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Single Molecule Translation Imaging Visualizes the Dynamics of Local β-Actin Synthesis in Retinal Axons.
Ströhl F
,
Lin JQ
,
Laine RF
,
Wong HH
,
Urbančič V
,
Cagnetta R
,
Holt CE
,
Kaminski CF
.
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Local mRNA translation occurs in growing axons enabling precise control of the proteome in response to signals. To measure quantitatively the spatiotemporal dynamics of protein synthesis in growth cones, we further developed a technique for single molecule translation imaging (SMTI). We report that Netrin-1 triggers a burst of β-actin synthesis at multiple non-repetitive sites, particularly in the periphery. The response is remarkably rapid starting within 20 seconds of cue application.
Figure 1. SMTI imaging procedure. (a) A reporter construct coding for Venus and a full-length β-actin sequence is electroporated into Xenopus eyeprimordia, which are dissected and cultured. (b) During imaging, a fluorescent image of a growth cone is acquired to segment the outline for later processing. Existing fluorescence is then photobleached using a brief pulse of laser irradiation. Subsequently, the translation of individual β-actin is recorded as individual Venus molecule emission. (c) Translation events are localized and processed to yield translation density maps of a representative RGC growth cone and a HEK293T cell. Scale bars are 5 μm.
Figure 2. Netrin-1 increases β-actin translation rate. (a) Translation density maps for RGC growth cones treated with culture medium, Netrin-1, or Netrin-1 with puromycin pre-incubation. (b) Respective translation rate time courses of examples shown in a. The apparent difference in the pre-treatment rate between the culture medium- and Netrin-1-treated growth cones can be attributed to biological variability; the average pre-treatment rates between the two groups (n = 16) did not show any significant difference (p = 0.63). (c) Cumulative event rates per growth cone *p < 0.05; **p < 0.001; two-way ANOVA. (d) Sholl analysis on culture medium- or Netrin-1-treated growth cone density maps revealing the spatial distributions of translational events. The center of 5 concentric circles is located at the base of the growth cone, with the outermost circle intersecting the furthest boundary of the growth cone. The radii are equidistant. A1 denotes the most central arc, while A5 indicates the most peripheral arc. Intracellular events within each arc are counted and the percentages within each arc are shown in the graph. **p < 0.001; Mann-Whitney Houston test; scale bars are 5 μm. Error bars indicate standard error of the mean. In c, n is the number of growth cones.
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