Myosin-Va function is required for transfer of RNA from Schwann cells to axons.

<p>Longitudinal 10-µm sections of transected sciatic nerves from null (d-l) <i>Myo5a</i> mutant mice have reduced axoplasmic levels of newly synthesized RNA. <b>A and C</b>, null mutant; <b>B and D</b>, wild-type control. RNA labeled by BrU is shown in green, the paranodal marker Caspr in red. Panels C and D show higher magnification views of boxed regions in panels A and B respectively. Arrows, nodes of Ranvier; arrowheads, bands of Cajal (compare to arrows in Fig. 6). Micrographs are single optical sections from Z-stacks imaged with a laser scanning confocal microscope. Bar = 5 µm. <b>E</b>, linescan quantitation of abundance of BrU-labeled RNA across fibers from d-l mutant and wild-type control mice. Edges are the outer wraps of Schwann cells; center approximates the location of the axon. Intensity measurements were normalized to the mean of each linescan. Bars represent standard deviations. <b>F</b>, Absolute BrU fluorescence intensities in edges (as shown in <b>E</b>, 4 bins at each end combined; n = 160) and centers (10 central bins combined; n = 200). Error bars represent standard errors.</p

Possible routes for transfer of newly-synthesized RNA from Schwann cells to axons.

<p>Diagram of a peripheral fiber showing a longitudinal section of parts of two adjacent Schwann cells and the axon they ensheath. This schematic depicts hypothesized routes (nodes of Ranvier and Schmidt-Lanterman incisures) of transport of BrU-labeled RNA (green dots) between the Schwann cell nucleus and the axon.</p

Actin depolymerization in injured sciatic nerves prevents transfer of RNA into axons.

<p><b>A, C, E, G, I</b>, representative confocal images of BrU labeling at nodes of Ranvier. Bar = 10 µm. <b>B, D, F, H, J</b>, quantification of BrU fluorescence from 10 or more line scans across perinodal regions normalized to the mean of each linescan. Error bars represent standard errors. <b>A and B</b>, control BrU labeling without Latrunculin A; <b>C and D</b>, 0.07 µg/ml Latrunculin A during BrU labeling; <b>E and F</b>, 0.2 µg/ml; <b>G and H</b>, 0.6 µg/ml; <b>I and J</b>, 1.8 µg/ml. <b>K</b>, absolute BrU fluorescence intensities for the 8 bins at each edge combined (n = 304), representing RNA in the outer Schwann cell wrap, and the 20 bins in the center of each linescan (n = 380), representing RNA in the axon, for the control untreated and highest latrunculin A concentration (1.8 µg/ml) nerves. Error bars represent standard errors.</p

Negative controls for observation of axonal BrU labeling.

<p>In all panels, newly-synthesized RNA is shown in green, with F-actin counterstaining shown in red. <b>A and A’</b>, experimental condition; fibers were incubated with BrU. <b>B and B’</b>, negative control incubated with medium without BrU. <b>C and C’</b>, negative control incubated in BrU, but primary anti-BrdU antibody was omitted. <b>D and D’</b>, negative control incubated with 10 mg/ml RNAse. Both BrU and F-actin channels at a single confocal plane are shown in <b>A</b>–<b>D</b>, whereas the BrU channel alone is shown in <b>A’</b>–<b>D’</b>.</p

Levels of newly-synthesized RNA decline as a function of distance from nerve injury.

<p><b>A</b>, low-magnification micrograph of transected end showing newly-synthesized RNA (green) and ribosomes detected by anti-P antibody (red). Bar = 100 µm. <b>B</b>, BrU-RNA signal plotted as a function of distance from the transection. Each point represents the mean of 10 nerve fragments with standard errors. <b>C–H</b>, series of images of a single fiber from the transected end, distal to proximal, showing newly-synthesized RNA labeled by BrU (green) and F-actin (red). <b>C</b>, transected end with a high concentration of newly-synthesized BrU-RNA. <b>D</b>, first proximal Schwann-cell nucleus from the tip. <b>E</b>, first node of Ranvier proximal from the tip. <b>F</b>, second Schwann cell nucleus. <b>G</b>, second node of Ranvier. <b>H</b>, third node of Ranvier. Bar = 10 µm.</p

Most newly-synthesized axonal RNA is not mitochondrial.

<p>Cryosections of injured BrU-labeled (green) sciatic nerve fragments were stained for BrU <b>(A, D, G)</b> and a monoclonal antibody against the mitochondrial Complex IV Subunit I <b>(B, E, H)</b>. A paranodal axon is shown in <b>A–C</b> and nodes of Ranvier are shown in <b>D–I</b>. Mitochondria corresponding to empty spaces in A and D are designated by arrows. Bar = 5 µm.</p

Newly synthesized RNA is present in axons and bands of Cajal.

<p><b>A</b>, confocal plane including a BrU-labeled axon. The myelin is unlabeled. The external border of the myelin is the outer wrap of Schwann cell cytoplasm that includes bands of Cajal. <b>B</b>, stack of confocal planes with the plane shown in A as the midpoint, showing the spiraling bands of Cajal (arrows). <b>C, D, E</b>, projected cross-sections boxed in the stack shown in panel B showing the separation between newly-synthesized RNA in the axon and band of Cajal. Bar = 10 µm.</p

Most of the newly-synthesized RNA is produced by RNA Polymerase II.

<p><b>A–E</b>, injured control nerves without α-amanitin (<b>A </b><b>and </b><b>C)</b>, and injured nerves treated with α-amanitin during the BrU labeling period <b>(B and D)</b> were stained for BrU (green) and F-actin with phalloidin (red). <b>A and </b><b>B</b>, Schwann cell nuclei; <b>C</b> and <b>D</b>, nodes of Ranvier. Bar = 10 µm. <b>E</b>, BrU-RNA fluorescence intensities plotted as a function of distance from the node of Ranvier for controls without α-amanitin (circles) and nerves treated with 10 µg/ml α-amanitin (triangles). Statistical significance at each distance was determined by Student's t-test. Error bars represent standard errors. <b>F</b>, Neurofilament L (NF-L) mRNA is found in both Schwann cells and axons by <i>in situ</i> hybridization (red) and BrU-RNA (green). Arrows are pointing to axons. <b>G</b>, negative control NF-L sense probe. Bar = 5 µm.</p

Colocalization of myosin-Va and newly-synthesized RNA in fibers of injured sciatic nerve axons.

<p><b>A, B, and C</b>, confocal micrographs of nodes of Ranvier showing newly-synthesized RNA detected by BrU incorporation (green) and myosin-Va detected by immunofluorescence (red); <b>D</b>, processed FRET (PFRET) image for the node shown in panel C. <b>E</b>, FRET efficiency (E%) image for the node shown in panels C and D. Scales below panels D and E show lookup tables. Bars = 5 µm.</p

Newly-synthesized RNA is transferred from Schwann cells to axons after sciatic nerve transection.

<p><b>A–D</b>, experimental procedure. <b>E–F</b>, single confocal planes of fibers at nodes of Ranvier showing BrU incorporation (green) and F-actin (red). <b>G</b>, Axonal BrU fluorescence intensity plotted as a function of distance from the node of Ranvier for uninjured control (open circles) and injured (closed circles) nerves. Statistical significance at each distance between injured and uninjured nerves was determined by Student's t-test. Error bars represent standard errors. <b>H–I</b>, single confocal planes showing BrU labeling (green) of F-actin-rich (red) Schmidt-Lanterman incisures (arrows). Bars = 5 µm.</p