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First published online 16 September 2003
doi: 10.1242/jcs.00745

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Organization and translation of mRNA in sympathetic axons

Sun-Kyung Lee and Peter J. Hollenbeck*

Department of Biological Sciences, Purdue University, West Lafayette, IN 47906, USA



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  		Fig. 1. In situ hybridization shows the punctate distribution of poly(A) mRNAs throughout chick sympathetic neurons. Fixed neurons were hybridized with an oligo d(T) probe and fluorescent signals were developed using alkaline-phosphatase enzymatic amplification. Phase-contrast images (A,C,E) and fluorescent images (B,D,F) are shown for each field. Bright, punctate signals were detected along the length of the axons, along with bright staining of the cell bodies (B), which are also phase-dense because of the accumulation of HNP/Fast-Red-Texas-Red fluorescent material. Neurons treated with RNase prior to hybridization (D) or hybridized with sense oligo d(A) probes (F) show no signal in the axons and only trace levels of background staining in cell bodies. Scale bar, 20 µm.

 


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  		Fig. 2. ß-Actin and ADF mRNAs are detected in axons by in situ hybridization with species-specific probes. Fixed neurons were hybridized with probes for the whole ß-actin sequence (A,B), the ß-actin 3' UTR (C,D) and the ADF partial sequence (E,F). Bright, punctate signals were detected in branch points, varicosities and growth cones, and also along the axonal shaft (Tables 1, 2). Neither cells treated with RNase prior to antisense probe hybridization (G,H) nor cells hybridized with sense probe (I,J) showed any signal. The controls shown were carried out with the full length ß-actin probe; separate control experiments for the ß-actin 3' UTR and ADF probes gave identical results. Scale bar, 20 µm.

 


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  		Fig. 3. Axons of ganglion explants can synthesize protein autonomously. Proliferation of non-neuronal cells was suppressed by culture conditions (A-D). When chick sympathetic ganglia were grown for 4-5 days on a poly-L-lysine substratum with intermittent Ara-C treatment, they produced a large radial halo of axons (A) that was virtually devoid of cell bodies or non-neuronal cells, as revealed by DAPI staining (B). The dotted line in (B) indicates the approximate position where the cell body mass would be removed for metabolic labeling experiments. When grown on a laminin substratum and in the absence of Ara-C treatment, axonal halos contained abundant non-neuronal cells, revealed by their nuclei (C,D). Normal ganglia grown under the conditions shown in (A,B) were incubated with [35S]-methionine/cysteine, subjected to emulsion autoradiography and viewed by dark-field microscopy. They revealed a bright signal in the cell body mass and a fainter one in the axonal halo (E). Axonal halos with the cell body mass meticulously removed prior to metabolic labeling also showed newly synthesized protein throughout the axons (F). This signal was eliminated by cycloheximide treatment (G) but was undiminished when chloramphenicol treatment accompanied metabolic labeling (H). Conditions of radiolabeling, autoradiography and photography were uniform, except that the photographic exposure of (E) was 25% that of F-H owing to the intensity of signal from the cell body mass. The dark area in the center of the cell body mass in E is an artefact of high radioactive signal damaging the emulsion. Scale bars, 200 µm (in D, for A-D; in H, for E-H).

 


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  		Fig. 4. Cycloheximide-sensitive axonal translation produces a complex population of proteins. (A) SDS-PAGE and autoradiography of newly synthesized proteins in the axonal halo vs entire ganglion. Lanes 1,3,5 contain protein from 17 axonal halos; lanes 2,4,6 contain protein from 0.25 whole ganglia. Protein synthesis in whole ganglia and in axon halos (lanes 1,2) was eliminated by treatment with cycloheximide (lanes 3,4) but not with chloramphenicol (lanes 5,6). (B) The protein composition of axonal halos is different from that of entire ganglia (lanes1,2, enlarged area at right). The arrow at left indicates a protein whose synthesis is highly enriched in the axon and the arrows at right indicate two proteins whose synthesis is highly enriched in the cell bodies. (C) Close examination using 4-15% gradient SDS-PAGE and autoradiography identifies the molecular weight of prominent proteins enriched in the axon (arrows at left) or cell bodies (arrowheads at right). 40 axonal halos (lane 1) and 1 ganglion (lane 2) were loaded.

 


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  		Fig. 5. ß-Actin, ADF and neurofilament protein are synthesized in axons separated from their cell bodies. Metabolically labeled whole ganglia (lanes 1,3,5) or axonal halos without cell bodies (lanes 2,4,6) were subjected to immunoprecipitation using the anti-ß-actin antibody AC-15 (lanes 1,2), antiserum against chick ADF (lanes 3,4) or the neurofilament antibody NR-4 (lanes 5,6). Newly synthesized 43 kDa ß-actin and 18.5 kDa ADF were precipitated not only from ganglia but also from axonal halos. Some actin was precipitated with ADF from the ganglia extract (denoted by filled circle). Newly synthesized neurofilament subunits NF-L, NF-M and NF-H were precipitated from whole ganglia (indicated by arrows to the right of lane 6) but axons synthesized mainly the NF-L subunit during the metabolic labeling period (lanes 5,6). For lanes 1 and 3, cell lysates from five ganglia were used after 5 hours incubation with [35S]-methionine/cysteine, and the exposure time of the autoradiogram was 1 hour for ß-actin and 3 hours for ADF. For lanes 2 and 4, approximately 40-50 axonal halos were used and the exposure time was 6 hours for ß-actin and 7 days for ADF. For lanes 5 and 6, lysates from four ganglia and 75 halos, respectively, were used and the exposure times were 6 hours and 7 days, respectively. In case of lane 6, metabolic labeling period was extended up to 10 hours.

 

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© The Company of Biologists Ltd 2003