Association of poly(A)RNA with microtubules in cultured neurons

Center for Neurologic Disease, Brigham and Women's Hospital, Boston, Massachusetts 02115.
Neuron (Impact Factor: 15.05). 04/1994; 12(3):571-82. DOI: 10.1016/0896-6273(94)90213-5
Source: PubMed


The structural basis for the synthesis of specific proteins within distinct intraneuronal compartments is unknown. We studied the distribution of poly(A) mRNA within cultured cerebrocortical neurons using high resolution in situ hybridization to identify cytoskeletal components that may anchor mRNA. After 1 day in culture, poly(A) mRNA was distributed throughout all of the initial neurites, including the axon-like process. At 4 days in culture, poly(A) mRNA was distributed throughout the cell body and dendritic processes, but confined to the proximal segment of the axon. Poly(A) mRNA was bound to the cytoskeleton as demonstrated by resistance to detergent extraction. Perturbation of microtubules with colchicine resulted in a major reduction of dendritic poly(A) mRNA; however, this distribution was unaffected by cytochalasin. Ultrastructural in situ hybridization revealed that poly(A) mRNA and associated ribosomes were excluded from tightly bundled microtubules.

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    • "Previously, two transcripts, Exoc3 and TC10, coding for components of the exocyst or exocyst complex associated proteins had been found in injured and naïve axons of cortical neurons, respectively9. In order to confirm their axonal localization in DRGs, we harvested total RNA from the axonal and cell body compartments of DRGs neurons grown in microfluidic chambers and performed RT-PCR for TC10, Exoc3, H1f0 (negative control; the cell body restricted transcript of a histone14), and β-actin (positive control for an axonally localized mRNA30). All mRNAs were detected in the cell body-derived material but only β-actin and TC10 mRNA were present the developing DRG axons (Fig. 4a). "
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    ABSTRACT: The surface of developing axons expands in a process mediated by the exocyst complex. The spatio-temporal regulation of the exocyst is only partially understood. Here we report that stimulated membrane enlargement in dorsal root ganglion (DRG) axons is triggered by intra-axonal synthesis of TC10, a small GTPase required for exocyst function. Induced membrane expansion and axon outgrowth are inhibited after axon-specific knockdown of TC10 mRNA. To determine the relationship of intra-axonal TC10 synthesis with the previously described stimulus-induced translation of the cytoskeletal regulator Par3, we investigate the signalling pathways controlling their local translation in response to NGF. Phosphoinositide 3-kinase (PI3K)-dependent activation of the Rheb-mTOR pathway triggers the simultaneous local synthesis of TC10 and Par3. These results reveal the importance of local translation in the control of membrane dynamics and demonstrate that localized, mTOR-dependent protein synthesis triggers the simultaneous activation of parallel pathways.
    Nature Communications 03/2014; 5:3506. DOI:10.1038/ncomms4506 · 11.47 Impact Factor
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    • "Thus, our data do not support a turnover mechanism, although it is possible that small increases in total camkII mRNA may have gone undetected. CamkII mRNA is thought to be loaded into storage granules in the soma and transported to dendrites via microtubule-based motors (Bassell et al., 1994; Knowles et al., 1996; Rook et al., 2000; Blichenberg et al., 2001). Instead of triggering a local redistribution of mRNA (Fig. 7), HFS might induce a long-range trafficking of granules from the soma. "

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    • "Some studies have identified mRNA in mature vertebrate neurons, including Mauthner cells [47], hypothalamic magnocellular neurons [42] and sensory neurons projecting to the olfactory bulb [48], but it is not clear whether axonally localized transcripts are a feature of most types of adult axons. Indeed, mRNA and rRNA levels disappear from axons of hippocampal neurons as they mature [49]. The mechanism by which mRNA and rRNA are lost is not currently understood. "
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    ABSTRACT: Much of our knowledge on the roles of intra-axonal translation derives from the characterization of a small number of individual mRNAs that were found to be localized in axons. However, two recent studies, using large-scale approaches to provide a more comprehensive characterization of the axonal transcriptome, have led to the discovery of thousands of axonal mRNAs. The apparent abundance of mRNAs in axons raises the possibility that local translation has many more functions than previously thought. Here, we review the recent studies that have profiled axonal mRNAs and discuss how the identification of axonal transcripts might point to unappreciated roles for local translation in axons.
    Open Biology 06/2012; 2(6):120079. DOI:10.1098/rsob.120079 · 5.78 Impact Factor
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