Article Figures & Data
Figures
- Figure 1.
Ribosomes and polyribosomes in axons. A, B, Intact (A) and Wlds (B) desomatized for 7 d. A, Ribosomes are scarce (arrowhead). B, A field of polyribosomes stands out in the axoplasm, which has not degenerated despite its protracted disconnection from the cell body. Inset, High magnification of an axonal polyribosome. M, Myelin. Scale bars: 200 nm; inset, 50 nm. C, Quantification of ribosomes in wild-type intact (WT int), Wlds intact (Wlds int) axons, and in desomatized Wlds axons crushed 7 d before (Wlds Crush). Ordinates: scale is logarithmic. Notice the impressive increase in ribosomal content, both in ribosomes per volume of axoplasm (*p < 0.03), as well as the number of axons containing ribosomes. (*p < 0.01). Student's t test.
- Figure 2.
Transfer of ribosomes from Schwann cell to axon. A, Western blot showing incorporation of the L4-eGFP fusion protein into ribosomes. HEK cells were infected with LV-L4-eGFP, fractionated, after which the blot was stained with anti-eGFP. First lane, Histidine-tagged eGFP; second lane, coomassie-stained supernatant of pellet fraction; third lane, supernatant of pellet fraction; fourth lane, sucrose gradient of purified ribosomal fraction. The antibody recognizes a single band that corresponds to the molecular weight of His-eGFP (first lane) and the fusion protein (fourth lane), respectively. In the supernatant (third lane), no signal is detected, indicating that the cytosol contains little if any L4-eGFP. Bottom image, The ribosomal pellet shows intense eGFP fluorescence after sucrose gradient purification. B, C, Wlds teased fibers, 7 d after crush; the distal segment of the nerve was injected immediately after the crush with LV-L4-eGFP (B), or with LV-eGFP (C). Color codes indicate the antibodies used. B, Top, Triple immunostaining and Z-projections at the corresponding numbers; in the neurofilament space, puncta are seen in which ribosomal and L4-eGFP fluorescent markers colocalize. Bottom, Higher magnification of boxed area shows in detail colocalization of signals in both Schwann cell cytoplasm and axoplasm (merged signals, left), ribosome signal (middle), and L4-eGFP signal (right). C, as in B, except that the encoded protein lacked the ribosomal protein L4. Ribosomal signals are present in Schwann cells and axoplasm, but eGFP is present only in the Schwann cell. These results suggest that the transfer mechanism between Schwann cells and axons is selective. Scale bars, 10 μm.
- Figure 3.
Putative intercellular pathway of ribosomal transfer. Wlds axons desomatized 7 d before. A, Teased fiber: triple immunostaining using anti-ribosome, neurofilament, and P0 antisera. Top, Ribosome immunoreactive puncta are clearly seen in the axoplasmic space outlined by the neurofilament staining; the arrow points to a structure penetrating through the myelin marked by the P0 staining, akin to a Schmidt-Lanterman incisure; bottom, Z-projections of the corresponding numbers of the top show ribosomal signal from the Schwann cell penetrating into the axoplasm (arrows in 2 and 3). Scale bar, 10 μm. B–D, EM micrographs of Wlds axons desomatized for 7 d. B, Polyribosomes (arrowheads) can be seen in enlarged adaxonal pockets of Schwann cell cytoplasm. At the arrow, a Schwann cell protrusion loaded with ribosomes appears to be in the process of disengaging itself from the Schwann cell. C, Vesicle with multiple membranes contains several polyribosomes (arrow), whereas many polyribosomes are present in the axoplasm (arrowhead). D, Vesicle with ruptured membranes, creating a continuum between the vesicular space and axoplasm [note a polyribosome at the interface between vesicle and axoplasm (arrow) and polyribosomes in the axoplasm (arrowheads)]. Scale bars, 300 nm. B1–D2 are high magnification images of areas indicated by arrows and arrowheads in B–D. Together, these images suggest the pathway followed by ribosomes from the Schwann cell to the axoplasm.
- Figure 4.
Proposed trajectory of ribosomes moving from the outer Schwann cell cytoplasm into the axoplasm. Ribosomes (Rib) move from the periaxonal Schwann cell cytoplasm through cytoplasmic channels that serve to supply the adaxonal Schwann cell cytoplasm. Here, ribosomes accumulate and protrusions of the Schwann cell cytoplasm invaginate into the axon and, during detachment from the Schwann cell, give rise to RVs. After rupture of RVs, the ribosomes gain access to the axoplasm. N, Schwann cell nuclei.
Additional Files
Supplemental Data
Files in this Data Supplement:
- supplemental material - Supplemental Legend
- supplemental material - Supplemental Figure 1
- supplemental material - Supplemental Figure 2
- supplemental material - Supplemental Figure 3
- supplemental material - Supplemental Figure 4
- supplemental material - Supplemental Figure 5
- supplemental material - Supplemental Figure 6
- supplemental material - Supplemental Figure 7
- supplemental material - Supplemental Figure 8
