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

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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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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The Plastid of Toxoplasma gondii Is Divided by Association with the Centrosomes

The Journal of Cell Biology ◽

10.1083/jcb.151.7.1423 ◽

2000 ◽

Vol 151 (7) ◽

pp. 1423-1434 ◽

Cited By ~ 163

Author(s):

Boris Striepen ◽

Michael J. Crawford ◽

Michael K. Shaw ◽

Lewis G. Tilney ◽

Frank Seeber ◽

...

Keyword(s):

Cell Division ◽

Toxoplasma Gondii ◽

Genetic Manipulation ◽

Fluorescent Proteins ◽

Recombinant Expression ◽

Molecular Genetic ◽

Time Lapse ◽

Microtubule Organization ◽

Apicomplexan Parasites ◽

Daughter Cells

Apicomplexan parasites harbor a single nonphotosynthetic plastid, the apicoplast, which is essential for parasite survival. Exploiting Toxoplasma gondii as an accessible system for cell biological analysis and molecular genetic manipulation, we have studied how these parasites ensure that the plastid and its 35-kb circular genome are faithfully segregated during cell division. Parasite organelles were labeled by recombinant expression of fluorescent proteins targeted to the plastid and the nucleus, and time-lapse video microscopy was used to image labeled organelles throughout the cell cycle. Apicoplast division is tightly associated with nuclear and cell division and is characterized by an elongated, dumbbell-shaped intermediate. The plastid genome is divided early in this process, associating with the ends of the elongated organelle. A centrin-specific antibody demonstrates that the ends of dividing apicoplast are closely linked to the centrosomes. Treatment with dinitroaniline herbicides (which disrupt microtubule organization) leads to the formation of multiple spindles and large reticulate plastids studded with centrosomes. The mitotic spindle and the pellicle of the forming daughter cells appear to generate the force required for apicoplast division in Toxoplasma gondii. These observations are discussed in the context of autonomous and FtsZ-dependent division of plastids in plants and algae.

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

Eukaryotic Cell ◽

10.1128/ec.00114-06 ◽

2006 ◽

Vol 5 (10) ◽

pp. 1622-1634 ◽

Cited By ~ 31

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.

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Blocking palmitoylation of Toxoplasma gondii myosin light chain 1 disrupts glideosome composition but has little impact on parasite motility

10.1101/2020.08.13.250399 ◽

2020 ◽

Author(s):

Pramod K. Rompikuntal ◽

Ian T. Foe ◽

Bin Deng ◽

Matthew Bogyo ◽

Gary E. Ward

Keyword(s):

Toxoplasma Gondii ◽

Current Model ◽

Severe Disease ◽

Gliding Motility ◽

The Body ◽

Phase Partitioning ◽

Apicomplexan Parasites ◽

Membrane Complex ◽

Animal Pathogens ◽

Parasite Motility

AbstractToxoplasma gondii is a widespread apicomplexan parasite that causes severe disease in immunocompromised individuals and the developing fetus. Like other apicomplexans, T. gondii uses an unusual form of gliding motility to invade cells of its hosts and to disseminate throughout the body during infection. It is well established that a myosin-based motor consisting of a Class XIVa heavy chain (TgMyoA) and two light chains (TgMLC1 and TgELC1/2) plays an important role in parasite motility. The ability of the motor to generate force at the parasite periphery is thought to be reliant upon its anchoring and immobilization within a peripheral membrane-bound compartment, the inner membrane complex (IMC). The motor does not insert into the IMC directly; rather, this interaction is believed to be mediated by the binding of TgMLC1 to the IMC-anchored protein, TgGAP45. The binding of TgMLC1 to TgGAP45 is therefore considered a key element in the force transduction machinery of the parasite. TgMLC1 is palmitoylated, and we show here that palmitoylation occurs on two N-terminal cysteine residues, C8 and C11. Mutations that block TgMLC1 palmitoylation disrupt the association of TgMLC1 with the membrane fraction of the parasite in phase partitioning experiments and completely block the binding of TgMLC1 to TgGAP45. Surprisingly, the loss of TgMLC1 binding to TgGAP45 in these mutant parasites has little effect on their ability to initiate or sustain movement. These results question a key tenet of the current model of apicomplexan motility and suggest that our understanding of gliding motility in this important group of human and animal pathogens is not yet complete.ImportanceGliding motility plays a central role in the life cycle of T. gondii and other apicomplexan parasites. The myosin motor thought to power motility is essential for virulence but distinctly different from the myosins found in humans. Consequently, an understanding of the mechanism(s) underlying parasite motility and the role played by this unusual myosin may reveal points of vulnerability that can be targeted for disease prevention and treatment. We show here that mutations that uncouple the motor from what is thought to be a key structural component of the motility machinery have little impact on parasite motility. This finding runs counter to predictions of the current, widely-held “linear motor” model of motility, highlighting the need for further studies to fully understand how apicomplexan parasites generate the forces necessary to move into, out of and between cells of the hosts they infect.

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Growth and single cell kinetics of the loricate choanoflagellate Diaphanoeca grandis

Scientific Reports ◽

10.1038/s41598-019-50998-0 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Niels Thomas Eriksen ◽

Jakob Tophøj ◽

Rasmus Dam Wollenberg ◽

Teis Esben Sondergaard ◽

Peter Funch ◽

...

Keyword(s):

Cell Division ◽

Single Cell ◽

Life Histories ◽

Daughter Cell ◽

Cell Kinetics ◽

Cell Formation ◽

Daughter Cells ◽

History Of ◽

A Cell ◽

Multiple Cell

Abstract Choanoflagellates are common members of planktonic communities. Some have complex life histories that involve transitions between multiple cell stages. We have grown the loricate choanoflagellate Diaphanoeca grandis on the bacterium Pantoea sp. and integrated kinetic observations at the culture level and at the single cell level. The life history of D. grandis includes a cell division cycle with a number of recognisable cell stages. Mature, loricate D. grandis were immobile and settled on the bottom substratum. Daughter cells were ejected from the lorica 30 min. after cell division, became motile and glided on the bottom substratum until they assembled a lorica. Single cell kinetics could explain overall growth kinetics in D. grandis cultures. The specific growth rate was 0.72 day−1 during exponential growth while mature D. grandis produced daughter cells at a rate of 0.9 day−1. Daughter cells took about 1.2 h to mature. D. grandis was able to abandon and replace its lorica, an event that delayed daughter cell formation by more than 2 days. The frequency of daughter cell formation varied considerably among individuals and single cell kinetics demonstrated an extensive degree of heterogeneity in D. grandis cultures, also when growth appeared to be balanced.

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Immobilization of the Type XIV Myosin Complex in Toxoplasma gondii

Molecular Biology of the Cell ◽

10.1091/mbc.e07-01-0040 ◽

2007 ◽

Vol 18 (8) ◽

pp. 3039-3046 ◽

Cited By ~ 50

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.

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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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