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Development of a heat-shock inducible gene expression system in the red alga Cyanidioschyzon merolae.

Sumiya N, Fujiwara T, Kobayashi Y, Misumi O, Miyagishima SY - PLoS ONE (2014)

Bottom Line: At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression.After the heat shock, the mRNA level decreases rapidly.Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure.

View Article: PubMed Central - PubMed

Affiliation: Center for Frontier Research, National Institute of Genetics, Mishima, Shizuoka, Japan; Japan Science and Technology Agency, CREST, Kawaguchi, Saitama, Japan.

ABSTRACT
The cell of the unicellular red alga Cyanidioschyzon merolae contains a single chloroplast and mitochondrion, the division of which is tightly synchronized by a light/dark cycle. The genome content is extremely simple, with a low level of genetic redundancy, in photosynthetic eukaryotes. In addition, transient transformation and stable transformation by homologous recombination have been reported. However, for molecular genetic analyses of phenomena that are essential for cellular growth and survival, inducible gene expression/suppression systems are needed. Here, we report the development of a heat-shock inducible gene expression system in C. merolae. CMJ101C, encoding a small heat shock protein, is transcribed only when cells are exposed to an elevated temperature. Using a superfolder GFP as a reporter protein, the 200-bp upstream region of CMJ101C orf was determined to be the optimal promoter for heat-shock induction. The optimal temperature to induce expression is 50°C, at which C. merolae cells are able to proliferate. At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression. After the heat shock, the mRNA level decreases rapidly. As an example of the system, the expression of a dominant negative form of chloroplast division DRP5B protein, which has a mutation in the GTPase domain, was induced. Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure. This result suggests that the dominant negative DRP5B inhibited the final scission of the chloroplast division site, but not the earlier stages of division site constriction. It is also suggested that cell cycle progression is not arrested by the impairment of chloroplast division at the final stage.

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Effect of the DRP5B K135A dominant negative mutation on chloroplast division and cell cycle progression in C. merolae.(A) Schematic diagram of the domain composition of the conventional dynamin protein which is involved in endocytosis. In the GTPase domain, four GTP binding motifs (G1, G2, G3 and G4) contribute to the GTP binding. Partial alignment of the DRP5B proteins showed the conservation of the G1 motif. The DRP5B amino acid sequences (Chlamydomonas reinhardtii, GI: 30349146; Chlorella variabilis, GI: 552813628; Ostreococcus tauri, GI: 308803420; Arabidopsis thaliana, GI: 30349146; Physcomitrella patens, GI: 224434564; Cyanidioschyzon merolae, GI: 544214467) were aligned using ClustalW [35], [36]. Alanine substitution for the conserved lysine residue within G1 of human dynamin 1 is known to result in a dominant negative effect by abolishing GTP-binding activity [32]. (B to F) GFP-DRP5B or GFP-DRP5B K135A cells cultured at 42°C under light were transferred to dark condition to stop cell growth and new entrance into S phase. Then cells were heat-shocked at 50°C for 1 h x two times. After cultivation under dark for 12 h, cells were transferred to light condition to resume cell growth (B). GFP fluorescence (along with the red the autofluorescence of chlorophyll) phase-contrast (PC) images are shown (C, GFP-DRP5B, D and E, GFP-DRP5B K135A). The triple arrowheads indicate the chloroplast before division site constriction. The double arrowheads indicates the chloroplast in the earlier stage of division site constriction. The arrowhead indicates the chloroplast in the final stage of division. Images by DAPI staining (blue fluorescence) for GFP-DRP5B K135A 24 h after the heat shock are also shown in (F). The scale bars represent 10 µm in (C, D) and 5 µm in (E, F). (G) Quantitative RT-PCR analyses showing the change in the transcript levels of an S-phase marker PCNA (CMS101C) and an M-phase marker CDC20 (CMA138C) in the GFP-DRP5B and GFP-DRP5B K135A expressing cells. DRP3 was used as the internal control. The expression levels at 0 h were defined as 1.0. The bars indicate the standard deviation (n = 3).
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pone-0111261-g008: Effect of the DRP5B K135A dominant negative mutation on chloroplast division and cell cycle progression in C. merolae.(A) Schematic diagram of the domain composition of the conventional dynamin protein which is involved in endocytosis. In the GTPase domain, four GTP binding motifs (G1, G2, G3 and G4) contribute to the GTP binding. Partial alignment of the DRP5B proteins showed the conservation of the G1 motif. The DRP5B amino acid sequences (Chlamydomonas reinhardtii, GI: 30349146; Chlorella variabilis, GI: 552813628; Ostreococcus tauri, GI: 308803420; Arabidopsis thaliana, GI: 30349146; Physcomitrella patens, GI: 224434564; Cyanidioschyzon merolae, GI: 544214467) were aligned using ClustalW [35], [36]. Alanine substitution for the conserved lysine residue within G1 of human dynamin 1 is known to result in a dominant negative effect by abolishing GTP-binding activity [32]. (B to F) GFP-DRP5B or GFP-DRP5B K135A cells cultured at 42°C under light were transferred to dark condition to stop cell growth and new entrance into S phase. Then cells were heat-shocked at 50°C for 1 h x two times. After cultivation under dark for 12 h, cells were transferred to light condition to resume cell growth (B). GFP fluorescence (along with the red the autofluorescence of chlorophyll) phase-contrast (PC) images are shown (C, GFP-DRP5B, D and E, GFP-DRP5B K135A). The triple arrowheads indicate the chloroplast before division site constriction. The double arrowheads indicates the chloroplast in the earlier stage of division site constriction. The arrowhead indicates the chloroplast in the final stage of division. Images by DAPI staining (blue fluorescence) for GFP-DRP5B K135A 24 h after the heat shock are also shown in (F). The scale bars represent 10 µm in (C, D) and 5 µm in (E, F). (G) Quantitative RT-PCR analyses showing the change in the transcript levels of an S-phase marker PCNA (CMS101C) and an M-phase marker CDC20 (CMA138C) in the GFP-DRP5B and GFP-DRP5B K135A expressing cells. DRP3 was used as the internal control. The expression levels at 0 h were defined as 1.0. The bars indicate the standard deviation (n = 3).

Mentions: DRP5B is a member of a eukaryotic dynamin family of self-assembling GTPases and localizes on the cytosolic side of the chloroplast division site, where it is involved in the division process [29], [30]. Conventional dynamin, which is involved in endocytosis, consists of five domains: an N-terminal GTPase domain, a middle domain, a pleckstrin homology domain, a GTPase effecter domain and a proline-rich domain (Reviewed in [31], Figure 8A). GTPase activity is essential for membrane fission in endocytosis and the K44A mutation in human dynamin 1 is known to abolish the activity of dynamin binding GTP, and the expression of the K44A mutant protein exhibits dominant-negative effects [32]. The GTP binding domain is well conserved in the DRP5B of algae and plants (Figure 8A). It has been shown that DRP5B is required for normal chloroplast division based on the impairment of chloroplast division in DRP5B knockout or mutants in Arabidopsis thaliana[30] and Physcomitrella patens[33]. In addition, DRP5B is recruited to the chloroplast division site before the onset of division site constriction and persists there throughout the division process [29]. However, it is not known how GTP-binding and/or GTP hydrolysis by DRP5B is involved in chloroplast division. To investigate the role of GTP-binding and/or hydrolysis by DRP5B in chloroplast division in C. merolae, we induced the expression of GFP-DRP5B or GFP-DRP5B K135A, the mutation of which corresponds to K44A of the human dynamin1 mutation, by the heat shock system in the stable transformants (Figure 8A).


Development of a heat-shock inducible gene expression system in the red alga Cyanidioschyzon merolae.

Sumiya N, Fujiwara T, Kobayashi Y, Misumi O, Miyagishima SY - PLoS ONE (2014)

Effect of the DRP5B K135A dominant negative mutation on chloroplast division and cell cycle progression in C. merolae.(A) Schematic diagram of the domain composition of the conventional dynamin protein which is involved in endocytosis. In the GTPase domain, four GTP binding motifs (G1, G2, G3 and G4) contribute to the GTP binding. Partial alignment of the DRP5B proteins showed the conservation of the G1 motif. The DRP5B amino acid sequences (Chlamydomonas reinhardtii, GI: 30349146; Chlorella variabilis, GI: 552813628; Ostreococcus tauri, GI: 308803420; Arabidopsis thaliana, GI: 30349146; Physcomitrella patens, GI: 224434564; Cyanidioschyzon merolae, GI: 544214467) were aligned using ClustalW [35], [36]. Alanine substitution for the conserved lysine residue within G1 of human dynamin 1 is known to result in a dominant negative effect by abolishing GTP-binding activity [32]. (B to F) GFP-DRP5B or GFP-DRP5B K135A cells cultured at 42°C under light were transferred to dark condition to stop cell growth and new entrance into S phase. Then cells were heat-shocked at 50°C for 1 h x two times. After cultivation under dark for 12 h, cells were transferred to light condition to resume cell growth (B). GFP fluorescence (along with the red the autofluorescence of chlorophyll) phase-contrast (PC) images are shown (C, GFP-DRP5B, D and E, GFP-DRP5B K135A). The triple arrowheads indicate the chloroplast before division site constriction. The double arrowheads indicates the chloroplast in the earlier stage of division site constriction. The arrowhead indicates the chloroplast in the final stage of division. Images by DAPI staining (blue fluorescence) for GFP-DRP5B K135A 24 h after the heat shock are also shown in (F). The scale bars represent 10 µm in (C, D) and 5 µm in (E, F). (G) Quantitative RT-PCR analyses showing the change in the transcript levels of an S-phase marker PCNA (CMS101C) and an M-phase marker CDC20 (CMA138C) in the GFP-DRP5B and GFP-DRP5B K135A expressing cells. DRP3 was used as the internal control. The expression levels at 0 h were defined as 1.0. The bars indicate the standard deviation (n = 3).
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pone-0111261-g008: Effect of the DRP5B K135A dominant negative mutation on chloroplast division and cell cycle progression in C. merolae.(A) Schematic diagram of the domain composition of the conventional dynamin protein which is involved in endocytosis. In the GTPase domain, four GTP binding motifs (G1, G2, G3 and G4) contribute to the GTP binding. Partial alignment of the DRP5B proteins showed the conservation of the G1 motif. The DRP5B amino acid sequences (Chlamydomonas reinhardtii, GI: 30349146; Chlorella variabilis, GI: 552813628; Ostreococcus tauri, GI: 308803420; Arabidopsis thaliana, GI: 30349146; Physcomitrella patens, GI: 224434564; Cyanidioschyzon merolae, GI: 544214467) were aligned using ClustalW [35], [36]. Alanine substitution for the conserved lysine residue within G1 of human dynamin 1 is known to result in a dominant negative effect by abolishing GTP-binding activity [32]. (B to F) GFP-DRP5B or GFP-DRP5B K135A cells cultured at 42°C under light were transferred to dark condition to stop cell growth and new entrance into S phase. Then cells were heat-shocked at 50°C for 1 h x two times. After cultivation under dark for 12 h, cells were transferred to light condition to resume cell growth (B). GFP fluorescence (along with the red the autofluorescence of chlorophyll) phase-contrast (PC) images are shown (C, GFP-DRP5B, D and E, GFP-DRP5B K135A). The triple arrowheads indicate the chloroplast before division site constriction. The double arrowheads indicates the chloroplast in the earlier stage of division site constriction. The arrowhead indicates the chloroplast in the final stage of division. Images by DAPI staining (blue fluorescence) for GFP-DRP5B K135A 24 h after the heat shock are also shown in (F). The scale bars represent 10 µm in (C, D) and 5 µm in (E, F). (G) Quantitative RT-PCR analyses showing the change in the transcript levels of an S-phase marker PCNA (CMS101C) and an M-phase marker CDC20 (CMA138C) in the GFP-DRP5B and GFP-DRP5B K135A expressing cells. DRP3 was used as the internal control. The expression levels at 0 h were defined as 1.0. The bars indicate the standard deviation (n = 3).
Mentions: DRP5B is a member of a eukaryotic dynamin family of self-assembling GTPases and localizes on the cytosolic side of the chloroplast division site, where it is involved in the division process [29], [30]. Conventional dynamin, which is involved in endocytosis, consists of five domains: an N-terminal GTPase domain, a middle domain, a pleckstrin homology domain, a GTPase effecter domain and a proline-rich domain (Reviewed in [31], Figure 8A). GTPase activity is essential for membrane fission in endocytosis and the K44A mutation in human dynamin 1 is known to abolish the activity of dynamin binding GTP, and the expression of the K44A mutant protein exhibits dominant-negative effects [32]. The GTP binding domain is well conserved in the DRP5B of algae and plants (Figure 8A). It has been shown that DRP5B is required for normal chloroplast division based on the impairment of chloroplast division in DRP5B knockout or mutants in Arabidopsis thaliana[30] and Physcomitrella patens[33]. In addition, DRP5B is recruited to the chloroplast division site before the onset of division site constriction and persists there throughout the division process [29]. However, it is not known how GTP-binding and/or GTP hydrolysis by DRP5B is involved in chloroplast division. To investigate the role of GTP-binding and/or hydrolysis by DRP5B in chloroplast division in C. merolae, we induced the expression of GFP-DRP5B or GFP-DRP5B K135A, the mutation of which corresponds to K44A of the human dynamin1 mutation, by the heat shock system in the stable transformants (Figure 8A).

Bottom Line: At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression.After the heat shock, the mRNA level decreases rapidly.Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure.

View Article: PubMed Central - PubMed

Affiliation: Center for Frontier Research, National Institute of Genetics, Mishima, Shizuoka, Japan; Japan Science and Technology Agency, CREST, Kawaguchi, Saitama, Japan.

ABSTRACT
The cell of the unicellular red alga Cyanidioschyzon merolae contains a single chloroplast and mitochondrion, the division of which is tightly synchronized by a light/dark cycle. The genome content is extremely simple, with a low level of genetic redundancy, in photosynthetic eukaryotes. In addition, transient transformation and stable transformation by homologous recombination have been reported. However, for molecular genetic analyses of phenomena that are essential for cellular growth and survival, inducible gene expression/suppression systems are needed. Here, we report the development of a heat-shock inducible gene expression system in C. merolae. CMJ101C, encoding a small heat shock protein, is transcribed only when cells are exposed to an elevated temperature. Using a superfolder GFP as a reporter protein, the 200-bp upstream region of CMJ101C orf was determined to be the optimal promoter for heat-shock induction. The optimal temperature to induce expression is 50°C, at which C. merolae cells are able to proliferate. At least a 30-min heat shock is required for the expression of a protein of interest and a 60-min heat shock yields the maximum level of protein expression. After the heat shock, the mRNA level decreases rapidly. As an example of the system, the expression of a dominant negative form of chloroplast division DRP5B protein, which has a mutation in the GTPase domain, was induced. Expression of the dominant negative DRP5B resulted in the appearance of aberrant-shaped cells in which two daughter chloroplasts and the cells are still connected by a small DRP5B positive tube-like structure. This result suggests that the dominant negative DRP5B inhibited the final scission of the chloroplast division site, but not the earlier stages of division site constriction. It is also suggested that cell cycle progression is not arrested by the impairment of chloroplast division at the final stage.

Show MeSH
Related in: MedlinePlus