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First published online 12 September 2006
doi: 10.1242/jcs.03103

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Dystroglycan loss disrupts polarity and ß-casein induction in mammary epithelial cells by perturbing laminin anchoring

¹ California Pacific Medical Center Research Institute, 475 Brannan Street, Suite 217, San Francisco, CA 94107, USA
² Howard Hughes Medical Institute, Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA 52242, USA
³ Division of Life Sciences, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

View larger version (91K): [in a new window]   Fig. 1. Generation of DG+/+ and partial-DG-/- MEC populations by adenoviral infection of immortalized mouse MECs. (A) Diagram of DG, including the extracellular -DG subunit, with central mucin domain, and the transmembrane ß-DG subunit. (B) Diagram of laminin-111 (LN), including the three subunits (, ß, ), and the five C-terminal LG domains, with respective receptor binding sites. (C) Western blot of cell extracts (5 µg protein) prepared on different days after infection of the immortalized, floxed DG mouse MEpG cell line with control or Cre-recombinase-expressing adenovirus to generate DG+/+ and partial-DG-/- cell populations, respectively. The first lane (far left) represents uninfected cells at time 0. Blots were incubated with antibodies specific for -DG, C-terminal ß-DG or E-cadherin (loading control), followed by HRP-conjugated secondary antibodies. Sizes of molecular mass markers are shown in kDa. (D) Vertically paired immunofluorescent images of DG+/+ and partial-DG-/- MEpG cell populations using primary antibodies specific for -DG or C-terminal ß-DG, followed by FITC-labeled secondary antibodies (upper panel). Nuclei were stained with propidium iodide (bottom panel). Bar, 60 µm.

View larger version (28K): [in a new window]   Fig. 2. Loss of polarity in DG-/- colonies grown in a 3D matrix of collagen I-laminin-111. DG+/+ and DG-/- MEpG cells were grown in a 3D matrix of collagen I or collagen I-laminin-111 and co-immunostained. Confocal immunofluorescent images were taken at colony centers. Bars, 10 µm. (A) Staining using anti-ZO-1 and anti-6 integrin antibodies, visualized with FITC-(green) and Cy5-(blue) labeled secondary antibodies, respectively, and propidium iodide to stain nuclei (red). (B) Staining using antibodies against 6 integrin and C-terminal ß-DG (insets), detected with Rhodamine-(red) and Cy5-(blue changed to white for easier visualization) labeled secondary antibodies, respectively. Actin was seen using Alexa Fluor-488-phalloidin (green). Overlap between actin and 6 integrin staining appeared yellow. (C) Quantification of polarity in DG+/+ and DG-/- colonies grown in collagen I (C) or collagen I-laminin-111 (C/L) using ZO-1 as a polarity marker. Results are shown as the mean ± s.e.m. of four to six independent experiments, each with triplicate or quadruplicate counts. *P<0.01, for all paired combinations.

View larger version (47K): [in a new window]   Fig. 3. Loss of laminin binding and DG colocalization on the surface of DG-/- cells grown in a 3D matrix of collagen I or collagen-I-laminin-111. (A) Vertically paired confocal immunofluorescent images of DG+/+ and DG-/- MEpG cells grown in collagen I or collagen-I-laminin-111. Samples were co-immunostained with laminin, 6 integrin and C-terminal ß-DG (insets) antibodies, followed by Rhodamine-(red), FITC-(green), and Cy5-(blue changed to white for easier visualization) labeled secondary antibodies, respectively. Images were taken at colony centers. (B) Confocal immunofluorescent images taken at the cell surface of DG+/+ colonies shown in A to reveal co-staining for laminin and ß-DG, and their extent of co-localization. Arrows point to arrays of laminin. Bars, 10 µm.

View larger version (76K): [in a new window]   Fig. 4. DG-/- cell monolayers failed to bind endogenous laminin or exogenous laminin-111-FITC. (A) Vertically paired immunofluorescent images of DG+/+ and partial-DG-/- MEpG cell populations co-stained using laminin and C-terminal ß-DG antibodies, followed by Rhodamine- and FITC-labelled secondary antibodies, respectively, all in the presence of Tween-20 (images above line). Arrows point to a DG-/- cell that retained staining for intracellular but not cell-surface laminin. Cells immunostained for laminin in the absence of Tween-20 are shown below the line, with corresponding phase images. (B) Immunofluorescent images of DG+/+ and partial-DG-/- cell populations treated with 10 nM exogenously added laminin-111-FITC for 4 hours. Samples were co-stained using C-terminal ß-DG antibody and Rhodamine-labeled secondary antibody. Corresponding phase images are shown in the bottom panel. Arrows point to a DG-/- cell lacking laminin-111-FITC staining. (C) Immunofluorescent images of DG+/+ cells treated with 10 nM exogenous laminin-111-FITC for 24 hours in the absence or presence of 6 and/or ß1 integrin function blocking antibodies (upper panel). Corresponding phase images are shown in the bottom panel. Insets show cells incubated only with 10 nM AEBSF-treated laminin-111 for 24 hours, followed by immunostaining for laminin as described for upper images in A. Bars, 10 µm.

View larger version (103K): [in a new window]   Fig. 5. Partial-DG+/+ colonies grown in a 3D matrix of collagen-I-laminin-111 retain laminin and DG colocalization on the surface of DG+/+ cells only, but fail to polarize. (A,B) Confocal immunofluorescent images taken at the center of partial-DG+/+ MEpG colonies grown in collagen-I-laminin-111 and co-immunostained as follows: (A) 6 integrin and C-terminal ß-DG antibodies were detected using Rhodamine-(red) and Cy5-(blue) labeled secondary antibodies, respectively. Actin was visualized with Alexa Fluor-488-phalloidin (green). (B) Laminin, 6 integrin and C-terminal ß-DG antibodies were detected using Rhodamine-(red), FITC-(green), and Cy5-(blue) labeled secondary antibodies, respectively. Arrows show part of colony surface lacking laminin and ß-DG staining. (C) Confocal immunofluorescent images were taken at the cell surface of the colony shown in B to reveal co-staining for laminin and ß-DG, and their extent of co-localization. Dotted outline represents outer edge of colony. Bar, 10 µm.

View larger version (117K): [in a new window]   Fig. 6. Loss of ß-casein production in response to laminin-111 in DG-/- cells. (A) Western blot of cell extracts (10 µg protein) prepared from DG+/+ and partial-DG-/- MEpL cell populations incubated with laminin-111 overlay in the absence (-) or presence (+) of prolactin and hydrocortisone. Blots were incubated with antibodies specific for ß-casein or E-cadherin (loading control), followed by HRP-conjugated secondary antibodies. Sizes of molecular mass markers are shown in kDa. Dotted lines separate non-adjacent lanes derived from the same blot. (B) Immunofluorescent images of DG+/+ and partial-DG-/- cell population treated with 10 nM exogenously added laminin-111-FITC for 4 hours. Samples were co-stained using C-terminal ß-DG antibody and Rhodamine-labeled secondary antibody. Corresponding phase images are shown in the bottom panel. Arrows point to a DG-/- cell lacking laminin-111-FITC binding.

View larger version (58K): [in a new window]   Fig. 7. Re-expression of full-length DG or DG mutants in a completely DG-/- cell line restored laminin-111 binding on monolayer cell surfaces. (A) Western blot of cell extracts (10 µg protein) prepared from DG+/+ cells and from a DG-/- cell line (derived from MEpG cells) infected with retroviral vector (VEC) or that encoding full-length DG (wtDG) or various ß-DG cytoplasmic deletions (DEL A, B and C). Blots were incubated with antibodies specific for -DG, N-terminal ß-DG (right panel), C-terminal ß-DG (left panel), or E-cadherin (loading control), followed by HRP-conjugated secondary antibodies. Sizes of molecular mass markers are shown in kDa. (B) Paired immunofluorescent images of cells in A, co-stained for -DG and nuclei, using FITC-labeled secondary antibody and propidium iodide, respectively. (C) Immunofluorescent images of cells in A, treated for 4 hours with 10 nM exogenously added laminin-111-FITC. Corresponding phase images are shown in the bottom panel. Bars, 10 µm.

View larger version (96K): [in a new window]   Fig. 8. Expression of full-length DG and DG mutants in a pure DG-/- MEpG cell line restored polarity and surface laminin in an 3D matrix of collagen-I-laminin-111. (A) Confocal immunofluorescent images taken at the center of colonies grown in collagen I (upper panel) or collagen-I-laminin-111 (middle panel). Samples were co-stained for ZO-1, 6 integrin and nuclei as described in Fig. 2A. Bottom panel shows laminin staining of a second group of colonies grown in collagen-I-laminin-111, visualized with Rhodamine-labeled secondary antibody (red). Cells are described in Fig. 7A. Bars, 10 µm. (B) Quantification of polarity in colonies grown in collagen I (C) or collagen-I-laminin-111 (C/L) using ZO-1 as a polarity marker. Results are shown as the average ± s.e.m. of 3-5 independent experiments, each with triplicate or quadruplicate counts. (^) or (*)=P<0.001 for all paired combinations except with each other.

View larger version (65K): [in a new window]   Fig. 9. Expression of full-length DG and DG mutants in a pure DG-/- MEpL cell line restored ß-casein protein expression in response to laminin-111. Western blot of cell extracts prepared from cells infected with retroviral vector (VEC) or that encoding full-length DG (wtDG) or various ß-DG cytoplasmic deletions (DEL A, B and C) and incubated with a laminin-111 overlay in the absence (-) or presence (+) of prolactin and hydrocortisone. Blots were incubated with antibodies specific for ß-casein or E-cadherin (loading control), followed by HRP-conjugated secondary antibodies. Sizes of molecular mass markers are shown in kDa.

View larger version (23K): [in a new window]   Fig. 10. The DG extracellular domain alone is crucial to laminin assembly. (A) Western blot of cell extracts prepared from a DG-/- MEpL cell line (entirely DG-/-) infected with retroviral vector (VEC) or that encoding full-length DG (wtDG), a fusion protein comprised of the extracellular DG sequences fused to the transmembrane region of TACE (DG-tmf), or deletions within the -DG mucin domain (DEL D and E). Blots were incubated with antibodies specific for -DG, N-terminal ß-DG, or E-cadherin (loading control). Sizes of molecular mass markers are shown in kDa. (B) Immunofluorescent images of cells in A, treated for 4 hours with 10 nM exogenously added laminin-111-FITC. Corresponding phase images are shown in the bottom panel. Bar, 10 µm.

View larger version (73K): [in a new window]   Fig. 11. Model for the role of DG as a MEC co-receptor in laminin-111 assembly and laminin-111-induced functions. -DG on the MEC surface serves as the initial anchoring site for laminin-111 (LN) monomers by interacting with their C-terminal LG domains (step 1). The laminin-111-DG complexes recruit ß1 integrin (INT) co-receptors, which contribute to laminin-111 polymerization (step 2). Subsequent activation of co-receptors, possibly integrins (INT), influences intracellular signaling pathways leading to polarity and ß-casein induction (step 3).

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