Supplemental Material

This article contains the following supporting material:

• Movie 1 - Behavior of GFP-APC in MDCK cells. Image is 21 X 24 μm. Time in min:sec is shown.

• Movie 2 - Mitosis in MDCK cells proceeds normally after microinjection of Ax568-N-APC mAb. Cells were synchronized by nocodazole treatment for 16 hr, released from nocodazole for 3 hours, microinjected with antibody, and subjected to time lapse filming during the cell cycle following release from the block. Cells marked with an arrow contain labeled mAb, and proceed through mitosis. Arrow disappears when mitosis is complete. Image is 145 X 108 μm. Time in hr:min is shown.

• Movie 3 - Endogenous APC (red) visualized with Ax568-N-APC mAb and MTs (green) visualized with Alexa488 tubulin in a living MDCK cell. Image is 22 X 19 μm. Time in min:sec is shown.

• Movie 4 - A magnified region from Movie 3 (white box in Figure 2A) of endogenous APC (red) visualized with Ax568-N-APC mAb and MTs (green) visualized with Alexa488 tubulin in an MDCK cell. Image is 15 X 17 μm. Time in min:sec is shown.

• Movie 5 - Endogenous APC (red) visualized with Ax568-N-APC mAb and MTs (green) visualized with Alexa488 tubulin in a living wild type mouse primary fibroblast. Image is 25 X 20 μm. Time in min:sec is shown

• Movie 6 - Endogenous APC (red) visualized with Ax568-N-APC mAb and MTs (green) visualized with Alexa488 tubulin in a living APC-deficient mouse primary fibroblast. No APC clusters are associated with MTs in the cell periphery. The spherical red clusters moving on MTs in central cell regions are likely fluorescent antibody undergoing degradation in lysosomes. Image is 25 X 20 μm. Time in min:sec is shown.

• Movie 7 - APC clusters can associate with MT plus ends during the shortening phase. High-speed image acquisition of endogenous APC (red) visualized with Ax568-N-APC mAb and MTs (green) visualized with Alexa488 tubulin by TIR-FM with an electron multiplier CCD camera. Image is 14 X 6 μm. Time in sec:msec is shown.

• Movie 8 - EB1-GFP (green) and endogenous APC (red) visualized with Ax568-N-APC mAb in a living MDCK cell visualized with TIR-FM microscopy. EB1 comets only very transiently co-localize with APC clusters in the cell periphery. Image is 16 X 16 ìm for each movie. Time in min:sec is shown.

• Movie 9 - Dynamic behavior of endogenous APC (red) visualized with Ax568-N-APC mAb and MTs (green) visualized with Alexa488 tubulin in a wild type mouse primary fibroblast expressing siRNAi against EB1. Following live cell imaging, the cell was fixed and processed for anti-EB1 immunofluorescence to confirm EB1 depletion (Figure 6C). Image is 26 X 17 μm. Time in min:sec is shown.

• Movie 10 - Application of low concentration (100nM) nocodazole to a living MDCK cell with endogenous APC (red) visualized with Ax568-N-APC mAb and MTs (green) visualized with Alexa488 tubulin. The arrow highlights an APC cluster that remains in tight association with the end of a MT that neither grows or shortens throughout the movie. Image is 15 X 20 μm. Time in min:sec is shown.

• Movie 11 - Rapid disappearance of EB1-GFP comets after nocodazole (100nM) wash-in. TIR-FM imaging of GFP-EB1 in an MDCK cell. In the beginning of the movie, EB1 comets move throughout the cytoplasm. The movie then goes out-of focus for several frames, during which time 100nM nocodazole-containing media was perfused on the cell. Image is 25 X 19 μm. Time in min:sec is shown.

• Table 1 - Time spent from nuclear envelope breakdown to metaphase for control (non-injected) and Ax568-N-APC mAb-microinjected MDCK cells.

• Table 2 - Cell motility of control (non-injected) and Ax568-N-APC mAb-microinjected MDCK cells. Microinjection of anti-N-APC did not change cell motility.

• Figure 1 - A, Immunoblotting of endogenous and EB1-GFP in MDCK cells stably expressing GFP-EB1. B, Immunoblotting of tubulin (upper) and EB1 (lower) in wild type mouse primary fibroblasts transfected with a vector expressing a EB1-siRNA construct. The cells were transfected with EB1-siRNA expression vector (see Material and Method) and lysates were prepared for immunoblotting two days later. C, Time course of EB1 depletion and its effect on the expression of an untargeted protein (tubulin). lane1, 2 days; lane2, 5 days

• Figure 2 - Immunostaining of APC (left), MTs (center), and an overlayed image (right) of MDCK cells treated 15 min with 10μM nocodazole. Many MTs depolymerize from the cell periphery, with nocodazole-resistant MTs remaining in the cell center. Note that most peripheral APC clusters disappeared concomitant with MT loss from the cell periphery. Bar = 15μm.