Figure 4. F-Actin Dynamics during Collateral Axon Branch Formation.
(A) Time-lapse images of layer II/III pyramidal neuron axon co-labeled with eGFP and the calponin homology domain of utrophin fused to mRFP, an f-actin biosensor. Images were acquired every 2.5 min. Representative images are displayed every 10 min. The eGFP signal was used to create a mask of the axon (red outline), and the mRFP signal corresponding to f-actin is black (see also Movie S5). Blue arrows denote a newly formed collateral axon branch, the green arrow marks a newly formed protrusion emerging from the primary axon, and a red arrow marks a protrusion that is lost.
(B and C) The 647 f-actin pools quantified were binned by size (B) and by duration (C).
(D) A significant correlation was found between the size of the f-actin pool and the duration of the f-actin pool (p value < 0.0001).
(E and F) We compared the average area and duration of f-actin pools associated with a protrusion (green) and f-actin pools that did not correspond with a protrusion (red; E and F, respectively).
(G) If a protrusion was lost, there was a decrease in the size of the f-actin pool (red). If a new protrusion was formed from a pre-existing f-actin pool, there was an increase in the size of the factin pool (green).
(H) When the f-actin pools were binned by size, there was a significant increase in the percentage of f-actin pools corresponding with protrusions only in f-actin pools greater than 0.5 µm2.
(I) When the f-actin pools were binned by duration, there was a significant increase in the percentage of f-actin pools corresponding with protrusions only in f-actin pools that persisted longer than 2.5 min.
* indicates a p value < 0.05. *** indicates a p value < 0.001. NS indicates no significance. Error bars denote SD (E–G) and 95% confidence intervals (H and I). See also Movie S5.