COMMENTARY Cell Adhesion & Migration 6:2, 1–7; March/April 2012; G 2012 Landes Bioscience

Small heat shock proteins in cellular adhesion and migration Evidence from Plasmodium genetics

Georgina N. Montagna,1 Kai Matuschewski1,2,* and Carlos A. Buscaglia3 1Max Planck Institute for Infection Biology; Berlin, Germany; 2Institute of Biology; Humboldt University; Berlin, Germany; 3Instituto de Investigaciones Biotecnológicas (IIB-INTECH); Universidad Nacional de San Martín; San Martín, Buenos Aires, Argentina

ellular locomotion and adhesion domain of ~90 amino acid residues C critically depend on regulated (PFAM: PF00011).1-4 Variable N- and turnover of filamentous actin. C- termini flank this central domain, Biochemical data from diverse model resulting in an overall small molecular systems support a role for the family of mass, ranging from 12 to 43 kDa. HSPB small heat shock proteins (HSPBs) in molecules (1) have the tendency to form microfilament regulation. The small cha- large oligomers,2-5 (2) display classical © 2012 Landesperones could either act directly, Bioscience. through chaperone activity and suppress protein competition with the motor myosin, or aggregation6,7 and (3) associate with lipid indirectly, through modulation of actin bilayers, potentially preserving membrane depolymerizing factor/cofilin activity. integrity.8,9 The physiological importance However, a direct link between HSPBs of HSPBs is underscored by their ubi- and actin-based cellular motility remained quitous presence in all three domains of Do notto be established. distribute. In a recent experimental life, i.e., archaea, bacteria and eukarya,10,11 genetics study, we provided evidence for and by their association with a variety of regulation of Plasmodium motility by severe diseases, e.g., cataracts, myopathies, HSPB6/Hsp20. The infectious forms of dystrophies and diverse cancers.12-14 For malaria parasites, termed sporozoites, dis- instance, the founding member of this play fast and continuous substrate-depend- group, a-crystallin/HSPB4, is a major ent motility, which is largely driven by component of the vertebrate lens,15 and turnover of actin microfilaments. HSPB4 polymorphisms are associated with Sporozoite gliding locomotion is essential congenital cataract, a common cause of 16,17 Keywords: gliding motility, adhesion sites, to avoid destruction by host defense blindness in infants. cellular migration, small heat shock mechanisms and to ultimately reach a In the human genome 10 HSPB mem- 18,19 proteins, Hsp20, Plasmodium, sporozoite, hepatocyte, the target cell, where to bers can be identified, and some multi- malaria transform and replicate. Genetic ablation cellular eukaryotes, such as Arabidopsis of Plasmodium HSP20 dramatically chan- thaliana, express as many as 19 variants.20 Submitted: 01/31/12 ged sporozoite speed and substrate adhe- The precise biological functions of indi- Revised: 03/20/12 sion, resulting in impaired natural malaria vidual HSPBs, which are often develop- Accepted: 03/22/12 transmission. In this article, we discuss the mentally regulated or tissue-specific, function of Hsp20 in this fast-moving remain largely unknown. For example, http://dx.doi.org/10.4161/cam.20101 unicellular protozoan and implications for human Hsp20/HSPB6 is expressed at high *Correspondence to: Kai Matuschewski; the roles of HSPBs in adhesion and levels in all three major types of muscle, Email: [email protected] migration of eukaryotic cells. i.e., skeletal, cardiac and vascular smooth muscle, and cardiac-specific overexpression Commentary to: Montagna GN, Buscaglia CA, Münter S, Goosmann C, Frischknecht F, confers protection against ischemia/reper- 21 Brinkmann V, et al. Critical role for heat shock Introduction fusion injury. protein 20 (HSP20) in migration of malarial One biochemical activity of HSPBs sporozoites. J Biol Chem 2012; 287:2410–22; The family of small heat shock proteins deserves particular attention, namely their PMID:22139844; http://dx.doi.org/10.1074/jbc. (sHSPs; HSPBs) includes molecular mini- potential to regulate dynamics of fila- M111.302109 chaperones with a signature a-crystallin mentous (F-) actin. Indeed, an avian

www.landesbioscience.com Cell Adhesion & Migration 1 ortholog of HSPB1 was isolated based on these findings establish an important in (MyoA), that are anchored to flattened its biochemical activity to suppress F-actin vivo role for HSPBs in cellular adhesion Golgi-derived cisternae, termed inner polymerization and viscosity in vitro, and and migration. membrane complex (IMC). The IMC is originally termed inhibitor of actin poly- a rigid structure, supported on its cyto- merization (IAP).22 This finding was Cell Migration of Malaria Parasites plasmic side by microtubules and a net- supported by subsequent studies with work of ill-defined filaments that extends mouse and human orthologs showing Cell migration is a highly spatially and beneath the entire surface of the parasite. that F-actin modulation is strictly depen- temporally coordinated process, typically According to the current model,36-39 dent on the phosphorylation status of involving (1) the formation of extended immobilized MyoA treads along short HSPB1.23,24 However, how HSPB1 and protrusions in the direction of migration, and dynamic F-actin transiently formed actin interact remains controversial, and which is driven by dynamic cytoskeleton in the narrow, so-called cortical, space mutually exclusive modes of action, rearrangements, and (2) the reversible between the IMC and the plasma mem- including barbed end capping of pre-exist- assembly of focal adhesion sites, which brane, which in turn interact with type I ing microfilaments,22,24 actin monomer depend on the physical and chemical transmembrane proteins belonging to the sequestration25 and chaperone-mediated nature of the substrate.32 In a variation of thrombospondin-related anonymous pro- prevention of polymerization26 have been substrate-dependent amoeboid cell move- tein (TRAP) family. TRAP family proteins proposed. Likewise, contradictory models, ment or crawling motility eukaryotic are stored in specialized secretory orga- i.e., direct inhibition of myosin/F-actin parasitic protozoa of the phylum nelles, so-called micronemes, and immedi- coupling via a troponin I motif27 and Apicomplexa display so-called gliding ately translocated onto the parasite surface indirectly via activation of actin depoly- motility.33 This rapid movement on solid in response to external stimuli. They bear a merizing factor/cofilin,28 were proposed substrates occurs without any apparent short cytoplasmic domain, which mediates to explain the efficient inhibition of flexing, undulation of the parasite body, indirect binding to the parasite cytoskele- ©smooth muscle2012 contraction by phosphory- Landesnor participation of appendages, Bioscience. such as ton, and a single or multiple copies of lated HSPB6. cilia or flagella. extracellular adhesive domains of varying An emerging issue, which is also of Apicomplexa include parasites of major specificities.36-39 Forward gliding loco- therapeutic relevance, is whether HSPB- medical and veterinary importance, e.g., motion is achieved by apical secretion, mediated actin reorganization also directly Plasmodium, Toxoplasma gondii—the etio- capping and backward translocation of shapes cellular motility. Work in neutro- logic agent of human toxoplasmosis34— TRAP family proteins, which link the phils, motile (~0.1–0.2Domm/sec) immune notand Babesia, distribute. which causes severe anemia, substrate or the target cell membrane to cells that migrate to sites of infection or cachexia, cerebral dysfunction and pulmo- the parasite surface. Recent data showed inflammation, showed that biochemical nary edema in infected cattle.35 They are that sporozoite motility is actually a interference with HSPB1 impaired che- obligate intracellular eukaryotes that discontinuous process, made up of small motaxis and exocytosis.29 However, largely actively invade host cells by developing parasite thrusts in between disengagement because of their short life-span neutrophils into highly polarized and usually elongated of focal adhesion sites and formation of are not amenable to genetic manipulation. cells. Cell migration is essential at several new ones.40 Hence, the rapid turnover rate Experimental genetics in an alternative stages during apicomplexan life cycles. In of these transient adhesion sites is directly highly motile eukaryote might therefore Plasmodium, for instance, these stages proportional to the speed of sporozoites. provide important insights into the in vivo permit parasite dissemination from the Although MyoA alone can supposedly roles of HSPBs. In a recent study, we mosquito inoculation site, breaching of provide enough force to drive motility, demonstrated that ablation of HSP20 in non-permissive biological barriers by inhibitor studies indicate that actin the unicellular eukaryote Plasmodium, the passing through the cytoplasm of host filament formation is a rate-limiting step causative agent of malaria,30 profoundly cells, host cell invasion and egress and in Plasmodium motility as well.40 Actin affected locomotion of highly motile pathogenesis.36 Extracellular gliding dynamics apparently also regulates parasite (~1–3 mm/sec) sporozoites.31 This extra- motility, cell traversal and cell invasion adhesion to substrate before and during cellular parasite stage is the contagious are thought to be strictly dependent gliding locomotion.40,42 Despite the critical form residing inside mosquito salivary on a conserved actin/myosin motor role of actin regulation, a paradoxically glands and is transmitted upon an infec- machinery36-39 and a set of adhesion limited repertoire of classical actin-binding tious mosquito bite. HSP20-deficient receptors, which remain anchored to the proteins, in comparison to other eukaryotes, sporozoites displayed aberrant in vitro substrate and can resist strong shear was described so far in Plasmodium.36,43-45 speed and trajectories, which correlated forces.40,41 Experiments using total internal with the presence of a single large adhesion reflection fluorescence (TIRF) microscopy Hsp20 is a Novel Plasmodium site spanning almost the entire parasite revealed that adhesion in gliding sporo- Motility Regulator length.31 This aberrant gliding translated zoites is a complex, sequential, and in defective parasite migration in the developmentally regulated process.42 Expression profiling and immunofluore- vertebrate dermis, thus impairing natural Apicomplexa express small, unconven- scence assays showed that Plasmodium malaria transmission. We suggest that tional class XIV myosins, e.g., myosin A Hsp20 is predominantly expressed in the

2 Cell Adhesion & Migration Volume 6 Issue 2 insect vector during the Plasmodium life remained entirely malaria-free upon infec- We speculate that transmission of traction cycle.31 The Hsp20 signal is progressively tious mosquito bite, whereas the other half forces by the molecular motor is most lost once the parasite encounters the was diagnosed with blood infections only important to propel the parasite in the mammalian host and undetectable after a substantial delay. Strikingly, in vitro dermis, where skin tissue tightly surrounds throughout the pathogenic blood infection invasion of cultured hepatoma cells the sporozoites. of the parasite. This expression pattern is was not affected in mutant sporozoites. Two high-resolution imaging techni- somewhat counterintuitive, since trans- Furthermore, in vivo infectivity of ques, reflection interference contrast ition from a poikilothermic mosquito to Hsp20-deficient sporozoites could be microscopy (RICM) and scanning electron the homeothermic mammal involves a fully restored to wild type (WT) levels by microscopy (SEM), lent support to these sudden increase in temperature of at least simply bypassing the skin passage through propositions (Fig. 1). In hsp20(-) sporo- 10°C and, hence, is expected to trigger a intravenous injection by syringe.31 zoites the presence of a single large heat shock response. However, this obser- Together, these findings indicate a critical adhesion site spanning almost the entire vation seems to fit other HSPBs, where and distinct role for Hsp20 in sporozoites parasite length can be observed.31 In only HSPB1 and HSPB4 are heat-indu- during the brief, but essential period of contrast, WT sporozoites form short-lived cible. In particular, human HSPB6/Hsp20 intra-dermal migration to reach the blood and discrete adhesion sites that determine is not upregulated upon a temperature circulation and finally enter the liver. A their typical stop-and-go motion.40 In line shift.46 It should be noted, however, that deeper understanding of many basic with this, distribution of TRAP, which is in other apicomplexan parasites Hsp20 aspects of sporozoite migration in the skin typically located at the focal adhesion sites proteins are expressed in blood-dwelling would certainly clarify why this phase of sporozoites, is aberrant in hsp20(-) forms such as merozoites from Babesia constitutes the Achilles heel of “motility sporozoites.31 Instead of its characteristic spp47 and in T. gondii tachyzoites.48.49 handicapped” hsp20(-) parasites. patchy distribution, TRAP signal is Targeted gene deletion in the murine detected over the entire ventral surface of ©model parasite 2012Plasmodium berghei using LandesHsp20(-) Sporozoites DisplayBioscience.hsp20(-) sporozoites, thus overlapping with cultured blood stages permits systematic Aberrant Cell Motility their single attachment site. Together with phenotyping of the resulting mutant recent in vitro data,52 the observed defects parasites throughout the complex life cycle In order to pinpoint a block in life cycle in sporozoites lacking a key regulator of in vivo. When applied to HSP20, viable progression in vivo, parasites can be adhesion and locomotion further empha- hsp20(-) parasites could be selected and no analyzed by surrogate assays, such as size the major impact of the extracellular apparent deficiency inDo the warm-blooded nottracking cellular distribute. locomotion on artificial environment on sporozoite motility. host could be detected, in good agreement substrates33,40 and quantifying traversal with the apparent paucity of Hsp20 and invasion of cultured cells (Fig. 1). In Spatial Regulation of Hsp20 expression during blood infection.31 support of a critical role in cell adhesion during Cellular Movement Intriguingly, an important role of and motility, Hsp20-mutant sporozoites Hsp20 was restricted to host switch, i.e., displayed a fundamentally different gliding Changes in mammalian Hsp20 expression when parasites are injected into the pattern as compared with WT sporozoites. were first observed upon addition of mammalian host during a blood meal.31 Irrespective of the nature of the substrate, various chemical stress signals and mecha- Transmission from a vertebrate to an serum albumin-coated glass slides or nical force.46 An additional layer of protein invertebrate host and back are arguably cultured fibroblast and hepatoma cell activity regulation appears to occur at the the most critical events in the pathogen life monolayers, hsp20(-) sporozoites displayed posttranslational level, by reversible phos- cycle, as reflected by dramatic population slow and uniform motility and followed phorylation. In heart and skeletal muscle, bottlenecks.50,51 In order to establish an a previously unrecognized pattern.31 In for instance, Hsp20 localizes predomi- infection in the new host the malarial contrast to WT parasites that glide in nantly to the cytoplasm under physio- parasite forms tailor-made extracellular characteristic, near-perfect circles mutant logical conditions, but it is immediately stages that display the above-mentioned parasites displayed elongated and linear phosphorylated and translocated to the gliding motility. Hsp20(-) parasites dis- trajectories (Fig. 1). This distinction, but myofilaments under ischemic stress condi- played reduced ookinete motility, that is not the reduction in speed, disappeared tions.14 Similar stimulus-induced relocali- parasites formed in the blood bolus inside when cell motility was tracked in vivo in zation was described for other HSPBs in the mosquito midgut move substantially the dermis.31 We interpret these findings various cell types and organisms.53,54 slower in the absence of Hsp20.31 But this as evidence (1) for a decisive requirement Previous work in T. gondii tachyzoites, deficiency did not impair establishment of for fast (. 1 mm/sec) motility to reach the the fast-replicating, pathogenic stage of a infection and overall parasite population bloodstream after being discharged into Toxoplasma infection, established that development inside the mosquito. the host dermis, (2) that apparent defects TgHsp20 localizes to the IMC,49 a finding In stark contrast, Hsp20 ablation criti- in in vitro motility patterns can be com- that could be confirmed in P. berghei cally impaired natural transmission to the pensated for when sporozoites move in ookinetes and sporozoites.31 Unexpectedly, mammalian host.31 Experimental infection natural, three-dimensional environments when PbHsp20 localization was analyzed assays showed that half of the mice and (3) for an independence of cell speed. in motile sporozoites, a large proportion

www.landesbioscience.com Cell Adhesion & Migration 3 Figure 1. Plasmodium sporozoites lacking Hsp20 display aberrant cell adhesion and gliding motility. (A) Time-lapse projections of WT (red) and hsp20(-) (green) sporozoites gliding on glass slides (left), on cultured fibroblasts (center) or in vivo in the skin of a mouse ear (right; z-projections). Note that WT parasites describe circular trajectories, while hsp20(-) parasites display more elongated trajectories, in vitro and on cultured cells. Irrespective of the substrate, hsp20(-) sporozoites display a significantly reduced speed (indicated in the upper right corner). (B) Adhesion of gliding parasites is strengthened in the absence of Hsp20. Shown are micrographs of WT and hsp20(-) sporozoites, using reflection interference contrast microscopy (RICM; left panels). Note discrete adhesion sites at both parasite tips, consistent with its stick-slip motion40 and a single elongated attachment site in WT ©and hsp20(-) 2012sporozoites, respectively. Black arrowsLandes indicate the adhesion sites. Scanning Bioscience. electron microscopy (SEM; center panels) exemplify that WT sporozoites can alternate gliding periods with non-adherent gliding phases, while hsp20(-) parasites remain continuously surface-attached during gliding. Immunofluorescence assay with anti-TRAP antiserum (right panels) illustrate the accumulation of surface adhesion molecules (red arrows)on the ventral site of an hsp20(-) sporozoite, consistent with its enlarged adhesion site.

(. 50%) displayed a unique, focal distri- exogenous addition of Hsp20-specific on residues lying outside the signature bution at the parasite tipsDo31 (Fig. 2). Itnot is antibodies inhibiteddistribute. cell invasion.55 Of a-crystallin domain, as already described worth noting that these PbHsp20 foci did note, Hsp20 in another parasitic pathogen, in other systems.23,24,57 Upon phosphoryla- not match sporozoite adhesion sites (see Leishmania, was shown to be antigenic tion, this protein could also bind to a below). Differential permeabilization tech- in natural infections and to elicit robust 14-3-3 protein,58 resulting in displacement niques, using gentle (saponin) or strong B-cell responses in infected dogs,56 of other binding partners of 14-3-3, such (Triton X-100) detergents indicated the indicating that translocation to the cell as the actin monomer sequesterer cofilin, presence of two distinct Hsp20 popula- periphery and eventual exposure of HSPBs or directly inhibit actin/myosin interac- tions in gliding sporozoites. On one hand, might not be restricted to apicomplexan tions. In all cases, only phosphorylated a fraction is distributed along the entire parasites. Hsp20 is capable of regulating cytoskele- parasite body and likely localizes to the Since Hsp20 apparently lacks a cleav- ton reorganization.27,28 Experimental test- cytoplasmic side of the cholesterol-rich, able signal peptide or transmembrane ing of these putative posttranslational Triton-resistant IMC membranes (Fig. 2). spanning domains, recruitment to the modifications can be done by site-directed This fraction could be clearly distin- IMC, followed by polarization upon loss-of- and gain-of-function mutagenesis guished from a second Hsp20 population sporozoite activation, most likely and complementation of hsp20(-) parasites that is accessible to antibodies upon involves posttranslational modifications. to either partially restore or exacerbate the fixation or gentle permeabilization, which Accordingly, PbHsp20 contains three observed adhesion and motility defects. corresponds to the one undergoing polari- cysteine residues positions at 3, 5 and 8, An alternative possibility is that variable zation upon parasite activation. This which are conserved across all Plasmodium spatial localization of PbHsp20 is not an fraction somehow traverses the IMC and species with the exception of Cys8 that is inherent property but, similar to vertebrate localizes to the cortical space either in a absent in P. falciparum, and which could HSPB1,22,23 the result of its interaction soluble form, anchored to the external face be potentially palmitoylated and, hence, with actin and/or another dynamic pro- of the IMC, or to the inner leaflet of the may mediate reversible insertion into tein. Relocalization of different parasite plasma membrane (see below). The strik- lipid bilayers. Alternatively, Plasmodium molecules, e.g., glycolytic enzymes and ing abundant presence close to the parasite Hsp20, as its human8 and T. gondii49 glycolipids has been shown in moving surface of this Hsp20 fraction is surprising, counterparts, might directly interact with T. gondii tachyzoites.59,60 Irrespective of but supported by data from Babesia membrane components. Such an inter- the precise molecular mechanisms, iden- divergens merozoites47 and T. gondii tachy- action could be further modulated by tification of proteins that share a transient, zoites,48,49 where it was shown that reversible phosphorylation or acetylation polarized localization to the apical IMC

4 Cell Adhesion & Migration Volume 6 Issue 2 Figure 2. Hsp20 distribution during gliding. (A) Immunofluorecescence assays of gliding sporozoites after mild or strong permeabilization conditions ©reveal two distinct2012 Hsp20 populations (left panels).Landes All assays were performed using parasites Bioscience. expressing Hsp20 fused to the fluorescent mCherry protein and antisera against the fluorescent tag Dsred, PbHsp20, or PbTRAP, a signature parasite microneme protein. Using mild permeabilization conditions (saponin; top panels), two distinct Hsp20 populations are discriminated, one at the tip of the parasite (anti-Dsred antibody, green), and one uniform (mCherry fluorescence, red), which remained inaccessible to the antibody. Upon harsh permeabilization conditions (Triton X100; center panels) the signals revealed by anti-Dsred and anti-Hsp20 antisera substantially overlapped. Under similar harsh permeabilization conditions, double staining with anti-Hsp20 (red) and anti-TRAP (green) antisera (bottom panels) revealed no substantial overlap of the signals. (B) High resolution scanning immunoelectron microscopyof the apical end of a gliding parasite revealed abundant Hsp20 at the sporozoite periphery, but exclusion from the attachment sites (feet-like protrusions). Sporozoites were fixed afterDo gliding, permeabilized not with saponin anddistribute. incubated with Hsp20 antiserum followed by 40-nm gold labeling (green dots).

with Hsp20 might reveal additional regu- likely to discharge secretory organelles disengagement of parasite attachment lators of parasite adhesion and motility. normally. Therefore, low gliding speed sites, for instance by aiding the release of and slow turnover of adhesion sites in TRAP-substrate complexes, which likely Putative Roles of Hsp20 hsp20(-) sporozoites are best explained by requires intramembrane cleavage by a in Actin-Myosin Based Motility inefficient turnover of actin microfila- Rhomboid protease. However, there is ments, which are challenging to visualize presently no indication for co-localization Evidence from other biological systems has in sporozoites, or a rigor state of actin/ of Hsp20 and TRAP in gliding parasites implicated Hsp20 in a number of diverse myosin interactions in the absence of (Fig. 2). Furthermore, high-resolution cellular pathways that involve cyclic Hsp20. In the former scenario, Hsp20 immuno-SEM revealed that Hsp20 is adenosine monophosphate (cAMP),61 cyc- would participate in actin regulation by suspiciously excluded from the margins lic guanosine monophosphate (cGMP)27 indirect competition with cofilins, as of parasite attachment sites, which and Ca2+ signaling.62 While the potential proposed for HSPB1 in smooth muscle resemble feet-like protrusions under the signaling pathways that trigger sporozoite contraction.28 The transient localization of fixation conditions, and the trails left activation remain uncertain, evidence for a subset of Hsp20 molecules at the cortical behind moving sporozoites (Fig. 2). similar mechanisms have been reported. space, where the motor operates, is A third possibility is that Hsp20, in this Intracellular Ca2+ mobilization correlates consistent with this hypothesis (Fig. 2). case the non-polarized fraction underneath with discharge of apical organelles and sur- We consider a filament capping activity, the IMC, participates in microtubule face translocation of adhesion molecules.63 as shown for HSPB1,22 less likely, since stabilization. Hsp20 mutants might cGMP-dependent signaling is also involved targeted deletion of the β-subunit of remain attached to the substrate simply in microneme secretion, at least in T. gondii Plasmodium barbed-end capping protein because of the reduction in traction forces. tachyzoites,64 and at least three Ca2+- (CP) revealed a vital function for sporo- In apicomplexan parasites microtubules dependent kinases (CDPKs) seem to contri- zoite motility,66 arguing against functional form a characteristically dynamic network bute to sporozoite motility and invasion.65 redundancy between Hsp20 and CP. that transmits tensile force. In support Parasites lacking Hsp20 are able to An alternative possibility is that Hsp20 of this hypothesis TgHsp20, but not translocate TRAP31 and, by inference, participates in the establishment and/or PbHsp20, is distributed in discontinuous

www.landesbioscience.com Cell Adhesion & Migration 5 stripes along the parasite body, following Hsp20 binding partners and post- Acknowledgments the microtubule trajectories.49 In muscle translational modifications, is warranted to We are grateful to our collaborators Dr. cells, mechanical stress, such as lengthen- validate or reject the proposed mechanisms. Sylvia Münter (Carl Zeiss MicroImaging ing contraction, induces translocation of Labs), Christian Goosmann (MPI-IB), cytosolic small HSPBs to the cytoskeleton Conclusions Dr. Friedrich Frischknecht (Heidelberg at structures prone to disruption.67 University), and Dr. Volker Brinkmann Although examples of HSPBs interacting Slow hsp20(-) malarial sporozoites pro- (MPI-IB) for our joint work on with microtubules are scarce, a recent vided genetic evidence for an important Plasmodium Hsp20. We also thank Drs. study showed that aB-crystallin could role of this HSPB in cellular adhesion and Volker Brinkmann (MPI-IB) and María interact with tubulin subunits to regulate motility. These unicellular eukaryotes Corvi and Sergio Angel (IIB-INTECH) the dynamics of microtubules.68 This display a remarkably fast substrate- for providing us with micrographs and interaction supposedly relies on a protein dependent migration, yet rely on an unpublished results, respectively. Financial domain shared between tubulin and aB- ancient, minimal actin-myosin based support from the Alexander von crystallin. Such a consensus sequence is motor. Extension of these findings to Humboldt Foundation to G.N.M., the however missing in Plasmodium HSPBs. other motile cells crucially depends on Max Planck Society and the European Finally, it should be considered that molecular genetic tools, such as siRNA- Commission (EviMalaR, #34) to K.M., transient interactions between Hsp20 and mediated knockdown of one or multiple the UNICEF/UNDP/World Bank/WHO certain lipids8,49 might directly affect the HSPBs. Because cellular motility is central Special Programme for Research and fluidity/integrity of the IMC membranes, to diverse physiological and pathological Training in Tropical Diseases (TDR), which, in turn, might influence the processes, ranging from embryogenesis Agencia Nacional de Promoción Científica topology and overall activity of the motor and wound healing to metastatic cancers, y Tecnológica (ANPCyT), and UNSAM to molecules anchored to the IMC. scrutinizing the contribution of HSPBs to C.A.B. is appreciated. C.A.B. is a career ©Clearly, 2012 additional work, including Landes a this process also has important Bioscience. medical investigator from the Argentinean Research detailed biochemical characterization of implications beyond infection biology. Council (CONICET).

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