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Differential mRNA localization in astroglial cells in culture
1Department of Neuroscience, University of Virginia, Charlottesville, Virginia 22908, USA.
Abstract:
Messenger RNA (mRNA) targeting to specific subcellular domains has been studied extensively in many cell types, and there is increasing evidence suggesting that mRNA sorting also occurs in astrocytes. As a step toward developing strategies to evaluate the signals that govern mRNA sorting in astrocytes, the authors studied the subcellular distribution of several representative mRNAs, poly(A) RNA and ribosomal RNA, in process-bearing (type-2) astroglial cells in culture. Nonradioactive in situ hybridization analysis revealed a gradual increase in the expression of glial fibrillary acidic protein (GFAP) mRNA as type-2 astrocytes differentiated in culture. In mature cells, labeling was present in both cell bodies and processes. GFAP mRNA labeling was granular in nature and was particularly concentrated at branch points and at the tips of the processes. Unlike GFAP mRNA, vimentin, beta-tubulin, and beta- and gamma-actin mRNAs were mainly confined to the cell bodies, with only occasional labeling seen in the processes. Nonradioactive and radioactive in situ hybridization analysis of poly(A) and ribosomal RNA, respectively, revealed labeling in cell bodies and processes of immature and differentiated astrocytes. Treatment with nocodazole, a microtubule depolymerizing agent, resulted in a substantial reduction of GFAP mRNA labeling in the processes, whereas treatment with cytochalasin D, a microfilament-disrupting agent, did not alter GFAP mRNA distribution. The results indicate that cultured type-2 astrocytes have the capacity to sort mRNAs to different subcellular domains and that the localization of GFAP mRNA to astrocyte processes requires intact microtubules.
Insights
Astrocyte messenger RNA (mRNA) sorting is crucial for cell function. Glial fibrillary acidic protein (GFAP) mRNA localizes to astrocyte processes, requiring intact microtubules for transport.
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- Messenger RNA (mRNA) localization to subcellular domains is vital for cellular function.
- Evidence suggests mRNA sorting occurs in astrocytes, but the underlying mechanisms are not fully understood.
Purpose of the Study:
- To investigate the subcellular distribution of specific mRNAs in cultured type-2 astrocytes.
- To identify signals governing mRNA sorting in astrocytes.
Main Methods:
- Nonradioactive and radioactive in situ hybridization were used to analyze mRNA and RNA distribution.
- Type-2 astrocytes were cultured and differentiated.
- Cells were treated with nocodazole (microtubule inhibitor) and cytochalasin D (microfilament inhibitor).
Main Results:
- Glial fibrillary acidic protein (GFAP) mRNA expression increased with astrocyte differentiation and localized to cell bodies and processes.
- GFAP mRNA was concentrated at process branch points and tips.
- Vimentin, beta-tubulin, and actin mRNAs were primarily in cell bodies.
- Poly(A) and ribosomal RNA were found in both cell bodies and processes.
- Nocodazole treatment reduced GFAP mRNA in processes, while cytochalasin D had no effect.
Conclusions:
- Cultured type-2 astrocytes can sort mRNAs to distinct subcellular locations.
- Intact microtubules are essential for the transport of GFAP mRNA to astrocyte processes.