Intramolecular autoinhibition regulates the selectivity of PRPF40A tandem WW domains for proline-rich motifs.

PRPF40A plays an important role in the regulation of pre-mRNA splicing by mediating protein-protein interactions in the early steps of spliceosome assembly. By binding to proteins at the 5´ and 3´ splice sites, PRPF40A promotes spliceosome assembly by bridging the recognition of the splices. The PRPF40A WW domains are expected to recognize proline-rich sequences in SF1 and SF3A1 in the early spliceosome complexes E and A, respectively. Here, we combine NMR, SAXS and ITC to determine the structure of the PRPF40A tandem WW domains in solution and characterize the binding specificity and mechanism for proline-rich motifs recognition. Our structure of the PRPF40A WW tandem in complex with a high-affinity SF1 peptide reveals contributions of both WW domains, which also enables tryptophan sandwiching by two proline residues in the ligand. Unexpectedly, a proline-rich motif in the N-terminal region of PRPF40A mediates intramolecular interactions with the WW tandem. Using NMR, ITC, mutational analysis in vitro, and immunoprecipitation experiments in cells, we show that the intramolecular interaction acts as an autoinhibitory filter for proof-reading of high-affinity proline-rich motifs in bona fide PRPF40A binding partners. We propose that similar autoinhibitory mechanisms are present in most WW tandem-containing proteins to enhance binding selectivity and regulation of WW/proline-rich peptide interaction networks.

Dishevelled2 activates WGEF via its interaction with a unique internal peptide motif of the GEF.

The Wnt-planar cell polarity (Wnt-PCP) pathway is crucial in establishing cell polarity during development and tissue homoeostasis. This pathway is found to be dysregulated in many pathological conditions, including cancer and autoimmune disorders. The central event in Wnt-PCP pathway is the activation of Weak-similarity guanine nucleotide exchange factor (WGEF) by the adapter protein Dishevelled (Dvl). The PDZ domain of Dishevelled2 (Dvl2PDZ) binds and activates WGEF by releasing it from its autoinhibitory state. However, the actual Dvl2PDZ binding site of WGEF and the consequent activation mechanism of the GEF have remained elusive. Using biochemical and molecular dynamics studies, we show that a unique "internal-PDZ binding motif" (IPM) of WGEF mediates the WGEF-Dvl2PDZ interaction to activate the GEF. The residues at P2, P0, P-2 and P-3 positions of IPM play an important role in stabilizing the WGEFpep-Dvl2PDZ interaction. Furthermore, MD simulations of modelled Dvl2PDZ-WGEFIPM peptide complexes suggest that WGEF-Dvl2PDZ interaction may differ from the reported Dvl2PDZ-IPM interactions. Additionally, the apo structure of human Dvl2PDZ shows conformational dynamics different from its IPM peptide bound state, suggesting an induced fit mechanism for the Dvl2PDZ-peptide interaction. The current study provides a model for Dvl2 induced activation of WGEF.

The NTE domain of PTENα/ß promotes cancer progression by interacting with WDR5 via its SSSRRSS motif.

PTENα/ß, two variants of PTEN, play a key role in promoting tumor growth by interacting with WDR5 through their N-terminal extensions (NTEs). This interaction facilitates the recruitment of the SET1/MLL methyltransferase complex, resulting in histone H3K4 trimethylation and upregulation of oncogenes such as NOTCH3, which in turn promotes tumor growth. However, the molecular mechanism underlying this interaction has remained elusive. In this study, we determined the first crystal structure of PTENα-NTE in complex with WDR5, which reveals that PTENα utilizes a unique binding motif of a sequence SSSRRSS found in the NTE domain of PTENα/ß to specifically bind to the WIN site of WDR5. Disruption of this interaction significantly impedes cell proliferation and tumor growth, highlighting the potential of the WIN site inhibitors of WDR5 as a way of therapeutic intervention of the PTENα/ß associated cancers. These findings not only shed light on the important role of the PTENα/ß-WDR5 interaction in carcinogenesis, but also present a promising avenue for developing cancer treatments that target this pathway.

A conserved motif in the immune-subdominant RAP-1 related antigen of Babesia bovis contains a B-cell epitope recognized by antibodies from protected cattle.

Introduction: Babesia bovis, a tick-borne apicomplexan parasite causing bovine babesiosis, remains a significant threat worldwide, and improved and practical vaccines are needed. Previous studies defined the members of the rhoptry associated protein-1 (RAP-1), and the neutralization-sensitive rhoptry associated protein-1 related antigen (RRA) superfamily in B. bovis, as strong candidates for the development of subunit vaccines. Both RAP-1 and RRA share conservation of a group of 4 cysteines and amino acids motifs at the amino terminal end (NT) of these proteins. Methods and results: Sequence comparisons among the RRA sequences of several B. bovis strains and other Babesia spp parasites indicate a high level of conservation of a 15-amino acid (15-mer) motif located at the NT of the protein. BlastP searches indicate that the 15-mer motif is also present in adenylate cyclase, dynein, and other ATP binding proteins. AlphaFold2 structure predictions suggest partial exposure of the 15-mer on the surface of RRA of three distinct Babesia species. Antibodies in protected cattle recognize a synthetic peptide representing the 15-mer motif sequence in iELISA, and rabbit antibodies against the 15-mer react with the surface of free merozoites in immunofluorescence. Discussion and conclusion: The presence of the 15-mer-like regions in dynein and ATP-binding proteins provides a rationale for investigating possible functional roles for RRA. The demonstrated presence of a surface exposed B-cell epitope in the 15-mer motif of the B. bovis RRA, which is recognized by sera from protected bovines, supports its inclusion in future subunit epitope-based vaccines against B. bovis.

1H, 13C, and 15N resonance assignments of the La Motif of the human La-related protein 1.

Human La-related protein 1 (HsLARP1) is involved in post-transcriptional regulation of certain 5' terminal oligopyrimidine (5'TOP) mRNAs as well as other mRNAs and binds to both the 5'TOP motif and the 3'-poly(A) tail of certain mRNAs. HsLARP1 is heavily involved in cell proliferation, cell cycle defects, and cancer, where HsLARP1 is significantly upregulated in malignant cells and tissues. Like all LARPs, HsLARP1 contains a folded RNA binding domain, the La motif (LaM). Our current understanding of post-transcriptional regulation that emanates from the intricate molecular framework of HsLARP1 is currently limited to small snapshots, obfuscating our understanding of the full picture on HsLARP1 functionality in post-transcriptional events. Here, we present the nearly complete resonance assignment of the LaM of HsLARP1, providing a significant platform for future NMR spectroscopic studies.

A conserved N-terminal motif of CUL3 contributes to assembly and E3 ligase activity of CRL3KLHL22.

The CUL3-RING E3 ubiquitin ligases (CRL3s) play an essential role in response to extracellular nutrition and stress stimuli. The ubiquitin ligase function of CRL3s is activated through dimerization. However, how and why such a dimeric assembly is required for its ligase activity remains elusive. Here, we report the cryo-EM structure of the dimeric CRL3KLHL22 complex and reveal a conserved N-terminal motif in CUL3 that contributes to the dimerization assembly and the E3 ligase activity of CRL3KLHL22. We show that deletion of the CUL3 N-terminal motif impairs dimeric assembly and the E3 ligase activity of both CRL3KLHL22 and several other CRL3s. In addition, we found that the dynamics of dimeric assembly of CRL3KLHL22 generates a variable ubiquitination zone, potentially facilitating substrate recognition and ubiquitination. These findings demonstrate that a CUL3 N-terminal motif participates in the assembly process and provide insights into the assembly and activation of CRL3s.

Modulating Polyalanine Motifs of Synthetic Spidroin for Controllable Preassembly and Strong Fiber Formation.

Spider dragline (major ampullate) silk is one of the toughest known fibers in nature and exhibits an excellent combination of high tensile strength and elasticity. Increasing evidence has indicated that preassembly plays a crucial role in facilitating the proper assembly of silk fibers by bridging the mesoscale gap between spidroin molecules and the final strong fibers. However, it remains challenging to control the preassembly of spidroins and investigate its influence on fiber structural and mechanical properties. In this study, we explored to bridge this gap by modulating the polyalanine (polyA) motifs in repetitive region of spidroins to tune their preassemblies in aqueous dope solutions. Three biomimetic silk proteins with varying numbers of alanine residues in polyA motif and comparable molecular weights were designed and biosynthesized, termed as N16C-5A, N15C-8A, and N13C-12A, respectively. It was found that all three proteins could form nanofibril assemblies in the concentrated aqueous dopes, but the size and structural stability of the fibrils were distinct from each other. The silk protein N15C-8A with 8 alanine residues in polyA motif allowed for the formation of stable nanofibril assemblies with a length of approximately 200 nm, which were not prone to disassemble or aggregate as that of N16C-5A and N13C-12A. More interestingly, the stable fibril assembly of N15C-8A enabled spinning of simultaneously strong (623.3 MPa) and tough (107.1 MJ m-3) synthetic fibers with fine molecular orientation and close interface packing of fibril bundles. This work highlights that modulation of polyA motifs is a feasible way to tune the morphology and stability of the spidroin preassemblies in dope solutions, thus controlling the structural and mechanical properties of the resulting fibers.

A Targetable N-Terminal Motif Orchestrates α-Synuclein Oligomer-to-Fibril Conversion.

Oligomeric species populated during α-synuclein aggregation are considered key drivers of neurodegeneration in Parkinson's disease. However, the development of oligomer-targeting therapeutics is constrained by our limited knowledge of their structure and the molecular determinants driving their conversion to fibrils. Phenol-soluble modulin α3 (PSMα3) is a nanomolar peptide binder of α-synuclein oligomers that inhibits aggregation by blocking oligomer-to-fibril conversion. Here, we investigate the binding of PSMα3 to α-synuclein oligomers to discover the mechanistic basis of this protective activity. We find that PSMα3 selectively targets an α-synuclein N-terminal motif (residues 36-61) that populates a distinct conformation in the mono- and oligomeric states. This α-synuclein region plays a pivotal role in oligomer-to-fibril conversion as its absence renders the central NAC domain insufficient to prompt this structural transition. The hereditary mutation G51D, associated with early onset Parkinson's disease, causes a conformational fluctuation in this region, leading to delayed oligomer-to-fibril conversion and an accumulation of oligomers that are resistant to remodeling by molecular chaperones. Overall, our findings unveil a new targetable region in α-synuclein oligomers, advance our comprehension of oligomer-to-amyloid fibril conversion, and reveal a new facet of α-synuclein pathogenic mutations.

Identification of a short sequence motif in the influenza A virus pathogenicity factor PB1-F2 required for inhibition of human NLRP3.

Influenza A virus infection activates the NLRP3 inflammasome, a multiprotein signaling complex responsible for the proteolytic activation and release of the proinflammatory cytokine IL-1ß from monocytes and macrophages. Some influenza A virus (IAV) strains encode a short 90-amino acid peptide (PB1-F2) on an alternative open reading frame of segment 2, with immunomodulatory activity. We recently demonstrated that contemporary IAV PB1-F2 inhibits the activation of NLRP3, potentially by NEK7-dependent activation. PB1-F2 binds to NLRP3 with its C-terminal 50 amino acids, but the exact binding motif was unknown. On the NLRP3 side, the interface is formed through the leucine-rich-repeat (LRR) domain, potentially in conjunction with the pyrin domain. Here, we took advantage of PB1-F2 sequences from IAV strains with either weak or strong NLRP3 interaction. Sequence comparison and structure prediction using Alphafold2 identified a short four amino acid sequence motif (TQGS) in PB1-F2 that defines NLRP3-LRR binding. Conversion of this motif to that of the non-binding PB1-F2 suffices to lose inhibition of NLRP3 dependent IL-1ß release. The TQGS motif further alters the subcellular localization of PB1-F2 and its colocalization with NLRP3 LRR and pyrin domain. Structural predictions suggest the establishment of additional hydrogen bonds between the C-terminus of PB1-F2 and the LRR domain of NLRP3, with two hydrogen bonds connecting to threonine and glutamine of the TQGS motif. Phylogenetic data show that the identified NLRP3 interaction motif in PB1-F2 is widely conserved among recent IAV-infecting humans. Our data explain at a molecular level the specificity of NLRP3 inhibition by influenza A virus. IMPORTANCE: Influenza A virus infection is accompanied by a strong inflammatory response and high fever. The human immune system facilitates the swift clearance of the virus with this response. An essential signal protein in the proinflammatory host response is IL-1b. It is released from inflammatory macrophages, and its production and secretion depend on the function of NLRP3. We had previously shown that influenza A virus blocks NLRP3 activation by the expression of a viral inhibitor, PB1-F2. Here, we demonstrate how this short peptide binds to NLRP3 and provide evidence that a four amino acid stretch in PB1-F2 is necessary and sufficient to mediate this binding. Our data identify a new virus-host interface required to block one signaling path of the innate host response against influenza A virus.

Functional Integrity of Radical SAM Enzyme Dph1•Dph2 Requires Non-Canonical Cofactor Motifs with Tandem Cysteines.

The Dph1•Dph2 heterodimer from yeast is a radical SAM (RS) enzyme that generates the 3-amino-3-carboxy-propyl (ACP) precursor for diphthamide, a clinically relevant modification on eukaryotic elongation factor 2 (eEF2). ACP formation requires SAM cleavage and atypical Cys-bound Fe-S clusters in each Dph1 and Dph2 subunit. Intriguingly, the first Cys residue in each motif is found next to another ill-defined cysteine that we show is conserved across eukaryotes. As judged from structural modeling, the orientation of these tandem cysteine motifs (TCMs) suggests a candidate Fe-S cluster ligand role. Hence, we generated, by site-directed DPH1 and DPH2 mutagenesis, Dph1•Dph2 variants with cysteines from each TCM replaced individually or in combination by serines. Assays diagnostic for diphthamide formation in vivo reveal that while single substitutions in the TCM of Dph2 cause mild defects, double mutations almost entirely inactivate the RS enzyme. Based on enhanced Dph1 and Dph2 subunit instability in response to cycloheximide chases, the variants with Cys substitutions in their cofactor motifs are particularly prone to protein degradation. In sum, we identify a fourth functionally cooperative Cys residue within the Fe-S motif of Dph2 and show that the Cys-based cofactor binding motifs in Dph1 and Dph2 are critical for the structural integrity of the dimeric RS enzyme in vivo.

14-3-3 binding motif phosphorylation disrupts Hdac4-organized condensates to stimulate cardiac reprogramming.

Cell fate conversion is associated with extensive post-translational modifications (PTMs) and architectural changes of sub-organelles, yet how these events are interconnected remains unknown. We report here the identification of a phosphorylation code in 14-3-3 binding motifs (PC14-3-3) that greatly stimulates induced cardiomyocyte (iCM) formation from fibroblasts. PC14-3-3 is identified in pivotal functional proteins for iCM reprogramming, including transcription factors and chromatin modifiers. Akt1 kinase and protein phosphatase 2A are the key writer and key eraser of the PC14-3-3 code, respectively. PC14-3-3 activation induces iCM formation with the presence of only Tbx5. In contrast, PC14-3-3 inhibition by mutagenesis or inhibitor-mediated code removal abolishes reprogramming. We discover that key PC14-3-3-embedded factors, such as histone deacetylase 4 (Hdac4), Mef2c, and Foxo1, form Hdac4-organized inhibitory nuclear condensates. PC14-3-3 activation disrupts Hdac4 condensates to promote cardiac gene expression. Our study suggests that sub-organelle dynamics regulated by a PTM code could be a general mechanism for stimulating cell reprogramming.

CORDAX web server: an online platform for the prediction and 3D visualization of aggregation motifs in protein sequences.

MOTIVATION: Proteins, the molecular workhorses of biological systems, execute a multitude of critical functions dictated by their precise three-dimensional structures. In a complex and dynamic cellular environment, proteins can undergo misfolding, leading to the formation of aggregates that take up various forms, including amorphous and ordered aggregation in the shape of amyloid fibrils. This phenomenon is closely linked to a spectrum of widespread debilitating pathologies, such as Alzheimer's disease, Parkinson's disease, type-II diabetes, and several other proteinopathies, but also hampers the engineering of soluble agents, as in the case of antibody development. As such, the accurate prediction of aggregation propensity within protein sequences has become pivotal due to profound implications in understanding disease mechanisms, as well as in improving biotechnological and therapeutic applications. RESULTS: We previously developed Cordax, a structure-based predictor that utilizes logistic regression to detect aggregation motifs in protein sequences based on their structural complementarity to the amyloid cross-beta architecture. Here, we present a dedicated web server interface for Cordax. This online platform combines several features including detailed scoring of sequence aggregation propensity, as well as 3D visualization with several customization options for topology models of the structural cores formed by predicted aggregation motifs. In addition, information is provided on experimentally determined aggregation-prone regions that exhibit sequence similarity to predicted motifs, scores, and links to other predictor outputs, as well as simultaneous predictions of relevant sequence propensities, such as solubility, hydrophobicity, and secondary structure propensity. AVAILABILITY AND IMPLEMENTATION: The Cordax webserver is freely accessible at https://cordax.switchlab.org/.

Discovery and Characterization of Linear Motif Mediated Protein-Protein Complexes.

There are myriads of protein-protein complexes that form within the cell. In addition to classical binding events between globular domains, many protein-protein interactions involve short disordered protein regions. The latter contain so-called linear motifs binding specifically to ordered protein domain surfaces. Linear binding motifs are classified based on their consensus sequence, where only a few amino acids are conserved. In this chapter we will review experimental and in silico techniques that can be used for the discovery and characterization of linear motif mediated protein-protein complexes involved in cellular signaling, protein level and gene expression regulation.

Evolution-guided engineering of trans-acyltransferase polyketide synthases.

Bacterial multimodular polyketide synthases (PKSs) are giant enzymes that generate a wide range of therapeutically important but synthetically challenging natural products. Diversification of polyketide structures can be achieved by engineering these enzymes. However, notwithstanding successes made with textbook cis-acyltransferase (cis-AT) PKSs, tailoring such large assembly lines remains challenging. Unlike textbook PKSs, trans-AT PKSs feature an extraordinary diversity of PKS modules and commonly evolve to form hybrid PKSs. In this study, we analyzed amino acid coevolution to identify a common module site that yields functional PKSs. We used this site to insert and delete diverse PKS parts and create 22 engineered trans-AT PKSs from various pathways and in two bacterial producers. The high success rates of our engineering approach highlight the broader applicability to generate complex designer polyketides.

The HEAT repeat protein HPO-27 is a lysosome fission factor.

Lysosomes are degradation and signalling centres crucial for homeostasis, development and ageing1. To meet diverse cellular demands, lysosomes remodel their morphology and function through constant fusion and fission2,3. Little is known about the molecular basis of fission. Here we identify HPO-27, a conserved HEAT repeat protein, as a lysosome scission factor in Caenorhabditis elegans. Loss of HPO-27 impairs lysosome fission and leads to an excessive tubular network that ultimately collapses. HPO-27 and its human homologue MROH1 are recruited to lysosomes by RAB-7 and enriched at scission sites. Super-resolution imaging, negative-staining electron microscopy and in vitro reconstitution assays reveal that HPO-27 and MROH1 self-assemble to mediate the constriction and scission of lysosomal tubules in worms and mammalian cells, respectively, and assemble to sever supported membrane tubes in vitro. Loss of HPO-27 affects lysosomal morphology, integrity and degradation activity, which impairs animal development and longevity. Thus, HPO-27 and MROH1 act as self-assembling scission factors to maintain lysosomal homeostasis and function.

Prediction of motif-mediated viral mimicry through the integration of host-pathogen interactions.

One of the mechanisms viruses use in hijacking host cellular machinery is mimicking Short Linear Motifs (SLiMs) in host proteins to maintain their life cycle inside host cells. In the face of the escalating volume of virus-host protein-protein interactions (vhPPIs) documented in databases; the accurate prediction of molecular mimicry remains a formidable challenge due to the inherent degeneracy of SLiMs. Consequently, there is a pressing need for computational methodologies to predict new instances of viral mimicry. Our present study introduces a DMI-de-novo pipeline, revealing that vhPPIs catalogued in the VirHostNet3.0 database effectively capture domain-motif interactions (DMIs). Notably, both affinity purification coupled mass spectrometry and yeast two-hybrid assays emerged as good approaches for delineating DMIs. Furthermore, we have identified new vhPPIs mediated by SLiMs across different viruses. Importantly, the de-novo prediction strategy facilitated the recognition of several potential mimicry candidates implicated in the subversion of host cellular proteins. The insights gleaned from this research not only enhance our comprehension of the mechanisms by which viruses co-opt host cellular machinery but also pave the way for the development of novel therapeutic interventions.

Fast and scalable querying of eukaryotic linear motifs with gget elm.

MOTIVATION: Eukaryotic linear motifs (ELMs), or Short Linear Motifs, are protein interaction modules that play an essential role in cellular processes and signaling networks and are often involved in diseases like cancer. The ELM database is a collection of manually curated motif knowledge from scientific papers. It has become a crucial resource for investigating motif biology and recognizing candidate ELMs in novel amino acid sequences. Users can search amino acid sequences or UniProt Accessions on the ELM resource web interface. However, as with many web services, there are limitations in the swift processing of large-scale queries through the ELM web interface or API calls, and, therefore, integration into protein function analysis pipelines is limited. RESULTS: To allow swift, large-scale motif analyses on protein sequences using ELMs curated in the ELM database, we have extended the gget suite of Python and command line tools with a new module, gget elm, which does not rely on the ELM server for efficiently finding candidate ELMs in user-submitted amino acid sequences and UniProt Accessions. gget elm increases accessibility to the information stored in the ELM database and allows scalable searches for motif-mediated interaction sites in the amino acid sequences. AVAILABILITY AND IMPLEMENTATION: The manual and source code are available at https://github.com/pachterlab/gget.

Heterotrimeric G protein signaling without GPCRs: The Gα-binding-and-activating (GBA) motif.

Heterotrimeric G proteins (Gαßγ) are molecular switches that relay signals from 7-transmembrane receptors located at the cell surface to the cytoplasm. The function of these receptors is so intimately linked to heterotrimeric G proteins that they are named G protein-coupled receptors (GPCRs), showcasing the interdependent nature of this archetypical receptor-transducer axis of transmembrane signaling in eukaryotes. It is generally assumed that activation of heterotrimeric G protein signaling occurs exclusively by the action of GPCRs, but this idea has been challenged by the discovery of alternative mechanisms by which G proteins can propagate signals in the cell. This review will focus on a general principle of G protein signaling that operates without the direct involvement of GPCRs. The mechanism of G protein signaling reviewed here is mediated by a class of G protein regulators defined by containing an evolutionarily conserved sequence named the Gα-binding-and-activating (GBA) motif. Using the best characterized proteins with a GBA motif as examples, Gα-interacting vesicle-associated protein (GIV)/Girdin and dishevelled-associating protein with a high frequency of leucine residues (DAPLE), this review will cover (i) the mechanisms by which extracellular cues not relayed by GPCRs promote the coupling of GBA motif-containing regulators with G proteins, (ii) the structural and molecular basis for how GBA motifs interact with Gα subunits to facilitate signaling, (iii) the relevance of this mechanism in different cellular and pathological processes, including cancer and birth defects, and (iv) strategies to manipulate GBA-G protein coupling for experimental therapeutics purposes, including the development of rationally engineered proteins and chemical probes.

Leishmania profilin interacts with actin through an unusual structural mechanism to control cytoskeletal dynamics in parasites.

Diseases caused by Leishmania and Trypanosoma parasites are a major health problem in tropical countries. Because of their complex life cycle involving both vertebrate and insect hosts, and >1 billion years of evolutionarily distance, the cell biology of trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is divergent when compared with other eukaryotes. To understand how actin dynamics are regulated in trypanosomatid parasites, we focused on a central actin-binding protein profilin. Co-crystal structure of Leishmania major actin in complex with L. major profilin revealed that, although the overall folds of actin and profilin are conserved in eukaryotes, Leishmania profilin contains a unique α-helical insertion, which interacts with the target binding cleft of actin monomer. This insertion is conserved across the Trypanosomatidae family and is similar to the structure of WASP homology-2 (WH2) domain, a small actin-binding motif found in many other cytoskeletal regulators. The WH2-like motif contributes to actin monomer binding and enhances the actin nucleotide exchange activity of Leishmania profilin. Moreover, Leishmania profilin inhibited formin-catalyzed actin filament assembly in a mechanism that is dependent on the presence of the WH2-like motif. By generating profilin knockout and knockin Leishmania mexicana strains, we show that profilin is important for efficient endocytic sorting in parasites, and that the ability to bind actin monomers and proline-rich proteins, and the presence of a functional WH2-like motif, are important for the in vivo function of Leishmania profilin. Collectively, this study uncovers molecular principles by which profilin regulates actin dynamics in trypanosomatids.