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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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(A and B) Distribution of a nonspecific mRNA in 70min cells. A PstI fragment of pcNg 3-28 (20) was labeled with  DIG, and the location of this mRNA was determined as in Fig. 1.  The same cells were observed using DIC optics at two different  focal planes to show the flagella. (C) 70-min cells were stained  with alkaline phosphatase–conjugated anti-DIG antibody after  hybridization in the absence of a DIG-labeled probe. Bar, 10 μm.
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Figure 4: (A and B) Distribution of a nonspecific mRNA in 70min cells. A PstI fragment of pcNg 3-28 (20) was labeled with DIG, and the location of this mRNA was determined as in Fig. 1. The same cells were observed using DIC optics at two different focal planes to show the flagella. (C) 70-min cells were stained with alkaline phosphatase–conjugated anti-DIG antibody after hybridization in the absence of a DIG-labeled probe. Bar, 10 μm.

Mentions: The specific localization of the DS mRNAs was not a general phenomenon of N. gruberi differentiation. We examined distribution of another Naegleria mRNA that is present at high concentrations both in amebas and in flagellates, a nonspecific mRNA (19, 20). Unlike the DS mRNAs, the nonspecific mRNA showed uniform distribution throughout the differentiation (Fig. 4, A and B; and data not shown). The specific localization of the DS mRNAs is further supported by the observed facts that the locations of flagellar calmodulin mRNA and the tubulin mRNAs are different in 70-min cells. When the cells were stained with the anti-DIG antibody without prior hybridization with the DIG-labeled probes or treated with RNase before the hybridization, no staining was observed (Fig. 4 C; and data not shown).


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)

(A and B) Distribution of a nonspecific mRNA in 70min cells. A PstI fragment of pcNg 3-28 (20) was labeled with  DIG, and the location of this mRNA was determined as in Fig. 1.  The same cells were observed using DIC optics at two different  focal planes to show the flagella. (C) 70-min cells were stained  with alkaline phosphatase–conjugated anti-DIG antibody after  hybridization in the absence of a DIG-labeled probe. Bar, 10 μm.
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Related In: Results  -  Collection

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

Figure 4: (A and B) Distribution of a nonspecific mRNA in 70min cells. A PstI fragment of pcNg 3-28 (20) was labeled with DIG, and the location of this mRNA was determined as in Fig. 1. The same cells were observed using DIC optics at two different focal planes to show the flagella. (C) 70-min cells were stained with alkaline phosphatase–conjugated anti-DIG antibody after hybridization in the absence of a DIG-labeled probe. Bar, 10 μm.
Mentions: The specific localization of the DS mRNAs was not a general phenomenon of N. gruberi differentiation. We examined distribution of another Naegleria mRNA that is present at high concentrations both in amebas and in flagellates, a nonspecific mRNA (19, 20). Unlike the DS mRNAs, the nonspecific mRNA showed uniform distribution throughout the differentiation (Fig. 4, A and B; and data not shown). The specific localization of the DS mRNAs is further supported by the observed facts that the locations of flagellar calmodulin mRNA and the tubulin mRNAs are different in 70-min cells. When the cells were stained with the anti-DIG antibody without prior hybridization with the DIG-labeled probes or treated with RNase before the hybridization, no staining was observed (Fig. 4 C; and data not shown).

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