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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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Related in: MedlinePlus

Localization of the β-tubulin mRNA in 40-min cells is  not a result of concentration in the nucleus. After in situ hybridization with DIG-labeled β-tubulin cDNA and immunological  detection of the mRNA with anti-DIG antibody as in Fig. 1, the  samples were stained with propidium iodide and observed under  DIC conditions and fluorescence microscopy. (A) Location of  β-tubulin mRNA and nucleus in 40-min cells. Cells were observed under DIC optics as in Fig. 1. Faint circles (n) represent  nuclei. (B) The same cells in A were observed using fluorescence  optics with a rhodamine filter. Faint circles in A were specifically  stained with the fluorescent dye. Bar, 10 μm.
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Figure 3: Localization of the β-tubulin mRNA in 40-min cells is not a result of concentration in the nucleus. After in situ hybridization with DIG-labeled β-tubulin cDNA and immunological detection of the mRNA with anti-DIG antibody as in Fig. 1, the samples were stained with propidium iodide and observed under DIC conditions and fluorescence microscopy. (A) Location of β-tubulin mRNA and nucleus in 40-min cells. Cells were observed under DIC optics as in Fig. 1. Faint circles (n) represent nuclei. (B) The same cells in A were observed using fluorescence optics with a rhodamine filter. Faint circles in A were specifically stained with the fluorescent dye. Bar, 10 μm.

Mentions: This localization of the DS mRNAs could be the result of active transcription of the DS genes in the nucleus, especially at early stages of differentiation when the genes are actively transcribed (19). To test this hypothesis, we stained the 40-min cells, in which the DS genes are being most actively transcribed, with propidium iodide after in situ hybridization with the DS cDNA probes. As shown in Fig. 3, A and B, the location of β-tubulin mRNA was clearly cytoplasmic, distinct from that of the nucleus. This result showed that the specific localization was not the result of active transcription and of concentration of the mRNAs in the nucleus.


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)

Localization of the β-tubulin mRNA in 40-min cells is  not a result of concentration in the nucleus. After in situ hybridization with DIG-labeled β-tubulin cDNA and immunological  detection of the mRNA with anti-DIG antibody as in Fig. 1, the  samples were stained with propidium iodide and observed under  DIC conditions and fluorescence microscopy. (A) Location of  β-tubulin mRNA and nucleus in 40-min cells. Cells were observed under DIC optics as in Fig. 1. Faint circles (n) represent  nuclei. (B) The same cells in A were observed using fluorescence  optics with a rhodamine filter. Faint circles in A were specifically  stained with the fluorescent dye. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Localization of the β-tubulin mRNA in 40-min cells is not a result of concentration in the nucleus. After in situ hybridization with DIG-labeled β-tubulin cDNA and immunological detection of the mRNA with anti-DIG antibody as in Fig. 1, the samples were stained with propidium iodide and observed under DIC conditions and fluorescence microscopy. (A) Location of β-tubulin mRNA and nucleus in 40-min cells. Cells were observed under DIC optics as in Fig. 1. Faint circles (n) represent nuclei. (B) The same cells in A were observed using fluorescence optics with a rhodamine filter. Faint circles in A were specifically stained with the fluorescent dye. Bar, 10 μm.
Mentions: This localization of the DS mRNAs could be the result of active transcription of the DS genes in the nucleus, especially at early stages of differentiation when the genes are actively transcribed (19). To test this hypothesis, we stained the 40-min cells, in which the DS genes are being most actively transcribed, with propidium iodide after in situ hybridization with the DS cDNA probes. As shown in Fig. 3, A and B, the location of β-tubulin mRNA was clearly cytoplasmic, distinct from that of the nucleus. This result showed that the specific localization was not the result of active transcription and of concentration of the mRNAs in the nucleus.

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