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First published online 11 April 2006
doi: 10.1242/jcs.02903

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Dynamic microtubules regulate the local concentration of E-cadherin at cell-cell contacts

Samantha J. Stehbens^1,*, Andrew D. Paterson^1,2,*, Matthew S. Crampton¹, Annette M. Shewan¹, Charles Ferguson^1,3, Anna Akhmanova⁴, Robert G. Parton^1,3 and Alpha S. Yap^1,

¹ Division of Molecular Cell Biology, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia
² School for Biomedical Science, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia
³ Centre for Microscopy and Microanalysis, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia
⁴ MGC Department of Cell Biology and Genetics, Erasmus Medical Center, 3000 DR Rotterdam, The Netherlands

View larger version (171K): [in a new window]   Fig. 1. A subpopulation of radially directed MTs extend into E-cadherin-based cell-cell contacts. MCF-7 cell monolayers were fixed at confluence and processed for epi-illumination immunofluorescence microscopy or electron microscopy. (A-D) Dual-label immunofluorescence imaging for E-cadherin (A) or ß-tubulin (B). A subpopulation of MTs appeared to radiate from the perinuclear area into sites of cadherin-rich cell-cell contacts (C,D); D shows the magnified boxed region in panel C. Distal tips of these MTs often appeared to terminate in concentrations (`puncta') of E-cadherin staining (D, arrowhead). Bar, 5 µM in panel D. (E-I). Gallery of electron microscopic images of junctional areas in MCF-7 cells and in hE-CHO cells. MTs (arrowheads in E), recognised by their characteristic morphology, run close to the adherens junction (between arrows), in some cases entering the dense microfilament network underlying the junctional complex. Panels F-I show lower magnification views of junctional areas in which MTs have been traced by hand and coloured to facilitate their recognition (E and F show the same image, F with MTs traced). Bars, 200 nm (F-I same magnification).

View larger version (70K): [in a new window]   Fig. 2. Orientation of MTs as MCF-7 cultures reassemble cell-cell contacts. MCF-7 cells were fixed at confluence (untreated), immediately after incubation with 4 mM EGTA (-Calcium), or 15-90 minutes after replenishment of 5 mM extracellular Ca2+. One set of cells were treated with nocodazole (100 nM, added 60 minutes prior to experiments and maintained in media for the duration of the experiments), whereas control cultures were grown in standard media supplemented with carrier (DMSO) alone. E-cadherin and ß-tubulin were identified by dual-label epi-illumination immunofluorescence microscopy. The right-hand panel of each pair represent high-power details of the regions marked by the boxes. The density of MTs at contacts was quantitated by counting the number of MTs found within 3 µm of the E-cadherin-stained contacts. Data are means ± s.e.m., n=40, in control (C) or nocodazole (N)-treated cells. Prominent (or pioneer) MTs appeared to terminate in these cadherin accumulations (arrow), which became more numerous as cell contacts extended. Treatment of MCF-7 cells with low-dose nocodazole (100 nM) inhibited the formation of these pioneer MTs at reforming contacts.

View larger version (38K): [in a new window]   Fig. 3. E-Cadherin homophilic ligation is sufficient to recruit MTs into adhesion sites. CHO cells stably expressing full-length human E-cadherin (hE-CHO) and E-cadherin lacking the p120 binding site (764-CHO) were grown to 80% confluency on glass coverslips. Latex beads (6 mm diameter) coated with either hE/Fc or ConA were allowed to adhere to the dorsal surfaces of cells for 90 minutes. Samples were immunolabelled for E-cadherin and tubulin, and visualised by laser scanning confocal microscopy. (A) Adhesion of hE/Fc-coated beads, but not ConA beads (asterisks), was associated with the accumulation of E-cadherin around the beads and projection of MTs into the sites of adhesion. (B) MT recruitment was quantified by counting the number of individual MTs that extended into contacts made between hE/Fc or ConA-coated beads and cells expressing full-length E-cadherin (hE-CHO) or CHO cells expressing the hE-Cad 764AAA mutant (764-CHO). We included all MTs that appeared to come into contact with the beads. Data are means ± s.e.m. (n=30 beads) and are representative of three separate experiments.

View larger version (95K): [in a new window]   Fig. 4. Radial MTs extend with plus-ends directed towards cadherin contacts. MCF-7 cells were triple labelled for E-cadherin, tubulin and CLIP-170 and imaged by confocal microscopy (A). A magnified boxed region of the triple label overlay identified CLIP-170 at the tips of MTs that extended into E-cadherin-based cell-cell contacts (A', arrows).

View larger version (92K): [in a new window]   Fig. 5. Nocodazole inhibits the accumulation of E-cadherin at cell-cell contacts without affecting cadherin traffic to the cell surface. (A) Low concentrations of nocodazole disrupt the morphology of cadherin-based cell-cell contacts. MCF-7 monolayers were incubated with nocodazole (100 nM, 240 minutes) or DMSO carrier (Control) before fixation and immunostaining for E-cadherin. (B) Nocodazole reduces E-cadherin accumulation in newly forming cell-cell contacts. Cells were incubated with nocodazole (100 nM, Noc) or DMSO vehicle alone (Control) for 60 minutes beforehand, and throughout the experiments. Cell-cell contacts between confluent MCF-7 cells were disrupted by chelation of extracellular Ca2+ (0 minutes) then allowed to reform by addition of Ca2+. At varying times thereafter, samples were fixed and stained for E-cadherin. E-cadherin fluorescence intensity at cell-cell contacts (E-cadherin intensity) was measured by digital image analysis. Representative images of control and nocodazole-treated cells at 30 minutes are shown. (C) Nocodazole does not affect total or surface expression levels of E-cadherin. Surface expression of E-cadherin was measured using surface trypsin protection assays in control cells or cells pre-incubated with nocodazole (100 nM, 90 minutes). Cells were lysed immediately (WCL) or after trypsinisation in the presence (+Ca) or absence (-Ca) of extracellular Ca2+. Total cellular levels of E-cadherin (E-cad) were unaffected by nocodazole and all the cadherin remained accessible to surface trypsinisation in the absence of Ca2+ in both the control and drug-treated cells. (D) Nocodazole does not affect transport of E-cadherin to the cell surface. Cells were studied at baseline (BL) or trypsinised to strip E-cadherin from the cell surface (0'), then incubated for 2-4 hours before surface biotinylation. Biotinylated proteins were separated by SDS-PAGE and immunoblotted for E-cadherin or transferrin receptor (TfR), as a loading control. Cultures were incubated in the presence of nocodazole (100 nM, pre-incubated for 1 hour, then for the duration of the experiments) or with vehicle alone (C). Results are representative of three independent experiments.

View larger version (38K): [in a new window]   Fig. 6. Mutant CLIP-170 perturbs the focal accumulation of E-cadherin at cell-cell contacts. (A) MCF-7 monolayers were transiently transfected with CLIP-170 -Tail (Tail) or with a control vector expressing GFP alone. After 12-24 hours, cells were fixed and immunolabelled for GFP and E-cadherin to identify the transfected cells and cell-cell contacts, respectively. E-cadherin accumulation was reduced at contacts between cells expressing CLIP-170 -Tail (A, arrow) in comparison with contacts between cells that were untransfected or expressing GFP alone (A, arrowhead). (B) Quantitative image analysis was used to measure E-cadherin accumulation at cell-cell contacts between transfected cells, both expressing either CLIP-170 -Tail or GFP alone (pEGFP). Data are means ± s.e.m. (n=60 contacts) and are representative of three separate experiments.

View larger version (21K): [in a new window]   Fig. 7. Recovery of E-cadherin-YFP fluorescence following photobleaching of cell-cell contacts is perturbed by manoeuvres that disrupt dynamic MTs. Cell-cell contacts between CHO cells stably expressing E-Cad-YFP were photobleached and fluorescence recovery imaged by epi-illumination microscopy as described in the Materials and Methods. (A) Cells were incubated with nocodazole (100 nM) or DMSO (Control). (B) Cells were co-microinjected with HA-tagged CLIP-170 -Tail and mRFP for identification (-Tail) or with mRFP alone (Control). Fluorescence intensity at cell-cell contacts following photobleaching was normalised to the fluorescence intensity at the contacts prior to photobleaching as described in the Materials and Methods.

View larger version (77K): [in a new window]   Fig. 8. Dynamic MTs are necessary for accumulation of myosin II at cell-cell contacts. MCF-7 cells were incubated with nocodazole (100 nM) for 1-3 hours, then fixed and stained for myosin IIA and E-cadherin. (A) Myosin IIA and E-cadherin displayed consistent co-localisation in control cells (arrowhead). By contrast, after treatment with nocodazole (Noc), many cadherin-containing contacts showed little myosin IIA staining (arrow). (B) Quantitation of myosin IIA localisation at cadherin-based cell-cell contacts was performed by counting the number of cadherin-positive contacts that also showed co-localised myosin IIA staining. Cell-cell contacts were scored as positive for myosin IIA if they showed clear co-localisation of myosin IIA staining with E-cadherin. All other contacts that failed to show co-localisation of myosin IIA and E-cadherin were scored as negative. Data are means ± s.e.m. (n=150).

View larger version (96K): [in a new window]   Fig. 9. Dynamic MTs are necessary for myosin II activation at cell-cell contacts. (A) Nocodazole blocks activation of MLC by E-cadherin homophilic ligation. Control MCF-7 cells or cells pretreated with nocodazole (100 nM, 60 minutes; noc) were allowed to adhere to hE/Fc- or poly-L-lysine (PLL)-coated substrata for 90 minutes before lysis. Western blots of cell lysates were probed for activation-specific phosphorylated MLC (ppMLC) or ß-tubulin as a loading control. (B,C) Nocodazole blocks accumulation of phosphorylated MLC at cell-cell contacts. Confluent MCF-7 monolayers were incubated with nocodazole (100 nM) for 1-3 hours, then processed and stained for phosphorylated MLC or E-cadherin. (B) Phosphorylated MLC staining was consistently observed at cadherin-based cell-cell contacts in control cells (arrowheads). By contrast, many contacts in nocodazole-treated cells showed little or no co-accumulation of phosphorylated MLC (arrows). (C) Co-accumulation of phosphorylated MLC at cadherin cell-cell contacts was quantitated by counting the number of cadherin-positive contacts that also showed phosphorylated MLC staining.