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. 2015 Aug 3;210(3):401–417. doi: 10.1083/jcb.201506110

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© 2015 Ganguly et al.

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Figure 7. — Presynaptic F-actin dynamics are Formin dependent. (A) Images (left) and kymographs (right) of boutons from neurons transfected with GFP:Utr-CH (to label F-actin) and synaptophysin:mRFP (SyPhy:mRFP to label synaptic vesicle clusters). Note that F-actin appears as dynamic patches, circumferentially organized around the synaptic vesicle cluster. (B) Same experiment as above, except GFP:Utr-CH kymographs are scaled to reveal actin trails terminating into boutons (small arrowheads). (C and D) Representative images from a FRAP assay to detect F-actin entry into boutons, quantified in D. Neurons were transfected with GFP:Utr-CH and F-actin–enriched boutons were identified; a single bouton (dashed circles) within a string of synapses was photobleached, and recovery of fluorescence was visualized over time (see Materials and methods for details). Note rapid recovery of F-actin that is attenuated upon formin inhibition by SMIFH2, indicating diminished entry of F-actin into boutons after formin inhibition. (E and F) Maximum intensity projection images from a representative synaptic bouton demonstrate reduction in total F-actin fluorescence upon SMIFH2. Graph in F shows that fluorescence intensities decrease from 1,859 ± 351.7 to 999.1 ± 146.7 arbitrary fluorescence units (n = 9 boutons paired) upon SMIFH2 treatment for 30 min. All values represent means ± SEM. Paired t test **, P < 0.01.