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Measuring tubulin content in Toxoplasma gondii: a comparison of laser-scanning confocal and wide-field fluorescence microscopy.

ABSTRACT: Toxoplasma gondii is an intracellular parasite that proliferates within most nucleated cells, an important human pathogen, and a model for the study of human and veterinary parasitic infections. We used a stable yellow fluorescent protein-alpha-tubulin transgenic line to determine the structure of the microtubule cytoskeleton in T. gondii. Imaging of living yellow fluorescent protein-alpha-tubulin parasites by laser-scanning confocal microscopy (LSCM) failed to resolve the 22 subpellicular microtubules characteristic of the parasite cytoskeleton. To understand this result, we analyzed sources of noise in the LSCM and identified illumination fluctuations on time scales from microseconds to hours that introduce significant amounts of noise. We confirmed that weakly fluorescent structures could not be imaged in LSCM by using fluorescent bead standards. By contrast, wide-field microscopy (WFM) did visualize weak fluorescent standards and the individual microtubules of the parasite cytoskeleton. We therefore measured the fluorescence per unit length of microtubule by using WFM and used this information to estimate the tubulin content of the conoid (a structure important for T. gondii infection) and in the mitotic spindle pole. The conoid contains sufficient tubulin for approximately 10 microtubule segments of 0.5-microm length, indicating that tubulin forms the structural core of the organelle. We also show that the T. gondii mitotic spindle contains approximately 1 microtubule per chromosome. This analysis expands the understanding of structures used for invasion and intracellular proliferation by an important human pathogen and shows the advantage of WFM combined with image deconvolution over LSCM for quantitative studies of weakly fluorescent structures in moderately thin living cells.

SUBMITTER: Swedlow JR

PROVIDER: S-EPMC122311 | biostudies-literature | 2002 Feb

REPOSITORIES: biostudies-literature

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Measuring tubulin content in Toxoplasma gondii: a comparison of laser-scanning confocal and wide-field fluorescence microscopy.

Swedlow Jason R JR Hu Ke K Andrews Paul D PD Roos David S DS Murray John M JM

Proceedings of the National Academy of Sciences of the United States of America 20020205 4

Toxoplasma gondii is an intracellular parasite that proliferates within most nucleated cells, an important human pathogen, and a model for the study of human and veterinary parasitic infections. We used a stable yellow fluorescent protein-alpha-tubulin transgenic line to determine the structure of the microtubule cytoskeleton in T. gondii. Imaging of living yellow fluorescent protein-alpha-tubulin parasites by laser-scanning confocal microscopy (LSCM) failed to resolve the 22 subpellicular micro ...[more]

PMID: 11830634

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Project description:Background:Live-cell fluorescence microscopy (LCFM) is a powerful tool used to investigate cellular dynamics in real time. However, the capacity to simultaneously measure DNA content in cells being tracked over time remains challenged by dye-associated toxicities. The ability to measure DNA content in single cells by means of LCFM would allow cellular stage and ploidy to be coupled with a variety of imaging directed analyses. Here we describe a widely applicable nontoxic approach for measuring DNA content in live cells by fluorescence microscopy. This method relies on introducing a live-cell membrane-permeant DNA fluorophore, such as Hoechst 33342, into the culture medium of cells at the end of any live-cell imaging experiment and measuring each cell's integrated nuclear fluorescence to quantify DNA content. Importantly, our method overcomes the toxicity and induction of DNA damage typically caused by live-cell dyes through strategic timing of adding the dye to the cultures; allowing unperturbed cells to be imaged for any interval of time before quantifying their DNA content. We assess the performance of our method empirically and discuss adaptations that can be implemented using this technique. Results:Presented in conjunction with cells expressing a histone 2B-GFP fusion protein (H2B-GFP), we demonstrated how this method enabled chromosomal segregation errors to be tracked in cells as they progressed through cellular division that were later identified as either diploid or polyploid. We also describe and provide an automated Matlab-derived algorithm that measures the integrated nuclear fluorescence in each cell and subsequently plots these measurements into a cell cycle histogram for each frame imaged. The algorithm's accurate assessment of DNA content was validated by parallel flow cytometric studies. Conclusions:This method allows the examination of single-cell dynamics to be correlated with cellular stage and ploidy in a high-throughput fashion. The approach is suitable for any standard epifluorescence microscope equipped with a stable illumination source and either a stage-top incubator or an enclosed live-cell incubation chamber. Collectively, we anticipate that this method will allow high-resolution microscopic analysis of cellular processes involving cell cycle progression, such as checkpoint activation, DNA replication, and cellular division.

Dataset Information

Measuring tubulin content in Toxoplasma gondii: a comparison of laser-scanning confocal and wide-field fluorescence microscopy.

Publications

Measuring tubulin content in Toxoplasma gondii: a comparison of laser-scanning confocal and wide-field fluorescence microscopy.

Similar Datasets

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