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First published online 17 October 2006
doi: 10.1242/jcs.03218

Journal of Cell Science 119, 4541-4553 (2006)
Published by The Company of Biologists 2006
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Podoplanin binds ERM proteins to activate RhoA and promote epithelial-mesenchymal transition

Ester Martín-Villar1, Diego Megías2, Susanna Castel3, Maria Marta Yurrita1, Senén Vilaró3,* and Miguel Quintanilla1,{ddagger}

1 Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad Autónoma de Madrid (UAM), 28029 Madrid, Spain
2 Centro Nacional de Investigaciones Oncológicas, 28029 Madrid, Spain
3 Departamento de Biología Celular, Universidad de Barcelona, 08028 Barcelona, Spain


Figure 1
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  		Fig. 1. Schematic representation of podoplanin fusion constructs used for transfection. Numbers indicate podoplanin amino acid sequences (Martín-Villar et al., 2005Go) conserved in the construct. CT, cytoplasmic domain; EC, ectodomain; FP, fluorescent protein (EYFP or EGFP) used for the experiments specified in the text; SP, signal peptide; TM, transmembrane domain. Basic amino acids (bold) within the CT domain were substituted by uncharged polar residues (bold and underlined).

 

Figure 2
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  		Fig. 2. Podoplanin binds to ERM proteins through a cluster of basic amino acids within its cytoplasmic tail. (A) Association of ezrin and moesin with podoplanin CT. GST and GST fusion proteins bound to Sepharose beads were incubated with purified recombinant full-length ezrin or moesin or their N-ERMADs. Proteins bound to the beads were fractionated by SDS-PAGE followed by western blotting using anti-ezrin or anti-moesin antibodies. CD44-CT was used as a positive control. (B) FRETeff values for cells coexpressing EYFP/Ezrin-ECFP (n=5), PWT-EYFP/Ezrin-ECFP (n=14), P{Delta}CT-EYFP/Ezrin-ECFP (n=7), PCTQN.N-EYFP/Ezrin-ECFP (n=7), PCTQN-EYFP/Ezrin-ECFP (n=6) and PCT.N-EYFP/Ezrin-ECFP (n=10). Note the significant reduction (**P<0.01) of FRETeff in PCT.N with respect to PWT and the absence of FRET in PCTQN.N, PCTQN and P{Delta}CT cell transfectants. (C-E) Confocal fluorescence images showing acceptor photobleaching FRET analysis in MDCK cell transfectants. Images of representative cells coexpressing Ezrin-ECFP with PWT-EYFP (C), P{Delta}CT-EYFP (D) or PCT.N-EYFP (E) are shown. ECFP and EYFP emission signals were collected before (left panels) and after (right panels) EYFP photobleaching in the boxed regions. An increased ECFP fluorescence signal after photobleaching indicates FRET. The FRETeff is represented in the bottom panel on a pseudocolor cell map with scale shown on the right.

 

Figure 3
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  		Fig. 3. MDCK cells expressing podoplanin undergo an EMT that depends on the cytoplasmic domain. (A) Western blot analysis of podoplanin proteins and of differentiation-related markers in MDCK-derived cell clones expressing EGFP (control) and PWT, P{Delta}CT, P{Delta}EC, PCTQN.N, PCTQN or PCT.N fusion proteins. (B) Expression of E-cadherin, Snail and Slug mRNA transcripts by RT-PCR. GAPDH was amplified as a control for the amount of cDNA present in each sample. The RT-lane shows the results of amplification in the absence of cDNA. The morphology of the cell transfectants is indicated below (E, epithelial; F, fibroblastic).

 

Figure 4
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  		Fig. 4. Subcellular localization of wild-type and mutant podoplanin proteins. Confocal images of horizontal (x-y) and vertical (x-z) sections of MDCK cells expressing the indicated podoplanin proteins fused to EGFP are shown. The exogenous proteins specifically expressed at the cell surface were detected by in vivo pre-embedding staining using antibodies either recognizing the podoplanin EC domain or EGFP (for detection of P{Delta}EC).

 

Figure 5
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  		Fig. 5. Podoplanin-induced EMT is associated with increased migratory and invasive abilities. (A) In vitro wound-healing assay. Confluent cell cultures were scratched with a pipette tip to produce a wound and analyzed by time-lapse video microscopy. Images of the wounded area immediately (0 hours) and 12 hours after the incision was made are shown. Arrows in P{Delta}CT indicate individual cells moving into the wound as fibroblasts. An animated sequence of these data is shown in supplementary material Movie 1. In the bottom panel a graph shows quantification of cell migration from three independent experiments. **P<0.01; ***P<0.001 vs control (EGFP) cells. (B) Matrigel invasion assay. Cell transfectants expressing EGFP-tagged wild-type and mutant podoplanin proteins were seeded on a 24-well Matrigel invasion chamber. FBS (5%) was used as a chemoattractant. Fluorescent cells that invaded the Matrigel-coated filter after 24 hours were detected by confocal microscopy. Images were acquired each 2 µm along the z-axis and quantified by fluorescence intensity profiles. Vertical (x-z) sections of the filter are shown below. Results are representative of two experiments.

 

Figure 6
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  		Fig. 6. Dynamics of podoplanin subcellular localization during cell locomotion. Isolated MDCK cells expressing EGFP-tagged PWT, P{Delta}EC and P{Delta}CT proteins were imaged by capturing sections of 0.5 µm every 10 minutes for 4 hours. Images in all panels correspond to 3D reconstructions. Arrowheads indicate podoplanin fluorescence signal concentrated at the leading edge of lamellipodial extensions and on the retracting tail, whereas asterisks indicate loss of fluorescence signal on lamellipodial extensions attached to the substratum. The direction of cell migration is indicated by arrows. Animated sequences of these data are shown in supplementary material Movies 3-5.

 

Figure 7
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  		Fig. 7. Podoplanin-induced EMT is associated with upregulation of RhoA activity. (A) Rho, Rac and Cdc42 bound to GTP affinity pull-down assays were used to determine the levels of active Rho GTPases. Levels of active RhoA, Rac1 and Cdc42 in MDCK and HaCaT cell clones transfected with the empty vector (EGFP) and with wild-type and mutant podoplanin constructs fused to EGFP. Quantification of RhoA-GTP expression level relative to total RhoA level was performed by densitometric analysis. Values below blots are relative to control (EGFP) cells, to which an arbitrary value of 1 was given. Results are representative of two experiments. (B) Western blot analysis of ERM phosphorylation relative to the total expression levels of ezrin and moesin. The levels of {alpha}-tubulin were determined as a control for protein loading. (C) Western blot analysis of phospho-ERM levels relative to the total expression levels of ezrin and moesin in MDCK cells transfected with the empty vector (EGFP) and PWT before and after treatment with the Rock inhibitor Y27632. (D) Inhibition of RhoA signaling blocks podoplanin-stimulated cell migration. In vitro wound-healing assays of control (EGFP) and PWT-MDCK cells were performed (as in Fig. 5A) in the absence or presence of either Y27632 (which inhibits Rock) or soluble C3 transferase (which inhibits RhoA, RhoB and RhoC). Y27632 and C3 transferase reduced PWT-MDCK cell migration to basal and below basal levels, respectively. *P<0.05; ***P<0.001 vs PWT-MDCK cells.

 

Figure 8
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  		Fig. 8. Dominant-negative effects of N19RhoA and of N-terminal domain of ezrin (N-ezrin) in podoplanin-induced EMT. (A) Phase-contrast micrographs and confocal fluorescence detection of podoplanin in MDCK cells cotransfected with EGFP-tagged PWT and N-ezrin or N19RhoA. Control cells expressing EGFP alone, EGFP/N-ezrin and EGFP/N19RhoA are also shown. Confocal images are maximum projections of horizontal optical sections through the whole depth of the transfectants. (B) Western blot analysis of N-ezrin (VSVG), N19RhoA (HA) and differentiation-related proteins in MDCK cell transfectants. N-ezrin and N19RhoA expression was determined by using antibodies against their respective tags. (C) N-ezrin and N19RhoA inhibits podoplanin-mediated RhoA activation and ERM phosphorylation. The levels of RhoA-GTP relative to the levels of total RhoA and the levels of phospho-ERM relative to the total expression levels of ezrin and moesin are shown. The expression of {alpha}-tubulin was determined as a control for protein loading.

 

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© The Company of Biologists Ltd 2006