Adenomatous polyposis coli on microtubule plus ends in cell extensions can promote microtubule net growth with or without EB1

Abstract

In interphase cells, the adenomatous polyposis coli (APC) protein accumulates on a small subset of microtubules (MTs) in cell protrusions, suggesting that APC may regulate the dynamics of these MTs. We comicroinjected a nonperturbing fluorescently labeled monoclonal antibody and labeled tubulin to simultaneously visualize dynamics of endogenous APC and MTs in living cells. MTs decorated with APC spent more time growing and had a decreased catastrophe frequency compared with non-APC-decorated MTs. Endogenous APC associated briefly with shortening MTs. To determine the relationship between APC and its binding partner EB1, we monitored EB1-green fluorescent protein and endogenous APC concomitantly in living cells. Only a small fraction of EB1 colocalized with APC at any one time. APC-deficient cells and EB1 small interfering RNA showed that EB1 and APC localized at MT ends independently. Depletion of EB1 did not change the growth-stabilizing effects of APC on MT plus ends. In addition, APC remained bound to MTs stabilized with low nocodazole, whereas EB1 did not. Thus, we demonstrate that the association of endogenous APC with MT ends correlates directly with their increased growth stability, that this can occur independently of its association with EB1, and that APC and EB1 can associate with MT plus ends by distinct mechanisms.

Figure 1.

Alexa Fluor 568-labeled anti-N-APC mAb (Ax568-N-APC) as a probe to visualize endogenous APC. (A) Domain map of APC protein showing binding sites for specific proteins and epitopes of the antibodies used in this study. Note that the epitope of anti-N-APC is not overlapped with any known functional domains of APC. (B) Fixed MDCK cells were stained with anti-tubulin mAb (green; MTs), Ax568-N-APC (red; anti-N-APC), and anti-M-APC polyclonal antibody (blue; anti-M-APC). Both antibodies colocalize specifically to clusters near MT ends primarily in the cell protrusion. Bar, 5 μm. (C) GFP-APC expressed in MDCK cells, which, like endogenous APC localizes to clusters in the cell protrusion, also localizes along MT lattices throughout the cell. Also see Movie 1. Bar, 10 μm. (D-F) An MDCK cell was fixed 3 h after microinjection with Ax568-N-APC (outlined cell) and then followed by immunostaining. Ax568-N-APC (D), anti-M-APC (E), and merged image (F). Bar, 10 μm.

Figure 2.

APC associates transiently with MT ends during all phases of dynamic instability. MTs (green) and endogenous APC (red) were visualized by microinjecting Alexa Fluor 488-tubulin and Alexa Fluor 568-anti-N-APC mAb together into MDCK cells, pairs of images of the two probes were captured over time, and the image pairs were color encoded and overlaid. (A) Spinning disk confocal image from the first frame of Movie 3; the inset shows the phase contrast picture of the same cell. Note that only a small subset of MTs concentrated near the leading edge have an APC cluster at their plus end. Bar, 8 μm. (B) Image series from the region delineated by the white box in A (Movie 4). APC clusters remain associated with growing MT plus ends, and the APC dissociates when the MT switches to shortening (arrowheads). APC clusters also remain stable in the cell protrusion in the absence of association with MT plus ends (small arrows). Bar, 2 μm. (C) Typical MT “life history plots” of the position of different MT plus ends in the same cell over time with and without bound APC. Time during APC-MT association is indicated by red symbols. (D) Total internal reflection fluorescence microscopy image series taken with a high-speed intensified CCD camera reveal the association of an APC cluster that remains associated with a shortening MT plus end (arrow). Also see Movie 5. Bar, 1 μm. (E) Dual-color kymograph of the region highlighted by a white line in D, showing the association of APC with a shortening MT over time.

Figure 3.

MTs (green) and Ax568-N-APC (red) in 341 (wild type; A) and 335 (APC deficient; B) mouse primary fibroblasts (Movies 6 and 7, respectively). Note that APC signal is neither detected at MT ends nor in peripheral clusters in the APC deficient cells in B. Red granule-like puncta in the cell center are likely nonspecific accumulation of the labeled mAb in lysosomes and can sometimes also be seen in MDCK cells in Figure 2A. Bars in A and B, 5 μm. (C) Magnified image sequence from the region delineated by the white box in A showing the association of APC on dynamic MT ends. Bar, 2 μm. (D) Typical life history plot of APC-decorated MTs (blue) versus non-APC-decorated MTs in wild-type (green) and APC-deficient (brown) cells. Time during APC-MT association marked by red symbols.

Figure 4.

EB1 and APC subcellular localizations are independent. (A-D) 335 (APC-deficient) primary mouse fibroblast. (E-H) 341 (wild-type) primary mouse fibroblast. (I-L) 341 fibroblasts expressing double-stranded RNA to inhibit translation of EB1. (A, E, and I) Anti-EB1 immunofluorescence. (B, F, and J) Anti-tubulin immunofluorescence. (C, G, and K) Anti-M-APC immunofluorescence. (D, H, and L) Color overlay with EB1 in green, MTs in red, and APC in blue. Insets in all images show the magnified region indicated by the boxed area in the same panel. APC deficiency does not affect the MT plus end localization of EB1 (compare A-D to E-H). RNAi inhibition of EB1 protein expression level does not change APC localization to clusters in cell protrusions (E-H and I-L). Note that the anti-M-APC antibody produces a nonspecific perinuclear staining that is present in wild-type, APC-deficient, and EB1-deficient fibroblasts. Bars in D, H, and L, 10 μm.

Figure 5.

APC and EB1 colocalization is a spatiotemporally restricted event. Ax568-N-APC mAb was microinjected into MDCK cells stably expressing EB1-GFP, and total internal reflection fluorescent images were collected 3 h after microinjection (Movie 8). Many EB1 comets lack associated APC, and many APC clusters lack associated EB1; however, a small population of the two structures colocalize (arrow for EB1 and arrowhead for APC) for a short time during the later phases of a MT growth excursion. Bar, 3 μm.

Figure 6.

APC dynamic behavior is independent of EB1. (A) Time-lapse spinning disk confocal image series of Ax568-N-APC (red) and Ax 488 MTs (green) in an EB1-RNAi-expressing wild-type mouse primary fibroblast (Movie 9). Endogenous APC exhibits dynamic interactions with MT plus ends in the absence of EB1. Bar, 5 μm. (B) Examples of MT life history plots for APC-decorated MTs in cells expressing EB1-RNAi. Times of APC association are indicated by red symbols. (C) After time-lapse filming of the cell in A, the cell was fixed and EB1 was immunolocalized. The outline of the cell is shown with a dotted white line. This cell lacks EB1 comets, whereas the neighboring cell (asterisk) that was not expressing the EB1-siRNA construct clearly displays EB1comets, indicating that the cell in A had reduced EB1 expression. Bar, 10 μm.

Figure 7.

EB1 requires MT growth for plus-end localization, whereas APC does not. (A-D) MDCK cell stably expressing GFP-EB1 (A) were treated with 100 nM nocodazole for 15 min to inhibit MT plus-end assembly/disassembly dynamics, fixed, and MTs (B) and APC (C) were immunolocalized. The density of MTs is indistinguishable from untreated cells (i.e., see Figure 4) and APC clusters still localize near the plus ends of MTs in cell protrusions; however, EB1 comets at MT plus ends are absent. Bar, 3 μm. (E) Spinning disk confocal image series of Ax568-N-APC (red) and Ax488 tubulin (green) in an MDCK cell treated with 100 nM nocodazole. MT plus ends neither grow nor shorten, yet APC clusters remain in tight association with plus ends (arrow) (Movie 10). Bar, 2 μm. (F) Total internal reflection fluorescence images of a GFP-EB1 expressing MDCK cell before (left) and after (right) perfusion of 100 nM nocdazole. EB1 comets quickly disappear after wash-in of nocodazole (Movie 11). Bar, 5 μm.