Article Figures & Data
Figures
- Fig. 1.
Schematic diagram of a compartmented culture.A, A Teflon divider provides a barrier that divides the culture dish into one central compartment and two side compartments.B, An enlargement of a single track shows the cell bodies and proximal axons restricted to the center compartment, whereas the distal axons cross under the barrier and extend into the side compartments.
- Fig. 2.
Comparison of the profiles of proteins labeled by incubation with [35S]met in left and right compartments or center compartments. Lanes 1,2, The center compartments of cultures were incubated for 4 hr with 250 μCi/ml [35S]met before harvesting the left and right (L+R) and center compartments (C). Extracts were analyzed by SDS-PAGE as described in Materials and Methods. Protein from three cultures was loaded on each lane. Lanes 3,4, To assess the possibility of protein synthesis in distal axons, left and right compartments of cultures were incubated with [35S]met for 4 hr, and the cultures were analyzed. Protein from 10 cultures was loaded on each lane. Lane 5, To determine whether the labeled proteins detected in axons incubated with [35S]met were transported there after synthesis in the cell bodies, the cell bodies were removed from cultures immediately before labeling the distal axons for 4 hr with [35S]met. Protein from 10 cultures was loaded on this lane.
- Fig. 3.
Inhibition of axonal protein synthesis. Cultures were preincubated 2 hr with culture medium in the distal axon compartments containing either cycloheximide (100 μg/ml;Cy), puromycin (120 μg/ml; Pu), or no drug as control (Co). Then, the distal compartment medium was replaced with [35S]met labeling medium containing the inhibitor or no inhibitor as appropriate, and the cultures were incubated for 4 hr. Proteins were harvested and analyzed by SDS-PAGE as described in Materials and Methods. Protein from six cultures was loaded on each lane. A, Lanes from the distal axons (DAx). B, Density of the individual lanes from A quantitated by phosphorimaging and expressed as a percentage of the control lane. C, Lanes from the cell bodies and proximal axons (CB/PAx).
- Fig. 4.
Inhibition of axonal protein synthesis by direct application of cycloheximide and puromycin in isolated axons. The center compartments were washed with distilled water to remove the cell bodies before labeling. Control axons (Co) were not treated with drugs. Application of cycloheximide (Cy) and puromycin (Pu), labeling of axons, and analysis were as in Figure 3. Protein from six cultures was loaded on each lane.
- Fig. 5.
Immunoprecipitation of actin and β-tubulin synthesized in distal axons. After removal of the cell bodies, axons were incubated in [35S]met for 4 hr. Cell lysates from six cultures were immunoprecipitated with either anti-actin or anti-β-tubulin antibodies as described in Materials and Methods. Immunoprecipitated proteins were analyzed by SDS-PAGE and phosphorimaging. Anti-actin antibodies precipitated actin at 43 kDa (filled arrow) and coprecipitated one major band at 56 kDa (open arrow), as well as several other minor bands of varying molecular weight. Anti-β-tubulin antibodies precipitated β-tubulin (55 kDa, filled arrow), as well as four other bands (open arrows) of molecular weight 85, 75, 14, and 12 kDa.
- Fig. 6.
Effect of protein synthesis inhibitors on axonal growth. Cycloheximide (100 μg/ml; A) or puromycin (120 μg/ml; B) was applied directly to axons in the right compartments of intact cultures, and medium without inhibitor was applied to axons in the left compartments as control. The growth of axons after introduction of the inhibitors was measured after 6 and 30 hr as described in Materials and Methods. The mean ± SE axonal extension for each time point is shown; n = 15–34 tracks.
Tables
mRNA for Animal Reference β-actin rat brain Bassell et al., 1998 β-actin chick neurons Olink-Coux and Hollenbeck, 1996 β-actin squid axon Kaplan et al., 1992 β-tubulin squid axon Kaplan et al., 1992 neurofilament gold fish brain Weiner et al., 1996 neurofilament squid axon Crispino et al., 1993 tau rat brain Litman et al., 1993 kinesin squid axon Gioio et al., 1994 neuropeptides rat brain Mohr et al., 1991 enolase squid axon Chun et al., 1995 pA6 squid axon Chun et al., 1997
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