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First published online 6 February 2007
doi: 10.1242/jcs.03383

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EHD1 regulates 1 integrin endosomal transport: effects on focal adhesions, cell spreading and migration

Marko Jovi¹, Naava Naslavsky¹, Debora Rapaport², Mia Horowitz² and Steve Caplan^1,*

¹ Department of Biochemistry and Molecular Biology and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA
² Department of Cell Research and Immunology, Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel

View larger version (42K): [in this window] [in a new window]   Fig. 1. EHD1-knockdown affects 1 integrin transport and localization in HeLa cells. HeLa cells on plates (A,B) or coverslips (C,D,F) were mock-treated (A,B,C,F) or treated with EHD1-RNAi (A,B,D) for 48 hours. RNAi efficacy was determined following sample calibration for protein content, and immunoblotting for EHD1 (A; top panel) or actin control (A; bottom panel). Recycling of anti-1-integrin–1-integrin complexes to the plasma membrane in HeLa cells was quantitatively measured by a flow cytometry recycling assay using mouse anti-human 12G10 antibodies that preferentially recognize ligand-bound 1 integrins (MCA2028; Serotec) (B). The numbers denoted in the graph represent mean levels of 1 integrin fluorescence (from 10,000 cells) reappearing at the cell surface because of recycling. A similar pulse-strip-chase experiment was done in HeLa cells on coverslips to follow the internalization and subcellular distribution of 1 integrins upon EHD1-RNAi treatment (C compared with D). LSM 5 Pascale software was used to quantitatively determine the mean level of remaining, non-recycled internal 1 integrins by measuring the mean level of fluorescence per cell (E). Mock cells (n=100) yielded a mean of 475 with a standard deviation of 121, whereas EHD1-RNAi-treated cells (n=82) yielded a mean of 1112 with a standard deviation of 256. One-tailed Student's t-tests showed significance at P<0.0001. HeLa cells transfected with GFP-EHD1 were pulsed for 1 hour with 12G10 anti-1 integrin antibodies and visualized by confocal microscopy (F). Serial z-sections were obtained every 0.4 µm, and the crosshairs and arrows depict common membrane structures on a representative micrograph containing both EHD1 (green) and 1 integrins (red). Bar, 10 µm.

View larger version (53K): [in this window] [in a new window]   Fig. 2. Impaired recycling in the absence of EHD1 leads to accumulation of 1 integrin in the endosomal recycling compartment. The intracellular localization of labeled transferrin and 1 integrins was compared using pulse-chase analysis in HeLa cells treated for 48 hours with mock-RNAi (A-C) or EHD1-RNAi (D-F). Cells were pulsed with 12G10 antibody that preferentially recognizes ligand-bound human 1 integrin (MCA2028; Serotec) for 2 hours, acid-stripped to remove non-internalized antibodies and chased for 30 minutes at 37°C. During this chase, transferrin–Alexa-Fluor-568 (Tf-568) was added for 15 minutes, and the Tf-568 was then chased in complete media for the remaining 15 minutes prior to fixation. Permeabilized cells were incubated with Alexa-Fluor-488-conjugated donkey anti-mouse antibody and analyzed by z-sectioning; confocal images were obtained every 0.4 µm. The representative images show the same z-sections through mock- and RNAi-treated cells, and crosshairs show colocalizations on x- and y-axes. Arrows highlight specific vesicular membrane structures containing both transferrin and 1 integrins. Bar, 10 µm.

View larger version (123K): [in this window] [in a new window]   Fig. 3. Loss of EHD1 in MEF cells and human fibroblasts causes accumulation of 1 integrins in internal vesicles. MEF cells were pulsed with 9EG7 monoclonal antibodies that preferentially recognize ligand-bound mouse 1 integrins for 1 hour at 37°C (A-H). The cells were then briefly acid-stripped to remove non-internalized antibody, and then either fixed/permeabilized (1-hour pulse; A,B) or chased for 1, 2 or 4 hours (C-H). At the end of every chase, cells were stripped again to remove any recycled 1 integrins from the plasma membrane and the remaining intracellular 1 integrins were detected by immunostaining with Alexa Fluor 568 anti-rat secondary antibody. Arrows depict accumulation of 1 integrins at the ERC in Ehd1–/– MEFs (F). Levels of intracellular 1 integrins in Ehd1+/+ MEF (G) and Ehd1–/– MEF cells (H) were quantified (graph in I) by the LSM 5 Pascale software using the Profile function. Representative fields (comprising more than 100 cells) were profiled by measuring the mean fluorescence in the field (80 µm) every 4 µm, and obtaining a mean value for all sections sampled. Standard deviations are depicted in the error bars, and the Student's t-test values for significance calculated at P<0.0001. MEF cells were grown in culture, lysed, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to immunoblot analysis (J). Human fibroblast cells growing on culture dishes (K) or coverslips (L,M) were treated with mock-RNAi or RNAi specific for EHD1 for 48 hours. Efficacy of RNAi was confirmed by immunoblotting with anti-EHD1 (K; top panel) and anti-actin as a control for equal protein loading (K; bottom panel). Mock-treated (L) and EHD1-RNAi-treated (M) human fibroblasts on coverslips were incubated with anti-human 1 integrin antibodies (MCA2028; Serotec) for 2 hours, briefly acid-stripped and chased with complete media for 2 hours. Intracellular 1 integrins were visualized by confocal microscopy following incubation with Alexa Fluor 568 anti-mouse secondary antibody under permeabilizing conditions. The micrographs shown are from a representative experiment from four independent experiments. Bars, 10 µm.

View larger version (25K): [in this window] [in a new window]   Fig. 4. Increased levels of activated 1 integrins but lower overall levels of 1 integrins are expressed on the plasma membrane of Ehd1–/– MEF cells. MEF cells grown on uncoated (A-C) or fibronectin-coated (A; FN) plates were harvested by brief trypsinization (A; Trypsin and B,C) or scraping (A; Scrape), fixed and subjected to flow cytometry analysis under non-permeabilizing conditions with secondary antibody only (A-C) or first with 9EG7 antibodies that preferentially recognize ligand-bound 1 integrins (BD Pharmingen) (A), conformation-independent 1 integrin antibodies (MB1.2, Chemicon) (B), anti-LAMP1 antibodies (C) or antibodies directed against the epidermal growth factor (EGF) receptor (C) prior to secondary antibodies. The graphs depicted are representative of 4-6 independent experiments. +/+, Ehd1+/+ MEF cells; –/–, Ehd1–/– MEF cells.

View larger version (85K): [in this window] [in a new window]   Fig. 5. The distribution of focal adhesion proteins in Ehd1–/– MEF cells is altered at steady-state. MEF cells were immunostained with antibodies to FAK (A,B), paxillin (C,D) or phospho-paxillin (E,F) and analyzed by confocal microscopy. Lysates containing an equal amount of protein from Ehd1+/+ and Ehd1–/– MEF cells were immunoblotted with antibodies against actin, FAK, phosphorylated FAK, paxillin or phosphorylated paxillin (G). Bar, 10 µm.

View larger version (66K): [in this window] [in a new window]   Fig. 6. Slower disassembly of focal adhesions in Ehd1–/– MEF cells. MEF cells were plated on fibronectin-coated coverslips, serum-starved and fixed (A,B; I,J) or treated with 10 µM nocodazole (NOC) for 15 minutes at 37°C (C-H; K-P). For some coverslips, NOC was washed out by incubating the cells in starvation media in the absence of the drug for an additional 15 minutes (E,F; M,N) or 30 minutes (G,H; O,P). After fixation, cells were immunostained with either mouse anti-tubulin (A-H) or mouse anti-paxillin (I-P). (Q) Quantification of focal-adhesion length in Ehd1–/– and Ehd+/+ cells was done by using Zeiss LSM 5 Pascale software. The number of focal adhesions measured is indicated, and Student's t-tests were applied to determine the significance of differences in mean focal adhesion lengths. Differences exhibiting a high degree of significance (P<0.0001) are noted with asterisks. This is a representative experiment from four independent experiments. Bars, 10 µm.

View larger version (132K): [in this window] [in a new window]   Fig. 7. Loss of EHD1 expression results in impaired cell motility. Equal numbers of Ehd1+/+ MEF (A,C,E) and Ehd1–/– MEF cells (B,D,F), were plated on fibronectin (FN)-coated coverslips (C,D) or vitronectin (VN)-coated coverslips (E,F). Scratches of 700 µm were introduced on the coverslips, and the cells were either fixed immediately (A,B) or after an additional 12-hour incubation period (C-F). Cells were visualized with fluorochrome-labeled wheat germ agglutinin and confocal microscopy. The micrographs depicted are representative ones from six independent experiments. Ehd1+/+ and Ehd1–/– MEF cells were plated on fibronectin-coated coverslides at sub-confluent concentrations (G,H) and scratched as in A-F. Eight hours after the scratch, cells were then pulsed with 9EG7 anti-1 integrin antibody for an additional hour to visualize 1 integrins in the migrating cells. Arrows indicate the direction of the migration, and the scratch region is indicated by broken lines. This experiment is a representative one of five independent experiments. Bars, 200 µm (A-F); 10 µm (G,H).

View larger version (49K): [in this window] [in a new window]   Fig. 8. Cell spreading on fibronectin is impaired in the absence of EHD1. Ehd1+/+ MEF (A), Ehd1–/– MEF (B), Ehd1–/– MEF cells transfected with GFP-EHD1 (C) or Ehd1–/– MEF cells transfected with GFP-EHD1 G65R (D) were plated on coverslips coated with 10 µg/ml fibronectin and allowed to spread for 3 hours in complete media. Cells were then fixed and stained with Cy3 wheat germ agglutinin (WGA; red) (A-D). Cell surface boundaries were outlined for 80-100 individual cells chosen randomly [Ehd1+/+ MEF cells, Ehd1–/– MEF cells and Ehd1–/– cells transfected with GFP-EHD1 (green)], and 40 cells for Ehd1–/– MEF cells transfected with GFP-EHD1 G65R (green). LSM 5 Pascal software was used to calculate the mean surface area of each population (see error bars for standard deviation) (E). One-tailed Student's t-tests were applied to test statistical significance of the data, with the P values for Ehd1+/+ versus Ehd1–/– MEF being P<0.001; Ehd1+/+ versus Ehd1–/– MEF transfected with GFP-EHD1 G65R being P<0.0001; Ehd1–/– versus Ehd1–/– MEF transfected with GFP-EHD1 being P<0.0001; and Ehd1–/– MEF transfected with GFP-EHD1 versus Ehd1–/– MEF transfected with GFP-EHD1 G65R being P<0.0001. This is a representative experiment from four independent experiments showing a similar trend. Bar, 10 µm.