Supporting Information legends
Figure S1. F-actin and myosin XI dynamics at the cell’s apex.
( a ) Distribution of 3xmEGFPmyosin XIa and Lifeact-mCherry by confocal microscopy. Average projections of the corresponding z-stacks (9 confocal slices) are shown in the top two panels and the merged image in the third one with F-actin in red and myosin XI in green. Images from an independent transgenic line from that shown in Figure 1 were acquired at 5 sec intervals for the complete time series. Selected images from a time series are shown with the seconds from the series indicated at the top. The intensity is shown as colors using a “Rainbow” look up table where the high intensity is represented by warm colors (maximum white, then red, etc) and the lower intensity is represented by cooler colors (minimum black, then blue, etc). The grey-value to color conversion is indicated on the right side of each panel. ( b ) Intensity changes of myosin XI and F-actin at the apical region of the growing cell. The intensity values were obtained by kymographic analysis
(Supporting Figure S2), de-trended by singular spectrum analysis (SSA) and normalized
(Supporting Figure S4 and Experimental Procedures). ( c ) Enlarged region indicated by the bracket at the top b.
The time segment corresponding to the images from Supporting Figure S1a is indicated by the bracket at the top of c.
Figure S2. Kymographic analysis of F-actin and myosin XI signals. ( a ) and ( b ) Maximal projections of the complete time series for F-actin and myosin XI after average projection of each time point z-stacks. This illustrates in one image, the positional dynamics of the apical spot from all time points. The white rectangle indicates the region that was used in the individual time points to construct the kymograph shown below. ( c ) and ( d ) The intensity of the box shown in a and b was averaged over 5.1 µm in the Y-axis as explained in the Experimental Procedures section and displayed as the first row of the images. This was repeated over all time points. The slope of the line flanking the signal at the tip was used to calculate growth rate. The dimensions of the regions used to quantify the fluorescent levels are as follows: 1) 9.1-25.8 µm, 2) 5.4 µm,
3)3.4 µm, 4) 2.5 µm, 5) 3.4 µm. The intensity within the limits of the 1) shank, 3) tip, and 5) background sections was used for quantification. Note that the variable distance in the shank region is due to variations in the total length of the cell imaged; in order to increase the averaged area the largest possible length was used.

Figure S3.
Analysis of intensity changes of myosin XI and F-actin at the apical region of the growing cell shown in Figure 1. ( a ) The intensity ratio values tip/shank were obtained by kymographic analysis (Supporting Figure S2), ( b ) de-trended by singular spectrum analysis
(SSA) and ( c ) normalized by subtraction of the mean intensity and division by the standard deviations (see Experimental Procedures).
Figure S4.
Analysis of intensity changes of myosin XI and F-actin at the apical region of the growing cell shown in Supporting Figure S1. ( a ) The intensity ratio values tip/shank were obtained by kymographic analysis (Supporting Figure S2), ( b ) de-trended by singular spectrum analysis (SSA) and ( c ) normalized by subtraction of the mean intensity and division by the standard deviations (see Experimental Procedures).
Figure S5.
VAMP and Myosin XI dynamics at the cell’s apex. ( a ) Distribution of 3xmEGFPmyosin XIa and 3xmCherry-VAMP by confocal microscopy. Average projections of the corresponding z-stacks (9 confocal slices) are shown in the top two panels and the merged image in the third one with VAMP in red and myosin XI in green. Images from an independent transgenic line from that shown in Figure 3 were acquired at 5 sec intervals for the complete time series. Selected images from a time series are shown with the seconds from the series indicated at the top. The intensity is shown as colors using a “Rainbow” look up table where the high intensity is represented by warm colors (maximum white, then red, etc) and the lower intensity is represented by cooler colors (minimum black, then blue, etc). The grey-value to color conversion is indicated on the right side of each panel. ( b ) Intensity changes of myosin XI and VAMP at the apical region of the growing cell. The intensity values were obtained by kymographic analysis
(Supporting Figure S2), de-trended by singular spectrum analysis (SSA) and normalized
(Supporting Figure S7 and Experimental Procedures). ( c ) Enlarged region indicated by the bracket at the top of b.
This region covers the time segment corresponding to the images from
Supporting Figure S5a.
Figure S6.
Analysis of intensity changes of myosin XI and VAMP at the apical region of the growing cell shown in Figure 3. ( a ) The intensity ratio values tip/shank were obtained by

kymographic analysis (Supporting Figure S2), ( b ) de-trended by singular spectrum analysis
(SSA) and ( c ) normalized by substraction of the mean intensity and divison by the standard deviations (See Experimental Procedures).
Figure S7.
Analysis of intensity changes of myosin XI and VAMP at the apical region of the growing cell shown in Supporting Figure S5. ( a ) The intensity ratio values tip/shank were obtained by kymographic analysis (Supporting Figure S2), ( b ) de-trended by singular spectrum analysis (SSA) to remove the base line and ( c ) normalized by substraction of the mean intensity and divison by the standard deviations (See Experimental Procedures).
Figures S 8. Myosin XI and VAMP72 dynamics at the apex of cells treated with low levels of latrunculin B. ( a ) Distribution of 3xmEGFP-myosin XIa and 3xmCherry-VAMP after treatment with 0.5 µM latrunculin B by confocal microscopy. Average projections of the corresponding Zstacks (6 confocal slices) are shown in the top two panels and the merged image in the bottom one with VAMP in red and myosin XI in green. Images from one transgenic line were acquired at 5 sec intervals for the complete time series. Selected images from a time series are shown with the seconds after the addition of latrunculin B indicated at the top. The intensity is shown as colors using a “Rainbow” look up table where the high intensity is represented by warm colors
(maximum white, then red, etc) and the lower intensity is represented by cooler colors (minimum black, then blue, etc). The grey-value to color conversion is indicated on the right side of each panel. ( b ) Average intensity of 3xmEGFP-myosin XIa and 3xmCherry-VAMP at the apex of the cell, before and after treatment with 0.5 µM latrunculin B (indicated). The mean and standard error are shown from the intensity values from three cells. Average intensity values at the cell apex were measured from average projections of the corresponding Z-stacks (6 confocal slices) and normalized against average fluorescence levels in the shank (see Experimental Procedures).
The asterisk denotes the time point compared in Figure 4, which is identical in this series but is significantly different between myosin XI and F-actin signals.
Figures S 9, S10, and S11.
Three other examples of F-actin and Myosin XI dynamics at the shank of cells treated with latrunculin B. ( a ) Distribution of Lifeact-mCherry and 3xmEGFPmyosin XIa after treatment with 0.5 µM latrunculin B by confocal microscopy. Average

projections of the corresponding z-stacks (6 confocal slices) are shown with F-actin in red and myosin XI in green. Images from the same transgenic line than that shown in Figure 5 were acquired at 5 sec intervals for the complete time series. Selected images from a time series are shown with the seconds after the addition of latrunculin B indicated at the top. Scale bar 2
 m in
S9 and S10, and 3
 m in S11. For consistent display, the image in S11 was rotated 90 degrees clockwise, as a result the tip of the cell is pointing downward. ( b ) Intensity changes of myosin
XI and F-actin at the area where the cluster starts. Intensity values were calculated from average projections of the corresponding z-stacks (6 confocal slices) and normalized to the lower intensity of the trace. The tracked area is shown with a white circle in Supporting Figure S9a,
S10a, and S11a. ( c ) Intensity changes of myosin XI and F-actin following the trajectory and speed of the myosin XI cluster. Intensity values were calculated from average projections of the corresponding z-stacks (6 confocal slices) and normalized to the lower intensity of the trace. The speed of the myosin XI cluster was calculated using an in-house developed ImageJ macro by tracking the displacement of the area shown with a blue circle in Supporting Figure S9a, S10a and S11a. Note that S11 was measured in a different cell.
Figures S 12 and S13.
Examples of VAMP72 and Myosin XI dynamics at the shank of cells treated with latrunculin B. ( a ) Distribution of 3xmCherry-VAMP72 and 3xmEGFP-myosin XIa after treatment with 0.5 µM latrunculin B by confocal microscopy. Average projections of the corresponding z-stacks (6 confocal slices) are shown with VAMP in red and myosin XI in green.
Selected images from a time series are shown with the seconds after the addition of latrunculin B indicated at the top. Scale bar 3
 m. ( b ) Intensity changes of myosin XI and VAMP at the area where the cluster starts. Intensity values were calculated from average projections of the corresponding z-stacks (6 confocal slices) and normalized to the lower intensity of the trace. The tracked area is shown with a white circle in Supporting Figure S12a and S13a. ( c ) Intensity changes of myosin XI and VAMP following the trajectory and speed of the myosin XI cluster.
Intensity values were calculated from average projections of the corresponding z-stacks (6 confocal slices) and normalized to the lower intensity of the trace. The speed of the myosin XI cluster was calculated using an in-house developed ImageJ macro by tracking the displacement of the area shown with a blue circle in Supporting Figure S12a and S13a. Note that each measurement is from a different cell.

Movie S1. Fluctuations of myosin XI and F-actin signals at the cell apex of the transgenic line analyzed in Figure 1.
Distribution of 3xmEGFP-myosin XIa and Lifeact-mCherry by confocal microscopy. Average projections of the corresponding z-stacks (9 confocal slices) are shown in the top two panels and the merged image in the third one with F-actin in red and myosin XI in green. Images were acquired at 2 sec intervals for the complete time series. The intensity is shown as colors using a “Rainbow” look up table where the high intensity is represented by warm colors (maximum white, then red, etc) and the lower intensity is represented by cooler colors (minimum black, then blue, etc).
Movie S2. Fluctuations of myosin XI and F-actin signals at the cell apex of the transgenic line analyzed in Supporting Figure S1. Distribution of 3xmEGFP-myosin XIa and Lifeact-mCherry by confocal microscopy. Average projections of the corresponding z-stacks (9 confocal slices) are shown in the top two panels and the merged image in the third one with F-actin in red and myosin XI in green. Images were acquired at 5 sec intervals for the complete time series. The intensity is shown as colors using a “Rainbow” look up table where the high intensity is represented by warm colors (maximum white, then red, etc) and the lower intensity is represented by cooler colors (minimum black, then blue, etc).
Movie S3. Fluctuations of myosin XI and VAMP signals at the cell apex of the transgenic line analyzed in Figure 3.
Distribution of 3xmEGFP-myosin XIa and 3xmCherry-VAMP by confocal microscopy. Average projections of the corresponding z-stacks (9 confocal slices) are shown in the top two panels and the merged image in the third one with VAMP in red and myosin XI in green. Images were acquired at 5 sec intervals for the complete time series. The intensity is shown as colors using a “Rainbow” look up table where the high intensity is represented by warm colors (maximum white, then red, etc) and the lower intensity is represented by cooler colors (minimum black, then blue, etc).
Movie S4. Fluctuations of myosin XI and VAMP signals at the cell apex of the transgenic line analyzed in Supporting Figure S5. Distribution of 3xmEGFP-myosin XIa and 3xmCherry-
VAMP by confocal microscopy. Average projections of the corresponding z-stacks (9 confocal

slices) are shown in the top two panels and the merged image in the third one with VAMP in red and myosin XI in green. Images were acquired at 5 sec intervals for the complete time series.
The intensity is shown as colors using a “Rainbow” look up table where the high intensity is represented by warm colors (maximum white, then red, etc) and the lower intensity is represented by cooler colors (minimum black, then blue, etc).
Movie S5. Uncoupling of myosin XI clusters and F-actin.
Distribution of Lifeact-mCherry and
3xmEGFP-myosin XIa after treatment with 0.5 µM latrunculin B by confocal microscopy.
Average projections of the corresponding z-stacks (6 confocal slices) are shown with F-actin in red and myosin XI in green. Images from the same transgenic line than that shown in Figure 5 were acquired at 5 sec intervals for the complete time series.
Movie S6. Ectopic myosin XI-rich clusters anticipate F-actin increase and become motile. The same cell of Supporting Movie S5 is shown, but the time series was enhanced to emphasize the formation of the ectopic myosin XI-rich clusters.
Movie S7. Myosin XI and VAMP72 dynamics at the cell apex are similar after and F-actin disruption.
Distribution of 3xmCherry-VAMP72 and 3xmEGFP-myosin XIa after treatment with
0.5 µM latrunculin B by confocal microscopy. Average projections of the corresponding z-stacks
(6 confocal slices) are shown with VAMP in red and myosin XI in green.
Movie S8. Ectopic myosin XI-rich clusters (green) coincide with VAMP72 signal (red), their signal increases simultaneously before becoming motile. The same cell of Supporting Movie S8 is shown, but the time series was enhanced to emphasize the formation of the ectopic myosin XIrich clusters and their co-localization with VAMP.