Figure 5.
CSPP1 binds to sites where MT lattices are damaged. (A) Kymographs of GMPCPP- (left) and Taxol-stabilized (right) MT seeds. 5 nM GFP-CSPP-L was flushed in during acquisition in absence of free taxol or tubulin. Scale bars, 2 μm (horizontal) and 30 s (vertical). (B) GFP-CSPP-L intensity profile of developing accumulation after flow-in of experiments done in A. (C) Schematic representation of laser damage of MT lattice at a region with no prior GFP-CSPP-L accumulation (left) and time-lapse images (right) of laser damage of a GMPCPP-stabilized MT seed in presence of 5 nM GFP-CSPP-L in absence of soluble tubulin. The MT region illuminated with the 532-nm pulsed laser is highlighted by a white arrowhead. The blue arrowhead indicates the damage inflicted on the coverslip. Scale bar, 2 µm. (D) Kymograph corresponding to time-lapse images shown in C. The laser-illuminated MT region is highlighted by a red lightning bolt. Scale bars, 2 μm (horizontal) and 30 s (vertical). (E) Averaged GFP-CSPP-L intensity profiles after photodamage (from kymographs as shown in D). Plots were aligned using half-maximum effective intensity values from nonlinear regression fits as reference points. Dashed lines represent SEM. Number of events analyzed, n = 15 from three independent experiments. (F) Kymograph illustrating laser damage of dynamic MT lattice in presence of 5 nM GFP-CSPP-L and 15 µM soluble tubulin. The laser-illuminated MT region is highlighted by a red lightning bolt. Scale bars, 2 μm (horizontal) and 30 s (vertical). (G and H) Mean intensity of GFP-CSPP-L (G) or tubulin (H) normalized to the intensity before damage (set at 100) over time at the photodamage site (from kymographs as shown in F). Dashed lines represent SEM. Number of events analyzed, n = 24 from four independent experiments. (I) Kymographs illustrating complete severing of dynamic MT lattice in presence of 15 µM soluble tubulin with or without 5 nM GFP-CSPP-L. The laser-illuminated MT region is highlighted by a red lightning bolt, the newly generated ends are indicated by white arrows. Scale bars, 2 μm (horizontal) and 30 s (vertical). (J) Plot showing the immediate fate of newly generated plus ends upon complete severing in presence of 15 µM soluble tubulin with or without 5 nM GFP-CSPP-L. Number of fragments analyzed; control, n = 46; CSPP-L, n = 56. Bars represent pooled data from four independent experiments. (K) Time lapse images of photodamage experiments in COS-7 cells overexpressing GFP-CSPP-L and β-tubulin-mCherry. Arrowheads indicate the events where MTs were damaged (white) or severed (blue). Imaging was performed using spinning disk microscopy and photodamage was induced with a 355-nm laser. Scale bars, 10 µm (left) and 4 µm (zoom). (L) Kymographs of the events shown in F. Scale bars, 1 μm (horizontal) and 20 s (vertical). See also Video 5.

CSPP1 binds to sites where MT lattices are damaged. (A) Kymographs of GMPCPP- (left) and Taxol-stabilized (right) MT seeds. 5 nM GFP-CSPP-L was flushed in during acquisition in absence of free taxol or tubulin. Scale bars, 2 μm (horizontal) and 30 s (vertical). (B) GFP-CSPP-L intensity profile of developing accumulation after flow-in of experiments done in A. (C) Schematic representation of laser damage of MT lattice at a region with no prior GFP-CSPP-L accumulation (left) and time-lapse images (right) of laser damage of a GMPCPP-stabilized MT seed in presence of 5 nM GFP-CSPP-L in absence of soluble tubulin. The MT region illuminated with the 532-nm pulsed laser is highlighted by a white arrowhead. The blue arrowhead indicates the damage inflicted on the coverslip. Scale bar, 2 µm. (D) Kymograph corresponding to time-lapse images shown in C. The laser-illuminated MT region is highlighted by a red lightning bolt. Scale bars, 2 μm (horizontal) and 30 s (vertical). (E) Averaged GFP-CSPP-L intensity profiles after photodamage (from kymographs as shown in D). Plots were aligned using half-maximum effective intensity values from nonlinear regression fits as reference points. Dashed lines represent SEM. Number of events analyzed, n = 15 from three independent experiments. (F) Kymograph illustrating laser damage of dynamic MT lattice in presence of 5 nM GFP-CSPP-L and 15 µM soluble tubulin. The laser-illuminated MT region is highlighted by a red lightning bolt. Scale bars, 2 μm (horizontal) and 30 s (vertical). (G and H) Mean intensity of GFP-CSPP-L (G) or tubulin (H) normalized to the intensity before damage (set at 100) over time at the photodamage site (from kymographs as shown in F). Dashed lines represent SEM. Number of events analyzed, n = 24 from four independent experiments. (I) Kymographs illustrating complete severing of dynamic MT lattice in presence of 15 µM soluble tubulin with or without 5 nM GFP-CSPP-L. The laser-illuminated MT region is highlighted by a red lightning bolt, the newly generated ends are indicated by white arrows. Scale bars, 2 μm (horizontal) and 30 s (vertical). (J) Plot showing the immediate fate of newly generated plus ends upon complete severing in presence of 15 µM soluble tubulin with or without 5 nM GFP-CSPP-L. Number of fragments analyzed; control, n = 46; CSPP-L, n = 56. Bars represent pooled data from four independent experiments. (K) Time lapse images of photodamage experiments in COS-7 cells overexpressing GFP-CSPP-L and β-tubulin-mCherry. Arrowheads indicate the events where MTs were damaged (white) or severed (blue). Imaging was performed using spinning disk microscopy and photodamage was induced with a 355-nm laser. Scale bars, 10 µm (left) and 4 µm (zoom). (L) Kymographs of the events shown in F. Scale bars, 1 μm (horizontal) and 20 s (vertical). See also Video 5.

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