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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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Localization of DS mRNAs during the differentiation of N. gruberi. Differentiating cells were taken at various stages of differentiation and fixed as described in Materials and Methods. The fixed cells were used for in situ hybridization with cDNA probes labeled  with DIG. Location of each mRNA probe was determined by using anti-DIG antibody conjugated with alkaline phosphatase. (A–H)  Location of β-tubulin mRNA. (A) 0 min; (B) 20 min; (C) 40 min; (D and E) the same cells at 70 min at different focal planes to show the  flagella; (F and G) the same cells at 85 min at different focal planes to show the flagella; (H) 120 min. (I–L) Location of α-tubulin  mRNA; (I) 40 min; (J and K) the same cells at 70 min at different focal planes to show the flagella; (L) 85 min. (M–P) Location of Class  I mRNA. (M) 40 min; (N and O) the same cells at 70 min at different focal planes; (P) 85 min. (Q–T) Location of flagellar calmodulin  mRNA. (Q) 40 min; (R and S) the same cells at 70 min at different focal planes; (T) 85 min. Black arrowheads indicate locations of the DS  mRNAs, and open arrowheads point to the flagella. The pictures were taken using a microscope (model Optiphot-2; Nikon, Inc.,  Melville, NY) under differential interference contrast (DIC) conditions. Bar, 10 μm.
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Figure 1: Localization of DS mRNAs during the differentiation of N. gruberi. Differentiating cells were taken at various stages of differentiation and fixed as described in Materials and Methods. The fixed cells were used for in situ hybridization with cDNA probes labeled with DIG. Location of each mRNA probe was determined by using anti-DIG antibody conjugated with alkaline phosphatase. (A–H) Location of β-tubulin mRNA. (A) 0 min; (B) 20 min; (C) 40 min; (D and E) the same cells at 70 min at different focal planes to show the flagella; (F and G) the same cells at 85 min at different focal planes to show the flagella; (H) 120 min. (I–L) Location of α-tubulin mRNA; (I) 40 min; (J and K) the same cells at 70 min at different focal planes to show the flagella; (L) 85 min. (M–P) Location of Class I mRNA. (M) 40 min; (N and O) the same cells at 70 min at different focal planes; (P) 85 min. (Q–T) Location of flagellar calmodulin mRNA. (Q) 40 min; (R and S) the same cells at 70 min at different focal planes; (T) 85 min. Black arrowheads indicate locations of the DS mRNAs, and open arrowheads point to the flagella. The pictures were taken using a microscope (model Optiphot-2; Nikon, Inc., Melville, NY) under differential interference contrast (DIC) conditions. Bar, 10 μm.

Mentions: When a probe for β-tubulin mRNA was used, no β-tubulin mRNA was detected in amebas (0 min, Fig. 1 A) as expected from the previous reports (19, 20). At 20 min after the initiation, β-tubulin mRNA is actively transcribed (19), and the amount of this mRNA has reached about 20 to 30% of the peak value (20). At this stage, the β-tubulin mRNA was detected in 76% of cells, and in 74% (56 out of 76) of the labeled cells, the β-tubulin mRNA was concentrated at one location (Fig. 1 B; Table I). In the rest of the labeled cells, the tubulin mRNA was concentrated at two, or rarely three, close locations (Fig. 1, B and C; Table I; also see Fig. 1 I for α-tubulin mRNA).


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 DS mRNAs during the differentiation of N. gruberi. Differentiating cells were taken at various stages of differentiation and fixed as described in Materials and Methods. The fixed cells were used for in situ hybridization with cDNA probes labeled  with DIG. Location of each mRNA probe was determined by using anti-DIG antibody conjugated with alkaline phosphatase. (A–H)  Location of β-tubulin mRNA. (A) 0 min; (B) 20 min; (C) 40 min; (D and E) the same cells at 70 min at different focal planes to show the  flagella; (F and G) the same cells at 85 min at different focal planes to show the flagella; (H) 120 min. (I–L) Location of α-tubulin  mRNA; (I) 40 min; (J and K) the same cells at 70 min at different focal planes to show the flagella; (L) 85 min. (M–P) Location of Class  I mRNA. (M) 40 min; (N and O) the same cells at 70 min at different focal planes; (P) 85 min. (Q–T) Location of flagellar calmodulin  mRNA. (Q) 40 min; (R and S) the same cells at 70 min at different focal planes; (T) 85 min. Black arrowheads indicate locations of the DS  mRNAs, and open arrowheads point to the flagella. The pictures were taken using a microscope (model Optiphot-2; Nikon, Inc.,  Melville, NY) under differential interference contrast (DIC) conditions. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 1: Localization of DS mRNAs during the differentiation of N. gruberi. Differentiating cells were taken at various stages of differentiation and fixed as described in Materials and Methods. The fixed cells were used for in situ hybridization with cDNA probes labeled with DIG. Location of each mRNA probe was determined by using anti-DIG antibody conjugated with alkaline phosphatase. (A–H) Location of β-tubulin mRNA. (A) 0 min; (B) 20 min; (C) 40 min; (D and E) the same cells at 70 min at different focal planes to show the flagella; (F and G) the same cells at 85 min at different focal planes to show the flagella; (H) 120 min. (I–L) Location of α-tubulin mRNA; (I) 40 min; (J and K) the same cells at 70 min at different focal planes to show the flagella; (L) 85 min. (M–P) Location of Class I mRNA. (M) 40 min; (N and O) the same cells at 70 min at different focal planes; (P) 85 min. (Q–T) Location of flagellar calmodulin mRNA. (Q) 40 min; (R and S) the same cells at 70 min at different focal planes; (T) 85 min. Black arrowheads indicate locations of the DS mRNAs, and open arrowheads point to the flagella. The pictures were taken using a microscope (model Optiphot-2; Nikon, Inc., Melville, NY) under differential interference contrast (DIC) conditions. Bar, 10 μm.
Mentions: When a probe for β-tubulin mRNA was used, no β-tubulin mRNA was detected in amebas (0 min, Fig. 1 A) as expected from the previous reports (19, 20). At 20 min after the initiation, β-tubulin mRNA is actively transcribed (19), and the amount of this mRNA has reached about 20 to 30% of the peak value (20). At this stage, the β-tubulin mRNA was detected in 76% of cells, and in 74% (56 out of 76) of the labeled cells, the β-tubulin mRNA was concentrated at one location (Fig. 1 B; Table I). In the rest of the labeled cells, the tubulin mRNA was concentrated at two, or rarely three, close locations (Fig. 1, B and C; Table I; also see Fig. 1 I for α-tubulin mRNA).

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