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doi: 10.1242/10.1242/jcs.00104

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Functional role of -actinin, PI 3-kinase and MEK1/2 in insulin-like growth factor I receptor kinase regulated motility of human breast carcinoma cells

¹ Department of Microbiology, University of Pennsylvania, 3610 Hamilton Walk, 211 Johnson Pavilion, Philadelphia PA 19104, USA
² MRC-Laboratory for Molecular Cell Biology and Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK
³ Department of Cell Biology, Building NC1, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195, USA

View larger version (48K): [in a new window]   Fig. 1. IGF-IR expression levels and signaling in MCF-7-derived cell lines. (a) Expression of the IGF-IR was detected in MCF-7, MCF-7/IGF-IR/WT and MCF-7/IGF-IR/DK cells by western blotting of 20 µg of total protein with an antibody against the C-terminus of the IGF-IR ß subunit. The arrows show the position of the ß subunit in the mature receptor (95 kDa) and its precursors: prepro-(190 kDa) and pro-receptor (185 kDa). (b) Top panel: phosphotyrosine blot of MCF-7/IGF-IR/WT and MCF-7/IGF-IR/DK cells stimulated with 50 ng/ml IGF-I. Cell lysates were collected from serum-starved cells before (0 min) and after stimulation with IGF-I for 5, 15 and 60 minutes. Arrows show the position of the IGF-IR ß subunit (95 kDa) and IRS-1 (175 kDa). Protein molecular weight markers are indicated in kDa on the left of the blot. Lower panel: quantification of sample loading by western blot for the IGF-IR.

View larger version (109K): [in a new window]   Fig. 2. (A) IGF-I induces membrane ruffling in separating MCF-7/IGF-IR/WT cells. Phase-contrast images of serum-starved MCF-7/IGF-IR/WT cells before and after stimulation with 50 ng/ml IGF-I for indicated times. The thin arrows show examples of apical ruffles. Thick arrows indicate nascent gaps at the sites of cell-cell detachment. Bar, 10 µm. (B) Inhibition of membrane ruffling and cell motility in dead-kinase expressing cells. Phase-contrast images of MCF-7/IGF-IR/WT (a,b) and MCF-7/IGF-IR/DK (d,e) cells. Cells were serum-starved (a,d) or stimulated with 50 ng/ml IGF-I for 60 minutes (b,e). Examples of sporadic lateral ruffles in serum-starved cells are indicated by arrows in a and d. As MCF-7/IGF-IR/WT cells moved apart, they extended long, fine cytoplasmic processes with the lamellipodia at the ends (arrows in b). Bar, 10 µm. (c,f) Quantification of cells with ruffles at free edges and over the cell apex, and migratory cells observed during time-lapse filming in MCF-7/IGF-IR/WT (c) and MCF-7/IGF-IR/DK (f) cells. Cells were incubated overnight in serum-free medium (SFM) and then stimulated with 50 ng/ml IGF-I (IGF-I).

View larger version (114K): [in a new window]   Fig. 3. (A) The effects of IGF-IR activation on redistribution of -catenin, -actinin and F-actin. Serum-starved (0 min) MCF-7/IGF-IR/WT cells were treated with 50 ng/ml IGF-I for 5, 15 and 60 minutes, then fixed in 3.7% formaldehyde and stained with a polyclonal antibody to -catenin (a-d), a monoclonal antibody to -actinin (e-h) or TRITC-phalloidin for F-actin (i-l). Arrows show examples of -catenin disappearance from cell-cell contacts (a-c), relocalization of -actinin to the cellular borders (e-h) and reorganization of F-actin and microspike development (i-l) induced by IGF-I. Inset in e, examples of -actinin localized at the tips of stress fibers seen at the free edge of the spread cell. Bar, 10 µm. (B) IGF-I-stimulated reorganization of -actinin and F-actin is blocked when the IGF-IR kinase is inactive. Localization of -actinin (staining with antibody to -actinin) (a-c) and F-actin (staining with TRITC-phalloidin) (d-f) was examined in MCF-7/IGF-IR/DK cells that were serum starved (0 min) and exposed to 50 ng/ml IGF-I for 5 and 60 minutes. Confocal images are representative of three experiments. Bar, 10 µm.

View larger version (105K): [in a new window]   Fig. 4. (A) Colocalization of -actinin with F-actin in microspikes during IGF-I-induced cell separation. MCF-7/IGF-IR/WT cells, untreated (a-c) or stimulated with 50 ng/ml IGF-I for 5 minutes (d-f), were fixed in 3.7% formaldehyde and co-stained for F-actin with TRITC-phalloidin and for -actinin with a monoclonal antibody to -actinin. The images of middle sections acquired by confocal laser-scanning microscopy illustrate staining for F-actin in red (a,d) and -actinin in green (b,e). In overlays (c,f), sites of -actinin and F-actin colocalization appear in yellow. Insets in d-f: examples of F-actin and -actinin-containing microspikes from the zone of the intercellular membrane ruffles pointed to by arrow (d-f). Bar, 10 µm. (B) Ultra-structural analysis of IGF-I-induced microspikes in MCF-7/IGF-IR/WT cells. (a) Examples of adherens junction (aj), desmosome (d) and intermediate filaments (if) proximate to desmosomes are indicated in confluent serum-starved cells. The longitudinal section of continuous contacts between lateral membranes is shown in the upper inset. (b) Cross-sections through microspikes (MS) formed in the cells stimulated with 50 ng/ml IGF-I for 15 minutes. Individual bundles of actin in the core of MS formed by opposing cells are pointed out by a row of arrows; gaps between single microspikes are labeled with asterisks. A membrane site resembling an immature or disassembling desmosome in close proximity to intermediate filaments is labeled d. In the inset, the horizontal section through apical zones of IGF-I-stimulated cells is shown. N, nucleus. Bar, 250 nm. In insets, bar, 5 µm.

View larger version (111K): [in a new window]   Fig. 5. (A) The development of -actinin motile projections in living MCF-7/IGF-IR/WT cells stimulated with IGF-I. Dynamics of EGFP--actinin in live MCF-7/IGF-IR/WT cells transiently transfected with WT -actinin-EGFP encoding plasmid. (A-C) The representative laser confocal fluorescence images taken at the apical, middle and basal focal planes in serum-starved cells (a,d,g) stimulated with 50 ng/ml IGF-I for 5 minutes (b,e,h) or for 9 minutes (c,f,i) are shown. Circled is an example of a cell that is flattening, separating and moving from the adjacent cells in response to IGF-I treatment. The optical section from the apical region was taken about 2.0 µm from the top of cells. The example of the lateral single projection that extended and folded over the cell apex is indicated by arrow in a-c. The middle optical sections were collected from the focal plane about 6.5 µm from the top of cells. Basal images were taken close to the substratum level (about 1.5 µm from the glass surface). Bar, 10 µm. (B) Reorganization of -actinin is blocked in living MCF-7/IGF-IR/DK cells treated with IGF-I. The images illustrate the localization of -actinin in live MCF-7/IGF-IR/DK cells transfected with EGFP-labeled -actinin before (0 min) and after IGF-I stimulation (shown for 1 and 5 minutes). The representative images derived by 3D projection of 20 consecutive apical and middle optical sections are shown. Bar, 10 µm.

View larger version (56K): [in a new window]   Fig. 6. (A) Schematic diagram of -actinin-EGFP fusion monomers. The WT, N and C -actinins are expressed as C-terminal chimeras with EGFP. The primary sequence of the WT -actinin consists of the N-terminal conservative actin-binding region (residues 1-245), a central domain of four spectrin-like repeats implicated in dimerization of -actinin and interaction with other proteins including -catenin (residues 246-712), and the C-terminal domain containing the vinculin-binding site (residues 713-749) and the two EF-hand binding Ca2+ motifs (residues 770-817). Numbering of residues includes the initiating methionine. (B) Expression levels of -actinin-EGFP fusion proteins in MCF-7/IGF-IR/WT cells. Expression of EGFP was detected in transiently transfected MCF-7/IGF-IR/WT cells by western blotting of 20 µg of total protein with an antibody against GFP. Upper panels, the representative western blot shows the levels of exogenous EGFP (27 kDa) and -actinin-EGFP chimeras (WT, 130 kDa, N, 104 kDa, C, 109 kDa) expressed as transgenes 48 hours after transfection (asterisks indicate the position of chimera). Lower panel, the same blot was stripped and re-probed with an antibody against -actinin to detect endogenous (100 kDa) and exogenous EGFP--actinins whose positions on the blot are indicated by asterisks. Note that the endogenous -actinin and N -actinin-EGFP overrun in a 100 kDa band. (C) Intracellular localization of WT, N, C -actinin-EGFP fusion proteins in serum-starved and IGF-I-stimulated MCF-7/IGF-IR/WT cells. MCF-7/IGF-IR WT cells were transfected with plasmid encoding WT or N or C -actinin-EGFP. 24 hours after transfection, serum-starved cells were either fixed in 3.7% formaldehyde (a-c) or stimulated with 50 ng/ml IGF-I for 15 minutes and then fixed (d-f). The representative images of the middle optical sections acquired by confocal laser-scanning microscopy show (top panel, a-c) the WT and C -actinins incorporated into the mature cell-cell junctions (pointed by arrow) and N -actinin stained in the cytoplasm and nucleus. Lower panel, d-f: the images show the formation of -actinin-EGFP-containing spikes (arrows in d); diffuse staining for N -actinin-EGFP (e), aggregation of C -actinin-EGFP and formation of the defective microspikes (f). Bar, 10 µm.

View larger version (28K): [in a new window]   Fig. 7. Effects of inhibitors on IGF-I-induced activation of the PI 3-kinase and MAP kinase signaling pathways. (a) Serum-starved cells (0). Cells not exposed to inhibitors (IGF-I) and pre-treated with 10 µM LY 294002 for 30 minutes (LY+IGF-I) or 40 µM PI analog for 3 hours (PI analog+IGF-I) were stimulated with 50 ng/ml IGF-I for the indicated times. Phosphorylation of PKB/Akt was detected with an antibody against phospho-Akt. For a loading control, the same blots were stripped and re-probed with an antibody against Akt (Materials and Methods). One of the re-probed blots is shown. (b). Serum-starved cells (0). Cells not exposed to inhibitors (IGF-I) and pre-treated with 50 µM PD 98059 (PD+IGF-I) or 50 µM UO 126 (UO+IGF-I) for 30 minutes were stimulated with 50 ng/ml IGF-I for the indicated times. The ERK1/2 MAPK phosphorylation was detected with specific phospho-MAPK antibodies. The same blot was stripped and re-probed with antibody against total ERK1/ERK2 (p44/p42) (Materials and Methods).

View larger version (70K): [in a new window]   Fig. 8. A. The development of -actinin/actin microspikes by IGF-I is blocked by the inhibitors of PI-3 kinase signaling. MCF-7/IGF-IR/WT cells were serum-starved (a), serum-starved and then stimulated with 50 ng/ml IGF-I for 15 minutes (b), pretreated for 30 minutes with 10 µM LY 294002 (c), for 3 hours with 40 µM D3-PI analog (d), 30 minutes with 50 µM PD 98059 (e) or 30 minutes with 50 µM UO 126 (f) and then stimulated with 50 ng/ml IGF-I for 15 minutes. (a-f) Overlays of confocal images of cells stained with TRITC-phalloidin (red) and antibody against -actinin (green). Examples of actin/-actinin microspikes are indicated by arrows. Images are representative of three experiments. Bar, 10 µm. (B) Differential effects of the PI analog and UO 126 on IGF-I-stimulated loss of adherens junctions and formation of microspikes. Serum-starved MCF-7/IGF-IR/WT cells were pretreated with 40 µM D3-PI analog for 3 hours, stimulated with 50 ng/ml IGF-I for 15 minutes in the presence of the inhibitor, fixed and stained with antibody to -catenin (a) or fascin (c) (Materials and Methods). In parallel, the same serum-starved cells were pretreated with 50 µM UO 126 for 30 minutes, stimulated with 50 ng/ml IGF-I for 15 minutes, then fixed and stained with an antibody to -catenin (b) or fascin (d). The relocation of -catenin from adherens junctions normally induced by IGF-I (compare with Fig. 3Aa-d) is blocked by the PI analog but not UO 126. Normal stimulation of fascin microspikes by IGF-I (inset in c, 50 ng/ml IGF-I, 15 minutes) is also blocked only by the PI analog. Confocal images are representative of four independent experiments. Bar, 10 µm. (C) The MEK1/2 inhibitor affects function of stress fibers in IGF-I-stimulated cells. Serum-starved cells not exposed to inhibitor (a) or pretreated with 50 µM UO 126 for 30 minutes (b) were stimulated with 50 ng/ml IGF-I for 15 minutes, then fixed and stained with TRITC-phalloidin. IGF-I normally promotes disassembly of stress fibers within 5 minutes followed by their re-assembly typically after 15 minutes. In cells pretreated with the MEK1/2 inhibitor, re-assembly of the prominent stress fibers is markedly inhibited. The representative images of optical sections collected at the basal level of the cells are shown. Bar, 10 µm.

View larger version (31K): [in a new window]   Fig. 9. (a) Scheme showing which IGF-IR signaling pathways were blocked using a molecular genetic approach (dead kinase IGF-IR) and the pharmacological compounds LY 294002, UO 126, PD 98059 and PI analog. *D3 phospholipids: PtdIns(3)P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(3,4,5)P2; (b) A model of biphasic motility responses regulated by the IGF-IR kinase in MCF-7 cells. Non-motile polarized epithelial cells develop mature adherens junctions and prominent stress fibers; apical ruffles and actin microspikes are not present. The initial stage of cell motility, active cell separation, is characterized by a loss of adherens junctions, disassembly of stress fibers, increased membrane ruffling and formation of actin microspikes; these processes are blocked by the inhibitors of the PI 3-kinase but not of MEK1/2 signaling. The subsequent transition of the cells from separating to migrating is characterized by re-assembly of stress fibers, development of the long membrane protrusions and translocation of the cell body over substratum. MEK1/2 inhibitor blocks all these processes.

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