites lacking introns in the profilin gene grew slightly more slowly in the blood stage ( Table 1 ) and reached slightly larger cell sizes of up to 45 μm3 ( S3C and S3D Fig ) .', 'The fluorescence intensity of Pfn+i mCh and Pfn-i mCh motile ookinetes also appeared slightly different ( p = 0 . 06 ) , yet the Pfn expression in sporozoites was not affected ( Fig 1A and 1B ) .', 'Curiously , while the speed of motile ookinetes was not affected ( Fig 1C ) , the speed of sporozoites expressing mCherry-tagged profilin both with and without introns was significantly reduced ( Fig 1D ) .', 'This suggests steric hindrance by the mCherry tag that only has an effect on motile sporozoites .', 'The speed of sporozoites could be restored to wild type levels in parasites that expressed a longer linker between profilin and mCherry ( Pfn // mCh ) ( Fig 1D ) .', 'However , in these parasites , the mCherry tag was largely cleaved off , leading to a majority of non-tagged profilin ( Fig 1E ) .', 'In all tagged lines , Pfn expression was under the control of the endogenous promoter and the 3’UTR from PbDHFR , suggesting that the 3’UTR did not influence the level of profilin expression ( S2A Fig ) .', 'These data highlight the importance of carefully evaluating the impact of large fluorescent tags on protein functionality .', 'As human profilin has previously been shown to partially compensate for P . berghei profilin depletion [33] , we hypothesized that P . falciparum profilin should also compensate for P . berghei profilin depletion .', 'We reasoned that this would enable detailed mutational analysis in a more straightforward fashion ( see methods ) and with the protein of a disease-relevant parasite species that has already been expressed and analyzed in vitro [33] .', 'Considering also the lack of striking differences in expression in the presence or absence of introns , we thus generated a replacement plasmid using P . falciparum cDNA and obtained a parasite line that showed no difference to wild type P . berghei parasites across the life cycle ( S2 Fig , Table 1 ) , suggesting that the two profilin orthologues are functionally interchangeable .', 'The structure of Pf Pfn suggested that the parasite-specific hairpin and in particular the acidic residues at the tip of this loop might be involved in interactions with actin , presenting a unique actin-profilin interface [33] .', 'The profilin-binding surface in Pf actin 1 is conserved with canonical actins .', 'However , the actin-binding surface in Pf Pfn is remarkably diverged .', 'To test whether Pf profilin binds to actin at the canonical interface , we expressed Pf profilin in E . coli and purified the protein ( see methods ) .', 'Incubation at a 1:1 ratio with pig muscle actin allowed investigation of complex formation by small-angle X-ray scattering .', 'We found that the shape of the complex corresponds to that of canonical profilin-actin complexes ( S4 Fig ) .', 'Furthermore , increasing concentrations of Pf Pfn increase the fluorescence signal of the pyrene-labelled Cys located in the C-terminal helix of actin at the canonical profilin binding site ( S5A and S5B Fig ) , indicating that Pf Pfn binds to the canonical binding site on actin .', 'To investigate the parasite actin-profilin interface in more detail , we constructed a homology model of the Pf Pfn-actin complex and used it for molecular dynamics simulations .', 'P . berghei actin and P . falciparum actin share 99 . 2% sequence identity , differing at only three residues: E3D , D5E and V11I , meaning that their electrostatic properties at the profilin binding face are very similar .', 'We first analyzed the electrostatic properties of the modelled interface between Plasmodium actin and P . falciparum profilin ( Pf Pfn ) .', 'The complex can be superimposed on the rabbit actin-human profilin complex ( PDB ID: 2PAV ) [44] , but there are remarkably large differences in the two profilin-actin complexes .', 'In addition to the arm-like β-hairpin insertion in Pf Pfn ( Fig 2A ) , the lack of the short α-helix 3 and the much longer β-strands 4 and 5 in Pf Pfn cause significant differences at the interface .', 'As hypothesized before , the acidic residues 64EDE66 , conserved across Plasmodium spp .', ', at the tip of the parasite-specific arm ( Fig 2B ) are in close contact with a positively charged surface patch on subdomain 3 of actin in our model , suggesting the possibility to form a strong ionic ( salt-bridge ) interaction ( Fig 2A , S1 Movie ) .', 'The actin residues contributing to this positive patch include K284 , R291 , K292 , Y295 and T325 ( numbering as per Pf actin 1 ) and are conserved between Plasmodium and vertebrate skeletal muscle α-actin , suggesting that Pf Pfn can engage in similar interactions with both actins .', 'To get more insight into how the acidic residues of the arm motif in Pf Pfn contribute to actin binding , we simulated actin-profilin complexes with two mutant versions , in which the residues 64EDE66 were exchanged to QNQ or AAA ( Fig 2B , S2 Movie ) .', 'These mutations were chosen in order to weaken ( QNQ ) or abolish ( AAA ) the interactions of the acidic arm motif with actin .', 'We analyzed the inter-protein hydrogen-bond occupancy during simulations for three interaction regions of the protein-protein interfaces: the arm-specific interaction ( shown in red in Fig 2A and 2C ) ; arm-neighbor interactions ( shown in green in Fig 2A right panel , C ) ; and the conventional actin-profilin interface interactions ( shown in blue in Fig 2A right panel , C ) .', 'At the tip of the arm motif , E66 of Pf Pfn makes two strong hydrogen bonds with R291 of actin with the highest occupancy ( 181% out of a possible 200% occupancy for 2 hydrogen bonds , individually 90% and 91% ) of all the hydrogen bonds at the actin-profilin interface ( Fig 2C ) .', 'In the QNQ mutant , Q66 lacks the negative charge and therefore loses the salt-bridge nature of this interaction ( S1 Movie ) .', 'Nevertheless , Q66 of the QNQ mutant profilin can form a single hydrogen bond with actin-R291 ( 52% occupancy ) ( Fig 2C ) .', 'On the other hand , the AAA mutant as predicted is unable to make any arm-mediated ionic or hydrogen-bonding interactions at the actin-profilin interface ( Fig 2C and 2D , S2 Movie ) .', 'In contrast to the profilin arm-mediated interactions , the hydrogen bonds in the vicinity of the Pfn arm are preserved in the two mutants .', 'Surprisingly , the hydrogen-bonding residue-pairs significantly differ at the interface formed by β-strand 4 of Pf Pfn ( Fig 2C ) , and the AAA mutant has the most divergent hydrogen-bonding pattern in this region .', 'For example , while E111 of Pf Pfn makes strong hydrogen bonds ( 103% ) with actin R373 , these have lower occupancy ( 80% ) in the QNQ mutant and are absent in the AAA mutant .', 'However , in the AAA mutant , E111 forms hydrogen bonds with K114 ( 79% ) and H372 ( 29% ) in actin .', 'The loss of these hydrogen bonds in the QNQ mutant is compensated by an additional stronger hydrogen bond: E107 with actin-K114 ( 56% ) ( S6 Fig ) .', 'The weakening of the interaction with actin for the AAA mutant , due to the lacking hydrogen bonds involving the tip of the β-hairpin loop , is also visualized by molecular dynamics simulations , during which the arm region in the AAA mutant is destabilized and moves away from actin ( Fig 2D , S2 Movie ) .', 'This destabilization further weakens the interactions at the conventional actin-profilin interface involving β-strand 4 ( Fig 2C , blue bars ) .', 'We finally computed the relative free energy values ( lacking the full entropic contribution ) for the wild type and mutant Pf Pfn-actin complexes ( Table 2 ) .', 'This suggests that wild type Pf Pfn binds most strongly to actin ( MM-PBSA free energy: -64 . 2 ± 11 . 2 kcal/mol; similar values and the same trends were obtained with the MM-GBSA method ) .', 'The QNQ mutant has a less favorable binding free energy ( -44 . 2 ± 12 . 8 kcal/mol ) , which is in line with its weaker H-bond interactions .', 'The AAA mutant is the least energetically stable ( -34 . 4 ± 11 . 1 kcal/mol ) of the complexes , which may be due to the instability of the arm region which further destabilizes other interactions at the actin-profilin interface .', 'To investigate whether the differences suggested by the simulations can lead to biochemically detectable differences in actin binding of the mutants , we next characterized the effect of recombinant P . falciparum profilin and both mutants on the polymerization kinetics of vertebrate skeletal muscle α-actin .', 'Like wild-type Pf Pfn , both QNQ and AAA mutants could be purified as monomeric , soluble , and folded proteins ( S7A and S7B Fig ) .', 'Whereas wild-type Pf Pfn drastically reduced the rate of canonical actin polymerization , both mutants showed a significantly reduced effect ( Fig 3 ) .', 'The weaker binding of the mutants to actin was also seen in the lack of increased initial fluorescence ( S5C and S5D Fig ) .', 'We next assessed , whether the same would be true for the biologically relevant binding partner , Pf actin 1 .', 'Using native PAGE , complex formation was observed for wild type but not for the mutant profilins ( Fig 4A ) .', 'However , intriguingly , the effects of Pf Pfn on canonical and parasite actin polymerization kinetics were different .', 'While Pf Pfn significantly reduced the rate of elongation for both actins , it had little effect on the steady-state of α-actin ( Fig 3A ) , but a clear monomer-sequestering activity on P . falciparum actin 1 ( Fig 4A and 4B ) .', 'The mutated profilins showed a weakened capacity for reducing the rate of polymerization as well as monomer sequestering ( Figs 3 and 4 ) .', 'However , there was no clear difference in the effects of the QNQ and AAA mutants , especially on parasite actin ( Fig 4 ) , suggesting that even the relatively subtle differences in the binding of the QNQ mutant compared to the wild type Pf Pfn are sufficient for significantly reducing the binding affinity to actin .', 'To address whether the mutations would lead to a detectable effect in vivo , we generated parasites expressing the respective P . falciparum profilins in place of the single endogenous P . berghei profilin gene ( S2 Fig ) and compared them to wild type P . berghei parasites .', 'Expression of the QNQ and AAA mutants in the parasite had no detrimental effect on life cycle progression ( Table 1 ) .', 'Whereas only minor differences in the speed of wild type and transgenic ookinetes were observed ( Fig 5A–5C ) , a striking effect was seen in sporozoites ( Fig 5D–5I ) .', 'We found no difference between the wild type and QNQ sporozoites in their capacity to glide on a flat substrate , but the AAA mutants largely failed to move progressively ( Fig 5D–5G ) .', 'Curiously , Pf Pfn-expressing sporozoites moved faster but less persistently than wild type P . berghei sporozoites , and this was also seen for the QNQ mutant ( Fig 5G and 5H ) .', 'The differences in speed were even more obvious when instantaneous speeds were compared ( Fig 5I ) .', 'Interestingly , also the AAA mutant ookinetes moved at lower speeds than the QNQ mutant ookinetes ( Fig 5B ) .', 'Together with the biochemical data and the molecular dynamics simulations , these data on sporozoite motility confirm that the parasite-specific arm in Pf Pfn is crucial for actin binding and , thereby , sporozoite motility .', 'It is interesting that both simulations and motility assays show differences between the QNQ and AAA mutants , but these are not obvious from the biochemical assays , suggesting that the subtle differences in the two mutants are more complicated than a simple effect on binding affinity .', 'Mutations of the unique profilin arm clearly emphasize the importance of this region in facilitating efficient sporozoite motility .', 'However , the phenotypic characterization described so far , does not yet lead to a functional understanding of how profilin-actin interactions modulate parasite motility .', 'To gain further functional understanding , we employed a recently-established laser trap assay [20] .', 'This allowed us to probe at defined forces the capacity of sporozoites to pull a micron-sized bead from an optical trap and to translocate it to the rear of the cell ( Fig 6 ) .', 'We presume that this pulling force corresponds to the force that sporozoites exert on their surrounding environment to move forward .', 'To measure this force , a bead was trapped in the laser focus and pushed onto the sporozoite surface , where it was captured by the sporozoite ( Fig 6A ) .', 'Subsequently , the sporozoite pulled the bead backwards , presumably due to the myosin-based translocation of actin filaments anchored via transmembrane adhesins [20] .', 'This translocation backwards thus most likely corresponds to the retrograde flow of actin filaments observed in fibroblasts and other cells and also assumed to occur in sporozoites [20 , 30 , 45] .', 'We probed our set of parasites with this assay and scored whether , at a given optical force , the sporozoite pulled the bead out of the trap or not .', 'In total , over 1 , 200 sporozoites were probed from 4 different parasite lines at 3 different optical forces ( Fig 6B–6D ) .', 'In this very sensitive assay , no difference was found between wild type P . berghei parasites and P . berghei parasites expressing P . falciparum profilin .', 'At an optical force of 70 pN , sporozoites from both lines were able to pull approximately 70% of the beads out of the trap , while at 130 pN , this percentage dropped to approximately 35% ( Fig 6B , [20] ) and at 190 pN to 20% [20] .', 'Both the AAA and the QNQ mutant were less efficient in pulling the bead from the trap at all forces ( Fig 6B ) .', 'At 70 pN , both mutant lines only managed to pull approximately 20% of beads out of the trap , and at 130 pN , this dropped further to approximately 10% .', 'To probe whether the mutations affect retrograde flow , we used the same laser trap setup .', 'However , in these experiments the force of the trap was set to approximately 10 pN , which was sufficiently small for the sporozoite to instantly move the bead towards the rear ( Fig 6A ) .', 'In all parasite lines , the beads were transported backwards with an increasing speed until a peak or plateau was reached ( Fig 6C ) .', 'To analyze potential differences in the transport speeds , we compared the maximum speed of over 170 sporozoites across the four different parasite lines .', 'We found no difference between the wild-type P . berghei and the line expressing wild type P . falciparum profilin ( Fig 6C and 6D ) .', 'However , parasites expressing the AAA and QNQ mutant profilins moved the beads at an increased speed , i . e . showed a faster retrograde flow ( Fig 6C and 6D ) .', 'Thus , while the QNQ mutant seems impaired in actin binding and force generation to a similar degree as the AAA mutant , there are subtle differences that lead to differences in the ability of the mutants to support fast , directional motility .', 'The molecular dynamics simulations suggest this could be due to differences in the off rates of the complexes , especially in the situation in vivo , where other proteins compete with profilin for actin binding .'], ['Profilins as ancient actin-binding proteins have typically low sequence identity but share a highly conserved 3D fold .', 'Apicomplexan profilins differ from all other profilins in containing a unique β-hairpin extension .', 'The exact length and sequence of this insertion is not fully conserved between different Apicomplexa , but they all share the acidic nature of the tip of this extension .', 'Here , we show that mutations in this unique motif in Plasmodium diminished actin monomer sequestration in vitro ( Figs 3 and 4 ) and led to impaired sporozoite motility ( Fig 5G–5I ) , while only slightly affecting ookinete motility ( Fig 5B and 5C ) .', 'This differential effect was already visible in the construction of profilin-mCherry fusions , which slightly impaired sporozoite motility while they had no effect on ookinete motility ( Fig 1C and 1D ) .', 'The defects were more accentuated in the mutant parasite lines analyzed .', 'This , together with previous data [31] suggests that for the evolution of the core motility machinery , the sporozoite presents the major constraining stage of the parasite , as it appears the most vulnerable to subtle changes .', 'This is likely due to the fact that the sporozoite has to migrate extremely fast , while passing several biologically very different tissue barriers .', 'Our data revealed an interesting dichotomy: while both QNQ and AAA mutants showed similar differences compared to wild type in our biochemical and biophysical assays , only the AAA mutant showed a defect in motility .', 'The molecular dynamics simulations might provide an answer for this intriguing difference .', 'They revealed a salt bridge with two hydrogen bonds that contributes to stabilizing the interaction between the arm-motif of profilin and actin ( Fig 2 , S1 Movie ) .', 'Interestingly the salt bridge is lost in both mutants , whereas one of the two hydrogen bonds is retained in the QNQ mutant but both hydrogen bonds are lost in the AAA mutant .', 'This suggests that the biochemical and biophysical assays were sensitive to the differences due to the loss of the salt bridge and one of the hydrogen bonds , whereas the motility assay revealed the difference upon losing the last hydrogen bond of the arm-motif .', 'This mechanistic insight would have not been revealed by simply studying defects in gliding motility and shows the power of the combination of biochemical with biophysical and computational assays .', 'The data suggests a threshold , such that a successively lower affinity between profilin and actin leads first to the loss of force generation ( concomitant with an increase in retrograde flow speed ) followed by a loss in the capacity to glide on a 2D substrate ( Table 3 ) .', 'Further disruption of actin dynamics or the organization of actin filaments would then lead to additional problems such as salivary gland invasion [30 , 31 , 35] .', 'We had previously hypothesized that integration of retrograde flow and force production produces motility [20] .', 'However , the near-normal motile behavior of the QNQ mutant could not have been predicted through these two parameters alone .', 'This leads us to postulate that an as yet unknown factor plays a role in producing sporozoite motility ( Fig 7 ) .', 'This factor could be a molecule , a molecular complex or the dynamic arrangement of proteins in complexes .', 'Considering the complexity of the interplay between many factors to generate motility , we favor the latter hypothesis .', 'Interesting in this respect and for the future direction of research into parasite motility that goes beyond the identification of molecular players are two recent studies on T . gondii tachyzoites .', 'The first study adapted our laser tweezer approach to T . gondii tachyzoites [47] .', 'This revealed surprising differences compared to sporozoites .', 'Most strikingly , retrograde flow in tachyzoites occurred after a delay of approximately one minute , while it was instantaneous in sporozoites .', 'Also , directed force production only occurred after this initial phase in tachyzoites .', 'This clearly suggests different kinetics to assemble a directional motor between the two parasites .', 'A second study investigating the essential role of many of the key proteins involved in motility showed that parasites can still be motile , albeit at a much reduced rate , in the absence of core motility proteins , including actin [48] .', 'This showed that even in the absence of actin , retrograde flow still moved beads towards the rear of the tachyzoite .', 'Yet , retrograde flow occurred in a manner of minutes and not seconds as observed with P . berghei sporozoites .', 'This suggests a much more complex picture and the existence of important differences between apicomplexan parasites that are currently not fully appreciated .', 'Sporozoite motility relies on a complex interaction of the formation and turnover of adhesion sites as well as the generation of force [19–21 , 49] .', 'It is currently not clear how this complex interplay is orchestrated .', 'Investigating motility thus relies on the determination of many different factors , such as the percentage of motile sporozoites , and their average and instantaneous speeds ( Fig 5 , Table 3 ) .', 'Previously , we have shown that low concentrations of jasplakinolide , which likely leads to longer or less dynamic actin filaments , cause higher retrograde flow speed but lower force [20] and less motility ( [46] , S1B Fig , Table 3 ) .', 'Cytochalasin D , which in contrast disrupts actin filaments , led to lower force and less motility , yet had only mild effects on retrograde flow ( [20] , S1B Fig , Table 3 ) .', 'Thus , longer and more stable filaments would lead to a higher retrograde flow , which is the case for both of our mutants ( Fig 6D ) .', 'Both longer filaments and disruption of filaments would lead to lower forces , which again is the case for both mutants ( Fig 6B ) .', 'Thus , the length of filaments and their dynamics do not scale linearly with the loss of force .', 'Rather , maximum force generation likely requires optimized filament length , dynamics , and orientation , which are achieved through the interplay of several proteins in vivo [18 , 20 , 30 , 50] .', 'Although both mutants have weakened actin-binding affinity , the QNQ mutant seems to be above a threshold required for gliding .', 'It seems likely that the AAA mutant releases actin monomers faster , so that in vivo , other monomer-binding proteins have the chance to bind actin .', 'These could be for example the ADFs , which promote filament formation by catalyzing nucleotide exchange [28 , 51] .', 'This also shows that sporozoite motility in vivo is robust enough to tolerate small changes in binding affinities of individual proteins , and that our in vitro assays are sensitive enough to now understand the fine-tuning of this incredibly efficient and fast machinery .', 'In summary , we present the first study using site-directed mutagenesis of a constitutively expressed protein involved in malaria parasite motility that uncovers a new type of interaction between actin and profilin .', 'We provide conclusive evidence from a combination of biochemical , biophysical , computational and molecular genetic studies that this actin-profilin interface is important for Plasmodium sporozoite migration .', 'We suggest that sporozoite entry into salivary glands is the key constraint for the evolution of the Plasmodium motility machinery .'], ['All animal experiments were performed according to the German Animal Welfare Act ( Tierschutzgesetz ) and were approved by the responsible German authorities ( Regierungspräsidium Karlsruhe , numbers G-3/11 , G-134/14 and G-310/14 ) .', 'Generation of parasite lines and infections of mosquitoes was performed using female NMRI mice ( Janvier ) .', 'Monitoring of parasite prepatency and blood stage growth rates was performed using female C57BL/6 mice ( Charles River ) .', 'A mutant Pf Pfn construct in which the sequence 64EDE66 was replaced by QNQ was obtained by gene synthesis from Mr . Gene and sub cloned into pETM-11 using NcoI and XhoI restriction sites .', 'This was used as a backbone to generate the AAA mutant using site-directed mutagenesis with the QuikChange Lightning mutagenesis kit ( Agilent Technologies ) with primer pair 12 .', 'Wild type [33] and mutant Pf Pfns were expressed and purified [37] and the correct folding of the mutants verified using synchrotron CD spectroscopy , as described before [40] ( S7 Fig ) .', 'Pig ( Sus scrofa ) skeletal muscle α-actin was purified as before [37 , 52] .', 'Pf actin 1 was expressed in Spodoptera frugiperda Sf21 cells ( Invitrogen ) as described earlier [37] .', 'After expression the cells were harvested and suspended in lysis buffer ( 10 mM HEPES pH 7 . 5 , 250 mM NaCl , 5 mM CaCl2 , 15 mM imidazole , 1 mM ATP and 7 mM β-mercaptoethanol ) .', 'Cells were lysed by sonication on ice , and the lysate was clarified by a 1-h centrifugation at 30600 g .', 'The supernatant was mixed with 1 ml of HisPur Ni-NTA resin ( Thermo Fisher Scientific ) , incubated for 1 h at 4°C , and 
['Introduction', 'Results', 'Discussion', 'Methods']
['Profilin is an actin monomer binding protein that provides ATP-actin for incorporation into actin filaments .', 'In contrast to higher eukaryotic cells with their large filamentous actin structures , apicomplexan parasites typically contain only short and highly dynamic microfilaments .', 'In apicomplexans , profilin appears to be the main monomer-sequestering protein .', 'Compared to classical profilins , apicomplexan profilins contain an additional arm-like β-hairpin motif , which we show here to be critically involved in actin binding .', 'Through comparative analysis using two profilin mutants , we reveal this motif to be implicated in gliding motility of Plasmodium berghei sporozoites , the rapidly migrating forms of a rodent malaria parasite transmitted by mosquitoes .', 'Force measurements on migrating sporozoites and molecular dynamics simulations indicate that the interaction between actin and profilin fine-tunes gliding motility .', 'Our data suggest that evolutionary pressure to achieve efficient high-speed gliding has resulted in a unique profilin-actin interface in these parasites .']
['The malaria parasite Plasmodium has two invasive forms that migrate across different tissue barriers , the ookinete and the very rapidly migrating sporozoite .', 'Previous work has shown that the motility of these and related parasites ( e . g . Toxoplasma gondii ) depends on a highly dynamic actin cytoskeleton and retrograde flow of surface adhesins .', 'These unusual actin dynamics are due to the divergent structure of protozoan actins and the actions of actin-binding proteins , which can have non-canonical functions in these parasites .', 'Profilin is one of the most important and most investigated actin-binding proteins , which binds ADP-actin and catalyzes ADP-ATP exchange to then promote actin polymerization .', 'Parasite profilins bind monomeric actin and contain an additional domain compared to canonical profilins .', 'Here we show that this additional domain of profilin is critical for actin binding and rapid sporozoite motility but has little impact on the slower ookinete .', 'Sporozoites of a parasite line carrying mutations in this domain cannot translate force production and retrograde flow into optimal parasite motility .', 'Using molecular dynamics simulations , we find that differences between mutant parasites in their capacity to migrate can be traced back to a single hydrogen bond at the actin-profilin interface .']
['chemical bonding', 'cell motility', 'parasite groups', 'actin filaments', 'plasmodium', 'cell processes', 'parasitology', 'developmental biology', 'apicomplexa', 'hydrogen bonding', 'physical chemistry', 'contractile proteins', 'polymer chemistry', 'actins', 'actin polymerization', 'proteins', 'life cycles', 'chemistry', 'polymerization', 'biochemistry', 'cytoskeletal proteins', 'cell biology', 'biology and life sciences', 'chemical reactions', 'physical sciences', 'sporozoites', 'parasitic life cycles']