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.
De Diego Otero
Yolanda
Y
CBG Department of Clinical Genetics. Department of Endocrinology and Reproduction, Erasmus University, Rotterdam, The Netherlands.
Severijnen
Lies-Anne
LA
van Cappellen
Gert
G
Schrier
Mariëtte
M
Oostra
Ben
B
Willemsen
Rob
R
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000737:chemistry / Q000378:metabolism*; D004318:Doxycycline / Q000494:pharmacology; D051860:Fragile X Messenger Ribonucleoprotein 1; D005600:Fragile X Syndrome / Q000378:metabolism; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D008607:Intellectual Disability; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016501:Neurites / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D016716:PC12 Cells; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
D000900:Anti-Bacterial Agents; D000970:Antineoplastic Agents; D015638:Cytochalasin D; D004318:Doxycycline; D051860:Fragile X Messenger Ribonucleoprotein 1; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; D015739:Nocodazole; D019384:Nucleic Acid Synthesis Inhibitors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C497683:FMR1 protein, human; C497685:Fmr1 protein, rat