7 results on '"Leann Tilley"'
Search Results
2. The knob protein KAHRP assembles into a ring-shaped structure that underpins virulence complex assembly.
- Author
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Oliver Looker, Adam J Blanch, Boyin Liu, Juan Nunez-Iglesias, Paul J McMillan, Leann Tilley, and Matthew W A Dixon
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Plasmodium falciparum mediates adhesion of infected red blood cells (RBCs) to blood vessel walls by assembling a multi-protein complex at the RBC surface. This virulence-mediating structure, called the knob, acts as a scaffold for the presentation of the major virulence antigen, P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1). In this work we developed correlative STochastic Optical Reconstruction Microscopy-Scanning Electron Microscopy (STORM-SEM) to spatially and temporally map the delivery of the knob-associated histidine-rich protein (KAHRP) and PfEMP1 to the RBC membrane skeleton. We show that KAHRP is delivered as individual modules that assemble in situ, giving a ring-shaped fluorescence profile around a dimpled disk that can be visualized by SEM. Electron tomography of negatively-stained membranes reveals a previously observed spiral scaffold underpinning the assembled knobs. Truncation of the C-terminal region of KAHRP leads to loss of the ring structures, disruption of the raised disks and aberrant formation of the spiral scaffold, pointing to a critical role for KAHRP in assembling the physical knob structure. We show that host cell actin remodeling plays an important role in assembly of the virulence complex, with cytochalasin D blocking knob assembly. Additionally, PfEMP1 appears to be delivered to the RBC membrane, then inserted laterally into knob structures.
- Published
- 2019
- Full Text
- View/download PDF
3. Disrupting assembly of the inner membrane complex blocks Plasmodium falciparum sexual stage development.
- Author
-
Molly Parkyn Schneider, Boyin Liu, Philipp Glock, Annika Suttie, Emma McHugh, Dean Andrew, Steven Batinovic, Nicholas Williamson, Eric Hanssen, Paul McMillan, Marion Hliscs, Leann Tilley, and Matthew W A Dixon
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Transmission of malaria parasites relies on the formation of a specialized blood form called the gametocyte. Gametocytes of the human pathogen, Plasmodium falciparum, adopt a crescent shape. Their dramatic morphogenesis is driven by the assembly of a network of microtubules and an underpinning inner membrane complex (IMC). Using super-resolution optical and electron microscopies we define the ultrastructure of the IMC at different stages of gametocyte development. We characterize two new proteins of the gametocyte IMC, called PhIL1 and PIP1. Genetic disruption of PhIL1 or PIP1 ablates elongation and prevents formation of transmission-ready mature gametocytes. The maturation defect is accompanied by failure to form an enveloping IMC and a marked swelling of the digestive vacuole, suggesting PhIL1 and PIP1 are required for correct membrane trafficking. Using immunoprecipitation and mass spectrometry we reveal that PhIL1 interacts with known and new components of the gametocyte IMC.
- Published
- 2017
- Full Text
- View/download PDF
4. The apical complex provides a regulated gateway for secretion of invasion factors in Toxoplasma.
- Author
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Nicholas J Katris, Giel G van Dooren, Paul J McMillan, Eric Hanssen, Leann Tilley, and Ross F Waller
- Subjects
Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
The apical complex is the definitive cell structure of phylum Apicomplexa, and is the focus of the events of host cell penetration and the establishment of intracellular parasitism. Despite the importance of this structure, its molecular composition is relatively poorly known and few studies have experimentally tested its functions. We have characterized a novel Toxoplasma gondii protein, RNG2, that is located at the apical polar ring--the common structural element of apical complexes. During cell division, RNG2 is first recruited to centrosomes immediately after their duplication, confirming that assembly of the new apical complex commences as one of the earliest events of cell replication. RNG2 subsequently forms a ring, with the carboxy- and amino-termini anchored to the apical polar ring and mobile conoid, respectively, linking these two structures. Super-resolution microscopy resolves these two termini, and reveals that RNG2 orientation flips during invasion when the conoid is extruded. Inducible knockdown of RNG2 strongly inhibits host cell invasion. Consistent with this, secretion of micronemes is prevented in the absence of RNG2. This block, however, can be fully or partially overcome by exogenous stimulation of calcium or cGMP signaling pathways, respectively, implicating the apical complex directly in these signaling events. RNG2 demonstrates for the first time a role for the apical complex in controlling secretion of invasion factors in this important group of parasites.
- Published
- 2014
- Full Text
- View/download PDF
5. The knob protein KAHRP assembles into a ring-shaped structure that underpins virulence complex assembly
- Author
-
Leann Tilley, Matthew W. A. Dixon, Oliver Looker, Paul J. McMillan, Adam J. Blanch, Boyin Liu, and Juan Nunez-Iglesias
- Subjects
Luminescence ,Erythrocytes ,Polymers ,Protozoan Proteins ,Spectrins ,KAHRP ,Biochemistry ,chemistry.chemical_compound ,Protein structure ,Contractile Proteins ,Medicine and Health Sciences ,Macromolecular Structure Analysis ,Electron Microscopy ,Malaria, Falciparum ,Biology (General) ,Materials ,Cytochalasin D ,0303 health sciences ,Microscopy ,Virulence ,Physics ,Electromagnetic Radiation ,030302 biochemistry & molecular biology ,Adhesion ,3. Good health ,Chemistry ,Membrane ,Macromolecules ,Physical Sciences ,Scanning Electron Microscopy ,Research Article ,Protein Structure ,Imaging Techniques ,QH301-705.5 ,Immunology ,Materials Science ,Plasmodium falciparum ,Research and Analysis Methods ,Microbiology ,Fluorescence ,Microbeads ,03 medical and health sciences ,Virology ,Fluorescence Imaging ,parasitic diseases ,Genetics ,Parasitic Diseases ,Humans ,Molecular Biology ,Actin ,030304 developmental biology ,Erythrocyte Membrane ,Actin remodeling ,Biology and Life Sciences ,Proteins ,RC581-607 ,Polymer Chemistry ,Actins ,Cytoskeletal Proteins ,Electron tomography ,chemistry ,Biophysics ,Microscopy, Electron, Scanning ,Parasitology ,Immunologic diseases. Allergy ,Peptides - Abstract
Plasmodium falciparum mediates adhesion of infected red blood cells (RBCs) to blood vessel walls by assembling a multi-protein complex at the RBC surface. This virulence-mediating structure, called the knob, acts as a scaffold for the presentation of the major virulence antigen, P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1). In this work we developed correlative STochastic Optical Reconstruction Microscopy–Scanning Electron Microscopy (STORM-SEM) to spatially and temporally map the delivery of the knob-associated histidine-rich protein (KAHRP) and PfEMP1 to the RBC membrane skeleton. We show that KAHRP is delivered as individual modules that assemble in situ, giving a ring-shaped fluorescence profile around a dimpled disk that can be visualized by SEM. Electron tomography of negatively-stained membranes reveals a previously observed spiral scaffold underpinning the assembled knobs. Truncation of the C-terminal region of KAHRP leads to loss of the ring structures, disruption of the raised disks and aberrant formation of the spiral scaffold, pointing to a critical role for KAHRP in assembling the physical knob structure. We show that host cell actin remodeling plays an important role in assembly of the virulence complex, with cytochalasin D blocking knob assembly. Additionally, PfEMP1 appears to be delivered to the RBC membrane, then inserted laterally into knob structures., Author summary The human malaria parasite Plasmodium falciparum causes severe disease, which is initiated by the adhesion of parasite-infected RBCs to receptors on the walls of the host’s capillaries. Adhesion is mediated by a structure called the knob, which acts as a scaffold for the presentation of the virulence protein, P. falciparum erythrocyte membrane protein-1 (PfEMP1). In this work we investigate the assembly of this complex at different stages of parasite development using a multimodal imaging approach that combines dSTORM localization microscopy and scanning electron microscopy (STORM-SEM). We show that the knob-associated histidine-rich protein (KAHRP) is delivered to the RBC membrane skeleton as individual protein modules that assemble into a ring-shaped complex. We provide evidence that host cell remodeling, driven by association of KAHRP with spectrin and the reorganization of actin, is required for assembly of the ring complex, which in turn supports a spiral scaffold that is required for correct knob morphology. Finally, we provide evidence that PfEMP1 is delivered to the RBC membrane before associating with knob complexes.
- Published
- 2019
6. Disrupting assembly of the inner membrane complex blocks Plasmodium falciparum sexual stage development
- Author
-
Eric Hanssen, Dean Andrew, Annika Suttie, Emma McHugh, Boyin Liu, Matthew W. A. Dixon, Marion Hliscs, Steven Batinovic, Molly Parkyn Schneider, Nicholas A. Williamson, Paul J. McMillan, Philipp Glock, and Leann Tilley
- Subjects
0301 basic medicine ,Plasmodium ,Cell Membranes ,Vacuole ,Gametocytes ,Microtubules ,Fluorescence Microscopy ,Animal Cells ,Biology (General) ,Cytoskeleton ,Microscopy ,biology ,Sexual Development ,Light Microscopy ,3. Good health ,Transport protein ,Cell biology ,Protein Transport ,Cellular Types ,Cellular Structures and Organelles ,Research Article ,Immunofluorescence Microscopy ,QH301-705.5 ,Plasmodium falciparum ,030106 microbiology ,Immunology ,Research and Analysis Methods ,Microbiology ,03 medical and health sciences ,Microtubule ,Virology ,Parasite Groups ,Genetics ,Gametocyte ,Animals ,Molecular Biology ,Inner membrane complex ,Biology and Life Sciences ,Membrane Proteins ,Cell Biology ,RC581-607 ,biology.organism_classification ,Microscopy, Electron ,Germ Cells ,030104 developmental biology ,Membrane protein ,Vacuoles ,Parasitology ,Immunologic diseases. Allergy ,Apicomplexa - Abstract
Transmission of malaria parasites relies on the formation of a specialized blood form called the gametocyte. Gametocytes of the human pathogen, Plasmodium falciparum, adopt a crescent shape. Their dramatic morphogenesis is driven by the assembly of a network of microtubules and an underpinning inner membrane complex (IMC). Using super-resolution optical and electron microscopies we define the ultrastructure of the IMC at different stages of gametocyte development. We characterize two new proteins of the gametocyte IMC, called PhIL1 and PIP1. Genetic disruption of PhIL1 or PIP1 ablates elongation and prevents formation of transmission-ready mature gametocytes. The maturation defect is accompanied by failure to form an enveloping IMC and a marked swelling of the digestive vacuole, suggesting PhIL1 and PIP1 are required for correct membrane trafficking. Using immunoprecipitation and mass spectrometry we reveal that PhIL1 interacts with known and new components of the gametocyte IMC., Author summary Transmission of the malaria parasite from humans to mosquitoes relies on the formation of the specialised blood stage gametocyte. Plasmodium falciparum gametocytes mature over about 10 days, during which time they undergo a remarkable morphological transformation, eventually adopting a characteristic crescent shape. The shape changes are thought to facilitate the mechanical sequestration of maturing gametocytes within the bone marrow and spleen, as well as the eventual release into the circulation. Failure to mature correctly leads to a failure to transmit. Despite the importance of this process, little is known about the molecular basis of elongation. In this work, we introduce 3D Electron Microscopy of P. falciparum gametocytes and use it, in a combination with super-resolution optical microscopy, to elucidate the genesis and expansion of the molecular structures that drive gametocyte elongation. We use protein interaction profiling to identify some of the proteins that help drive the shape change and employ inducible gene knockdown strategies to show that these proteins play a role in remodeling membranes, and are needed for gametocyte elongation. This work points to potential targets for the development of transmission-blocking therapies.
- Published
- 2017
7. The apical complex provides a regulated gateway for secretion of invasion factors in Toxoplasma
- Author
-
Nicholas J. Katris, Paul J. McMillan, Ross F. Waller, Leann Tilley, Eric Hanssen, and Giel G. van Dooren
- Subjects
lcsh:Immunologic diseases. Allergy ,Cell division ,Immunology ,Protozoan Proteins ,Biology ,Microbiology ,Cell Line ,Cell Signaling ,Virology ,Cell polarity ,Molecular Cell Biology ,Genetics ,Humans ,Secretion ,Cell Cycle and Cell Division ,Conoid ,Cytoskeleton ,lcsh:QH301-705.5 ,Molecular Biology ,Cyclic GMP ,Biology and Life Sciences ,Cell Biology ,Cell cycle ,Cell biology ,lcsh:Biology (General) ,Centrosome ,Cell Processes ,Gene Knockdown Techniques ,Parasitology ,Apical complex ,Cellular Structures and Organelles ,lcsh:RC581-607 ,Toxoplasma ,Toxoplasmosis ,Signal Transduction ,Research Article - Abstract
The apical complex is the definitive cell structure of phylum Apicomplexa, and is the focus of the events of host cell penetration and the establishment of intracellular parasitism. Despite the importance of this structure, its molecular composition is relatively poorly known and few studies have experimentally tested its functions. We have characterized a novel Toxoplasma gondii protein, RNG2, that is located at the apical polar ring—the common structural element of apical complexes. During cell division, RNG2 is first recruited to centrosomes immediately after their duplication, confirming that assembly of the new apical complex commences as one of the earliest events of cell replication. RNG2 subsequently forms a ring, with the carboxy- and amino-termini anchored to the apical polar ring and mobile conoid, respectively, linking these two structures. Super-resolution microscopy resolves these two termini, and reveals that RNG2 orientation flips during invasion when the conoid is extruded. Inducible knockdown of RNG2 strongly inhibits host cell invasion. Consistent with this, secretion of micronemes is prevented in the absence of RNG2. This block, however, can be fully or partially overcome by exogenous stimulation of calcium or cGMP signaling pathways, respectively, implicating the apical complex directly in these signaling events. RNG2 demonstrates for the first time a role for the apical complex in controlling secretion of invasion factors in this important group of parasites., Author Summary Apicomplexan parasites comprise major human pathogens, including the malaria-causing parasites Plasmodium spp., and Toxoplasma gondii that causes birth defects and neurological disorders. Key to the success of this group was the evolution of the apical complex, a structure at the focus of the events of host cell invasion. This structure was recently shown to derive from elements of the flagellar apparatus, and rudiments of an apical complex are used for feeding in related protists. Evolution of host cell invasion in Apicomplexa has entailed development of a coordinated secretion of invasion factors from the cell apex. Little is known, however, of the behaviour or function of the components of the apical complex during invasion. We have characterized a new protein, RNG2, that forms a ring at the heart of the apical complex in T. gondii. This is a dynamic ring that links the mobile conoid with the apical polar ring, and is assembled as one of the first structures in replicating parasites. When RNG2 is artificially depleted, cells become insensitive to the molecular cues for secretion, and invasion of host cells is blocked. This reveals that the apical complex participates directly in regulating secretion, and controlling the events of invasion.
- Published
- 2013
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