Immobilization of the Type XIV Myosin Complex in Toxoplasma gondii

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.200311137 ◽

2004 ◽

Vol 165 (3) ◽

pp. 383-393 ◽

Cited By ~ 185

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.

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

mBio ◽

10.1128/mbio.03622-20 ◽

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.

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

European Journal of Cell Biology ◽

10.1016/j.ejcb.2020.151149 ◽

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

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Toxoplasma gondii myosins B/C

The Journal of Cell Biology ◽

10.1083/jcb.200012116 ◽

2001 ◽

Vol 155 (4) ◽

pp. 613-624 ◽

Cited By ~ 66

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.

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

PLoS Pathogens ◽

10.1371/journal.ppat.1005403 ◽

2016 ◽

Vol 12 (2) ◽

pp. e1005403 ◽

Cited By ~ 33

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

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

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ◽

10.1016/j.bbamcr.2013.02.022 ◽

2013 ◽

Vol 1833 (6) ◽

pp. 1329-1337 ◽

Cited By ~ 14

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

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

Eukaryotic Cell ◽

10.1128/ec.00064-08 ◽

2008 ◽

Vol 7 (9) ◽

pp. 1500-1512 ◽

Cited By ~ 20

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.

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Daughter Cell Assembly in the Protozoan ParasiteToxoplasma gondii

Molecular Biology of the Cell ◽

10.1091/mbc.01-06-0309 ◽

2002 ◽

Vol 13 (2) ◽

pp. 593-606 ◽

Cited By ~ 120

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.

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