Spatial Localisation of Actin Filaments across Developmental Stages of the Malaria Parasite

Actin dynamics have been implicated in a variety of developmental processes during the malaria parasite lifecycle. Parasite motility, in particular, is thought to critically depend on an actomyosin motor located in the outer pellicle of the parasite cell. Efforts to understand the diverse roles actin plays have, however, been hampered by an inability to detect microfilaments under native conditions. To visualise the spatial dynamics of actin we generated a parasite-specific actin antibody that shows preferential recognition of filamentous actin and applied this tool to different lifecycle stages (merozoites, sporozoites and ookinetes) of the human and mouse malaria parasite species Plasmodium falciparum and P. berghei along with tachyzoites from the related apicomplexan parasite Toxoplasma gondii. Actin filament distribution was found associated with three core compartments: the nuclear periphery, pellicular membranes of motile or invasive parasite forms and in a ring-like distribution at the tight junction during merozoite invasion of erythrocytes in both human and mouse malaria parasites. Localisation at the nuclear periphery is consistent with an emerging role of actin in facilitating parasite gene regulation. During invasion, we show that the actin ring at the parasite-host cell tight junction is dependent on dynamic filament turnover. Super-resolution imaging places this ring posterior to, and not concentric with, the junction marker rhoptry neck protein 4. This implies motor force relies on the engagement of dynamic microfilaments at zones of traction, though not necessarily directly through receptor-ligand interactions at sites of adhesion during invasion. Combined, these observations extend current understanding of the diverse roles actin plays in malaria parasite development and apicomplexan cell motility, in particular refining understanding on the linkage of the internal parasite gliding motor with the extra-cellular milieu

Killer cell immunoglobulin-like receptor 3DL1-mediated recognition of human leukocyte antigen B [Letter]

Members of the killer cell immunoglobulin-like receptor (KIR) family, a large group of polymorphic receptors expressed on natural killer (NK) cells, recognize particular peptide-laden human leukocyte antigen (pHLA) class I molecules and have a pivotal role in innate immune responses1. Allelic variation and extensive polymorphism within the three-domain KIR family (KIR3D, domains D0–D1–D2) affects pHLA binding specificity and is linked to the control of viral replication and the treatment outcome of certain haematological malignancies1, 2, 3. Here we describe the structure of a human KIR3DL1 receptor bound to HLA-B*5701 complexed with a self-peptide. KIR3DL1 clamped around the carboxy-terminal end of the HLA-B*5701 antigen-binding cleft, resulting in two discontinuous footprints on the pHLA. First, the D0 domain, a distinguishing feature of the KIR3D family, extended towards β2-microglobulin and abutted a region of the HLA molecule with limited polymorphism, thereby acting as an ‘innate HLA sensor’ domain. Second, whereas the D2–HLA-B*5701 interface exhibited a high degree of complementarity, the D1–pHLA-B*5701 contacts were suboptimal and accommodated a degree of sequence variation both within the peptide and the polymorphic region of the HLA molecule. Although the two-domain KIR (KIR2D) and KIR3DL1 docked similarly onto HLA-C4, 5 and HLA-B respectively, the corresponding D1-mediated interactions differed markedly, thereby providing insight into the specificity of KIR3DL1 for discrete HLA-A and HLA-B allotypes. Collectively, in association with extensive mutagenesis studies at the KIR3DL1–pHLA-B*5701 interface, we provide a framework for understanding the intricate interplay between peptide variability, KIR3D and HLA polymorphism in determining the specificity requirements of this essential innate interaction that is conserved across primate species

Allosteric regulation of the 20S proteasome by the Catalytic Core Regulators (CCRs) family

Abstract Controlled degradation of proteins is necessary for ensuring their abundance and sustaining a healthy and accurately functioning proteome. One of the degradation routes involves the uncapped 20S proteasome, which cleaves proteins with a partially unfolded region, including those that are damaged or contain intrinsically disordered regions. This degradation route is tightly controlled by a recently discovered family of proteins named Catalytic Core Regulators (CCRs). Here, we show that CCRs function through an allosteric mechanism, coupling the physical binding of the PSMB4 β-subunit with attenuation of the complex’s three proteolytic activities. In addition, by dissecting the structural properties that are required for CCR-like function, we could recapitulate this activity using a designed protein that is half the size of natural CCRs. These data uncover an allosteric path that does not involve the proteasome’s enzymatic subunits but rather propagates through the non-catalytic subunit PSMB4. This way of 20S proteasome-specific attenuation opens avenues for decoupling the 20S and 26S proteasome degradation pathways as well as for developing selective 20S proteasome inhibitors

Plasmodium falciparum coronin organizes arrays of parallel actin filaments potentially guiding directional motility in invasive malaria parasites

Background: Gliding motility in Plasmodium parasites, the aetiological agents of malaria disease, is mediated by an actomyosin motor anchored in the outer pellicle of the motile cell. Effective motility is dependent on a parasite myosin motor and turnover of dynamic parasite actin filaments. To date, however, the basis for directional motility is not known. Whilst myosin is very likely orientated as a result of its anchorage within the parasite, how actin filaments are orientated to facilitate directional force generation remains unexplained. In addition, recent evidence has questioned the linkage between actin filaments and secreted surface antigens leaving the way by which motor force is transmitted to the extracellular milieu unknown. Malaria parasites possess a markedly reduced repertoire of actin regulators, among which few are predicted to interact with filamentous (F)-actin directly. One of these, PF3D7_1251200, shows strong homology to the coronin family of actin-filament binding proteins, herein referred to as PfCoronin. Methods: Here the N terminal beta propeller domain of PfCoronin (PfCor-N) was expressed to assess its ability to bind and bundle pre-formed actin filaments by sedimentation assay, total internal reflection fluorescence (TIRF) microscopy and confocal imaging as well as to explore its ability to bind phospholipids. In parallel a tagged PfCoronin line in Plasmodium falciparum was generated to determine the cellular localization of the protein during asexual parasite development and blood-stage merozoite invasion. Results: A combination of biochemical approaches demonstrated that the N-terminal beta-propeller domain of PfCoronin is capable of binding F-actin and facilitating formation of parallel filament bundles. In parasites, PfCoronin is expressed late in the asexual lifecycle and localizes to the pellicle region of invasive merozoites before and during erythrocyte entry. PfCoronin also associates strongly with membranes within the cell, likely mediated by interactions with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) at the plasma membrane. Conclusions: These data suggest PfCoronin may fulfil a key role as the critical determinant of actin filament organization in the Plasmodium cell. This raises the possibility that macro-molecular organization of actin mediates directional motility in gliding parasites. Electronic supplementary material The online version of this article (doi:10.1186/s12936-015-0801-5) contains supplementary material, which is available to authorized users

Concentration of actin labelling in the nucleus and around the nuclear periphery.

<p>Widefield IFA of representative <i>P. berghei </i><b>A</b>) ookinetes and <b>B</b>) sporozoites that show pronounced nuclear labelling using rabbit anti-Act_239–253 (Green) surface markers Pbs28 or PbCSP (Red) and DAPI (Blue). Scale bar = 5 µm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032188#pone.0032188.s009" target="_blank">Movie S5</a>. <b>C</b>) Widefield IFA of <i>P. falciparum</i> rings labelled with rabbit anti-Act_239–253 (Red) and DAPI (Blue). <b>D</b>) As <b>C</b> but following 6 hour JAS treatment. <b>E</b>) Two colour widefield IFA using rabbit anti-Act_239–253 (Red), rat anti-ERD2 (Green) and DAPI (Blue) in absence or presence of 1 µM JAS. All scale bars = 5 µm.</p

An apicomplexan parasite-specific anti-actin antibody.

<p><b>A</b>) Sequence comparison between human non-muscle actin amino acids 237–251 (the basis of anti-Gly₂₄₅<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032188#pone.0032188-Varma1" target="_blank">[39]</a>) and apicomplexan actin I orthologues over the amino acids 239–253 (the basis for anti-Act_239–253). <b>B</b>) Surface representation of the structures of rabbit G-actin (PDB:1J6Z; A) and a protomer in rabbit F-actin (PDB:3G37; B) showing anti-Gly₂₄₅ epitope. Residues in yellow indicate polymorphisms between mammalian and <i>P. falciparum</i> actin. <b>C</b>) Representative immunoblot showing reactivity of rabbit® anti-Act_239–253 serum with human erythrocytes (hRBC), asexual <i>P. falciparum</i> (3D7), mouse erythrocytes (mRBC), asexual <i>P. berghei</i> (ANKA), human foreskin fibroblasts (HFF) and <i>T. gondii</i> tachyzoites (RH). Lower panel shows same hRBC and 3D7 sample probed with mouse (m) anti-Act_239–253 serum. <b>D</b>) As C but using generic anti-actin monoclonal C4.</p