Supplemental Material

This article contains the following supporting material:

• Movie S1 - Time lapse of EB1-GFP comets in control (no receptor) 293 cells

  Images were collected at 3 second intervals. This movie contains to the image sequence presented in figure 6A (no receptor). Movies run at 1 frame per second. One pixel = 0.13 μm.

• Movie S2 - Time lapse of EB1-GFP comets in 293 cells stably expressing APC^1-1450.

  Images were collected at 3 second intervals.This movie contains the image sequence presented in figure 6A (APC^1-1450). Movies run at 1 frame per second. One pixel = 0.13 μm.

• Movie S3 - Time lapse of EB1-GFP comets in 293 cells stably expressing APC^58-1450.

  Images were collected at 3 second intervals. This movie contains the image sequence presented in figure 6A (APC^58-1450). Movies run at 1 frame per second. One pixel = 0.13 μm.

• Figure S1 - siRNA of APC or EB1

  (A) Human 293 cells were transfected with siRNA directed against APC, EB1, or GAPDH (control) and extracts were prepared 48 hours post-transfection. Immunoblots were performed with antibodies against EB1 or APC and a titration of extract (15, 30 and 60 μg) is shown for each knockdown. (B) An example image demonstrating APC levels are reduced upon APC siRNA treatment relative to GAPDH siRNA (48hrs). Tubulin is also shown to illustrate that the reported phenotype occurs upon APC knockdown (DAPI (blue), Tubulin (red), APC (green)). (C) An example of EB1 levels in 293 cells treated with siRNA against GAPDH or EB1 (DAPI (blue), EB1 (Red)). Bar (B, C) = 5 μm.

• Figure S2 - Inhibition of LIS1 or CLIP170 results in distinct chromosome alignment and mitotic spindle phenotypes.

  (A) Human 293 cells were transfected with siRNA directed against LIS1, CLIP170, or GAPDH (control), extracts were prepared 24 and 48 hours post-transfection, and immunoblots were performed with antibodies against LIS1, CLIP170 or tubulin (load control). (B) Cells were fixed at 48 hrs post-transfection, stained with antibodies against tubulin (red) and DAPI (blue), and images were collected. Projections of z-sections containing the central spindle are presented for several CLIP170 and LIS1 siRNA treated cells. Bar (B) = 5 μm.

• Figure S3 - APC fragments localize to the mitotic spindle with different distribution patterns.

  (A) Stable cell lines were grown for 72 hours in the presence of ponasterone to induce expression of APC fragments, fixed and stained to visualize microtubules (tubulin), kinetochores (ACA), APC fragments (myc) and chromosomes (DAPI). Chromosomes (blue) and myc (red) staining are presented in the left panels. Areas indicated by the insets (enlarged 2-fold) are shown with either tubulin (green) and myc (red) staining or ACA (green) and myc (red) staining. Projections of 15 Z-sections selected to include the middle of the spindle are presented. Arrowheads (APC^1-1450) show myc staining at the kinetochore. Arrow (APC^1-768) shows myc dotted along spindle microtubules. Bar (B) = 5 μm.

• Figure S4 - EB1-GFP positive microtubule dynamics in interphase cells expressing dominant negative APC^1-1450.

  Stable 293 cell lines expressing APC^1-1450, APC^58-1450 or the no receptor control were transfected with EB1-GFP and interphase cells expressing minimal amounts of EB1-GFP were imaged for (2-5 minutes). (A) Example time lapse sequence of plus-end GFP-EB1 tracking at 2 second intervals. Inset shows 1.5-fold enlargement of a growing GFP-EB1 microtubule for each cell line. Arrow denotes the growing EB1-GFP microtubule tracked in the inset. (B) Example of EB1-GFP comet life histories for each cell line analyzed. Bar (A) = 5 μm.

• Table S1
• Simulation