scholarly journals Toxoplasma ERK7 defends the apical complex from premature degradation

2021 ◽  
Author(s):  
William S O'Shaughnessy ◽  
Xiaoyu Hu ◽  
Sarah Ana Henriquez ◽  
Michael L Reese

Accurate cellular replication balances the biogenesis and turnover of complex structures. Apicomplexan parasites such as Plasmodium and Toxoplasma replicate by forming daughter cells within an intact mother cell, creating additional challenges to ensuring fidelity of division. Critical to these parasites' infectivity is an intricate cytoskeleton structure called the apical complex. Before the daughter apical complex can be inserted into the plasma membrane, the maternal material must be turned over. We previously identified the kinase ERK7 as required for the maturation of the apical complex in Toxoplasma gondii. Here we define the Toxoplasma ERK7 interactome, and identify a putative E3 ligase, CSAR1, as the downstream effector responsible for the phenotype. Genetic disruption of CSAR1 fully suppresses loss of the apical complex upon ERK7 knockdown. Furthermore, we show that CSAR1 is normally responsible for turnover of maternal cytoskeleton during cytokinesis, and that its aberrant function is driven by a mislocalization from the parasite residual body to the maternal and daughter apical complexes. These data identify a protein homeostasis pathway critical for Toxoplasma replication and fitness and suggest an unappreciated role for the parasite residual body in compartmentalizing processes that threaten the fidelity of parasite development.

2021 ◽  
Author(s):  
Alana Burrell ◽  
Virginia Marugan-Hernandez ◽  
Flavia Moreira-Leite ◽  
David J P Ferguson ◽  
Fiona M Tomley ◽  
...  

The apical complex of apicomplexan parasites is essential for host cell invasion and intracellular survival and as the site of regulated exocytosis from specialised secretory organelles called rhoptries and micronemes. Despite its importance, there is little data on the three-dimensional organisation and quantification of these organelles within the apical complex or how they are trafficked to this specialised region of plasma membrane for exocytosis. In coccidian apicomplexans there is an additional tubulin-containing hollow barrel structure, the conoid, which provides a structural gateway for this specialised secretion. Using a combination of cellular electron tomography and serial block face-scanning electron microscopy (SBF-SEM) we have reconstructed the entire apical end of Eimeria tenella sporozoites. We discovered that conoid fibre number varied, but there was a fixed spacing between fibres, leading to conoids of different sizes. Associated apical structures varied in size to accommodate a larger or smaller conoid diameter. However, the number of subpellicular microtubules on the apical polar ring surrounding the conoid did not vary, suggesting a control of apical complex size. We quantified the number and location of rhoptries and micronemes within cells and show a highly organised gateway for trafficking and docking of rhoptries, micronemes and vesicles within the conoid around a set of intra-conoidal microtubules. Finally, we provide ultrastructural evidence for fusion of rhoptries directly through the parasite plasma membrane early in infection and the presence of a pore in the parasitophorous vacuole membrane, providing a structural explanation for how rhoptry proteins (ROPs) may be trafficked between the parasite and the host cytoplasm


2001 ◽  
Vol 155 (4) ◽  
pp. 613-624 ◽  
Author(s):  
Frédéric Delbac ◽  
Astrid Sänger ◽  
Eva M. Neuhaus ◽  
Rolf Stratmann ◽  
James W. Ajioka ◽  
...  

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.


2000 ◽  
Vol 151 (7) ◽  
pp. 1423-1434 ◽  
Author(s):  
Boris Striepen ◽  
Michael J. Crawford ◽  
Michael K. Shaw ◽  
Lewis G. Tilney ◽  
Frank Seeber ◽  
...  

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.


1961 ◽  
Vol s3-102 (58) ◽  
pp. 273-292
Author(s):  
BHUPINDER N. SUD

The chromatoid body was discovered by von Brunn (1876) in the cytoplasm of the young spermatid in the white rat. It was first described in a marsupial by KorfT (1902), in a vertebrate other than mammals by the Schreiners (1905, 1908), and in an invertebrate by Bösenberg (1905). The word chromatoide was first used in connexion with spermatogenesis by Benda (1891), who called this cytoplasmic inclusion der chromatoide Nebenkörper. The German authors generally call it der chromatoide Körper, the French authors corps chromatoïde. Wilson (1913) referred to it as the chromatoid body and it is generally given this name in papers written in English, though the expression ‘chromatic body’ is sometimes used. It is suggested that the ‘residual body’ described by Gresson and Zlotnik (1945) is identical with the chromatoid body of other authors. In most species the chromatoid body is spherical or ovoid but in some it assumes other forms as well and in a few it is never spherical or ovoid. The chromatoid body is usually single in each cell, but sometimes there are 2 or 3 and in a few there are many. In living cell the chromatoid body generally gives a low phase-change, and is invisible or almost invisible when studied by direct microscopy. In the Mammalia, however, it gives a higher phase-change. The chromatoid body is highly resistant to acetic acid. It is deeply stained by basic dyes and basic dye-lakes. It is also stained intensely by acid dyes. The chromatoid body cannot in most cases be blackened by silver or long osmication techniques. The histochemical reactions show that the chromatoid body consists mainly of RNA and basic proteins rich in arginine. There is little or no tyrosine. Lipid, carbohydrates, DNA, alkaline phosphatase, and calcium are not shown by histochemical techniques. As a rule the chromatoid body is homogeneous but in some cases it has a cortex and a medulla. In many cases it is surrounded by a clear, vacuole-like space. Under the electron microscope it has been seen as an opaque irregular body, as an irregular mass of closely aggregated, dense, osmiophil granules, or as a faintly electron-opaque body. The chromatoid body has so far been recorded in certain species of mammals, a bird, reptiles, cyclostomes, Crustacea, insects, and arachnids. In most cases it appears for the first time during the growth of the primary spermatocyte. Its presence in the spermatid has been recorded in practically all cases. With a few exceptions it has not been found to take any obvious part in the final make-up of the spermatozoon. The chromatoid body in most cases seems to disappear at the metaphases of meiosis and to be later reconstructed in the daughter cells. The chromatoid body probably originates from the ground cytoplasm. On the basis of histochemical studies it is tentatively suggested that the function of the chromatoid body may be to provide basic proteins for the final maturation of the chromatin in the nucleus of late spermatids. Certain authors have considered that a cytoplasmic inclusion occurring in the young (and in some cases mature) spermatozooids of certain liverworts, mosses, and a gymnosperm is to be regarded as the homologue of the chromatoid body. Reasons are given for denying this supposed homology.


2016 ◽  
Vol 27 (25) ◽  
pp. 4021-4032 ◽  
Author(s):  
Irene Arcones ◽  
Carlos Sacristán ◽  
Cesar Roncero

The major chitin synthase activity in yeast cells, Chs3, has become a paradigm in the study of the intracellular traffic of transmembrane proteins due to its tightly regulated trafficking. This includes an efficient mechanism for the maintenance of an extensive reservoir of Chs3 at the trans-Golgi network/EE, which allows for the timely delivery of the protein to the plasma membrane. Here we show that this intracellular reservoir of Chs3 is maintained not only by its efficient AP-1–mediated recycling, but also by recycling through the retromer complex, which interacts with Chs3 at a defined region in its N-terminal cytosolic domain. Moreover, the N-terminal ubiquitination of Chs3 at the plasma membrane by Rsp5/Art4 distinctly labels the protein and regulates its retromer-mediated recycling by enabling Chs3 to be recognized by the ESCRT machinery and degraded in the vacuole. Therefore the combined action of two independent but redundant endocytic recycling mechanisms, together with distinct labels for vacuolar degradation, determines the final fate of the intracellular traffic of the Chs3 protein, allowing yeast cells to regulate morphogenesis, depending on environmental constraints.


2017 ◽  
Vol 37 (7) ◽  
Author(s):  
Françoise M. Roelants ◽  
Kristin L. Leskoske ◽  
Ross T. A. Pedersen ◽  
Alexander Muir ◽  
Jeffrey M.-H. Liu ◽  
...  

ABSTRACT Depending on the stress, plasma membrane alterations activate or inhibit yeast target of rapamycin (TOR) complex 2, which, in turn, upregulates or downregulates the activity of its essential downstream effector, protein kinase Ypk1. Through phosphorylation of multiple substrates, Ypk1 controls many processes that restore homeostasis. One such substrate is protein kinase Fpk1, which is negatively regulated by Ypk1. Fpk1 phosphorylates and stimulates flippases that translocate aminoglycerophospholipids from the outer to the inner leaflet of the plasma membrane. Fpk1 has additional roles, but other substrates were uncharacterized. We show that Fpk1 phosphorylates and inhibits protein kinase Akl1, related to protein kinases Ark1 and Prk1, which modulate the dynamics of actin patch-mediated endocytosis. Akl1 has two Fpk1 phosphorylation sites (Ark1 and Prk1 have none) and is hypophosphorylated when Fpk1 is absent. Conversely, under conditions that inactivate TORC2-Ypk1 signaling, which alleviates Fpk1 inhibition, Akl1 is hyperphosphorylated. Monitoring phosphorylation of known Akl1 substrates (Sla1 and Ent2) confirmed that Akl1 is hyperactive when not phosphorylated by Fpk1. Fpk1-mediated negative regulation of Akl1 enhances endocytosis, because an Akl1 mutant immune to Fpk1 phosphorylation causes faster dissociation of Sla1 from actin patches, confers elevated resistance to doxorubicin (a toxic compound whose entry requires endocytosis), and impedes Lucifer yellow uptake (a marker of fluid phase endocytosis). Thus, TORC2-Ypk1, by regulating Fpk1-mediated phosphorylation of Akl1, adjusts the rate of endocytosis.


1992 ◽  
Vol 118 (5) ◽  
pp. 1041-1056 ◽  
Author(s):  
R Bowser ◽  
H Müller ◽  
B Govindan ◽  
P Novick

The SEC8 and SEC15 genes are essential for exocytosis in the yeast Saccharomyces cerevisiae and exhibit strong genetic interactions with SEC4, a gene of the ras superfamily. The SEC8 gene encodes a hydrophilic protein of 122 kD, while the temperature-sensitive sec8-9 allele encodes a protein prematurely truncated at 82 kD by an opal stop codon. The Sec8p sequence contains a 202 amino acid region that is 25% identical to the leucine rich domain of yeast adenylate cyclase that has been implicated in ras responsiveness. Fractionation, stability, and cross-linking studies indicate that Sec8p is a component of a 19.5S particle that also contains Sec15p. This particle is found both in the cytosol and peripherally associated with the plasma membrane, but it is not associated with secretory vesicles. Gel filtration studies suggest that a portion of Sec4p is in association with the Sec8p/Sec15p particle. We propose that this particle may function as a downstream effector of Sec4p, serving to direct the fusion of secretory vesicles with the plasma membrane.


2008 ◽  
Vol 7 (9) ◽  
pp. 1500-1512 ◽  
Author(s):  
Jennifer L. Gordon ◽  
Wandy L. Beatty ◽  
L. David Sibley

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.


2015 ◽  
Vol 26 (17) ◽  
pp. 3001-3012 ◽  
Author(s):  
Silvia Haase ◽  
Dennis Zimmermann ◽  
Maya A. Olshina ◽  
Mark Wilkinson ◽  
Fabio Fisher ◽  
...  

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.


2016 ◽  
Vol 214 (5) ◽  
pp. 507-515 ◽  
Author(s):  
Isabelle Tardieux ◽  
Jake Baum

The capacity to migrate is fundamental to multicellular and single-celled life. Apicomplexan parasites, an ancient protozoan clade that includes malaria parasites (Plasmodium) and Toxoplasma, achieve remarkable speeds of directional cell movement. This rapidity is achieved via a divergent actomyosin motor system, housed within a narrow compartment that lies underneath the length of the parasite plasma membrane. How this motor functions at a mechanistic level during motility and host cell invasion is a matter of debate. Here, we integrate old and new insights toward refining the current model for the function of this motor with the aim of revitalizing interest in the mechanics of how these deadly pathogens move.


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