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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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Specific colocalization of the DS mRNAs. In situ hybridizations were carried out as in Fig. 1 except that two different  DIG-labeled cDNA probes were added simultaneously to each  hybridization. (A and B) Location of β-tubulin mRNA and α-tubulin mRNA in 20- and 40-min cells, respectively. (C and D) Location of α-tubulin mRNA and Class I mRNA in 20- and 40-min  cells, respectively. (E and F) Location of β-tubulin mRNA and  flagellar calmodulin mRNA in 40- and 70-min cells, respectively.  Bar, 10 μm.
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Figure 2: Specific colocalization of the DS mRNAs. In situ hybridizations were carried out as in Fig. 1 except that two different DIG-labeled cDNA probes were added simultaneously to each hybridization. (A and B) Location of β-tubulin mRNA and α-tubulin mRNA in 20- and 40-min cells, respectively. (C and D) Location of α-tubulin mRNA and Class I mRNA in 20- and 40-min cells, respectively. (E and F) Location of β-tubulin mRNA and flagellar calmodulin mRNA in 40- and 70-min cells, respectively. Bar, 10 μm.

Mentions: The distribution of the three DS mRNAs (α-tubulin mRNA, β-tubulin mRNA, and Class I mRNA) at 70 and 85 min suggested that they are colocalized during the differentiation. To test this possibility, we performed two sets of in situ hybridization experiments. In one set of the experiments, locations of β-tubulin mRNA and α-tubulin mRNA were examined simultaneously by adding the respective DIG-labeled cDNA probes in one hybridization reaction. If the two mRNAs were not colocalized, we would expect several (at least two) distinctly stained regions. As shown in Fig. 2, A (20 min) and B (40 min), the staining pattern was similar to that of Fig. 1, B and C, where only the β-tubulin cDNA probe was used. These results were summarized in Table III. We observed one stained region in 58% of stained cells at 20 min, 74% of the cells at 40 min, and 91% of the flagellated cells at 70 min. When the locations of αtubulin mRNA and Class I mRNA were examined in the same way, we obtained similar results (Fig. 2, C and D). These data suggest that the three DS mRNAs are colocalized during differentiation.


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)

Specific colocalization of the DS mRNAs. In situ hybridizations were carried out as in Fig. 1 except that two different  DIG-labeled cDNA probes were added simultaneously to each  hybridization. (A and B) Location of β-tubulin mRNA and α-tubulin mRNA in 20- and 40-min cells, respectively. (C and D) Location of α-tubulin mRNA and Class I mRNA in 20- and 40-min  cells, respectively. (E and F) Location of β-tubulin mRNA and  flagellar calmodulin mRNA in 40- and 70-min cells, respectively.  Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139841&req=5

Figure 2: Specific colocalization of the DS mRNAs. In situ hybridizations were carried out as in Fig. 1 except that two different DIG-labeled cDNA probes were added simultaneously to each hybridization. (A and B) Location of β-tubulin mRNA and α-tubulin mRNA in 20- and 40-min cells, respectively. (C and D) Location of α-tubulin mRNA and Class I mRNA in 20- and 40-min cells, respectively. (E and F) Location of β-tubulin mRNA and flagellar calmodulin mRNA in 40- and 70-min cells, respectively. Bar, 10 μm.
Mentions: The distribution of the three DS mRNAs (α-tubulin mRNA, β-tubulin mRNA, and Class I mRNA) at 70 and 85 min suggested that they are colocalized during the differentiation. To test this possibility, we performed two sets of in situ hybridization experiments. In one set of the experiments, locations of β-tubulin mRNA and α-tubulin mRNA were examined simultaneously by adding the respective DIG-labeled cDNA probes in one hybridization reaction. If the two mRNAs were not colocalized, we would expect several (at least two) distinctly stained regions. As shown in Fig. 2, A (20 min) and B (40 min), the staining pattern was similar to that of Fig. 1, B and C, where only the β-tubulin cDNA probe was used. These results were summarized in Table III. We observed one stained region in 58% of stained cells at 20 min, 74% of the cells at 40 min, and 91% of the flagellated cells at 70 min. When the locations of αtubulin mRNA and Class I mRNA were examined in the same way, we obtained similar results (Fig. 2, C and D). These data suggest that the three DS mRNAs are colocalized during differentiation.

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