Characterization of the Toxoplasma Cell Cycle'

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Characterization of the Toxoplasma Cell Cycle.

• Michael N. Guerini
• Department of Veterinary Molecular Biology
• Montana State University, Bozeman MT

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• Tachyzoite Cell cycle analysis (random and
  synchronous populations).
• DNA content by propidium iodide/Flow Cytometry
  provides estimates of
• the length of G1, S, and G2M.

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Blocked Released CTK11
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Tachyzoite Cell cycle analysis (random and
synchronous populations). 1. DNA content by
propidium iodide/Flow Cytometry provides
estimates of the length of G1, S, and G2M. 2.
Nuclear morphology by radiolabeling (thymidine
index) and TgPCNA in situ fluorescence
(replication foci assembly) confirms the length
of S-phase.
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Thymidine Index
RHTK Tachyzoites were pulsed with 3H-thymidine
15 min 30 min RH/Hx- control
32 cells incorporate the radiolabel
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Time-lapse Microscopy of tachyzoites expressing
TgPCNA1-GFP.
Time Lapse
Replication Foci
Deconvolution Microscopy
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Tachyzoite Cell cycle analysis (random and
synchronous populations). 1. DNA content by
propidium iodide/Flow Cytometry provides
estimates of the length of G1, S, and G2M. 2.
Nuclear morphology by radiolabeling (thymidine
index) and TgPCNA in situ fluorescence
(replication foci assembly) confirms the length
of S-phase. 3. Mitotic forms as visualized by in
situ fluorescence of daughter parasites
confirms length of mitosis, nuclear division,
and the initiation and completion of
cytokinesis.
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Time-lapse Microscopy of tachyzoites expressing
TgPCNA1-GFP.
Mitosis - 10 minutes Nuclear Division 20
minutes Cytokinesis 20 minutes
Dividing Nuclei
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Daughter Cell Formation --- Cytokinesis
Phase Fluorescence
C4F3-antibody
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Fraction of daughters slightly exceeds the FACS
estimates of 2N parasites.
11 daughters
28 daughters
Blue Histogram - Asynchronous RH tachyzoites,
8 2N parasites Red overlay - RHTK -
tachyzoites 4 hour post release from
thymidine block, 25 2N parasites
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Summary of the Tachyzoite Cell Cycle

• Distinct Features
• G1 Most dominant phase gt50
• S-phase
• bimodal
• 1.5 hours
• No apparent G2
• Similar to cell cycles of
• Theileria macroschizont
• Paramecium micronucleus
• Apicomplexan schizonts
• Mitosis
• endomitotic
• Cytokinesis
• Begins in late S-phase and ends 20 minutes
  post- nuclear division

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Clincal Importance of Toxoplasma. Life-long
chronic disease Central Nervous System Disease
in immuno-compromised individuals---- AIDS
patients transplant patients elderly cancer
patients
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Sporozoite Initiated Development
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Developmental associated cell cycle changes
G1 S phase G2 M/C BAG1 Day 95 5 0 0
0 0 61 30 0 9 0
3 64 29 0 8 2 7 71 22 5
2 12 9 84 gt1 15 gt1 51 15 76
3 21 gt1 99 pH shift
Sporozoite
Tachyzoite
Tachy/Brady
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FACS analysis of the DNA Content of
Differentiated Populations.
SAG-1 antibody
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Cell Cycle Model for Toxoplasma Development
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Acknowledgements
Montana State University University of
Georgia Michael W. White Boris Streipen Maria
Jerome Jay R. Radke Chris Kvaal Trina
Larson University of Pennsylvania Stanford
University David Roos John Boothroyd Naomi
Morrissette Univsersity of Gottingen Wolfgang
Bohne