Langford KJ et al. (2006), Examination of actin and microtubule dependent ...

XB-ART-812

BMC Cell Biol 2006 Jan 19;7:3. doi: 10.1186/1471-2121-7-3.

Show Gene links Show Anatomy links

Examination of actin and microtubule dependent APC localisations in living mammalian cells.

Langford KJ , Askham JM , Lee T , Adams M , Morrison EE .

Abstract
The trafficking of the adenomatous polyposis coli (APC) tumour suppressor protein in mammalian cells is a perennially controversial topic. Immunostaining evidence for an actin-associated APC localisation at intercellular junctions has been previously presented, though live imaging of mammalian junctional APC has not been documented. Using live imaging of transfected COS-7 cells we observed intercellular junction-associated pools of GFP-APC in addition to previously documented microtubule-associated GFP-APC and a variety of minor localisations. Although both microtubule and junction-associated populations could co-exist within individual cells, they differed in their subcellular location, dynamic behaviour and sensitivity to cytoskeletal poisons. GFP-APC deletion mutant analysis indicated that a protein truncated immediately after the APC armadillo repeat domain retained the ability to localise to adhesive membranes in transfected cells. Supporting this, we also observed junctional APC immunostaining in cultures of human colorectal cancer cell line that express truncated forms of APC. Our data indicate that APC can be found in two spatially separate populations at the cell periphery and these populations can co-exist in the same cell. The first localisation is highly dynamic and associated with microtubules near free edges and in cell vertices, while the second is comparatively static and is closely associated with actin at sites of cell-cell contact. Our imaging confirms that human GFP-APC possesses many of the localisations and behaviours previously seen by live imaging of Xenopus GFP-APC. However, we report the novel finding that GFP-APC puncta can remain associated with the ends of shrinking microtubules. Deletion analysis indicated that the N-terminal region of the APC protein mediated its junctional localisation, consistent with our observation that truncated APC proteins in colon cancer cell lines are still capable of localising to the cell cortex. This may have implications for the development of colorectal cancer.

PubMed ID: 16423286
PMC ID: PMC1386658
Article link: BMC Cell Biol

Species referenced: Xenopus
Genes referenced: actl6a ctnnb1 mapre1 nrk

Article Images: [+] show captions

	Figure 1. Schematic of full-length and deletion GFP-APC constructs used in this study (not to scale). Panel A. Full-length GFP-APC including known APC domains. Panel B. pEGFP-APCΔC construct lacking the last 54 amino acids of APC. Panel C. pEGFP-APCΔN+ΔC construct lacking the first 206 and last 54 amino acids of APC. Panel D. pEGFP-APC-C construct consisting of the last 170 amino acids of APC. Panel E. pEGFP-APC-N consisting of the first 746 amino acids of APC.
	Figure 2. Western blotting of endogenous APC and GFP-APC constructs expressed in COS-7 cells. Panel A: Western blot of cell extracts from COS-7 (lane 1), HeLa (lane 2), MDCK (lane 3), NRK-52E (lane 4) and Caco-2 (lane 5), 10 μg total protein was loaded for each extract and the membrane probed with the ALI 12-28 antibody specific for the APC N-terminus. Full-length APC (310 kDa) can be detected in all cell lines except CaCo-2 cells that possess a much smaller truncated APC protein (not shown). Panel B: Western blot of COS-7 cell extracts transfected with plasmids directing the expression of GFP-APC (lane 1), GFP-APC-ΔN (lane 2), GFP-APC-ΔN+ΔC (lane 3), and untransfected COS-7 cells (lane 4). Prior to western blotting extracts were immunoprecipitated with a polyclonal anti-GFP antibody. The immunoprecipitation from each extract was loaded onto a gradient gel, subjected to SDS-PAGE, transferred to nitrocellulose and the membrane probed with a polyclonal anti-GFP antibody. Panel C: Western blot of the COS-7 cell extract transfected with the plasmid directing the expression of GFP-APC-N construct (lane 1) or a non-transfected COS-7 cell extract (lane 2). 20 μg of each protein extract was loaded onto a 10% non-gradient gel and the membrane probed with a polyclonal anti-GFP antibody.
	Figure 3. Junction formation and APC localisation in COS-7 cells. Panels A-C. COS-7 cells contain cadherins and endogenous APC (arrowheads) at sites of cell-cell contact, Cadherin green and APC red, panel C is a merged image. Panels D-F. COS-7 cells also have the junctional protein β-catenin localised along with endogenous APC (arrowheads) at adhesive membranes. β-catenin green, APC red and DAPI blue, panel F is a merged image. Panels G-I. Cortical actin and APC (arrowheads) are associated with sites of cell-cell contact in COS-7 cells. Actin red, APC green and DAPI blue, Panel I is a merged image. Bars = 10 μm.
	Figure 4. Detailed imaging of full-length GFP-APC in living COS-7 cells. Panel A. In subconfluent cells GFP-APC decorates the distal tips of microtubules at cell vertices (arrows, additional file 1, 5s time lapse). Panels B-D. GFP-APC clusters decorating distal tips of microtubules in fixed GFP-APC expressing cells. GFP-APC green, tubulin red and DAPI blue, panel D is a merged image. Panels E-H. GFP-APC puncta can be deposited at the cell cortex (arrows, additional file 2). Panels I-L. GFP-APC puncta could be seen undergoing retrograde movements at the cell periphery (arrowhead, additional file 3). Panels M-P. Part of an original GFP-APC puncta is lost from a shrinking tip while the remaining attached portion continues retrograde movement (arrowheads, additional file 4). Panels Q-T. Some shrinking microtubules tipped with GFP-APC puncta undergo re-growth after periods of shrinkage (arrowhead, move 5). Bars = 10 μm.
	Figure 5. Additional localisations seen in GFP-APC expressing COS-7 cells. Panel A. In some cells GFP-APC can be seen at the centrosome (arrowhead). Panels B-D. Localisation of GFP-APC to the centrosome was confirmed in immunostained cells using the centrosomal marker γ-tubulin. GFP-APC green, γ-tubulin red, panel D is a merged image. Panel E. A minor population of transfected cells had a comet-like GFP-APC distribution (additional file 6). Panels F- H. Co-staining of GFP-APC expressing COS-7 cells with EB1 confirmed the two proteins co-localise at growing microtubule tips in some cells. GFP-APC green, EB1 red, Panel H is a merged image. Panels I-L. Occasional cells contained GFP-APC puncta that were rapidly transported through the cytoplasm (arrowhead, additional file 7). Bars = 10 μm
	Figure 6. Localisation of GFP-APC to junctional membranes in COS-7 cells. Panel A. In highly confluent cells GFP-APC showed a purely junctional localisation (additional file 8,). Panels B-D. GFP-APC at the cortex in COS-7 cells is located close to the junctional protein α-catenin. GFP-APC green, α-catenin red, panel D is a merged image. Panels E-G. GFP-APC is also closely associated with cortical actin at sites of cell-cell adhesion. GFP-APC green, actin red, Panel G is a merged image. Panel H. Both junctional (arrowheads) and microtubule-associated (arrow) GFP-APC populations can co-exist in the same cell (additional file 9). Bars = 10 μm.
	Figure 7. Effects of microtubule depolymerisation on GFP-APC distribution in COS-7 cells. Panels A-C. GFP-APC expressing COS-7 cells were incubated for 60 min in 5 μg/ml Nocodazole. GFP-APC associated with remnant microtubules (arrows) can be found in Nocodazole treated cells. However, junctional GFP-APC (arrowheads) is not associated with microtubules and appears unaffected by Nocodazole treatment. GFP-APC green, tubulin red, panel C is a merged image. Panels D-G. Nocodazole treatment of living cells possessing microtubule-associated and junctional-associated GFP-APC (additional file 10). Microtubule-associated GFP-APC (arrow) disperses over time but junctional GFP-APC puncta (arrowheads) are unaffected.
	Figure 8. Effect of actin poison on GFP-APC localisation in COS-7 cells. Panels A-D. Treatment of GFP-actin expressing COS-7 cells with 1 μg/ml Cytochalasin D gradually perturbed the integrity of the cortical actin ribbon. With increasing time of exposure to drug the cortical actin ribbon thins, snaps and contracts (arrowheads, additional file 11 arrows). Bars = 20 μm. Panels E-H. Cytochalasin D treatment (1 μg/ml) of cells possessing both microtubule-associated and junctional GFP-APC (additional file 12). Following drug addition cell-cell contacts decorated with GFP-APC puncta can be seen to break (black arrow), followed by movement of the GFP-APC puncta along the cell edge towards cell vertices (white arrow shows direction of movement). The dynamic behaviour of microtubule-associated GFP-APC continues within the constraints imposed by retraction of the free cell edge. Bars = 10 μm. Panels I-K. Paraformaldehyde fixed, phalloidin-stained cells expressing GFP-APC after 30 min treatment with Cytochalasin D (1 μg/ml). Co-localization of GFP-APC with actin aggregates in the cytoplasm (arrows) and with cortical actin (arrowheads) is seen. GFP green, phalloidin red, panel K is a merged image. Bars = 20 μm.
	Figure 9. Distribution of truncated APC proteins. Panels A and B. GFP-APCΔC and GFP-APCΔNΔC localize to both the cortex (arrowheads) and to microtubules (data not shown) in a manner similar to that previously seen for GFP-APC. Panel C. The short C-terminal fragment GFP-APC-C1 has a cytoplasmic localization similar to that seen with GFP alone (data not shown). Panel D. GFP-APC-N localizes to the centrosome (arrow), cytoplasmic puncta and the cortex (arrowheads, additional file 13). Panels E-G. GFP-APC-N expressing cells co-immunostained for the centrosomal marker γ-tubulin show GFP-APC-N localises to the centrosome (arrow). GFP-APC-N green, γ-tubulin red, panel G is a merged image. Panels H-J. GFP-APC-N expressing cells co-immunostained for β-catenin confirm the GFP-APC-N junctional localisation (arrowheads). GFP-APC-N green, β-catenin red. Panel J is a merged image. Bars = 10 μm.
	Figure 10. Junctional staining of the colon cancer cell line Caco-2. Panels A-C. Confluent Caco-2 cells were co-immunostained with the polyclonal M-APC antibody and for microtubules. A microtubule-independent junctional localisation is seen. Tubulin green, APC red and DAPI blue. Panel C is a merged image. Panels D-F. Confluent Caco-2 cells immunostained using the monoclonal antibody ALI 12-28. Tubulin red, APC green and DAPI blue. Panel F is a merged image. Panels G-I. A monoclonal antibody specific for the APC C-terminus shows no specific APC localisation, as would be expected with the truncated APC expressed in Caco-2 cells. Tubulin red, APC green and DAPI blue, panel I is a merged image. Panels J-L. Actin and APC co-staining in Caco-2 cells confirms the localisation of APC to junctional membranes. Actin green, M-APC red, panel L is a merged image. Bars = 10 μm.

References [+] :

Ballestrem, Actin dynamics in living mammalian cells. 1998, Pubmed

Ballestrem, Actin dynamics in living mammalian cells. 1998, Pubmed
Banks, Adenomatous polyposis coli associates with the microtubule-destabilizing protein XMCAK. 2004, Pubmed , Xenbase
Barth, Dissecting interactions between EB1, microtubules and APC in cortical clusters at the plasma membrane. 2002, Pubmed
Bienz, The subcellular destinations of APC proteins. 2002, Pubmed
Bienz, Spindles cotton on to junctions, APC and EB1. 2001, Pubmed
Bright-Thomas, APC, beta-Catenin and hTCF-4; an unholy trinity in the genesis of colorectal cancer. 2003, Pubmed
Brocardo, Redefining the subcellular location and transport of APC: new insights using a panel of antibodies. 2005, Pubmed
Dayanandan, Dynamic properties of APC-decorated microtubules in living cells. 2003, Pubmed
Etienne-Manneville, Cdc42 regulates GSK-3beta and adenomatous polyposis coli to control cell polarity. 2003, Pubmed
Faux, Restoration of full-length adenomatous polyposis coli (APC) protein in a colon cancer cell line enhances cell adhesion. 2004, Pubmed
Hamada, A Drosophila APC tumour suppressor homologue functions in cellular adhesion. 2002, Pubmed
Jimbo, Identification of a link between the tumour suppressor APC and the kinesin superfamily. 2002, Pubmed , Xenbase
Joslyn, Dimer formation by an N-terminal coiled coil in the APC protein. 1993, Pubmed
Kawasaki, Asef, a link between the tumor suppressor APC and G-protein signaling. 2000, Pubmed
Louie, Adenomatous polyposis coli and EB1 localize in close proximity of the mother centriole and EB1 is a functional component of centrosomes. 2004, Pubmed
Matsumine, Binding of APC to the human homolog of the Drosophila discs large tumor suppressor protein. 1996, Pubmed
Mimori-Kiyosue, Adenomatous polyposis coli (APC) protein moves along microtubules and concentrates at their growing ends in epithelial cells. 2000, Pubmed , Xenbase
Mogensen, The adenomatous polyposis coli protein unambiguously localizes to microtubule plus ends and is involved in establishing parallel arrays of microtubule bundles in highly polarized epithelial cells. 2002, Pubmed
Moore, MCAK associates with the tips of polymerizing microtubules. 2005, Pubmed
Morrison, The cellular distribution of the adenomatous polyposis coli tumour suppressor protein in neuroblastoma cells is regulated by microtubule dynamics. 1997, Pubmed
Morrison, EB1, a protein which interacts with the APC tumour suppressor, is associated with the microtubule cytoskeleton throughout the cell cycle. 1998, Pubmed
Munemitsu, The APC gene product associates with microtubules in vivo and promotes their assembly in vitro. 1994, Pubmed
Nakamura, Critical role for the EB1 and APC interaction in the regulation of microtubule polymerization. 2001, Pubmed
Newton, MCAK, a Kin I kinesin, increases the catastrophe frequency of steady-state HeLa cell microtubules in an ATP-dependent manner in vitro. 2004, Pubmed
Näthke, The adenomatous polyposis coli tumor suppressor protein localizes to plasma membrane sites involved in active cell migration. 1996, Pubmed
Rosin-Arbesfeld, Actin-dependent membrane association of the APC tumour suppressor in polarized mammalian epithelial cells. 2001, Pubmed
Su, Association between wild type and mutant APC gene products. 1993, Pubmed
Tighe, Aneuploid colon cancer cells have a robust spindle checkpoint. 2001, Pubmed
Watanabe, Interaction with IQGAP1 links APC to Rac1, Cdc42, and actin filaments during cell polarization and migration. 2004, Pubmed
Wen, EB1 and APC bind to mDia to stabilize microtubules downstream of Rho and promote cell migration. 2004, Pubmed
Zumbrunn, Binding of the adenomatous polyposis coli protein to microtubules increases microtubule stability and is regulated by GSK3 beta phosphorylation. 2001, Pubmed