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Organizational changes of the daughter basal complex during the parasite replication of Toxoplasma gondii.

Hu K - PLoS Pathog. (2008)

Bottom Line: This study focuses on key events during the biogenesis of the basal complex using high resolution light microscopy, and reveals that daughter basal complexes are established around the duplicated centrioles independently of the structural integrity of the daughter cortical cytoskeleton, and that they are dynamic "caps" at the growing ends of the daughters.This correlates with the constriction of the basal complex, a process that can be artificially induced by increasing cellular calcium concentration.The basal complex is therefore likely to be a new kind of centrin-based contractile apparatus.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Indiana University, Bloomington, Indiana, United States of America. kehu@indiana.edu

ABSTRACT
The apicomplexans are a large group of parasitic protozoa, many of which are important human and animal pathogens, including Plasmodium falciparum and Toxoplasma gondii. These parasites cause disease only when they replicate, and their replication is critically dependent on the proper assembly of the parasite cytoskeletons during cell division. In addition to their importance in pathogenesis, the apicomplexan parasite cytoskeletons are spectacular structures. Therefore, understanding the cytoskeletal biogenesis of these parasites is important not only for parasitology but also of general interest to broader cell biology. Previously, we found that the basal end of T. gondii contains a novel cytoskeletal assembly, the basal complex, a cytoskeletal compartment constructed in concert with the daughter cortical cytoskeleton during cell division. This study focuses on key events during the biogenesis of the basal complex using high resolution light microscopy, and reveals that daughter basal complexes are established around the duplicated centrioles independently of the structural integrity of the daughter cortical cytoskeleton, and that they are dynamic "caps" at the growing ends of the daughters. Compartmentation and polarization of the basal complex is first revealed at a late stage of cell division upon the recruitment of an EF-hand containing calcium binding protein, TgCentrin2. This correlates with the constriction of the basal complex, a process that can be artificially induced by increasing cellular calcium concentration. The basal complex is therefore likely to be a new kind of centrin-based contractile apparatus.

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The Basal Complex Is a Dynamic “Cap”Time-lapse experiment tracking the exchange of EGFP-TgMORN1 in the basal complexes of growing daughters using Fluorescence Recovery After Photo-bleaching (FRAP). The fluorescence of EGFP-TgMORN1 in the basal complexes of two daughters was partially bleached (arrowheads, insets) at time 0, and the recovery was well underway at ∼90 s.Insets: 2× magnification of regions indicated by the arrowheads.The merged image of EGFP-TgMORN1 (green) and mCherryFP-TgTubA1 (red) is the maximum intensity projection of a deconvolved 3D stack. All the gray scale images are non-deconvolved single optical planes.
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ppat-0040010-g006: The Basal Complex Is a Dynamic “Cap”Time-lapse experiment tracking the exchange of EGFP-TgMORN1 in the basal complexes of growing daughters using Fluorescence Recovery After Photo-bleaching (FRAP). The fluorescence of EGFP-TgMORN1 in the basal complexes of two daughters was partially bleached (arrowheads, insets) at time 0, and the recovery was well underway at ∼90 s.Insets: 2× magnification of regions indicated by the arrowheads.The merged image of EGFP-TgMORN1 (green) and mCherryFP-TgTubA1 (red) is the maximum intensity projection of a deconvolved 3D stack. All the gray scale images are non-deconvolved single optical planes.

Mentions: How is the basal complex able to remain at the constantly growing ends of the daughter cortical cytoskeletons? Studies in mammalian cells have shown that microtubule plus-end (MT-plus-end) binding proteins probably maintain their position at the growing ends of microtubules by rapid association and dissociation [20]. Fluorescence Recovery After Photobleaching (FRAP) analysis of daughter basal complexes in T. gondii reveals constant protein exchange between the daughter basal complex and the cytoplasm (Figure 6). Although it is difficult to calculate an exact t1/2 for the fluorescence recovery because of the noise introduced by constant fluctuation of the basal complex position due to daughter cell movement, the recovery is clearly underway by ∼90 sec after photobleaching. This result indicates that the basal complex is intrinsically a dynamic “cap”, thus suggesting a mechanism similar to microtubule association of MT-plus-end binding protein is possibly involved in retaining the basal complex at the growing ends of the daughter cortical cytoskeletons. However, the growth of the daughter cortical cytoskeleton is likely not to be the pre-requisite for the protein exchange in the basal complex, as the fluorescence in the mature basal complex also partially recovers after photobleaching (Figure S2).


Organizational changes of the daughter basal complex during the parasite replication of Toxoplasma gondii.

Hu K - PLoS Pathog. (2008)

The Basal Complex Is a Dynamic “Cap”Time-lapse experiment tracking the exchange of EGFP-TgMORN1 in the basal complexes of growing daughters using Fluorescence Recovery After Photo-bleaching (FRAP). The fluorescence of EGFP-TgMORN1 in the basal complexes of two daughters was partially bleached (arrowheads, insets) at time 0, and the recovery was well underway at ∼90 s.Insets: 2× magnification of regions indicated by the arrowheads.The merged image of EGFP-TgMORN1 (green) and mCherryFP-TgTubA1 (red) is the maximum intensity projection of a deconvolved 3D stack. All the gray scale images are non-deconvolved single optical planes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211554&req=5

ppat-0040010-g006: The Basal Complex Is a Dynamic “Cap”Time-lapse experiment tracking the exchange of EGFP-TgMORN1 in the basal complexes of growing daughters using Fluorescence Recovery After Photo-bleaching (FRAP). The fluorescence of EGFP-TgMORN1 in the basal complexes of two daughters was partially bleached (arrowheads, insets) at time 0, and the recovery was well underway at ∼90 s.Insets: 2× magnification of regions indicated by the arrowheads.The merged image of EGFP-TgMORN1 (green) and mCherryFP-TgTubA1 (red) is the maximum intensity projection of a deconvolved 3D stack. All the gray scale images are non-deconvolved single optical planes.
Mentions: How is the basal complex able to remain at the constantly growing ends of the daughter cortical cytoskeletons? Studies in mammalian cells have shown that microtubule plus-end (MT-plus-end) binding proteins probably maintain their position at the growing ends of microtubules by rapid association and dissociation [20]. Fluorescence Recovery After Photobleaching (FRAP) analysis of daughter basal complexes in T. gondii reveals constant protein exchange between the daughter basal complex and the cytoplasm (Figure 6). Although it is difficult to calculate an exact t1/2 for the fluorescence recovery because of the noise introduced by constant fluctuation of the basal complex position due to daughter cell movement, the recovery is clearly underway by ∼90 sec after photobleaching. This result indicates that the basal complex is intrinsically a dynamic “cap”, thus suggesting a mechanism similar to microtubule association of MT-plus-end binding protein is possibly involved in retaining the basal complex at the growing ends of the daughter cortical cytoskeletons. However, the growth of the daughter cortical cytoskeleton is likely not to be the pre-requisite for the protein exchange in the basal complex, as the fluorescence in the mature basal complex also partially recovers after photobleaching (Figure S2).

Bottom Line: This study focuses on key events during the biogenesis of the basal complex using high resolution light microscopy, and reveals that daughter basal complexes are established around the duplicated centrioles independently of the structural integrity of the daughter cortical cytoskeleton, and that they are dynamic "caps" at the growing ends of the daughters.This correlates with the constriction of the basal complex, a process that can be artificially induced by increasing cellular calcium concentration.The basal complex is therefore likely to be a new kind of centrin-based contractile apparatus.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Indiana University, Bloomington, Indiana, United States of America. kehu@indiana.edu

ABSTRACT
The apicomplexans are a large group of parasitic protozoa, many of which are important human and animal pathogens, including Plasmodium falciparum and Toxoplasma gondii. These parasites cause disease only when they replicate, and their replication is critically dependent on the proper assembly of the parasite cytoskeletons during cell division. In addition to their importance in pathogenesis, the apicomplexan parasite cytoskeletons are spectacular structures. Therefore, understanding the cytoskeletal biogenesis of these parasites is important not only for parasitology but also of general interest to broader cell biology. Previously, we found that the basal end of T. gondii contains a novel cytoskeletal assembly, the basal complex, a cytoskeletal compartment constructed in concert with the daughter cortical cytoskeleton during cell division. This study focuses on key events during the biogenesis of the basal complex using high resolution light microscopy, and reveals that daughter basal complexes are established around the duplicated centrioles independently of the structural integrity of the daughter cortical cytoskeleton, and that they are dynamic "caps" at the growing ends of the daughters. Compartmentation and polarization of the basal complex is first revealed at a late stage of cell division upon the recruitment of an EF-hand containing calcium binding protein, TgCentrin2. This correlates with the constriction of the basal complex, a process that can be artificially induced by increasing cellular calcium concentration. The basal complex is therefore likely to be a new kind of centrin-based contractile apparatus.

Show MeSH
Related in: MedlinePlus