Figure 3.
Figure 3. F-actin distribution and dynamics in G15S and tsr-RNAi neurons. (A) Images show dendritic trees in control (ppk-GAL4/+; ppk-CD4-td-Tom/+), G15S-expressing, and tsr-RNAi–knockdown neurons. (B) Bar graph shows average numbers of dendritic endpoints present in the posterior dorsal region of the ddaC dendritic field. The tsr-RNAi2 effect is also included. Numbers of neurons: 12 (control), 10 (G15S), 10 (tsr-RNAi), and 10 (tsr-RNAi2). (C) Images show LifeAct distributions (green) in the CD4-tdTomato–labeled dendrites (magenta) of G15S-expressing (top) and tsr-RNAi–knockdown (bottom) neurons. Merged images of two channels are at right. Arrows indicate high LifeAct signals in proximal dendrites, and arrowheads indicate these in terminal dendrites. For control, see Fig. 1 A. (D) Bar graphs show quantifications of LifeAct intensities normalized to CD4-tdTomato intensities in proximal dendrites (top) as indicated by arrows in C as well as terminal dendrites (bottom) as indicated by arrowheads in C. Each dot represents the average value from one neuron. Numbers of neurons: 9 (control), 8 (G15S), and 7 (tsr-RNAi) from five to six experiments. (E) LifeAct signals are dynamic (Video 10) in control terminal dendrites (top) and remain static in both G15S-expressing (middle) and tsr-RNAi–knockdown (bottom) terminal dendrites. Images are shown in time series of frames separated by 2-min intervals for a total of 10 min. (F) Line graphs show changes of LifeAct intensities in representative segments of dendrites over a 10-min live-imaging period with 2-min intervals for control (blue), G15S-expressing (green), and tsr-RNAi–knockdown (red) neurons. The intensities were assayed from linearized dendrites. x axis, μm; y axis, LifeAct gray value. Representative images were chosen from observations in 39 dendrites for control, 28 dendrites for tsr-RNAi, and 39 dendrites for G15S in six neurons from six experiments for each genotype. (G) Bar graphs show quantifications for actin blob numbers per 10 µm recorded in 10 min (top) as well as actin blob velocity in μm/min (bottom) in control (94 actin blobs in 39 dendrites), G15S-expressing (23 actin blobs in 29 dendrites), and tsr-RNAi–knockdown (26 actin blobs in 28 dendrites) neurons from six neurons in six experiments for each genotype. Genotypes for control: UAS-lacZ-RNAi/+; ppk-GAL4, UAS-LifeAct/+; ppk-CD4-td-Tom/+; for G15S: +/+; ppk-GAL4, UAS-LifeAct/UAS-ActinG15S; ppk-CD4-td-Tom/+; and for tsr-RNAi: +/+; ppk-GAL4, UAS-LifeAct/UAS-tsr-RNAi; ppk-CD4-td-Tom/+. Significance in comparison with control was determined by Student’s t test. ***, P < 0.001. Error bars represent SEM.

F-actin distribution and dynamics in G15S and tsr-RNAi neurons. (A) Images show dendritic trees in control (ppk-GAL4/+; ppk-CD4-td-Tom/+), G15S-expressing, and tsr-RNAi–knockdown neurons. (B) Bar graph shows average numbers of dendritic endpoints present in the posterior dorsal region of the ddaC dendritic field. The tsr-RNAi2 effect is also included. Numbers of neurons: 12 (control), 10 (G15S), 10 (tsr-RNAi), and 10 (tsr-RNAi2). (C) Images show LifeAct distributions (green) in the CD4-tdTomato–labeled dendrites (magenta) of G15S-expressing (top) and tsr-RNAi–knockdown (bottom) neurons. Merged images of two channels are at right. Arrows indicate high LifeAct signals in proximal dendrites, and arrowheads indicate these in terminal dendrites. For control, see Fig. 1 A. (D) Bar graphs show quantifications of LifeAct intensities normalized to CD4-tdTomato intensities in proximal dendrites (top) as indicated by arrows in C as well as terminal dendrites (bottom) as indicated by arrowheads in C. Each dot represents the average value from one neuron. Numbers of neurons: 9 (control), 8 (G15S), and 7 (tsr-RNAi) from five to six experiments. (E) LifeAct signals are dynamic (Video 10) in control terminal dendrites (top) and remain static in both G15S-expressing (middle) and tsr-RNAi–knockdown (bottom) terminal dendrites. Images are shown in time series of frames separated by 2-min intervals for a total of 10 min. (F) Line graphs show changes of LifeAct intensities in representative segments of dendrites over a 10-min live-imaging period with 2-min intervals for control (blue), G15S-expressing (green), and tsr-RNAi–knockdown (red) neurons. The intensities were assayed from linearized dendrites. x axis, μm; y axis, LifeAct gray value. Representative images were chosen from observations in 39 dendrites for control, 28 dendrites for tsr-RNAi, and 39 dendrites for G15S in six neurons from six experiments for each genotype. (G) Bar graphs show quantifications for actin blob numbers per 10 µm recorded in 10 min (top) as well as actin blob velocity in μm/min (bottom) in control (94 actin blobs in 39 dendrites), G15S-expressing (23 actin blobs in 29 dendrites), and tsr-RNAi–knockdown (26 actin blobs in 28 dendrites) neurons from six neurons in six experiments for each genotype. Genotypes for control: UAS-lacZ-RNAi/+; ppk-GAL4, UAS-LifeAct/+; ppk-CD4-td-Tom/+; for G15S: +/+; ppk-GAL4, UAS-LifeAct/UAS-ActinG15S; ppk-CD4-td-Tom/+; and for tsr-RNAi: +/+; ppk-GAL4, UAS-LifeAct/UAS-tsr-RNAi; ppk-CD4-td-Tom/+. Significance in comparison with control was determined by Student’s t test. ***, P < 0.001. Error bars represent SEM.

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