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The Trypanosoma cruzi nucleic acid binding protein Tc38 presents changes in the intramitochondrial distribution during the cell cycle.

Duhagon MA, Pastro L, Sotelo-Silveira JR, Pérez-Díaz L, Maugeri D, Nardelli SC, Schenkman S, Williams N, Dallagiovanna B, Garat B - BMC Microbiol. (2009)

Bottom Line: However, we found that Tc38 predominantly localizes into the mitochondrion.In epimastigotes, Tc38 is found only in association with kDNA in G1 phase.In non-replicating parasite stages such as trypomastigotes, the protein is found only surrounding the entire kinetoplast structure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratorio de Interacciones Moleculares, Facultad de Ciencias, Montevideo, Uruguay. mduhagon@fcien.edu.uy

ABSTRACT

Background: Tc38 of Trypanosoma cruzi has been isolated as a single stranded DNA binding protein with high specificity for the poly [dT-dG] sequence. It is present only in Kinetoplastidae protozoa and its sequence lacks homology to known functional domains. Tc38 orthologues present in Trypanosoma brucei and Leishmania were proposed to participate in quite different cellular processes. To further understand the function of this protein in Trypanosoma cruzi, we examined its in vitro binding to biologically relevant [dT-dG] enriched sequences, its expression and subcellular localization during the cell cycle and through the parasite life stages.

Results: By using specific antibodies, we found that Tc38 protein from epimastigote extracts participates in complexes with the poly [dT-dG] probe as well as with the universal minicircle sequence (UMS), a related repeated sequence found in maxicircle DNA, and the telomeric repeat. However, we found that Tc38 predominantly localizes into the mitochondrion. Though Tc38 is constitutively expressed through non-replicating and replicating life stages of T. cruzi, its subcellular localization in the unique parasite mitochondrion changes according to the cell cycle stage. In epimastigotes, Tc38 is found only in association with kDNA in G1 phase. From the S to G2 phase the protein localizes in two defined and connected spots flanking the kDNA. These spots disappear in late G2 turning into a diffuse dotted signal which extends beyond the kinetoplast. This later pattern is more evident in mitosis and cytokinesis. Finally, late in cytokinesis Tc38 reacquires its association with the kinetoplast. In non-replicating parasite stages such as trypomastigotes, the protein is found only surrounding the entire kinetoplast structure.

Conclusions: The dynamics of Tc38 subcellular localization observed during the cell cycle and life stages support a major role for Tc38 related to kDNA replication and maintenance.

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Tc38 patterns in T. cruzi epimastigotes synchronized with hydroxyurea. Tc38-Alexa 488 signal is shown in green and DAPI nucleic acid staining in blue. "N" indicates the nucleus and "k" indicates the kinetoplast. (A) Single confocal sections (~0.3 μm thick) of selected parasites that show the most frequent patterns seen in the cell cycle progression after hydroxyurea removal, at the indicated times. Upper panels show the DAPI blue signal, middle panels the Tc38 signal and bottom panels the merge of both. The same patterns were observed in three different synchronization experiments. (B) Z projection of 31 optical sections (~0.3 μm thick) of three selected parasites at 6 h after HU removal. Only the merge of the DAPI and Alexa-488 signals is shown. (C) Western blot of total protein extract using purified anti-Tc38 antibody. M: molecular weight markers, A: protein extracts of asynchronous epimastigote cultures in exponential growth phase. Remaining lanes correspond to protein extracts of epimastigote cultures after removal of HU at the times (hours) indicated above each lane. 1 × 107 cells were loaded onto each lane. Molecular weights of the protein ladder are indicated on the left of the gel (kDa).
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Figure 6: Tc38 patterns in T. cruzi epimastigotes synchronized with hydroxyurea. Tc38-Alexa 488 signal is shown in green and DAPI nucleic acid staining in blue. "N" indicates the nucleus and "k" indicates the kinetoplast. (A) Single confocal sections (~0.3 μm thick) of selected parasites that show the most frequent patterns seen in the cell cycle progression after hydroxyurea removal, at the indicated times. Upper panels show the DAPI blue signal, middle panels the Tc38 signal and bottom panels the merge of both. The same patterns were observed in three different synchronization experiments. (B) Z projection of 31 optical sections (~0.3 μm thick) of three selected parasites at 6 h after HU removal. Only the merge of the DAPI and Alexa-488 signals is shown. (C) Western blot of total protein extract using purified anti-Tc38 antibody. M: molecular weight markers, A: protein extracts of asynchronous epimastigote cultures in exponential growth phase. Remaining lanes correspond to protein extracts of epimastigote cultures after removal of HU at the times (hours) indicated above each lane. 1 × 107 cells were loaded onto each lane. Molecular weights of the protein ladder are indicated on the left of the gel (kDa).

Mentions: We also studied Tc38 localization in cultures synchronized with hydroxyurea (HU). HU inhibits the enzyme ribonucleotide reductase and the resulting depletion of deoxyribonucleotides arrests DNA replication in late G1/early S phase [26]. Previous reports on the effects of HU treatment on the T. cruzi cell cycle phases considered S phase to occur between 3–6 h and G2 at 9 h after HU removal [27,28]. Progression of the cell cycle was followed using the same time schedule. We obtained images that are in agreement with the Tc38 sub-cellular localization determined in asynchrony (Figure 6). At time 0 we found that cells generally show a homogeneous signal over the kDNA (Figure 6A). Among them, a small percentage of the cells present two intense signals generally associated with the kinetoplast DNA. At 3–6 h, cultures largely present two defined spots flanking the kDNA disk and the images at 10 h also exhibit a signal connecting them. Further quantitative analyses are required to determine the significance of each distinct pattern contribution. Interestingly, as indicated above, the Tc38 staining at 6 h after HU removal does not co-localize with the DAPI staining, being mainly adjacent to the kDNA disk. In fact, higher resolution confocal images of cultures indicate that Tc38 localizes near but not on the kDNA (Figure 6B). Images of either non-synchronized or HU synchronized cells show quite similar patterns in more than 200 parasites.


The Trypanosoma cruzi nucleic acid binding protein Tc38 presents changes in the intramitochondrial distribution during the cell cycle.

Duhagon MA, Pastro L, Sotelo-Silveira JR, Pérez-Díaz L, Maugeri D, Nardelli SC, Schenkman S, Williams N, Dallagiovanna B, Garat B - BMC Microbiol. (2009)

Tc38 patterns in T. cruzi epimastigotes synchronized with hydroxyurea. Tc38-Alexa 488 signal is shown in green and DAPI nucleic acid staining in blue. "N" indicates the nucleus and "k" indicates the kinetoplast. (A) Single confocal sections (~0.3 μm thick) of selected parasites that show the most frequent patterns seen in the cell cycle progression after hydroxyurea removal, at the indicated times. Upper panels show the DAPI blue signal, middle panels the Tc38 signal and bottom panels the merge of both. The same patterns were observed in three different synchronization experiments. (B) Z projection of 31 optical sections (~0.3 μm thick) of three selected parasites at 6 h after HU removal. Only the merge of the DAPI and Alexa-488 signals is shown. (C) Western blot of total protein extract using purified anti-Tc38 antibody. M: molecular weight markers, A: protein extracts of asynchronous epimastigote cultures in exponential growth phase. Remaining lanes correspond to protein extracts of epimastigote cultures after removal of HU at the times (hours) indicated above each lane. 1 × 107 cells were loaded onto each lane. Molecular weights of the protein ladder are indicated on the left of the gel (kDa).
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Figure 6: Tc38 patterns in T. cruzi epimastigotes synchronized with hydroxyurea. Tc38-Alexa 488 signal is shown in green and DAPI nucleic acid staining in blue. "N" indicates the nucleus and "k" indicates the kinetoplast. (A) Single confocal sections (~0.3 μm thick) of selected parasites that show the most frequent patterns seen in the cell cycle progression after hydroxyurea removal, at the indicated times. Upper panels show the DAPI blue signal, middle panels the Tc38 signal and bottom panels the merge of both. The same patterns were observed in three different synchronization experiments. (B) Z projection of 31 optical sections (~0.3 μm thick) of three selected parasites at 6 h after HU removal. Only the merge of the DAPI and Alexa-488 signals is shown. (C) Western blot of total protein extract using purified anti-Tc38 antibody. M: molecular weight markers, A: protein extracts of asynchronous epimastigote cultures in exponential growth phase. Remaining lanes correspond to protein extracts of epimastigote cultures after removal of HU at the times (hours) indicated above each lane. 1 × 107 cells were loaded onto each lane. Molecular weights of the protein ladder are indicated on the left of the gel (kDa).
Mentions: We also studied Tc38 localization in cultures synchronized with hydroxyurea (HU). HU inhibits the enzyme ribonucleotide reductase and the resulting depletion of deoxyribonucleotides arrests DNA replication in late G1/early S phase [26]. Previous reports on the effects of HU treatment on the T. cruzi cell cycle phases considered S phase to occur between 3–6 h and G2 at 9 h after HU removal [27,28]. Progression of the cell cycle was followed using the same time schedule. We obtained images that are in agreement with the Tc38 sub-cellular localization determined in asynchrony (Figure 6). At time 0 we found that cells generally show a homogeneous signal over the kDNA (Figure 6A). Among them, a small percentage of the cells present two intense signals generally associated with the kinetoplast DNA. At 3–6 h, cultures largely present two defined spots flanking the kDNA disk and the images at 10 h also exhibit a signal connecting them. Further quantitative analyses are required to determine the significance of each distinct pattern contribution. Interestingly, as indicated above, the Tc38 staining at 6 h after HU removal does not co-localize with the DAPI staining, being mainly adjacent to the kDNA disk. In fact, higher resolution confocal images of cultures indicate that Tc38 localizes near but not on the kDNA (Figure 6B). Images of either non-synchronized or HU synchronized cells show quite similar patterns in more than 200 parasites.

Bottom Line: However, we found that Tc38 predominantly localizes into the mitochondrion.In epimastigotes, Tc38 is found only in association with kDNA in G1 phase.In non-replicating parasite stages such as trypomastigotes, the protein is found only surrounding the entire kinetoplast structure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratorio de Interacciones Moleculares, Facultad de Ciencias, Montevideo, Uruguay. mduhagon@fcien.edu.uy

ABSTRACT

Background: Tc38 of Trypanosoma cruzi has been isolated as a single stranded DNA binding protein with high specificity for the poly [dT-dG] sequence. It is present only in Kinetoplastidae protozoa and its sequence lacks homology to known functional domains. Tc38 orthologues present in Trypanosoma brucei and Leishmania were proposed to participate in quite different cellular processes. To further understand the function of this protein in Trypanosoma cruzi, we examined its in vitro binding to biologically relevant [dT-dG] enriched sequences, its expression and subcellular localization during the cell cycle and through the parasite life stages.

Results: By using specific antibodies, we found that Tc38 protein from epimastigote extracts participates in complexes with the poly [dT-dG] probe as well as with the universal minicircle sequence (UMS), a related repeated sequence found in maxicircle DNA, and the telomeric repeat. However, we found that Tc38 predominantly localizes into the mitochondrion. Though Tc38 is constitutively expressed through non-replicating and replicating life stages of T. cruzi, its subcellular localization in the unique parasite mitochondrion changes according to the cell cycle stage. In epimastigotes, Tc38 is found only in association with kDNA in G1 phase. From the S to G2 phase the protein localizes in two defined and connected spots flanking the kDNA. These spots disappear in late G2 turning into a diffuse dotted signal which extends beyond the kinetoplast. This later pattern is more evident in mitosis and cytokinesis. Finally, late in cytokinesis Tc38 reacquires its association with the kinetoplast. In non-replicating parasite stages such as trypomastigotes, the protein is found only surrounding the entire kinetoplast structure.

Conclusions: The dynamics of Tc38 subcellular localization observed during the cell cycle and life stages support a major role for Tc38 related to kDNA replication and maintenance.

Show MeSH
Related in: MedlinePlus