Supplemental Material

Figure 1: Movies of GFP-CLIP-170 and GFP-H1 microtubule plus-end tracking behavior in vivo. Adherent cultures of Cos7 cells were grown on glass coverslips, transiently transfected with plasmids expressing the indicated CLIP- 170 constructs, and visualized 24-30 hours after transfection. Coverslips were inverted onto slides, sealed with VALAP, and imaged by taking streaming video at 0.2 second intervals on a Cascade 512B CCD camera driven by MetaMorph software (60x 1.4NA objective, 1.3x optivar).

Figure 2: Fitting of cosedimentation data to a quadratic binding curve To demonstrate that calculating [free tubulin] from [bound CLIP-170] does not introduce significant artifacts, the binding of H1 to Taxol microtubules was fit to a quadratic binding curve CT/C₀=[(K_d+T₀+C₀)-[ (K_d+T₀+C₀)2-4C₀T₀]^1/2]/2C₀ Where CT is the CLIP-170:tubulin complex; C0 is concentration of CLIP-170 added; and T₀ is the concentration of polymerized tubulin added.

This article contains the following supporting material:

• Movie 1a - GPP-CLIP-170 in COS7
• Movie 1b - GFP-CLIP-170 H1(head) fragment in COS7
• Movie 1c - GFP-CLIP-170 H1 in COS7, higher expression
• Movie 1d - Same cell as in movie 1c, with notable occurrences boxed.

  Box1: The microtubule in figure 2G

  Box2: Example of de novo CLIP-170 appearance that is unsuitable for kymograph analysis because the microtubule bends.

  Box3: A treadmilling microtubule that is polymerizing at the front end and depolymerizing at the back end. Because this is an unusual observation in our experiments, we did not point it out in the text.

  Box4: A pair of microtubules at the edge that appear to flicker. Our interpretation is that these microtubules are undergoing repeated cycles of pause/short catastrophe followed by short growth phases. CLIP-170is only present during the short growth phases and therefore gives the appearance of flickering. This behavior is very common at the edges of cells. Note that this behavior will give the appearance of pause or capture if movies are taken at low frame rates.

  Unlabeled: These boxes show examples that are intermediate between the clear rescues in boxes 1 and 2 and the flickering in box 4.