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

Protein aggregates in Huntington's disease

Huntington’s disease (HD) is an incurable neurodegenerative disease characterized by abnormal motor movements, personality changes, and early death. HD is caused by a mutation in the IT-15 gene that expands abnormally the number of CAG nucleotide repeats. As a result, the translated protein huntingtin contains disease-causing expansions of glutamines (polyQ) that make it prone to misfold and aggregate. While the gene and mutations that cause HD are known, the mechanisms underlying HD pathogenesis are not. Here we will review the state of knowledge of HD, focusing especially on a hallmark pathological feature—intracellular aggregates of mutant Htt called inclusion bodies (IBs). We will describe the role of IBs in the disease. We speculate that IB formation could be just one component of a broader coping response triggered by misfolded Htt whose efficacy may depend on the extent to which it clears toxic forms of mutant Htt. We will describe how IB formation might be regulated and which factors could determine different coping responses in different subsets of neurons. A differential regulation of IB formation as a function of the cellular context could, eventually, explain part of the neuronal vulnerability observed in HD

Actin regulation in Plasmodium falciparum: towards understanding the elusive nature of malarial actin filaments

© 2015 Dr. Maya Avital OlshinaMalaria disease, caused by the unicellular parasites from the genus Plasmodium, is a major cause of morbidity and mortality in many developing countries throughout the world. While there have been many improvements in intervention strategies in recent years, parasite resistance to front-line therapeutics is on the rise, highlighting the need for new and improved treatments and vaccines. To this end, a greater understanding of the biological mechanisms underpinning the disease will be crucial in the push towards malaria eradication. Across the malaria life cycle the parasite must traverse tissues and invade host cells in order to establish an infection and replicate. A conserved acto-myosin motor, anchored at the parasite periphery, generates the requisite force to drive the parasite forward, facilitating both invasion and motility. The actin at the heart of this motor is extremely divergent, forming filaments that are highly dynamic and unstable. Tightly controlled regulation of malaria actin is therefore necessary to direct the formation and disassembly of filaments in an appropriate spatio-temporal manner. However, malaria parasites possess a markedly reduced repertoire of actin regulators, of which coronin is one of the only predicted filament regulators. Much of the current literature surrounding Plasmodium actin biology relies on the production of actin from recombinant sources. In this study I investigate the various published methods for purifying recombinant malaria actin, and determine that the unusual characteristics previously reported for this actin are likely artifacts driven by incomplete protein folding in heterologous expression systems. This finding lead to the identification of the key actin folding chaperonin CCT in the Plasmodium genome, an essential protein complex required for producing native, functional actin in the cell. In parallel, characterization of the filament regulator, coronin, revealed its critical role in the organization of actin filaments. Using in vitro observations from recombinant Plasmodium falciparum coronin (PfCoronin), I have demonstrated that PfCoronin binds to actin filaments and bundles them together in parallel arrays. Furthermore, in vivo observations revealed PfCoronin to be located at the periphery of the parasite, consistent with the pellicular space in which the actin-myosin motor is housed. This localization is likely mediated by peripheral interactions with PI(4,5)P2 at the plasma membrane. These data identify PfCoronin as a potentially key regulator of actin filament recruitment and bundling at the cell cortex of motile Plasmodium parasites. Taken together, the identification of Plasmodium CCT and the characterization of PfCoronin have opened up new avenues for further development of these as potential drug targets, with the eventual aim of potentially crippling the motile malaria parasite and halting the progression of disease

Actin regulation in Plasmodium falciparum: towards understanding the elusive nature of malarial actin filaments

© 2015 Dr. Maya Avital OlshinaMalaria disease, caused by the unicellular parasites from the genus Plasmodium, is a major cause of morbidity and mortality in many developing countries throughout the world. While there have been many improvements in intervention strategies in recent years, parasite resistance to front-line therapeutics is on the rise, highlighting the need for new and improved treatments and vaccines. To this end, a greater understanding of the biological mechanisms underpinning the disease will be crucial in the push towards malaria eradication. Across the malaria life cycle the parasite must traverse tissues and invade host cells in order to establish an infection and replicate. A conserved acto-myosin motor, anchored at the parasite periphery, generates the requisite force to drive the parasite forward, facilitating both invasion and motility. The actin at the heart of this motor is extremely divergent, forming filaments that are highly dynamic and unstable. Tightly controlled regulation of malaria actin is therefore necessary to direct the formation and disassembly of filaments in an appropriate spatio-temporal manner. However, malaria parasites possess a markedly reduced repertoire of actin regulators, of which coronin is one of the only predicted filament regulators. Much of the current literature surrounding Plasmodium actin biology relies on the production of actin from recombinant sources. In this study I investigate the various published methods for purifying recombinant malaria actin, and determine that the unusual characteristics previously reported for this actin are likely artifacts driven by incomplete protein folding in heterologous expression systems. This finding lead to the identification of the key actin folding chaperonin CCT in the Plasmodium genome, an essential protein complex required for producing native, functional actin in the cell. In parallel, characterization of the filament regulator, coronin, revealed its critical role in the organization of actin filaments. Using in vitro observations from recombinant Plasmodium falciparum coronin (PfCoronin), I have demonstrated that PfCoronin binds to actin filaments and bundles them together in parallel arrays. Furthermore, in vivo observations revealed PfCoronin to be located at the periphery of the parasite, consistent with the pellicular space in which the actin-myosin motor is housed. This localization is likely mediated by peripheral interactions with PI(4,5)P2 at the plasma membrane. These data identify PfCoronin as a potentially key regulator of actin filament recruitment and bundling at the cell cortex of motile Plasmodium parasites. Taken together, the identification of Plasmodium CCT and the characterization of PfCoronin have opened up new avenues for further development of these as potential drug targets, with the eventual aim of potentially crippling the motile malaria parasite and halting the progression of disease

Disassembly activity of actin depolymerization factor (ADF) is associated with distinct cellular processes in apicomplexan parasites.

Proteins of the actin-depolymerizing factor (ADF)/cofilin family have been shown to be crucial for the motility and survival of apicomplexan parasites. However, the mechanisms by which ADF proteins fulfill their function remain poorly understood. In this study, we investigate the comparative activities of ADF proteins from Toxoplasma gondii and Plasmodium falciparum, the human malaria parasite, using a conditional T. gondii ADF-knockout line complemented with ADF variants from either species. We show that P. falciparum ADF1 can fully restore native TgADF activity, demonstrating functional conservation between parasites. Strikingly, mutation of a key basic residue (Lys-72), previously implicated in disassembly in PfADF1, had no detectable phenotypic effect on parasite growth, motility, or development. In contrast, organelle segregation was severely impaired when complementing with a TgADF mutant lacking the corresponding residue (Lys-68). Biochemical analyses of each ADF protein confirmed the reduced ability of lysine mutants to mediate actin depolymerization via filament disassembly although not severing, in contrast to previous reports. These data suggest that actin filament disassembly is essential for apicomplexan parasite development but not for motility, as well as pointing to genus-specific coevolution between ADF proteins and their native actin