Elsevier

Protist

Volume 158, Issue 2, 18 April 2007, Pages 147-157
Protist

ORIGINAL PAPER
Morphological Events during the Trypanosoma cruzi Cell Cycle

https://doi.org/10.1016/j.protis.2006.10.002Get rights and content

The replication and segregation of organelles producing two identical daughter cells must be precisely controlled during the cell cycle progression of eukaryotes. In kinetoplastid flagellated protozoa, this includes the duplication of the single mitochondrion containing a network of DNA, known as the kinetoplast, and a flagellum that grows from a cytoplasmic basal body through the flagellar pocket compartment before emerging from the cell. Here, we show the morphological events and the timing of these events during the cell cycle of the epimastigote form of Trypanosoma cruzi, the protozoan parasite that causes Chagas’ disease. DNA staining, flagellum labeling, bromodeoxyuridine incorporation, and ultra-thin serial sections show that nuclear replication takes 10% of the whole cell cycle time. In the middle of the G2 stage, the new flagellum emerges from the flagellar pocket and grows unattached to the cell body. While the new flagellum is still short, the kinetoplast segregates and mitosis occurs. The new flagellum reaches its final size during cytokinesis when a new cell body is formed. These precisely coordinated cell cycle events conserve the epimastigote morphology with a single nucleus, a single kinetoplast, and a single flagellum status of the interphasic cell.

Introduction

The eukaryotic cell cycle is characterized by the progression of linked events that allow segregation of identical genetic material into progeny (Murray 2004). The mechanisms that ensure correct segregation have been extensively studied with respect to the control of cell growth, DNA replication, and mitosis, including the establishment of networks of interacting molecules expressed at different times of the cell cycle (Tyers 2004). However, much less is known about the relationship between the duplication and segregation of organelles and cell cycle progression. One reason for this is that most eukaryotes contain multi-copy organelles in each cell. Some protists, therefore, are very useful systems in this regard because they have single-copy and specialized organelles. Trypanosomes are flagellated protists that contain a single Golgi complex (He et al. 2004) as well as a large mitochondrion that hosts a single kinetoplast formed by a network of minicircle and maxicircle DNA molecules. A single flagellum connected to the kinetoplast at its basal body emerges from a flagellar pocket, an invagination of the plasma membrane (Gull 2003; Robinson and Gull 1991).

In Trypanosoma brucei, the agent of African trypanosomiasis, the morphological events occurring during the cell cycle are precisely determined (Woodward and Gull 1990). However, despite that T. brucei is considered an archetypal organism among trypanosomes, particularly when descriptions of structural organization are provided, morphological alterations that occur during the cell cycle of other species of trypanosomes must be different as they have distinct cell shapes, kinetoplast position, and flagellum insertion. Crithidia and Leishmania species, for instance, do not replicate their nuclear and kinetoplast DNA in the same order as T. brucei (Bhattacharya and Ghosh 1985; Cosgrove and Skeen 1970). In most cases, there is no migration of the basal body and kinetoplast segregation occurs close to the nuclear mitosis. In addition, although no detailed description has been provided, the new flagellum does not attach to the old one (Briggs et al. 2004).

Much less is known about the morphological alterations and the timing of the cell division cycle of Trypanosoma cruzi, the agent of Chagas’ disease. T. cruzi cell division has been characterized previously (de Souza and Meyer 1974; O’Daly and Bretana 1976) and reviewed in (de Souza, 1999, de Souza, 2002; Tyler and Engman 2001), but a clear relationship between the generation of organelles and the cell cycle has not yet been established. Here, we describe the morphological events that occur during T. cruzi epimastigote cell division cycle, in particular, the replication of DNA, mitosis as well as kinetoplast and flagellum pocket division, and growth of the new flagellum.

Section snippets

Morphological Patterns of the Trypanosoma cruzi Cell Cycle

To understand the morphological alterations that occur during the Trypanosoma cruzi cell cycle, exponentially growing epimastigote cells (Fig. 1 A) were searched for the presence of cells with differing numbers of organelles. After propidium iodide staining, these cells were analyzed by FACS (fluorescence-activated cell sorting) and no unusual ploidy was found (data not shown; Elias et al. 2002). A monoclonal antibody (mAb 25) that recognizes a 24 kDa flagellar calcium-binding protein found

Discussion

Here, we have established the cytological events that occur during the Trypanosoma cruzi cell cycle. Figure 5 illustrates a sequential description of these events that includes DNA synthesis in the nucleus and kinetoplast, growth of a new flagellum, segregation of the kinetoplast, nuclear mitosis, formation of a new flagellar pocket, and finally, cytokinesis. The timing of these events is also indicated in Figure 5. The duration of kinetoplast replication and kinetoplast G2 could not be

Methods

Cell growth and cell synchronization: Trypanosoma cruzi epimastigotes (Y strain) were cultured in liver-infusion tryptose medium supplemented with 10% fetal bovine serum at 28 °C (Camargo 1964). For cell cycle arrest, an exponentially growing cell culture was treated for 24 h with 20 mM HU (Sigma). Release was done by 3 washes with phosphate-buffered saline (PBS) followed by addition of cells in a fresh medium (Elias et al. 2002).

BrdU labeling and immunofluorescence: Exponentially growing cells

Acknowledgements

We thank Dr. Kevin Tyler for helpful suggestions. We also thank Evânia Barbosa Azevedo and Claudio Rogério de Oliveira for maintaining the parasites and help in the immunofluorescence experiments, and Marcia F.A. Tanakay for technical support in the EM studies. This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de S. Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

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