Journal: Molecular Biology of the Cell
Article Title: Establishing epithelial glandular polarity: interlinked roles for ARF6, Rac1, and the matrix microenvironment
doi: 10.1091/mbc.E12-03-0246
Figure Lengend Snippet: The inversion of MDCK ARF6si cysts disrupts the localization of laminins and β1 integrin. (A) The distribution of β1 integrin in MDCK and MDCK ARF6si cysts. Cysts were fixed and labeled for actin (red) and β1 integrin (green). In both MDCK and MDCK ARF6si cysts, β1 integrin is appropriately targeted to the plasma membrane. However, in the MDCK ARF6si inverted cysts, the basolateral domain is not in contact with the matrix, and it is likely that β1 integrin is not occupied by the ECM. For quantifying β1-integrin labeling, the outer, luminal, and lateral domains of 25 cells on representative images were quantified, and the signal intensity was measured using the Image J program. The mean signal intensity was determined, and the averages of these intensities was graphed ± SD. β1 integrin was predominantly at the outer and lateral surfaces of parental cysts and at the lateral and luminal surfaces of inverted MDCK ARF6si cysts. (B) The distribution of laminin-332 in MDCK and MDCK ARF6si cysts. Cysts were fixed and labeled for actin (red) and laminin-332 (green). In parental cysts, laminin-332 is assembled at the cyst–matrix interface; in inverted MDCK ARF6si cysts, laminin-332 remains unassembled; and in scrambled MDCK ARF6si cysts, laminin-332 is deposited at sites at which the basolateral domain is in contact with the matrix. (C) Distribution of laminin-111 in MDCK and MDCK ARF6si cysts. Cysts were fixed and stained for laminin-111 (green), gp135 (red), and nuclei (blue). The exogenous laminin provides support around the parental cyst. In the MDCK ARF6si cultures, the inverted apical domains appear to be evading the confines of the exogenous matrix and are assembled at the free surface, clear of supportive laminin-111. (D) Activation of Rac1 is sufficient to rescue laminin-111 organization around the cyst. Representative images of EGF/CN02-treated cysts labeled for actin (red), laminin-111 (green), and nuclei (blue) are shown. Treatment with CN02 recovered normal cyst phenotypes in MDCK ARF6si cysts and the supportive assembly of laminin-111. The recovery was most effective when MDCK ARF6si cysts were treated with CN02 at day 2 of cyst development. When the recovery of the MDCK ARF6si phenotypes was not completely penetrant, the exogenous laminin continued to be disassociated from the apical domains of the scrambled or inverted cysts. (E) Activation of Rac1 is sufficient to rescue laminin-332 deposition in support of the cyst. MDCK ARF6si cysts were labeled for actin (red) and laminin-332 (green). Treatment with CN02 recovered normal cyst phenotypes in MDCK ARF6si cysts, as well as the deposition of laminin-332 at the cyst–matrix interface.
Article Snippet: For activation of Rac1, cells were serum-starved for 6 h prior to addition of 0.25 U/ml of EGF (commercially available as CN02; Cytoskeleton, Denver, CO), to the culture medium.
Techniques: Labeling, Membrane, Staining, Activation Assay
Cytoskeleton Inc is a verified supplier 
Cytoskeleton Inc manufactures this product 
![( A ) Orthogonal projections of two-photon volumetric images of Müller glia expressing membrane-GFP (mGFP, green) in sparsely recombined Slc1a3- CreER;mTmG retinas at P7 (left) and P47 (right). Cells without Cre-mediated recombination express membrane-tdTomato (magenta). Scale bar: 10 µm. IPL: inner plexiform layer. ( B ) Orthogonal projections of mGFP-expressing Müller glia from P8 to P23. Scale bar: 10 µm. ( C ) Temporally color-coded projections of two-photon Z-stack time series showing motile processes at P12 (top) and stable processes at P23 (bottom). The location and dynamics of lateral processes at each time point can be determined by referencing the color time scale at bottom. Images on right are enlarged insets from images on left to highlight individual processes. Left scale bar: 10 µm; right scale bar: 5 µm. See for enlarged images of these cells in sublayers S1, S3, and S5. ( D ) Distribution of lateral processes across IPL sublayers through development. See for summary statistics and goodness-of-fit test statistics, and for tests for independent proportions across age groups. ( E ) Proportion of lateral processes in each sublayer that underwent extension (blue), sprouting (light blue), retraction (red), elimination (light red), a combination (purple), or that were stable (gray). ( F ) Proportion of total stable processes across development. See for statistical comparisons. ( G ) Proportion of total processes that were motile (left) and stable (right) in the absence and presence of blockers of cytoskeletal turnover at P11/12. cyto-D: cytochalasin-D (5 µM); noco: nocodazole (10 µM). See for statistical comparisons. ( H ) Proportion of total processes that were motile (left) and stable (right) in the absence and presence <t>of</t> <t>epidermal</t> growth factor <t>(EGF,</t> 1 unit [100 ng]/ml) at P8 and P17. See for statistical comparisons. * corresponds to p < 0.05 for all comparisons. Source data available in and . Figure 1—source data 1. Proportions and counts of motile and stable lateral processes across sublayer and age. Figure 1—source data 2. Proportions and counts of motile and stable lateral processes in the presence and absence of cytoskeletal blockers or EGF.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6189/pmc08806189/pmc08806189__elife-73202-fig1.jpg)
