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mRNAs for microtubule proteins are specifically colocalized during the sequential formation of basal body, flagella, and cytoskeletal microtubules in the differentiation of Naegleria gruberi.

Han JW, Park JH, Kim M, Lee J - J. Cell Biol. (1997)

Bottom Line: At 70 min after the initiation of differentiation, these mRNAs were relocalized to the base of the growing flagella, adjacent to the basal bodies and microtubule organizing center for the cytoskeletal microtubules.Within an additional 15 min, the mRNAs were translocated to the posterior of the flagellated cells, and by the end of differentiation (120 min), very low levels of the mRNAs were observed.Cytochalasin D inhibited stage-specific localization of the mRNAs, demonstrating that RNA localization was actin dependent.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Yonsei University, Seoul, Korea 120-749.

ABSTRACT
We have examined the distribution of four mRNAs-alpha-tubulin, beta-tubulin, flagellar calmodulin, and Class I mRNA-during differentiation of Naegleria gruberi amebas into flagellates by in situ hybridization. Three of the four mRNAs-alpha-tubulin, beta-tubulin, and Class I mRNA-began to be colocalized at the periphery of the cells as soon as transcription of the respective genes was activated and before any microtubular structures were observable. At 70 min after the initiation of differentiation, these mRNAs were relocalized to the base of the growing flagella, adjacent to the basal bodies and microtubule organizing center for the cytoskeletal microtubules. Within an additional 15 min, the mRNAs were translocated to the posterior of the flagellated cells, and by the end of differentiation (120 min), very low levels of the mRNAs were observed. Cytochalasin D inhibited stage-specific localization of the mRNAs, demonstrating that RNA localization was actin dependent. Since cytochalasin D also blocked differentiation, this raises the possibility that actin-dependent RNA movement is an essential process for differentiation.

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Effect of cytochalasin D on the differentiation of Naegleria gruberi. (A) Dose-dependent inhibition of Naegleria differentiation by cytochalasin D. Cytochalasin D (5 mg/ml in dimethylsulfoxide) was added at the beginning of differentiation to  make a final concentration of 20, 50, or 100 μg/ml. The differentiation was monitored as described in Materials and Methods. •,  control; ▾, 20 μg/ml; ▪, 50 μg/ml ▴, 100 μg/ml. (B), Effects of cytochalasin D on accumulation of β-tubulin mRNA. RNA was  prepared from the control and cytochalasin D–treated cells, and  the amount of β-tubulin mRNA was estimated by RNA slot blot  hybridization (5 μg RNA/slot) using 32P-labeled β-tubulin cDNA  as a probe (2). •, control; ▪, 50 μg/ml; ▴, 100 μg/ml.
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Figure 5: Effect of cytochalasin D on the differentiation of Naegleria gruberi. (A) Dose-dependent inhibition of Naegleria differentiation by cytochalasin D. Cytochalasin D (5 mg/ml in dimethylsulfoxide) was added at the beginning of differentiation to make a final concentration of 20, 50, or 100 μg/ml. The differentiation was monitored as described in Materials and Methods. •, control; ▾, 20 μg/ml; ▪, 50 μg/ml ▴, 100 μg/ml. (B), Effects of cytochalasin D on accumulation of β-tubulin mRNA. RNA was prepared from the control and cytochalasin D–treated cells, and the amount of β-tubulin mRNA was estimated by RNA slot blot hybridization (5 μg RNA/slot) using 32P-labeled β-tubulin cDNA as a probe (2). •, control; ▪, 50 μg/ml; ▴, 100 μg/ml.

Mentions: Addition of cytochalasin D at the beginning of differentiation inhibited the differentiation in a dose-dependent manner. In 20 μg/ml of cytochalasin D, 60% of the cells formed flagella. In 50 or 100 μg/ml of cytochalasin D, the differentiation was strongly inhibited (Fig. 5 A). In these experiments, most of the cells changed their shape into spheres less than 30 min after the initiation of differentiation and remained as spheres until the end of the differentiation (Fig. 6).


mRNAs for microtubule proteins are specifically colocalized during the sequential formation of basal body, flagella, and cytoskeletal microtubules in the differentiation of Naegleria gruberi.

Han JW, Park JH, Kim M, Lee J - J. Cell Biol. (1997)

Effect of cytochalasin D on the differentiation of Naegleria gruberi. (A) Dose-dependent inhibition of Naegleria differentiation by cytochalasin D. Cytochalasin D (5 mg/ml in dimethylsulfoxide) was added at the beginning of differentiation to  make a final concentration of 20, 50, or 100 μg/ml. The differentiation was monitored as described in Materials and Methods. •,  control; ▾, 20 μg/ml; ▪, 50 μg/ml ▴, 100 μg/ml. (B), Effects of cytochalasin D on accumulation of β-tubulin mRNA. RNA was  prepared from the control and cytochalasin D–treated cells, and  the amount of β-tubulin mRNA was estimated by RNA slot blot  hybridization (5 μg RNA/slot) using 32P-labeled β-tubulin cDNA  as a probe (2). •, control; ▪, 50 μg/ml; ▴, 100 μg/ml.
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Related In: Results  -  Collection

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Figure 5: Effect of cytochalasin D on the differentiation of Naegleria gruberi. (A) Dose-dependent inhibition of Naegleria differentiation by cytochalasin D. Cytochalasin D (5 mg/ml in dimethylsulfoxide) was added at the beginning of differentiation to make a final concentration of 20, 50, or 100 μg/ml. The differentiation was monitored as described in Materials and Methods. •, control; ▾, 20 μg/ml; ▪, 50 μg/ml ▴, 100 μg/ml. (B), Effects of cytochalasin D on accumulation of β-tubulin mRNA. RNA was prepared from the control and cytochalasin D–treated cells, and the amount of β-tubulin mRNA was estimated by RNA slot blot hybridization (5 μg RNA/slot) using 32P-labeled β-tubulin cDNA as a probe (2). •, control; ▪, 50 μg/ml; ▴, 100 μg/ml.
Mentions: Addition of cytochalasin D at the beginning of differentiation inhibited the differentiation in a dose-dependent manner. In 20 μg/ml of cytochalasin D, 60% of the cells formed flagella. In 50 or 100 μg/ml of cytochalasin D, the differentiation was strongly inhibited (Fig. 5 A). In these experiments, most of the cells changed their shape into spheres less than 30 min after the initiation of differentiation and remained as spheres until the end of the differentiation (Fig. 6).

Bottom Line: At 70 min after the initiation of differentiation, these mRNAs were relocalized to the base of the growing flagella, adjacent to the basal bodies and microtubule organizing center for the cytoskeletal microtubules.Within an additional 15 min, the mRNAs were translocated to the posterior of the flagellated cells, and by the end of differentiation (120 min), very low levels of the mRNAs were observed.Cytochalasin D inhibited stage-specific localization of the mRNAs, demonstrating that RNA localization was actin dependent.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Yonsei University, Seoul, Korea 120-749.

ABSTRACT
We have examined the distribution of four mRNAs-alpha-tubulin, beta-tubulin, flagellar calmodulin, and Class I mRNA-during differentiation of Naegleria gruberi amebas into flagellates by in situ hybridization. Three of the four mRNAs-alpha-tubulin, beta-tubulin, and Class I mRNA-began to be colocalized at the periphery of the cells as soon as transcription of the respective genes was activated and before any microtubular structures were observable. At 70 min after the initiation of differentiation, these mRNAs were relocalized to the base of the growing flagella, adjacent to the basal bodies and microtubule organizing center for the cytoskeletal microtubules. Within an additional 15 min, the mRNAs were translocated to the posterior of the flagellated cells, and by the end of differentiation (120 min), very low levels of the mRNAs were observed. Cytochalasin D inhibited stage-specific localization of the mRNAs, demonstrating that RNA localization was actin dependent. Since cytochalasin D also blocked differentiation, this raises the possibility that actin-dependent RNA movement is an essential process for differentiation.

Show MeSH
Related in: MedlinePlus