J Cell Biol 15 February ; 4 : — We have identified and cloned a novel essential myosin I in Aspergillus nidulans called myoA. The 1,amino acid predicted polypeptide encoded by myoA is most similar to the amoeboid myosins I. Using affinity-purified antibodies against the unique myosin I carboxyl terminus, we have determined that MYOA is enriched at growing hyphal tips.
Disruption of myoA by homologous recombination resulted in a diploid strain heterozygous for the myoA gene disruption. We can recover haploids with an intact myoA gene from these strains, but never haploids that are myoA disrupted. These data indicated that myoA encodes an essential myosin I, and this has allowed us to use a unique approach to studying myosin I function.
We have developed conditionally null myoA strains in which myoA expression is regulated by the alcA alcohol dehydrogenase promoter. A conditionally lethal strain germinated on inducing medium grows as wild type, displaying polarized growth by apical extension. However, growth of the same myoA mutant strain on repressing medium results in enlarged cells incapable of hyphal extension, and these cells eventually die.
Under repressing conditions, this strain also displays reduced levels of secreted acid phosphatase. The mutant phenotype indicates that myoA plays a critical role in polarized growth and secretion. Sign In or Create an Account. Search Dropdown Menu. Advanced Search.
User Tools Dropdown. Sign In. Skip Nav Destination Article Navigation. Article February 15 This Site. More discrete sub-compartments have been recently visualized in T. In addition, Coccidians possess a conoid, a motile organelle composed of tubulin fibers arranged in spiral at the apical pole  , .
At the opposite pole, the basal complex remains more enigmatic but is composed of a basal polar ring where the membrane occupation and recognition nexus protein 1 MORN1 localizes and a posterior cup where centrin 2 is found . In Toxoplasma , both apical and basal complexes originate close to the centrosomes very early in the cell division process, and during the development of the daughter cells their basal complex appears as a ring structure that migrates to the basal pole and constricts in the mature parasite .
Most invasive apicomplexan zoites exhibit a unique substrate-dependent motion referred to as gliding motility, which allows parasites to cross non-permissive biological barriers and assists host cell invasion and egress from infected cells. The force generated by the parasite to propel itself inside a target cell originates from a conserved actomyosin machinery termed the glideosome that is located at the pellicle, in the limited space between the PM and the IMC .
The glideosome sustains the forward movement of the parasite by rearward translocation of adhesins that are apically secreted by the micronemes and bound to host cell receptors . In Toxoplasma , the molecular motor complex is composed of the myosin heavy chain A MyoA and two associated light chains, the myosin light chain 1 MLC1 and the essential light chain 1 ELC1  — . The central sequence of GAP45 adopts an extended coiled-coil conformation that critically maintains the cohesion between the PM and the IMC during glideosome function, holding the two membranes at an optimal and constant distance .
Moreover the genes coding for actin ACT1 , MLC1 and GAP45 were conditionally excised but similarly, the parasites failed to be cloned, indicative of their essentiality . GAP70 is a protein closely related to GAP45, which is found only in Coccidians and localizes exclusively to the apical cap . While GAP45 is essential for the lytic cycle of the parasite, GAP70 can be deleted without noticeable phenotype, likely due to a compensatory effect of the abundant GAP45 .
Coccidians possess a third member of this family, GAP80, which is shown here to localize to the posterior pole of T. Characterization of the partners interacting with GAP80 led to the identification of IMC-associated protein 1 IAP1 , a key determinant for the assembly of the MyoC-glideosome at the posterior polar ring. While this complex is dispensable for parasite survival, disruption of its individual components was strikingly compensated by the assembly of a chimeric glideosome composed of components of the MyoA- and MyoC-glideosomes.
These findings shed light on the complexity and versatility of the gliding machine in the coccidian subgroup of Apicomplexa. Stable parasite lines confirmed that both genes are expressed in the tachyzoite stage Figure 1A. Epitope tagging of GAP70 at the endogenous locus confirmed localization to the apical cap of the parasite previously reported based on expression of a second epitope-tagged copy .
In sharp contrast, GAP80 localized exclusively to the basal pole of mature parasites and showed a ring-shaped staining corresponding to the posterior polar ring Figure 1B. As a control, expression of a second copy of GAP80Ty was found mainly targeted to the basal pole, opposite to the apical microneme staining of MIC4, and also slightly at the parasite periphery due to overexpression Figure 1D. Profilin PRF was used as a loading control.
The star indicates a degradation product. To ascertain the association of GAP80 with the membrane, fractionation experiments were completed. GAP80Ty expressed as a second copy was more readily extracted in the various conditions, likely due to a looser IMC interaction caused by the overexpressed fraction that localizes to the periphery of the parasite and lacks basal-specific anchor s. Their distribution in different fractions was assessed by western blot using anti-Ty antibodies and the soluble catalase CAT as control for the correct fractionation.
Eluted proteins were visualized by autoradiography. The black circles correspond to the respective bait. The asterisks indicate the Ig heavy chains that cross-react with the antibodies. Localization of the endogenous MyoC in a ring-like structure at the basal pole of mature parasites and late stage daughter cells using anti-Ty and anti-IMC1 antibodies. Arrowheads point to the basal end of the parasites that are also presented in the magnifications.
To validate that most of the glideosome components were shared between the two complexes, a reverse co-IP experiment was carried out on metabolically labeled parasites expressing a second tagged copy of the shared MLC1 MLC1Ty. All the components of the glideosome were again present in the bound fraction including the protein migrating at around kDa Figure 2D. Taken together these findings identified a new coccidian-specific glideosome named the MyoC-glideosome, which shares the anchoring components to the IMC with the MyoA-glideosome, broadly conserved across the phylum of Apicomplexa.
Two bands, one below 40 kDa protein 1 and one above 35 kDa protein 2 , were cut out and 8 and 9 proteins were identified by mass spectrometry, respectively Table S2. Besides obvious contaminants corresponding to the abundant surface protein SAG1, heat shock and ribosomal proteins, peptides corresponding to MLC1 and GAP50 were also found.
More interestingly, peptides corresponding to three hypothetical genes present only in Coccidians and exhibiting a similar cell cycle transcription profile as MyoC were identified and investigated further by epitope tag knock-in at the endogenous locus.
No transmembrane spanning domain was apparent for IAP1 but instead five cysteine residues were predicted to be palmitoylated with a high probability  , supporting the strong interaction with the IMC Figures 3D and S3B. In addition, acylation at multiple sites could explain why IAP1 migrated higher than its expected size, a shift that was even more pronounced when one Figure S3C or three acidic Ty-tags Figure 3B were added.
Eluted proteins were boiled left panel or not right panel before migration on a SDS-page gel and then visualized by autoradiography. Stars indicate the Myc-tagged proteins. Proteins 1 and 2 are the candidates analyzed by mass spectrometry.
Schematic representation of the IAP1 constructs used in this study and highlighting the position of the cysteine residues. In red are the cysteines predicted to be palmitoylated by CSS-Palm 3. The asterisks indicate the Ig heavy and light chains that cross-react with the antibodies. Their distribution in different fractions was assessed by western blot using anti-Ty antibodies and the soluble catalase CAT was used as control for the correct fractionation. To unravel how IAP1 associates with the IMC, we examined the contribution of four out of the five predicted palmitoylated cysteine residues lying in the N-terminal part of the protein Figure 3D.
In addition, this mutant was completely soluble in PBS while the wild type protein endogenous or second copy was fully extracted only in the presence of detergent Figure 3H. Taken together, these data established that IAP1 is a component of the MyoC-glideosome that contributes to its basal polar ring localization most likely via N-terminal palmitoylation. In addition, no noticeable phenotype has been observed during the lytic cycle by plaque assay Figure 4C.
This mutant showed no defect in intracellular growth and no impairment in egress Figure S5B. No defect in the lytic cycle was observed. Given the position of MyoC at the basal polar ring, we monitored by time-lapse microscopy the ability of MyoC-KO parasites to perform twirling during an induced egress assay and observed no defect compared to the wild type strain Videos S1 and S2. Importantly, the interaction of MyoA with GAP80 suggested that this motor had the potential to substitute for the absence of MyoC at the posterior polar ring.
Since GAP80 level is lower in the absence of MyoC, it was not possible to make a quantitative comparison of the co-IPs between the two parasite lines. The absence of phenotype by plaque assay indicated that GAPKO parasites were able to accomplish their lytic cycle normally Figure 5A and indeed the individual steps including intracellular growth and egress were not altered Figure S5 G, H.
Little arrows point to the apical pole of the parasites while arrowheads point to the posterior pole. Since IAP1 is involved in the recruitment of the MyoC-glideosome to the basal polar ring, it has no known counterpart in the MyoA-glideosome to rescue its deletion.
However, in the absence of IAP1, GAP80 was no longer detectable at the basal polar ring or elsewhere in the parasite Figure 5E but remained detectable by western blot even though it appeared less abundant, likely due to its reduced stability in the absence of the complex Figure 5F. MyoC was also absent from the basal polar ring and instead localized to the cytoplasm, at the periphery and also concentrated at the apical polar ring Figure 5E.
Individual deletion of the components of the MyoC-glideosome for which counterparts exist in the MyoA-glideosome are compensated for by the formation of a chimeric glideosome attesting to the adaptability and versatility of T. To tackle MyoC function, it was necessary to hamper MyoA incorporation into the basal glideosome to avoid a compensatory effect. To achieve that, we thought of introducing a non-functional point mutation in the ATP-binding site of endogenous MyoC.
To ensure the integration of the mutation, the N-terminal MyoC fragment was synthetized with a different codon usage up to and including the mutated ATP-binding site and the homologous region was lying downstream Figure S4C. Surprisingly MyoC-KE failed to localize to the basal polar ring of mature parasites as previously described  Figure 6A. Instead, MyoC-KE was clearly visible in all the late stage developed daughter cells identified with IMC1 Figure 6A implying that the protein is expressed and targeted to the basal polar ring during division but is not incorporated in this structure in the mature parasites.
MyoC-KE is not detectable by western blot Figure 6B suggesting that it is destabilized when not incorporated into the basal pole of mature parasites. Parasites expressing a non-functional MyoC have no phenotype in intracellular growth, invasion or egress Figure S4 E-G. These data suggest that functional MyoC might be necessary for its integration in the basal pole.
The expression of a non-functional MyoC led to a situation similar to the deletion of MyoC, leaving again physical room for a compensatory mechanism. The mutated MyoC is never detected with anti-Myc antibodies. Given the overall similarities in the architecture and composition of the two glideosomes, it appeared legitimate to assume that the MyoC-glideosome participates in some aspects of the gliding function possibly exemplified by the stationary twirling where the parasite rotates, contacting the substrate via its posterior pole .
To support this notion we first anticipated that GAP80 could functionally complement the depletion of GAP45 when expressed at a suitable level. This correlated with the partial complementation seen by plaque assay Figure 7C and the normal intracellular growth curve Figure 7D . Intracellular: invaded parasites, extracellular: attached parasites.
For E and F, the significance of the results was assessed using a parametric paired t-test and the two-tailed p-values are written on the graphs. In agreement with these observations, the two mutants showed severe defects in gliding Figure 8B and in egress assays following ATc treatment Figure 8C and Table 1.
These results establish that the MyoC-glideosome contributes to an efficient invasion process in T. The significance of the data was evaluated using a parametric paired t-test and the two-tailed p-value is written on the graph.
Two exposures are presented for MycMyoC localization. The loading control was done at the same time with anti-PRF and fluorescent secondary antibodies on the same membrane as MLC1 for the upper panel and as Myc for the lower panel. To further investigate the behavior of the MyoC-glideosome in the absence of MyoA, we freshly excised the gene from the loxP-MyoA strain  and cloned the parasites. GAP45 and MLC1 were previously shown to remain localized at the periphery of the parasite in the absence of MyoA  and hence antibodies raised against these two proteins were used for co-IP experiments to assess the composition of the complex Figures 8E and S7.
Stable parasites were obtained and 3 independent clones were sequenced for the mutations introduced in the MyoA locus to repair the double-stranded break generated by the CAS9 at the specific target sequence Figure S7D. In the absence of MyoA, MyoC is localized not only to the basal polar ring of mature parasites but also relocalized peripherally up to the apical polar ring Figure 8F. This study reports the identification and characterization of a new glideosome in T.
The overall arrangement of the three glideosomes is similar and centered around a GAP45 family member that recruits a myosin motor complex to a sub-compartment of the IMC Figure 9A. The three proteins are predicted to be N-terminally acylated at the plasma membrane and exhibit an extended central region predicted to form a coiled-coil domain or short alpha helices that vary significantly in length.
Further characterization of the complex identified IAP1, which is the necessary determinant to restrict its localization to the posterior polar ring. Given the absence of a TMD, the localization of IAP1 could either be mediated by palmitoylation that would stabilize the protein in the membrane bilayer, or by interaction with an un-identified protein. Alanine substitution of the three N-terminal cysteine residues predicted to be palmitoylated resulted in a cytoplasmic localization of the mutated IAP1.
This result strongly suggests that palmitoylation at one or more sites is involved in the attachment of IAP1 to the lowest sub-compartment of the IMC and basal polar ring. In this context, one of the two recently characterized IMC-located protein S-acyl transferases might play an instrumental role in targeting .
Interestingly, the truncated version of IAP1 that encompasses aa including three of the four predicted palmitoylated cysteine residues was associated with the basal sub-compartment of the IMC but not anymore to the polar ring. The same relocalization was observed for GAP80 indicating that these two proteins are interacting together and implicates the N-terminus of IAP1.
The C-terminus of IAP1 is therefore associated with the polar ring either directly by palmitoylation or possibly by interaction with an integral membrane protein that remains to be identified. Localization and composition of the three glideosomes in T. Illustration of the composition of the basal glideosome according to the component that has been targeted for deletion. Illustration of the composition of the glideosomes upon deletion of MyoA. MyoC and MORN1 are the only two proteins identified so far at the posterior polar ring of the growing daughter cells.
MORN1 emerges much earlier than MyoC in dividing parasites where is also detected as dots at the extremities of the nascent IMC and at the centrocone  ,  , . Here we show that parasites lacking MyoC still assemble the basal complex as observed by the localization of GAP80 and also divide normally. In contrast, disruption of MORN1 has been achieved using two different strategies that led to a defect in basal complex assembly, cytokinesis and apicoplast segregation  , .
While the three GAP45 family members are expressed in tachyzoites, only GAP45 appears to compensate for the loss of the two others probably due to its high level of expression. Although GAP45 might not be optimally tailored to function at the apical and posterior sub-compartments of the IMC, the compensation in the absence of GAP70 or GAP80 is sufficient to sustain gliding, invasion and egress.
MyoC-glideosome harbors two other specific components, MyoC and IAP1 that in principle offer an opportunity to address directly the function of the basal glideosome upon individual deletion of these two genes however significant plasticity and compensatory mechanisms were observed as well Figure 9B. Deletion of MyoC showed no significant impact on the parasite lytic cycle, as also recently reported .
Importantly, even a non-functional form of MyoC failed to integrate into the glideosome of the mature posterior pole, offering again the possibility for a compensatory effect to replace the defective MyoC. Importantly, in the absence of MyoA, MyoC clearly relocalized to the parasite periphery and at the apical pole, providing evidence that MyoC could partially replace MyoA in the peripheral and apical glideosomes Figure 9C.
RNA was isolated from tachyzoites using Trizol Invitrogen. Sph I was then used to linearize the plasmid before transfection. The promoter region was removed by digestion with Kpn I and Xho I restriction enzymes. Finally, the plasmid was linearized with Hind III for transfection. A truncation of the endogenous IAP1 aa 1 to was created by knock-in.
Carruthers,  using the ligation independent cloning strategy . Around 1. A truncated version of MyoC aa 1 to was generated by introducing 3 Myc-tags after the head domain in the endogenous locus by knock-in. The plasmid was cut with Kpn I and Not I restriction enzymes before transfection. Parasite transfections were performed by electroporation as previously described .
In both cases, 24 hours after transfections, parasites were sorted by flow cytometry and cloned into well plates using a Moflo Astrios Beckman Coulter. The fusion protein was expressed into E. Fixed cells were then processed as previously described . Stacks of sections were processed with ImageJ and projected using the maximum projection tool.
Parasites were lysed by freeze and thaw followed by sonication on ice. The solubility of the catalase CAT was checked in the different conditions as control. The suspension was either boiled or subjected to sonication on ice. Giemsa staining was then performed as described in Plattner et al. The number of parasites per vacuole was determined by counting the parasites in vacuoles in duplicate for three independent experiments. This assay was performed as previously described  with the following specificities.
The number of intracellular and extracellular parasites was determined by counting parasites in duplicate for four independent experiments. The average number of egressed vacuoles was determined by counting vacuoles for each condition for four independent experiments.
Anti-SAG1 antibody was used without permeabilization to visualize the trails and the parasites. Three independent experiments have been performed. HFF cells were heavily infected with freshly egressed parasites and washed several hours later. Complexes were then washed three times in CoIP buffer. Finally, beads were resuspended in SDS loading buffer under reducing conditions.
Bands of interest were excised from the gel and sent to the Proteomics Core Facility Faculty of Medicine, Geneva, Switzerland for analysis according to their standard protocols for protein identification. The fragments were generated with trypsin and the peaklist files were searched against the Toxoplasma gondii GT1 database Toxoplasma Genomics Resource, release 8. GAPrelated proteins. Multiple alignments of the T.
Identical residues are in red, strongly similar residues in green and weakly similar residues in blue. The myristoylated glycine 2 and palmitoylated cysteine were predicted using myristoylator  and CSS-Palm 3. The coiled-coil domain predicted with Coils  in GAP45 and GAP70 proteins is indicated by a blue spring and the conserved C-terminal part is depicted by a green box.
Scheme of the knock-in strategy used to introduce a Ty-tag in the endogenous loci of gap70 or gap
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