Transport of fragile X mental retardation protein via granules in neurites of PC12 cells
Lack of fragile X mental retardation protein (FMRP) causes fragile X syndrome, a common form of inherited mental retardation. FMRP is an RNA binding protein thought to be involved in translation efficiency and/or trafficking of certain mRNAs. Recently, a subset of mRNAs to which FMRP binds with high affinity has been identified. These FMRP-associated mRNAs contain an intramolecular G-quartet structure. In neurons, dendritic mRNAs are involved in local synthesis of proteins in response to synaptic activity, and this represents a mechanism for synaptic plasticity. To determine the role of FMRP in dendritic mRNA transport, we have generated a stably FMR1-enhanced green fluorescent protein (EGFP)-transfected PC12 cell line with an inducible expression system (Tet-On) for regulated expression of the FMRP-GFP fusion protein. After doxycycline induction, FMRP-GFP was localized in granules in the neurites of PC12 cells. By using time-lapse microscopy, the trafficking of FMRP-GFP granules into the neurites of living PC12 cells was demonstrated. Motile FMRP-GFP granules displayed two types of movements: oscillatory (bidirectional) and unidirectional anterograde. The average velocity of the granules was 0.19 micro m/s with a maximum speed of 0.71 micro m/s. In addition, we showed that the movement of FMRP-GFP labeled granules into the neurites was microtubule dependent. Colocalization studies further showed that the FMRP-GFP labeled granules also contained RNA, ribosomal subunits, kinesin heavy chain, and FXR1P molecules. This report is the first example of trafficking of RNA-containing granules with FMRP as a core constituent in living PC12 cells.
|Keywords||Animals, Anti-Bacterial Agents/pharmacology, Antineoplastic Agents/pharmacology, Cytochalasin D/pharmacology, Cytoplasmic Granules/chemistry/*metabolism, Doxycycline/pharmacology, Fragile X Mental Retardation Protein, Fragile X Syndrome/metabolism, Gene Expression Regulation, Green Fluorescent Proteins, Humans, Indicators and Reagents/metabolism, Luminescent Proteins/genetics/metabolism, Mental Retardation, Microscopy, Confocal, Nerve Tissue Proteins/genetics/*metabolism, Neurites/*metabolism, Nocodazole/pharmacology, Nucleic Acid Synthesis Inhibitors/pharmacology, PC12 Cells, Protein Transport, RNA, Messenger/metabolism, RNA-Binding Proteins/metabolism, Rats, Recombinant Fusion Proteins/genetics/metabolism, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S., Time Factors|
|Persistent URL||dx.doi.org/10.1128/MCB.22.23.8332-8341.2002, hdl.handle.net/1765/10010|
|Journal||Molecular and Cellular Biology|
de Diego Otero, Y, Severijnen, E.A.W.F.M, van Cappellen, W.A, Schrier, M, Willemsen, R, & Oostra, B.A. (2002). Transport of fragile X mental retardation protein via granules in neurites of PC12 cells. Molecular and Cellular Biology, 22(23), 8332–8341. doi:10.1128/MCB.22.23.8332-8341.2002