scholarly journals Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii

2004 ◽  
Vol 165 (3) ◽  
pp. 383-393 ◽  
Author(s):  
Elizabeth Gaskins ◽  
Stacey Gilk ◽  
Nicolette DeVore ◽  
Tara Mann ◽  
Gary Ward ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Complex ◽  
Integral Membrane Protein ◽  
Gliding Motility ◽  
Host Cells ◽  
Inner Membrane ◽  
Apicomplexan Parasites ◽  
Membrane Complex ◽  
Inner Membrane Complex ◽  
Two Stages

Apicomplexan parasites exhibit a unique form of substrate-dependent motility, gliding motility, which is essential during their invasion of host cells and during their spread between host cells. This process is dependent on actin filaments and myosin that are both located between the plasma membrane and two underlying membranes of the inner membrane complex. We have identified a protein complex in the apicomplexan parasite Toxoplasma gondii that contains the class XIV myosin required for gliding motility, TgMyoA, its associated light chain, TgMLC1, and two novel proteins, TgGAP45 and TgGAP50. We have localized this complex to the inner membrane complex of Toxoplasma, where it is anchored in the membrane by TgGAP50, an integral membrane glycoprotein. Assembly of the protein complex is spatially controlled and occurs in two stages. These results provide the first molecular description of an integral membrane protein as a specific receptor for a myosin motor, and further our understanding of the motile apparatus underlying gliding motility in apicomplexan parasites.

scholarly journals Toxoplasma gondii myosins B/C

2001 ◽  
Vol 155 (4) ◽  
pp. 613-624 ◽  
Author(s):  
Frédéric Delbac ◽  
Astrid Sänger ◽  
Eva M. Neuhaus ◽  
Rolf Stratmann ◽  
James W. Ajioka ◽  
...  
Keyword(s):  
Cell Division ◽  
Toxoplasma Gondii ◽  
Daughter Cell ◽  
Gliding Motility ◽  
Apicomplexan Parasites ◽  
Daughter Cells ◽  
Membrane Complex ◽  
Alternatively Spliced ◽  
Butanedione Monoxime ◽  
Inner Membrane Complex

In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.

scholarly journals Identification of PhIL1, a Novel Cytoskeletal Protein of the Toxoplasma gondii Pellicle, through Photosensitized Labeling with 5-[125I]Iodonaphthalene-1-Azide

2006 ◽  
Vol 5 (10) ◽  
pp. 1622-1634 ◽  
Author(s):  
Stacey D. Gilk ◽  
Yossef Raviv ◽  
Ke Hu ◽  
John M. Murray ◽  
Con J. M. Beckers ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Membrane Proteins ◽  
Transmembrane Domain ◽  
Protozoan Parasite ◽  
Integral Membrane Protein ◽  
Apicomplexan Parasites ◽  
Membrane Complex ◽  
Inner Membrane Complex ◽  
Complex Integral ◽  
Host Cell Recognition

ABSTRACT The pellicle of the protozoan parasite Toxoplasma gondii is a unique triple bilayer structure, consisting of the plasma membrane and two tightly apposed membranes of the underlying inner membrane complex. Integral membrane proteins of the pellicle are likely to play critical roles in host cell recognition, attachment, and invasion, but few such proteins have been identified. This is in large part because the parasite surface is dominated by a family of abundant and highly immunogenic glycosylphosphatidylinositol (GPI)-anchored proteins, which has made the identification of non-GPI-linked proteins difficult. To identify such proteins, we have developed a radiolabeling approach using the hydrophobic, photoactivatable compound 5-[125I]iodonaphthalene-1-azide (INA). INA can be activated by photosensitizing fluorochromes; by restricting these fluorochromes to the pellicle, [125I]INA labeling will selectively target non-GPI-anchored membrane-embedded proteins of the pellicle. We demonstrate here that three known membrane proteins of the pellicle can indeed be labeled by photosensitization with INA. In addition, this approach has identified a novel 22-kDa protein, named PhIL1 (photosensitized INA-labeled protein 1), with unexpected properties. While the INA labeling of PhIL1 is consistent with an integral membrane protein, the protein has neither a transmembrane domain nor predicted sites of lipid modification. PhIL1 is conserved in apicomplexan parasites and localizes to the parasite periphery, concentrated at the apical end just basal to the conoid. Detergent extraction and immunolocalization data suggest that PhIL1 associates with the parasite cytoskeleton.

scholarly journals Immobilization of the Type XIV Myosin Complex in Toxoplasma gondii

2007 ◽  
Vol 18 (8) ◽  
pp. 3039-3046 ◽  
Author(s):  
Terezina M. Johnson ◽  
Zenon Rajfur ◽  
Ken Jacobson ◽  
Con J. Beckers
Keyword(s):  
Toxoplasma Gondii ◽  
Large Fraction ◽  
Complex Surface ◽  
Membrane Domains ◽  
Inner Membrane ◽  
Apicomplexan Parasites ◽  
Motor Complex ◽  
Membrane Complex ◽  
Inner Membrane Complex ◽  
Cytoskeletal Elements

The substrate-dependent movement of apicomplexan parasites such as Toxoplasma gondii and Plasmodium sp. is driven by the interaction of a type XIV myosin with F-actin. A complex containing the myosin-A heavy chain, a myosin light chain, and the accessory protein GAP45 is attached to the membranes of the inner membrane complex (IMC) through its tight interaction with the integral membrane glycoprotein GAP50. For the interaction of this complex with F-actin to result in net parasite movement, it is necessary that the myosin be immobilized with respect to the parasite and the actin with respect to the substrate the parasite is moving on. We report here that the myosin motor complex of Toxoplasma is firmly immobilized in the plane of the IMC. This does not seem to be accomplished by direct interactions with cytoskeletal elements. Immobilization of the motor complex, however, does seem to require cholesterol. Both the motor complex and the cholesterol are found in detergent-resistant membrane domains that encompass a large fraction of the inner membrane complex surface. The observation that the myosin XIV motor complex of Toxoplasma is immobilized within this cholesterol-rich membrane likely extends to closely related pathogens such as Plasmodium and possibly to other eukaryotes.

scholarly journals Identification and Molecular Dissection of IMC32, a Conserved Toxoplasma Inner Membrane Complex Protein That Is Essential for Parasite Replication

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Juan A. Torres ◽  
Rebecca R. Pasquarelli ◽  
Peter S. Back ◽  
Andy S. Moon ◽  
Peter J. Bradley
Keyword(s):  
Toxoplasma Gondii ◽  
Coiled Coil ◽  
Inner Membrane ◽  
Content Type ◽  
Apicomplexan Parasites ◽  
Conserved Regions ◽  
Membrane Complex ◽  
Parasite Survival ◽  
Parasite Replication ◽  
Inner Membrane Complex

ABSTRACT The inner membrane complex (IMC) is a unique organelle of apicomplexan parasites that plays critical roles in parasite motility, host cell invasion, and replication. Despite the common functions of the organelle, relatively few IMC proteins are conserved across the phylum and the precise roles of many IMC components remain to be characterized. Here, we identify a novel component of the Toxoplasma gondii IMC (IMC32) that localizes to the body portion of the IMC and is recruited to developing daughter buds early during endodyogeny. IMC32 is essential for parasite survival, as its conditional depletion results in a complete collapse of the IMC that is lethal to the parasite. We demonstrate that localization of IMC32 is dependent on both an N-terminal palmitoylation site and a series of C-terminal coiled-coil domains. Using deletion analyses and functional complementation, we show that two conserved regions within the C-terminal coiled-coil domains play critical roles in protein function during replication. Together, this work reveals an essential component of parasite replication that provides a novel target for therapeutic intervention of T. gondii and related apicomplexan parasites. IMPORTANCE The IMC is an important organelle that apicomplexan parasites use to maintain their intracellular lifestyle. While many IMC proteins have been identified, only a few central players that are essential for internal budding have been described and even fewer are conserved across the phylum. Here, we identify IMC32, a novel component of the Toxoplasma gondii IMC that localizes to very early daughter buds, indicating a role in the early stages of parasite replication. We then demonstrate that IMC32 is essential for parasite survival and pinpoint conserved regions within the protein that are important for membrane association and daughter cell formation. As IMC32 is unique to these parasites and not present in their mammalian hosts, it serves as a new target for the development of drugs that exclusively affect these important intracellular pathogens.

scholarly journals Raft microdomain localized in the luminal leaflet of inner membrane complex of living Toxoplasma gondii

2021 ◽  
Vol 100 (2) ◽  
pp. 151149
Author(s):  
Rikako Konishi ◽  
Yuna Kurokawa ◽  
Kanna Tomioku ◽  
Tatsunori Masatani ◽  
Xuenan Xuan ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Inner Membrane ◽  
Membrane Complex ◽  
Inner Membrane Complex

scholarly journals Gliding Associated Proteins Play Essential Roles during the Formation of the Inner Membrane Complex of Toxoplasma gondii

2016 ◽  
Vol 12 (2) ◽  
pp. e1005403 ◽  
Author(s):  
Clare R. Harding ◽  
Saskia Egarter ◽  
Matthew Gow ◽  
Elena Jiménez-Ruiz ◽  
David J. P. Ferguson ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Inner Membrane ◽  
Membrane Complex ◽  
Associated Proteins ◽  
Inner Membrane Complex

scholarly journals N-terminal palmitoylation is required for Toxoplasma gondii HSP20 inner membrane complex localization

2013 ◽  
Vol 1833 (6) ◽  
pp. 1329-1337 ◽  
Author(s):  
M.G. De Napoli ◽  
N. de Miguel ◽  
M. Lebrun ◽  
S.N.J. Moreno ◽  
S.O. Angel ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Inner Membrane ◽  
Membrane Complex ◽  
Inner Membrane Complex

scholarly journals A Novel Actin-Related Protein Is Associated with Daughter Cell Formation in Toxoplasma gondii

2008 ◽  
Vol 7 (9) ◽  
pp. 1500-1512 ◽  
Author(s):  
Jennifer L. Gordon ◽  
Wandy L. Beatty ◽  
L. David Sibley
Keyword(s):  
Cell Division ◽  
Toxoplasma Gondii ◽  
Daughter Cell ◽  
Cell Formation ◽  
The Body ◽  
Apicomplexan Parasites ◽  
Daughter Cells ◽  
Membrane Complex ◽  
Transport Vesicles ◽  
Inner Membrane Complex

ABSTRACT Cell division in Toxoplasma gondii occurs by an unusual budding mechanism termed endodyogeny, during which twin daughters are formed within the body of the mother cell. Cytokinesis begins with the coordinated assembly of the inner membrane complex (IMC), which surrounds the growing daughter cells. The IMC is compiled of both flattened membrane cisternae and subpellicular filaments composed of articulin-like proteins attached to underlying singlet microtubules. While proteins that comprise the elongating IMC have been described, little is known about its initial formation. Using Toxoplasma as a model system, we demonstrate that actin-like protein 1 (ALP1) is partially redistributed to the IMC at early stages in its formation. Immunoelectron microscopy localized ALP1 to a discrete region of the nuclear envelope, on transport vesicles, and on the nascent IMC of the daughter cells prior to the arrival of proteins such as IMC-1. The overexpression of ALP1 under the control of a strong constitutive promoter disrupted the formation of the daughter cell IMC, leading to delayed growth and defects in nuclear and apicoplast segregation. Collectively, these data suggest that ALP1 participates in the formation of daughter cell membranes during cell division in apicomplexan parasites.

scholarly journals Daughter Cell Assembly in the Protozoan ParasiteToxoplasma gondii

2002 ◽  
Vol 13 (2) ◽  
pp. 593-606 ◽  
Author(s):  
Ke Hu ◽  
Tara Mann ◽  
Boris Striepen ◽  
Con J. M. Beckers ◽  
David S. Roos ◽  
...  
Keyword(s):  
Cell Division ◽  
Fluorescent Protein ◽  
Molecular Genetic ◽  
Inner Membrane ◽  
Apicomplexan Parasites ◽  
Membrane Complex ◽  
Ideal System ◽  
Animal Pathogens ◽  
Inner Membrane Complex

The phylum Apicomplexa includes thousands of species of obligate intracellular parasites, many of which are significant human and/or animal pathogens. Parasites in this phylum replicate by assembling daughters within the mother, using a cytoskeletal and membranous scaffolding termed the inner membrane complex. Most apicomplexan parasites, including Plasmodium sp. (which cause malaria), package many daughters within a single mother during mitosis, whereas Toxoplasma gondii typically packages only two. The comparatively simple pattern of T. gondii cell division, combined with its molecular genetic and cell biological accessibility, makes this an ideal system to study parasite cell division. A recombinant fusion between the fluorescent protein reporter YFP and the inner membrane complex protein IMC1 has been exploited to examine daughter scaffold formation in T. gondii.Time-lapse video microscopy permits the entire cell cycle of these parasites to be visualized in vivo. In addition to replication via endodyogeny (packaging two parasites at a time), T. gondii is also capable of forming multiple daughters, suggesting fundamental similarities between cell division in T. gondii and other apicomplexan parasites.

scholarly journals Structural role of essential light chains in the apicomplexan glideosome

2019 ◽  
Author(s):  
Samuel Pazicky ◽  
Karthikeyan Dhamotharan ◽  
Karol Kaszuba ◽  
Haydyn Mertens ◽  
Tim Gilberger ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Light Chain ◽  
Structural Information ◽  
Host Cells ◽  
Light Chains ◽  
Apicomplexan Parasites ◽  
Membrane Complex ◽  
C Terminus ◽  
Inner Membrane Complex

AbstractApicomplexan parasites, such as Plasmodium falciparum and Toxoplasma gondii, traverse the host tissues and invade the host cells exhibiting a specific type of motility called gliding. The molecular mechanism of gliding lies in the actin-myosin motor localized to the intermembrane space between the plasma membrane and inner membrane complex (IMC) of the parasites. Myosin A (MyoA) is a part of the glideosome, a large multi-protein complex, which is anchored in the outer membrane of the IMC. MyoA is bound to the proximal essential light chain (ELC) and distal myosin light chain (MLC1), which further interact with the glideosome associated proteins GAP40, GAP45 and GAP50. Whereas structures of several individual glideosome components and small dimeric complexes have been solved, structural information concerning the interaction of larger glideosome subunits and their role in glideosome function still remains to be elucidated. Here, we present structures of a T. gondii trimeric glideosome sub complex composed of a myosin A light chain domain with bound MLC1 and TgELC1 or TgELC2. Regardless of the differences between the secondary structure content observed for free P. falciparum PfELC and T. gondii TgELC1 or TgELC2, the proteins interact with a conserved region of TgMyoA to form structurally conserved complexes. Upon interaction, the essential light chains undergo contraction and induce α-helical structure in the myosin A C-terminus, stiffening the myosin lever arm. The complex formation is further stabilized through binding of a single calcium ion to T. gondii ELCs. Our work provides an important step towards the structural understanding of the entire glideosome and uncovering the role of its members in parasite motility and invasion.Author summaryApicomplexans, such as Toxoplasma gondii or the malaria agent Plasmodium falciparum, are small unicellular parasites that cause serious diseases in humans and other animals. These parasites move and infect the host cells by a unique type of motility called gliding. Gliding is empowered by an actin-myosin molecular motor located at the periphery of the parasites. Myosin interacts with additional proteins such as essential light chains to form the glideosome, a large protein assembly that anchors myosin in the inner membrane complex. Unfortunately, our understanding of the glideosome is insufficient because we lack the necessary structural information. Here we describe the first structures of trimeric glideosome sub complexes of T. gondii myosin A bound to two different light chain combinations, which show that T. gondii and P. falciparum form structurally conserved complexes. With an additional calcium-free complex structure, we demonstrate that calcium binding does not change the formation of the complexes, although it provides them with substantial stability. With additional data, we propose that the role of the essential light chains is to enhance myosin performance by inducing secondary structure in the C-terminus of myosin A. Our work represents an important step in unveiling the gliding mechanism of apicomplexan parasites.

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