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First published online 4 March 2008
doi: 10.1242/jcs.016121

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PAK is required for the disruption of E-cadherin adhesion by the small GTPase Rac

Encarnación Lozano^1,2,*, Marieke A. M. Frasa¹, Katarzyna Smolarczyk¹, Ulla G. Knaus³ and Vania M. M. Braga^1,

¹ Molecular Medicine Section, NHLI, Faculty of Medicine, Imperial College London, London, SW7 2AZ, UK
² Ecology and Evolution Research Section, Faculty of Life Sciences, Imperial College London, London, SW7 2AZ, UK
³ Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA

View larger version (112K): [in this window] [in a new window]   Fig. 1. Destabilization of E-cadherin–catenin complexes from keratinocyte junctions. Normal human keratinocytes were microinjected with (a) Rac1Q61L and (b) Rac3G12V cDNA, expressed for different time points (2-8 hours) and stained with anti-Myc and anti-E-cadherin antibodies. Arrows indicate absence of E-cadherin. Arrowheads show thickening of E-cadherin staining in the middle of cell-cell contact areas. Scale bars, 20 µm.

View larger version (91K): [in this window] [in a new window]   Fig. 2. Rac1b activation does not affect E-cadherin localization at junctions. (a) The constitutively active Rac1b mutant (Rac1bQ61L) was expressed for up to 18 hours and stained with Myc-tagged and E-cadherin. Arrow indicates absence of E-cadherin staining; arrowheads show concentration of E-cadherin at cell-cell contact sites. (b) Quantification of the ratio of cadherin staining over junction length after an 8-hour expression of activated Rac1b, Rac1 and Rac3 (see Materials and Methods for details). Values are expressed as the percentage of the total number of junctions found in expressing cells. (c) Rac1b protein is not degraded after microinjection in keratinocytes. Active forms of Rac1 and Rac1b were expressed for 6 hours and detected using anti-Myc antibody (recognizes only exogenous Rac) or an anti-Rac monoclonal antibody [recognizes an epitope in both exogenous and endogenous Rac1 and Rac1b (total Rac)]. Junctions were visualized with -catenin staining. Asterisks show cells with low expression of Rac1Q61L in which disruption of cell-cell contacts was achieved. (d) Keratinocytes were transfected with activated forms of Rac1, Rac1b and Rac3. Protein lysates were obtained and probed with antibodies against Rac1, Rac1b, Rac3, Myc and actin; arrows indicate endogenous Rac. Scale bars, 20 µm.

View larger version (63K): [in this window] [in a new window]   Fig. 3. PAK is necessary for Rac1-induced destabilization of E-cadherin complexes from junctions. (a, left panels) Rac1Q61L was co-expressed with kinase-dead PAK1K299R or with the auto-inhibitory domain Myc-PAKAID. Seven hours post-injection, cells were fixed and immunostained for epitope tags and -catenin. (Right panels) As controls, Rac1Q61L, PAKK299R or Myc-tagged PAKAID were microinjected alone and stained as above. Arrow indicates absence of -catenin. Arrowheads show the presence of -catenin at cell-cell contacts. Scale bars, 20 µm. (b) Effects of co-expression of constitutively active Rac1 and PAK mutants were quantified by measuring the ratio of the length in cadherin staining to the length of the cell-cell contact (corner to corner of neighbouring expressing cells). Controls were arbitrarily set as 100% (length in cadherin staining = length of the cell-cell contact). *P<0.05; **P<0.01. (c) Keratinocytes express PAK1 mRNA, but not PAK2 or PAK3 mRNAs. Keratinocyte and monocyte RNAs were subjected to RT-PCR using primers specific for members of the class I PAK family.

View larger version (73K): [in this window] [in a new window]   Fig. 4. (a) siRNA targeting PAK1 or control siRNA oligos were transfected into keratinocytes. Following expression of constitutively active Rac1 (0.5 mg/ml) for 7 hours, siRNA-treated keratinocytes were stained for E-cadherin and the FLAG-tag. Scale bar, 20 µm. (b) Lysates were obtained and probed with antibodies against PAK1, actin and E-cadherin. (c) The rescue of Rac-dependent junction disruption by depletion of endogenous PAK was quantified using the ratio between cadherin staining and junction length (see Materials and Methods). *P<0.05.