Induction of Malaria Parasite Migration by Synthetically Tunable

Sep 12, 2011 - unique form of locomotion, termed gliding motility, to penetrate tissues and ... the skin, entering of blood vessels and infection of h...
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LETTER pubs.acs.org/NanoLett

Induction of Malaria Parasite Migration by Synthetically Tunable Microenvironments Nadine Perschmann,†,§ Janina Kristin Hellmann,‡,§ Friedrich Frischknecht,*,‡,§ and Joachim P. Spatz*,†,§ †

Department of New Materials and Biosystems, Max Planck Institute for Intelligent Systems, and Department of Biophysical Chemistry, University of Heidelberg, Heisenbergstrasse 3, 70569 Stuttgart, Germany ‡ Parasitology, Department of Infectious Diseases, Hygiene Institute, University of Heidelberg Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany

bS Supporting Information ABSTRACT: Interaction of Plasmodium sporozoites, the forms of the malaria parasite transmitted by the mosquito, with its microenvironment in form of adhesion and migration is essential for the successful establishment of infection. Myosin-based sporozoite migration relies on short and dynamic actin filaments. These are linked to transmembrane receptors, which in turn bind to the matrix microenvironment. In this work, we are able to define the characteristics that determine whether a matrix is favorable or adverse to sporozoite adhesion and motility using a specifically tunable hydrogel system decorated with gold nanostructures of defined interparticle spacing each equipped with molecules acting as receptor adhesion sites. We show that sporozoites migrate most efficiently on substrates with adhesion sites spaced between 55 and 100 nm apart. Sporozoites migrating on such substrates are more resilient toward disruption of the actin cytoskeleton than parasites moving on substrates with smaller and larger interparticle spacings. Plasmodium sporozoites adhesion and migration was also more efficient on stiff, bonelike interfaces than on soft, skinlike ones. Furthermore, in the absence of serum albumin, previously thought to be essential for motility, sporozoite movement was comparable on substrates functionalized with RGD- and RGE-peptides. This suggests that adhesion formation is sufficient for activating migration, and that modulation of adhesion formation and turnover during migration is efficiently controlled by the material parameters of the microenvironment, that is, adhesion site spacing and substrate stiffness. Our results and approaches provide the basis for a precise dissection of the mechanisms underlying Plasmodium sporozoites migration. KEYWORDS: Nanostructures, elastic substrates, hydrogels, Plasmodium sporozoites, motility, adhesion

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alaria is a devastating disease caused by human-infecting pathogens of the genus Plasmodium. This genus belongs to the phylum Apicomplexa, a group of obligate intracellular parasites. Infective forms of apicomplexan parasites often use a unique form of locomotion, termed gliding motility, to penetrate tissues and enter host cells.1 For the infectious forms of Plasmodium transmitted by the mosquito, named sporozoites, gliding is essential for entering mosquito salivary glands, migration within the skin, entering of blood vessels and infection of hepatocytes.2 The ability to manipulate sporozoite motility is clearly important for our understanding of pathogenesis and could yield insights for the future development of drugs or vaccines to combat malaria. Observations utilizing inverted microscopes,3,4 revealed that during ex vivo gliding on solid substrates sporozoites maintain a fixed shape and move in a mostly counterclockwise circular manner. In order to glide, sporozoites first have to attach to the substrate, which occurs in several steps. Floating parasites adhere with one end often followed by so-called waving.3 During waving the parasite is attached at one end, typically the rear end, and rotates around this attachment point in an active, actin-dependent way.3 5 Waving usually leads to the attachment of the other end and to gliding.3 5 Initiation of gliding motility has been r 2011 American Chemical Society

linked to the interaction of albumin with the surface of the sporozoite.5 Albumin was suggested to specifically trigger a signaling cascade via Ca2+ and cAMP that leads to secretion of proteins necessary for gliding motility including TRAP (thrombospondinrelated anonymous protein) family adhesins.5 Studies looking at the cellular and molecular levels of sporozoite gliding motility have shown some similarities and some differences to cell migration of higher eukaryotic cells, which utilize focal adhesions (FAs) for cell-substrate adhesion during actin-based locomotion. FAs serve to anchor the cell to the extracellular matrix (ECM) via integrins and thereby enable the transmission of mechanical force between the substrate and cellular actin.6 Cellular integrins, in turn, recognize and bind to short amino acid sequences, such as the tripeptide arginineglycine-aspartic acid (RGD) contained in many ECM proteins.7,8 Furthermore, FAs mediate bidirectional signaling across the cell membrane9 and are necessary for recognizing and responding to environmental parameters such as pliability, dimensionality, topography, and ligand density.6,10 Similarities between gliding Received: August 11, 2011 Revised: September 6, 2011 Published: September 12, 2011 4468

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Table 1. Sample Characterization: Molecular Weight of Poly(ethyleneglycol)-diacrylate (PEG-DA) in g/mol, the UVIllumination Time in Minutes, and Elasticity of the Respective Hydrogel are Listed molecular weight (g/mol)

UV illumination time (min)

elasticity (kPa)

700

60

6000

700

45

6000

700

35

6000

700

20

6000

10k

20

70

20k

20

10

35k

20

1

motility of sporozoites and migration of metazoan cells include the following: (i) utilization of an actin-myosin motor,1 (ii) formation of specific adhesion sites to the substrate,11 (iii) actin filaments are linked to the substrate by transmembrane adhesins,12 and (iv) the presence of one or more adhesive domains for host cell binding in the extracellular regions of these TRAP family adhesins.12 Contained in the adhesive domains of TRAP family adhesions are highly conserved adhesion site motifs, such as the MIDAS motif in the integrin-like A-domain12 This metal-ion dependent motif has been shown to stabilize integrin binding to RGD peptides under force13 and is crucial for TRAP-mediated host cell entry by sporozoites.14 Despite these similarities, Plasmodium actin and the involved adhesive proteins differ in many ways from their counterparts in metazoan cells. In contrast to actin of higher eukaryotic cells or yeast, actin filaments in malaria parasites are elusive15 and biochemical evidence suggests that they are likely to be very short and turn over rapidly.16 In addition, actin filaments are probably only formed at points of adhesion to the substrate15 and might be linked to the TRAP family adhesins solely via the glycolytic enzyme aldolase.12 Observations have made apparent that sporozoite speed is tissue-dependent. Sporozoites move slowly (about 0.1 μm/s) within the salivary duct system,17 but speed up to 1 2 μm/s after transmission into the skin of the host where they migrate to invade blood or lymphatic vessels.18 Because little is known about the substrate characteristics required by Plasmodium sporozoites for adhesion and migration as well as the details of the molecular mechanisms involved in these processes, we created synthetic cell environments with the aim of dissecting the mechanistic aspects of sporozoites migration. The created cell environments are unusual in their variability regarding (i) pliability (ranging from stiff, bonelike to soft, skinlike), (ii) the distance between adhesive ligands distributed on the surface (termed ligand spacing), and (iii) the ability to immobilize individual ligands such as peptides, antibodies or other proteins to the nanoparticles.19 On the basis of previously applied methods for the production of synthetic nanostructured cell environments,19,20 we first created nanopatterned substrates decorated with gold particles spaced apart in a user-defined way.21 Second, the gold particles were transferred to an elastic, cell- and protein-repellent PEGDA hydrogel.20 Lastly, we utilized gold nanoparticles as anchor points for active biomolecules, in this case cell-recognition peptides RGD and RGE a nonintegrin binding motif, commonly used as a negative control in cell adhesion studies. Utilizing these cell environments we investigated and quantified the role of matrix

Figure 1. Sporozoite motility on glass and PEG-hydrogels. (A) Schematic of the imaging chamber, where sporozoites (marked by red arrows) in medium are sandwiched between a glass coverslip (top) and the PEG hydrogel (bottom). (B) Circular motility of a Plasmodium berghei sporozoite expressing cytoplasmic GFP on a glass slide. The arrowheads indicate the counterclockwise direction of movement; individual frames are 2 s apart. “Max” indicates maximum intensity projection over 30 frames of a region of interest. (C) Maximum intensity projection over 30 frames of a region of interest showing sporozoite gliding motility (ccw), active waving motion (w) or passive floating of sporozoites in the medium (na). (D) Maximum intensity projections showing diverse sporozoite movements on PEG hydrogels depending on the UV illumination time as indicated during gel production. Red arrowheads indicate typical migration patterns for the certain condition. (E) Percentage of na and ccw moving sporozoites on PEG-700-DA gels at different UV-illumination times. (F) Percentage of na sporozoites as a function of UV-illumination time PEG gel and different PEG gels (PEG700-DA, PEG-10k-DA, PEG-20k-DA, PEG-35k-DA).

stiffness and adhesion ligand spacing (density) on adhesion and gliding of Plasmodium berghei sporozoites, a rodent malaria model organism. The poly(ethyleneglycol)diacrylate (PEG-DA) substrates (Table 1) that we employed can be divided in two groups: those without gold particles and those equipped with a nanopattern of gold particles while gold particles were nonfunctionalized or functionalized by ligands. The first group included PEG-DA hydrogels with variable elasticity from 1 kPa to 6 MPa. The surfaces in the second group varied in gold particle (ligand) spacing from 40 to 270 nm particle-to-particle distance, elasticity of the PEG-DA hydrogel surrounding the ligands from 1 kPa to 6 MPa and the type of ligand that was biofunctionalized to the gold particles (BSA - added to the medium; RGD and RGE - covalently bound to the gold particles via thiol linker). 4469

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Figure 2. Adhesion of sporozoites on nanopatterned surfaces. (A) Quantitative readout of the different movement patterns of sporozoites (na, nonadherent; a, attached; w, waving; ccw, counter-clockwise gliding) moving on nanopatterned hydrogels where gold nanoparticles are not specifically functionalized (no BSA present) or in the presence of BSA/functionalized with RGD or RGE. (B) Scanning electron micrographs of critical point dried sporozoites on a nanopatterned PEGpassivated glass slide with 270 nm (left) or 70 nm (right) particle-toparticle spacing. The red arrowheads point to direct contact points between parasites and gold particles. Scale bars correspond to 300 nm.

To enable a comparison of sporozoite adhesion and motility characteristic in the different synthetic environments, we commenced by defining and quantifying typical sporozoite movements by fluorescent optical microscopy. Therefore, an imaging chamber was used as schematically shown in Figure 1A. We imaged sporozoites on the PEG-DA hydrogel or, as a control, glass surface in the presence of BSA and finally classified them according to their different motile behaviors.4 Figure 1B show a sequence of the same Plasmodium berghei sporozoite expressing cytoplasmic GFP with its typical crescent shape on glass, indicating the counter clockwise circular movement of the sporozoite. Normally, on glass slides around 90% of the observed motile sporozoites turned in such a counterclockwise circular manner. Figure 1C demonstrates the different observed sporozoite movement patterns on glass by the maximum intensity projection (30 subsequent frames). These are sporozoite gliding motility (counterclockwise = ccw), active waving motion (waving = w), or passive floating of nonadherent sporozoites in the medium (nonadherent = na). In the experiments depicted in Figure 1D, we focused at adhesion and movement (waving and gliding motility) on surfaces without cell-recognition peptides with the aim of identifying a surface that inhibits sporozoite adhesion and locomotion. We investigated PEG-hydrogels of different crosslinking (PEG-700-DA with 20, 35, 45, and 60 min UV exposures). As expected, no attachment and gliding was observed

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in the absence of BSA on the different cross-linked gels. In the presence of BSA, these experiments revealed that sporozoites attached and glided best on the hydrogel that was cross-linked for 35 min (Figure 1E, Supporting Information, Figure S1A). Both the most and the least cross-linked surface restricted adhesion and gliding, but the inhibitory effect of the least cross-linked hydrogel (20 min. UV exposure) was slightly stronger (Figure 1E,F). On this hydrogel, none of the sporozoites were attached and idle and less than 7% were actively gliding. Instead, the vast majority of sporozoites were found wobbling above the substrate unable to attach. The same was the case hydrogels of different stiffness, that is, PEG-10k-DA, PEG-20k-DA, and PEG35k-DA. All showed inhibition of sporozoite adhesion and motility when the UV exposure time was 20 min. From these experiments, it can be concluded that the term “biologically inert”, which is commonly used to describe PEG-modified surfaces, does not apply to the interaction of PEG-DA hydrogels with sporozoites in general. It is known that cells cultured on tissue-culture plastic or glass coverslips can attach via adsorbed matrix proteins,22 and we presume attachment of sporozoites to some PEG-DA hydrogels to proceed in much the same way by binding proteins, for example, albumin that in turn bind to diacrylates. The successful identification of a surface that can be considered inert regarding sporozoite adhesion and motility allowed us to next produce synthetic cell environments that present a uniform pattern of biofunctionalized gold particles on a sporozoite-repellent background. To elucidate the role of specific interactions between sporozoites and surface ligands during adhesion and gliding motility, we covalently linked gold particles (spaced 70 nm apart) to inert PEG-DA hydrogels (PEG-700, 20 min UV exposure). Figure 2A shows a comparison of data for sporozoite adhesion and motility on nanopattern surfaces without any additive protein or on nanopattern in presence of BSA, the integrin recognition peptide RGD, or the control nonintegrin binding peptide RGE (Supporting Information, Figure S1B). The gold nanopattern allowed sporozoites to overcome the inability to attach (Figure 2A, Supporting Information, Figure S1C). Without albumin the parasites mostly remained attached to the substrate with only a few displaying waving motions. The addition of albumin increased the number of waving and even more so the percentage of gliding sporozoites. In the presence of albumin, 60% of sporozoites were gliding at an average speed of around 1.7 μm/s on the goldpresenting hydrogel. Since BSA was previously considered to be an essential signal for sporozoite motility,5 we prepared the parasites in the next step in the absence of BSA and added them to RGD-coated nanopatterned hydrogels. Sporozoites were able to attach to and additionally move on these hydrogels in the same way than in the presence of BSA. This strongly suggests that contact formation and not albumin per se induces signaling and that the role of albumin for gliding on nonfunctionalized substrates is merely to allow adhesion to proceed. Scanning electron micrographs of sporozoites on a ligand-coated surface further revealed contacts of the sporozoite plasma membrane to the individual adhesive gold particles spaced by 270 and 70 nm (Figure 2B) biofunctionalized with RGD, similar to observations with fibroblasts.20 These experiments show that if sporozoites are unable to attach to a surface, they are also unable to initiate gliding motility. The ability to adhere to a surface was unaffected by the presence of albumin, which confirms the observation that albumin does not influence initial adhesion to uncoated glass coverslips.5 Concerning gliding motility, we could confirm that 4470

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Figure 3. Sporozoites move best on rigid surfaces with intermediately spaced ligands. (A) Quantitative readout of the percentage of gliding sporozoites on glass (g.), homogeneous gold (h.g.), and nanopatterend substrates with the indicated interparticle distances between 40 to 270 nm. (B) Average speeds of the motile sporozoites from (A). (C) Quantification of the numbers of sporozoites on 70 nm RGD functionalized nanopatterned hydrogels with elasticities ranging from 6 MPa to 1 kPa (a, attached; w, waving; ccw, gliding). (D) Average speeds of counterclockwise gliding sporozoites on hydrogels of different rigidity.

on a surface where sporozoites can adhere, a ligand is necessary for the initiation of gliding motility but not specifically BSA. Furthermore, we could show that the ligand RGD or BSA are not specifically required for motility. To analyze the significance of ligand (BSA, RGD, RGE) spacing on sporozoite adhesion and gliding we varied the distance between ligands starting at a minimum of 40 nm up to a maximum of 270 nm (Figure 3). To ensure specific binding, the biofunctionalized gold particles were attached to the surface of a PEG-700-DA hydrogel with UV exposure time of 20 min. The uniformity of the nanopattern was confirmed by scanning electron microscopy.19 Fibroblasts adhere best to nanopatterned substrates where ligands are spaced closer than the threshold value of around 60 nm and progressively lose the ability to adhere to substrates with ligands spaced further apart.23 In contrast, sporozoites adhered to and moved very well on substrates with interparticle spacing in a range of 55 100 nm (Figure 3A) irrespective of the choice of ligand. Around 40% of sporozoites were found gliding on plain glass coverslips, homogeneous gold surfaces, and hydrogels with ligands spaced 40 nm apart. On the surfaces with ligands spaced 55 nm apart, the number of motile parasites increased to around 60%. Motility was decreased on substrates with an interligand spacing of 240 and 270 nm. The percentage of motile parasites was 38 and 25%, respectively. Sporozoite speed followed the same pattern (Figure 3B). Motile sporozoites moved with an average speed of ∼1.4 μm/s on glass or homogeneous gold and ∼1.8 μm/s on substrates with 55 nm spaced ligands. On substrates with 240 and 270 nm spaced ligands, the average speed of the motile sporozoites was ∼1.4 and 1.2 μm/s, respectively. Reversely, an increase in motile parasites led to a decrease in the number of attached or immobile sporozoites and vice versa (Supporting Information, Figure S1D). These trends and numbers were similar for patterned substrates functionalized with RGD and RGE, decreased on substrates in the presence of albumin (Figure 3B). The latter finding might be explained by the noncovalent bond between BSA and the gold particle, which could make for a comparatively less rigid contact point.

These findings most likely can be ascribed to the involvement of adhesion receptors of the TRAP family, which, only when spaced apart the right distance, provide optimal support for speedy locomotion. Considering that sporozoites move relatively fast in comparison to other cells, they must be able to rapidly form and disrupt adhesion sites to reach maximum speed.11 Thus, an abundance of adhesive ligands (such as on homogeneous gold and 40 nm spaced apart ligands) could lead to more adhering surface, resulting in slower movement, while an absence of ligands could result in more frequent detachment of sporozoites. The following calculation demonstrates how the different ligand spacings we applied in our experiments relate to the body size of a sporozoite, and illustrates how manipulating the distance between ligands influences the sporozoites ability to perform gliding motility. Considering that the average P. berghei sporozoite is about 12 μm long and just 1 μm wide, we estimate the sporozoite’s body surface area to be ∼12 μm2 on homogeneous gold or glass. Thus, on a substrate with ligands spaced apart 55 nm about 2700 gold dots functionalized with ligands are available for adhesion. Each gold dot has an approximate diameter of 10 nm (equivalent to ∼75 nm2 surface). Added together, the sum of all gold particle surfaces available for adhesion is ∼0.2 μm2 in size. In comparison, the surface with ligands spaced apart 100 nm has only approximately one-quarter of the surface mediating adhesion, but nevertheless, both the number of motile sporozoites and their average speed is identical. On the substrate with a ligand distance of 270 nm (equivalent to ∼100 available gold particles and less than 0.008 μm2 available adhesive surface), the number of motile cells decreases and those that are moving move at lower speed than on homogeneous gold (Figure 3B). These estimates allow for a calculation of the number of receptors possibly involved in generating traction for sporozoite motility. We assume the parasites adhesions to be similar in dimension to integrin, which is about 8 12 nm in diameter.24 From this, we deduce that between one and three individual TRAP sporozoite surface molecules can attach to a ligand biofunctionalized to a ∼10 nm large gold particle.8 4471

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Figure 4. Sporozoite motility on different RGD coated nanopatterned substrates is affected by an actin depolymerizing drug (cyto D = 10, 50, 100 nM). (A) Absolute values of the quantitative readout of the percentage of ccw gliding sporozoites under the influence of cyto D on glass and nanopatterend substrates with interligand distances between 40 to 270 nm. (B,C) Gliding (data from (A)) are normalized to 0 nM cyto D which is defined as 100% for the respective substrates (B), or normalized to the value of sporozoites on glass treated with the same concentration of cyto D (C).

Considering that we assume the presence of about 100 available gold particles on the entire substrate surface, this means that a total of just 100 300 adhesion molecules participate in linking actin filaments on the 270 nm spaced surface. But, because during rapid motility the sporozoite is only attached to the substrate with a fraction of its surface,11 a few dozen linkages appear to suffice for locomotion. Furthermore, the rigidity of the surrounding substrate is crucial for gliding motility even in the presence of adhesive ligands. Examples of how rigidity influences cells are numerous, including observations that cells cultured on elastic substrates with a rigidity gradient align their shape, cytoskeletal structures, cell adhesions through integrins and their traction force in the direction of increasing stiffness and flee from the softer substrate regions.25 27 Also, it has been shown that focal adhesion maturation, and as a result acto-myosin contraction, is influenced by substrate rigidity.28 Regarding the PEG-DA hydrogel system, substrate rigidity can be tuned over 4 orders of magnitude, including the rigidities for all the different human tissues.19 Employing a constant RGD ligand spacing of 70 nm (maximum motility), we next varied the flexibility of the hydrogel substrates. To mimic a wide range of surfaces that sporozoites might encounter during the course of

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their life we produced hydrogels with the following elasticities (Table 1): PEG-700-DA (6 MPa, nearly as hard as cell culture plastic), PEG-10k-DA (70 kPa, comparable to precalcified bone), PEG-20k-DA (10 kPa, similar to muscle), PEG-35k-DA (1 kPa, like brain). On the hardest hydrogel (PEG-700-DA, 6 MPa), large numbers of sporozoites moved readily and in the typical circular fashion (Figure 3C,D). On the softer substrates, sporozoites were found waving or simply hanging on (Figure 3C). Decreasing numbers of gliding sporozoites correlated with an increase in weakly attached sporozoites. Thus, the softer the hydrogel, the fewer motile sporozoites can be observed and the slower they move. Figure 3D shows the reduction of locomotion speed of sporozoites with softer surroundings. Interestingly, adhesion is still possible on very soft hydrogels equipped with ligands where sporozoites struggle to locomote. This suggests that rigid surfaces trigger a pleiotropic cellular response necessary for initiating locomotion, while soft surfaces do not. The process of integrin-mediated cell adhesion to a soft surface has been described as predominantly resulting in substrate deformation.29 On a rigid surface, in contrast, the pulling force is greater and adhesion leads to deformation of the adhesion complex and to conformational changes of its proteins.26,29 31 Such conformational changes may trigger processes that lead to an increase in acto-myosin contractility (namely the growth of focal adhesions and Ca2+-influx from cytoskeleton-coupled, stress-activated channels).29 Yet, it is unclear if similar processes could mediate sporozoite adhesion and gliding. It was previously shown that transient forces correlate with the formation and rupture of distinct substrate contact sites of Plasmodium sporozoites and that they are dependent on actin dynamics.11 This raises the question how Plasmodium actin can function during locomotion, considering its evident lack of length in comparison to actin of metazoan cells.19,20 In focal adhesions of fibroblasts, the actin filaments are bundled by actin filament cross-linkers including the contractile myosin. In sporozoites, however actin can only be arranged nearly parallel to the plasma membrane due to the narrow supra-alveolar space.15 It thus seems possible that actin filaments connect individual TRAP family adhesins for adhesion and gliding. Therefore, the observation that sporozoites move slower on substrates with densely spaced ligands could be linked to an accumulation and clustering of actin, which could cause enhanced adhesion. Indeed, enlarged adhesion sites were shown for slow sporozoites using RICM while rapidly moving sporozoites showed smaller adhesion sites. However, formed adhesions were insensitive to the actin depolymerizing drug cytochalasin D and persisted for several minutes.11 It is thus unclear if the concentration or length of actin filaments changes depending on the different ligand patterns. To better understand the role of actin in adhesion and gliding motility, we investigated how cytochalasin D (cyto D) influences parasite motility on the different nanopatterned substrates and glass (Figure 4). Sporozoites incubated with increasing concentrations of cyto D moved less frequently (Figure 4A) and slower (Supporting Information, Figure S1E). Surprisingly, a smaller reduction of moving sporozoites on substrates with 55, 70, and 100 nm spacing compared to other substrates of smaller or wider gold particle spacing was observed (Figure 4A,B). The IC50 of cyto D was nearly 3-fold higher on 70 nm spaced hydrogels with around 65 nM compared to glass controls with an IC50 around 20 nM (Figure 4B,C). Compared to untreated (-cyto D) parasites on the same respective gels we observed a 30% reduction of 4472

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Figure 5. Dual role of adhesion formation during sporozoite gliding. Different hypothesis of how ligand spacing could result either in a change in actin filaments (more or less actin than optimal for gliding, 1) or not (2). Seventy nanometer interligand spacing provides best conditions for sporozoite gliding as the optimal number (2 in the cartoon) of substrate-bound adhesins are linked with an actin filament (center). Sporozoite adhesion is likely increased (more adhesion) on 40 nm spaced gels and adhesion is decreased (less adhesion) on 240 nm conditions leading in both cases to less gliding (decreased from +++ to ++). Hypothesis 1 predicts that at larger ligand distance (e.g., 240 nm) shorter actin filaments could not connect two adhesins with the substrate. At shorter distances longer filaments could lead to too much substrate contact and thus more adhesion but less gliding. This would predict that if actin filaments could be shorted by cytochalasin D, sporozoites would be gliding more efficiently. In contrast, hypothesis 2 postulates that ligand distance does not influence actin filaments, which fits with the data from Figure 4.

gliding parasites on the most densely patterned substrate after administration of 10 nM cyto D. However, under the same drug conditions only 5% fewer parasites moved on substrates with 55, 70, or 100 nm spacing. On surfaces with a larger interparticle spacing motility was also reduced. This suggests that optimal spacing can “protect” sporozoite motility from the effects of actin depolymerization. Increasing cyto D concentrations resulted in an increase of motility inhibition. Importantly, normalization of drug treated parasites on nanopatterned gels to parasites treated with the same concentration of cyto D on glass surfaces showed the same distribution of motile sporozoites within the respective substrates independent from the cyto D concentration (Figure 4C). Thus the relative increase of sporozoite motility caused by an optimal ligand spacing is independent of the concentration or length of the actin filaments as modulated by cyto D. Moreover, the cyto D mediated inhibition of motility was similar for parasites moving on substrates with 40 or 240 nm gold particle spacing (Figures 4 and 5) as well as for parasites moving on substrates with 55, 70, and 100 nm spacing’s over all inhibitor concentrations (Figure 4A). This suggests that actin filaments are of similar lengths in sporozoites moving on the different substrates and that filament length plays no role in the modulation of motility by surface patterning (Figure 5). If there would be more filaments leading to tighter adhesion and thus less gliding on substrates with 40 nm gold particle spacing, we would have expected to see an increase in motility as small concentration of cyto D are employed. However, this was not the case. Thus we favor a model, where the same concentration and length of actin filaments are present independently on the ligand spacing and that ligand spacing solely leads to modulation of gliding by shifting the balance between stronger and weaker adhesion (Figure 5). Strikingly, actin filaments from Plasmodium and Toxoplasma are largely between 50 and 150 nm in length.32,33 Thus one could speculate (Figure 5) that the equally spaced adhesion sites lead to an optimal assembly of actin filaments, maybe within a twodimensional actin-aldolase raft34 in turn leading to optimal adhesion formation and turnover as required for optimal gliding

motility. Adhesins spaced too sparsely would not be able to connect the filaments together thus loosing grip to the substrate (Figure 5). Those spaced too densely could lead to more adhesion and thus less motility (Figure 5). Whether these findings translate directly to the in vivo situation where sporozoites migrate in 3D through dense tissue and cells at similar speeds18,35 remains to be shown directly. In conclusion, we provide experimental evidence that Plasmodium sporozoite motility is initiated by surface contact and not serum albumin mediated signaling and is optimal on substrates with an intermediate spacing (55 100 nm) of ligands on rigid substrates. This revealed that surprisingly few contact points and thus parasite adhesins are necessary for achieving the rapid motility of sporozoites. The tool set reported here will further be instrumental for dissecting the molecular basis and spatiotemporal regulation of sporozoite motility.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additonal figures and information. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*(J.P.S.) E-mail: [email protected]. Tel: 49-711-689-3610. Fax: 49-711-689-3612. (F.F.) E-mail: [email protected]. Tel: 49-6221-566537. Fax: 49-6221-564643. Author Contributions §

These authors contributed equally.

’ ACKNOWLEDGMENT We thank Yin Cai for help with tracking and data analysis, Iris Arnold and Katarina Abramovic for mosquito infections, and Jake Baum, Christian Boehm, Kai Matuscheswki, Christine Selhuber-Unkel, 4473

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Nano Letters and Ulrich Schwarz for discussions and reading the manuscript. The work was funded by grants from the German Federal Ministry of Education and Research (BMBF, Biofuture, and NGFN). We gratefully acknowledge support from the Max Planck Society, the Medical School and the Cluster of Excellence Cell Networks at the University of Heidelberg as well as the Chica and Heinz Schaller Foundation. J.K.H. is a member of the HBIGS graduate school at the University of Heidelberg. F.F. is a member of the European Network of Excellence EviMalaR. J.S. is a Weston Visiting Professorship at the Weizmann Institute of Science, Israel.

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dx.doi.org/10.1021/nl202788r |Nano Lett. 2011, 11, 4468–4474