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First published online 13 March 2007
doi: 10.1242/jcs.003913

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Microtubule-targeting-dependent reorganization of filopodia

¹ Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
² Department of Cell Biology and Genetics, Erasmus Medical Center, Rotterdam, The Netherlands

View larger version (45K): [in this window] [in a new window]   Fig. 1. Microtubules target filopodia in B16F1 melanoma cells. Fixed cells were immunostained with monoclonal anti-tubulin antibodies followed by Cy5-labeled secondary antibodies and Alexa Fluor-488-phalloidin. (A) Combined image of microtubules (green) and F-actin (red) shows a typical lamellipodium with embedded filopodia. A line connecting the bases of all filopodia (baseline) defines the peripheral region of the lamellipodium. (B) Enlarged image of upper region (boxed in A) of lamellipodium with three peripheral microtubules. One microtubule is targeted to a filopodium (+) and two are not targeted (-). (C) Enlarged image of lower region with two peripheral microtubules targeted to filopodia (+). (D) Lamellipodia were divided into five equal zones as indicated in the inset image. Zone 1 is the lamellipodium center and zones 3 are the lamellipodium wings. Zone 2 is the transition between the lamellipodium center and wings. The relative number of targeting events, peripheral microtubules and filopodia for each zone is plotted. Bar, 2 µm (A-C). Error bars indicate standard error (*P<0.01, unpaired t-test, n=28 cells).

View larger version (63K): [in this window] [in a new window]   Fig. 2. Microtubule targeting events correlate with filopodia turning and merging. B16F1 cells were co-transfected with YFP-fascin to mark filopodia and YFP--tubulin to highlight microtubules. (A) The time-lapse series shows two microtubules (MT1 and MT2) targeting a filopodium (FL1) in a lamellipodium wing. The targeted filopodium (FL1) turns toward the lamellipodium midline. A nearby nontargeted filopodium (FL2) serves as a reference. (B) The targeted filopodium (FL1) turns and completely merges with a neighboring filopodium (FL2) at 50 seconds (asterisk). A nearby nontargeted filopodium (FL3) remains about its original position. (C) Time course of turning expressed as angle change in degrees of filopodia FL1 and FL2 shown in A. (D) Time course of turning and merging of the filopodia shown in B. A positive value denotes filopodia turning towards the lamellipodium midline. (E) Time course of average difference in angle between targeted and reference filopodia pairs after the targeting event begins (n=15 targeting events). (F) Time course of cumulative percentage of the targeted filopodia that merge with neighboring filopodia after the targeting event begins (n=15 targeting events). Bars, 2 µm. Error bars indicate standard error.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Nocodazole uncouples targeting, decreases filopodia merging and increases filopodia in lamellipodia wings. B16F1 cells were allowed to spread on laminin for 30 minutes and then treated with 3 µM nocodazole for the indicated times. Cell were fixed and stained with anti-tubulin antibodies and phalloidin. (A) Time course of number of peripheral microtubules in cells treated with nocodazole. The number of peripheral microtubules rapidly decreases within 0.5 minutes. (B) Number of filopodia merging events in each lamellipodium zone in cells treated with nocodazole for 0, 0.5, 1.0, or 2.0 minutes. Merging is decreased in zones 3 at 1.0 and 2.0 minutes (compared with 0 minutes; *P<0.01, unpaired t-test, n=25 cells). (C) Number of filopodia in each zone in cells treated with nocodazole. Filopodia are increased in zones 3 at 1.0 and 2.0 minutes (compared with 0 minutes; *P<0.01, unpaired t-test, n=25 cells). Error bars indicate standard error.

View larger version (63K): [in this window] [in a new window]   Fig. 4. Total internal reflection fluorescence (TIRF) microscopy of targeting events. (A) Fixed B16F1 cells were stained with monoclonal anti-tubulin antibodies followed by Alexa Fluor-488-labeled secondary antibodies and TRITC-phalloidin. The representative wide-field and TIRF microscopy images show three targeted filopodia (arrowheads), one nontargeted filopodium (asterisk) and microtubules. The nontargeted filopodium, one targeted filopodium and all the microtubules are clearly visible by TIRF microscopy. Two targeted filopodia are not visible by TIRF. (B) Percentage of microtubules (MT) and filopodia (FL) visible by TIRF microscopy during targeting events (targeted) and nontargeting (nontargeted). Fewer targeted filopodia are visible by TIRF compared with nontargeted filopodia (*P<0.01, unpaired t-test). (C) Comparison of wide-field and TIRF microscopy of targeting events in live cells co-transfected with YFP-fascin and YFP--tubulin. As shown in the wide-field time series, a microtubule (MT1) targets a filopodium (FL3) at 00:00 seconds and another microtubule (MT2) targets a filopodium (FL2) at 00:20 seconds. As shown in the TIRF time series, FL3 and FL2 disappear after targeting. By contrast, the nontargeted filopodium (FL1) remains visible by TIRF throughout the sequence. Bars, 2 µm. Errors bars indicate standard error.

View larger version (61K): [in this window] [in a new window]   Fig. 5. Adhesion markers are not present at sites of microtubule targeting. B16F1 cells were incubated on laminin for 30 minutes, fixed in buffer containing 0.5% glutaraldehyde and 1% Triton X-100, and stained with monoclonal anti-tubulin antibodies and phalloidin. (A) Cells were stained with monoclonal anti-FAK antibodies. Cells were transfected with YFP-paxillin (B) or mRFP-VASP (C). Combined image shows adhesion sites (blue), actin (red) and microtubules (green). Insets (a-c) show combined adhesion site (blue) and actin (red) images of regions indicated by the boxes. Arrowheads indicate position of targeting sites. Graphs show percentage of targeted and nontargeted filopodia bases positive for the adhesion sites markers. Bars, 2 µm. Error bars indicate standard error.

View larger version (27K): [in this window] [in a new window]   Fig. 6. Polymerization and alignment of microtubule plus-ends at target sites. B16F1 cells were co-transfected with GFP-CLIP170 to track polymerization of microtubule plus-ends (green) and mRFP-actin (red) to visualize filopodia. A polymerizing microtubule end (arrowhead) approaches a filopodium base and then aligns with the filopodium at 00:15 seconds. Bars, 2 µm.

View larger version (45K): [in this window] [in a new window]   Fig. 7. Brief taxol treatment increases filopodia targeting. (A) B16F1 cells were treated with 3 µM taxol for 30 seconds, 1 minute or 2 minutes, and fixed in buffer containing 1% glutaraldehyde and 1% Triton X-100. Fixed cells were stained with monoclonal anti-tubulin antibodies and phalloidin. Microtubule (green) and actin (red) images are combined. Bars, 2 µm. (B) Time course of the number of peripheral microtubules in cells treated with taxol. The number of peripheral microtubules peaks at 30 seconds and rapidly declines. (C) Percentage filopodia targeted in cells treated with taxol for 30 seconds (*P<0.01 unpaired t-test). (D) Percentage positive microtubules in cells treated with taxol for 30 seconds. Error bars indicate standard error.