Figure 5.

Heat shock and heat shock-like proteins and encoding mRNAs in regenerating axons. A, RT-PCR was performed on axonal versus cell-body RNA isolates using primers specific for αB crystallin, HSP27, HSP60, grp75, HSP70, and HSP90 mRNAs. Purity of the axonal preparation was determined by RT-PCR for β- and γ-actin mRNAs as shown in Figure 1 B. Note that each of these mRNAs was detected in the axonal RNA templates, but the relative amounts vary. HSP27 and grp75 are more abundant in the cell body than in the axon. Approximately equivalent amounts of HSP60 and HSP90 mRNAs are amplified from the cell body and axons, whereas αB crystallin HSP70 mRNA is relatively more abundant in the axons than in the cell-body isolates compared with the other mRNAs examined. B, Injury-conditioned DRG cultures were labeled for HSP60. This representative image displays are constructed three-dimensional projection of HSP60 (red; 15 optical XY planes taken at 0.2 μm intervals) merged with a single differential interference contrast (DIC) image. Arrows show HSP60 immunoreactivity in proximal portions of the DRG axons. C, DRG cultures stained for grp75 signal (red) show intra-axonal grp75 immunoreactivity along the shaft of the axons (arrows) in this three-dimensional projection of grp75 signal (12 optical XY planes taken at 0.3 μm intervals) merged with a single DIC image. D, A single optical XY plane of distal axon from DRG cultures colabeled for HSP70 (green) and peripherin (red). Arrows indicate HSP70 immunoreactivity that extends beyond the peripherin immunoreactivity into the growth cone. E, Colabeling cultures of injury-conditioned DRG neurons for HSP90 (green) and neurofilament (red) shows that HSP90 extends into the distal axon (arrows), similar to HSP70 shown in D, in this three-dimensional projection (12 optical XY planes taken at 0.2 μm intervals). F, A reconstructed three-dimensional projection shows terminal axons of cultures stained for αB crystall in (red) and neurofilament (green) (projection is from 10 optical XY planes taken at ∼0.15 μmintervals). Note that similar to HSP70 and HSP90, αB crystallin immunoreactivity extends into the growth cone beyond the neurofilament signal (arrows). G, Section of sciatic nerve as in F was colabeled for HSP70 (green) and peripherin (red). The full panel shows a longitudinal section where intra-axonal HSP70 signal is visible in an optically isolated axon (arrows) in this projected three-dimensional image. HSP70 signal separate from the peripherin immunoreactivity is also seen in Schwann cell cytoplasm of the myelin sheath adjacent to this axon (arrow-heads). The inset shows a cross section of nerve where intra-axonal HSP70 signal is clearly discerned from the bright signal in the surrounding Schwann cells.