Article Figures & Data
Figures
- Figure 1.
Relationship between adhesion plaque formation, FAK/ERK signaling, and proliferation. A, FAK/ERK signaling and proliferation. Two different short hairpin RNA (shRNA) sequences targeting FAK and MEK1 mutants (dominant-negative MEK1 K97M; constitutively active MEK1-DD) were tested for their effects on proliferation and ERK phosphorylation under various conditions. D2A1 cells expressing FLAG-tagged ERK2 (FLAG-ERK) were used to determine ERK phosphorylation levels in cells disseminated to the lungs. Following tail vein injection, lungs were harvested and lysed, from which FLAG-ERK was immunoprecipitated (IP) and analyzed. ns, P > 0.5; *, P < 0.02. In “pERK blot,” values represent the ratio of band intensities relative to that of the control sample. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B, FAK/ERK signaling and lung colonization. D2A1-GFP cells, in which FAK/ERK signaling was manipulated as indicated, were injected into mice via the tail vein. The numbers of macrometastases in the left upper lobe of the lungs (bottom) and representative lobe images (top) are presented. The red bars represent the mean values. MEK1-DD expression restored the proliferation of FAK-knockdown D2A1 cells (see A), allowing them to form abundant macrometastases; this substantiated the role of ERKs as major effectors of FAK in regulating colonization. ns, P > 0.5; *, P < 0.001. C, formation of integrin β1–containing elongated adhesion plaques. D2 cells growing in MoT cultures were stained for integrin β1 (red; with an active conformation–specific antibody, 9EG7) and nuclei [4′,6-diamidino-2-phenylindole (DAPI): blue], top left. D2 cells expressing integrin α5-YPet (green) and α-actinin-TagRFP-T (red) were injected into mice via the tail vein, and the distributions of these fluorescent fusion proteins and nuclei (Hoechst 33342; blue) were determined on lung sections (bottom left). The presence of elongated adhesion plaques was quantified (right). D, FAK phosphorylation levels associated with different integrin β1–containing structures. MoT-cultured D2A1 cells were dually stained for pFAK (or total FAK) and integrin β1. Representative images of “elongated adhesion plaques” and “peripheral accumulations” with pFAK (Y397) (green)/integrin β1 (red) staining as well as pseudocolored images representing the ratio of fluorescence intensities are presented (top). The ratio of fluorescence intensities within the region drawn around each of these structures was plotted (n = 50; bottom). *, P < 1 × 10−9. Scale bars, 2 mm (B); 10 μm (D). Values = means ± SD (n = 3; A and C).
- Figure 2.
FLPs that precede adhesion plaque assembly. A, temporal development of elongated adhesion plaques. The localization of integrin β1 (red), F-actin (phalloidin; green), and nuclei (blue) was determined at indicated time points in the MoT-cultured D2A1 cells (left). Integrin β1–containing protrusions (orange arrowheads), elongated adhesion plaques (pink arrows), protrusions of F-actin (blue arrowheads), and actin stress fibers (green arrows) are indicated. The presence of these structures was quantified (right). B, up- and downward projections of FLPs. Top and side views of the MoT-cultured D2A1 cells with F-actin staining (green) are presented with indication of FLPs by the pink arrowheads. C, localization of integrin subunits to FLPs. MoT-cultured D2A1 cells were stained for integrin subunits (red), F-actin (green), and nuclei (blue; left). The accumulation of these subunits to FLPs is indicated (blue arrowheads). The probability with which the length of FLP shaft was covered by the integrin staining was plotted (right). D, FLP formation in MoT-cultured D2 cells. Integrin β1–containing FLPs are indicated by the yellow arrowheads (top). The number of FLPs per cell was plotted (bottom). E, in vivo FLP formation. D2 cells expressing lifeact-Tag-RFP-T (red) and integrin α5-YPet (green) were injected into mice via the tail vein. Blood vessels (PECAM-1 staining; white) and nuclei (blue) were visualized together with these fluorescent proteins on lung sections, where FLPs are indicated (blue arrowheads). These cells are not surrounded by the PECAM-1 staining and therefore are likely to have extravasated into the lung parenchyma. The number of FLPs per cell was plotted (bottom right). Scale bars, 10 μm. Values = means ± SD (n = 3; A and C) or means ± SEM (n = 100; D and E). DAPI, 4′,6-diamidino-2-phenylindole.
- Figure 3.
Formation of FLPs and elongated adhesion plaques by various human breast cancer cell lines. A and B, in vitro formation of FLPs and adhesion plaques. Cells were cultured under MoT conditions to quantify the formation of FLPs (A) and elongated adhesion plaques (B). In B, integrin β1 (red) was stained with an active conformation–specific antibody, HUTS-21. *, P = 0.004; **, P = 0.02. C and D, in vivo formation of FLPs and adhesion plaques Cells were engineered to express integrin α5-YPet (green) and either of lifeact-Tag-RFP-T (red; C) or α-actinin-Tag-RFP-T (red; D). These cells were injected into mice via the tail vein to test the formation of FLPs (C) and elongated adhesion plaques (D) within the lung parenchyma. *, P = 0.0005; **, P = 0.0008. Scale bars, 10 μm. Values = means ± SEM (n = 100; A and C) or means ± SD (n = 3; B and D). DAPI, 4′,6-diamidino-2-phenylindole.
- Figure 4.
FLP formation as a prerequisite for adhesion plaque assembly. A–C, comparison of filopodia (monolayer) and FLPs (MoT). Cells growing in either monolayer or MoT culture were stained for F-actin (green; A and B), integrin β1 (red; A) and α-actinin (red; B, bottom). Fascin1 localization was determined by the use of TagRFP-T-fascin1 fusion protein (red; B, top). A, the fluorescence intensity along the line [root (R) −; tip (T)] was plotted (right), and integrin β1 distribution within each protrusion type was scored (bottom). B, fascin1 was localized along the length of filopodia but not to FLPs; α-actinin was detected at the root of filopodia and along the length of FLPs. C, the kinetics of the assembly and disassembly of these protrusions are represented by the Kaplan–Meier survival curves. D2A1 cells expressing the lifeact-YPet actin marker were analyzed by time-lapse microscopy. *, P < 0.0001 (by log-rank test). D, initial accumulation of adhesion plaque proteins to FLPs. The localization of F-actin (green), nuclei (blue), and FAK (red) was determined at indicated time points (top). The pseudocolored images represent the profiles of FAK staining intensity. The accumulation of integrin β1 and various adhesion plaque proteins to FLPs and elongated adhesion plaques was scored (bottom). E and F, functional connection between FLPs and elongated adhesion plaques. Integrin α5-YPet–expressing D2A1 cells growing under the MoT conditions were analyzed by time-lapse microscopy. E, cells were classified by the presence or absence of FLPs (purple) and elongated adhesion plaques (green) at the beginning of observation. Initially adhesion plaque–negative cells (113 + 122 cells) were analyzed for the subsequent development of FLPs and elongated adhesion plaques. F, an example of progressive inward movement of FLP-associated integrin α5-YPet clumps (arrowheads; 0–80 minutes) and subsequent outward extension of the plasma membrane, which cooperatively converted these integrin clumps into the ones constituting elongated adhesion plaques (arrowheads; 140–160 minutes), is presented. The pink dotted lines represent cell edges. Scale bars, 10 μm. Values = means ± SD (n = 3; A) or means ± SEM (n = 30; D). DAPI, 4′,6-diamidino-2-phenylindole.
- Figure 5.
Identification of the molecular regulators of FLP formation. A and B, screening of filopodium regulators for their involvement in FLP formation and proliferation. D2A1 cells, manipulated to block the expression or function of filopodium-associated proteins, were tested for FLP formation under MoT culture conditions (A). Two different short hairpin RNA (shRNA) sequences were tested for each knockdown target. The effects of these manipulations on the cell number after 10 days of monolayer or MoT cultures were also tested (B). ns, P > 0.2; *, P < 0.02 (vs. control). C, localization of Rif and mDia2 to FLPs. YPet–Rif fusion protein was used for analyzing Rif distribution, whereas mDia2 localization was determined by direct immunostaining (green; mDia2 localization at FLP tips is indicated by pink arrowheads). F-Actin (red) and nuclei (blue) were also visualized. D, role of Rif/mDia2 signaling and Ena/VASP proteins in adhesion plaque formation. Some of the engineered D2A1 cells described in A were cultured under MoT conditions. The presence of integrin β1–containing elongated adhesion plaques was quantified (right). ns, P > 0.05; *, P < 0.0005. E, role of Rif/mDia2 signaling in FAK/ERK activation. Lysates from the indicated cell types were immunoprecipitated (IP) with an anti-total FAK antibody and analyzed for FAK phosphorylation levels. These lysates were also analyzed by direct immunoblotting for ERK phosphorylation levels. F, rescue of proliferation defects by CD2-FAK expression. The control, Rif-, and mDia2-knockdown D2A1 cells were further engineered to express the constitutively active CD2–FAK fusion protein. Cell numbers after 10-day culture were plotted. ns, P > 0.1; *, P < 0.005. Scale bars, 10 μm. Values = means ± SEM (n = 100; A) or means ± SD (n = 3; B, D, and F). DAPI, 4′,6-diamidino-2-phenylindole.
- Figure 6.
Rif/mDia2 signaling and lung colonization. A and B, Rif/mDia2 signaling and in vivo formation of FLPs and elongated adhesion plaques. D2A1 cells expressing integrin α5-YPet (green) and either lifeact-Tag-RFP-T (red; A) or α-actinin-Tag-RFP-T (red; B) were further engineered as indicated and tested for the formation of FLPs (blue arrowheads; A) and elongated adhesion plaques (pink arrowheads; B) within the lung parenchyma. The number of FLPs per cell (A, right) and the presence of elongated adhesion plaques (B, right) were quantified (right). ns, P > 0.1; *, P < 1 × 10−8; **, P < 0.01. C, Rif/mDia2 signaling and in vivo FAK activation. D2A1 cells expressing hemagglutinin (HA)-tagged FAK (FAK-HA), with or without Rif- or mDia2-knockdown, were injected into mice via the tail vein. Lysates were prepared from the lungs, from which FAK-HA was immunoprecipitated and analyzed. D and E, Rif/mDia2 signaling and lung colonization following tail vein injection. D, the D2A1-GFP cells, engineered as in A, were tested for macrometastasis formation and proliferation in the lungs. ns, P > 0.5; *, P < 0.0002; **, P < 0.05; ***, P < 0.005. E, lung sections were prepared 10 days after the injection of the engineered D2A1 cells, also expressing membrane-targeted tdTomato (tdTomato membrane). The numbers of macroscopic (>20 cells per colony) and microscopic (≤20 cells per colony) metastases were plotted (bottom). “M”-labeled regions indicate macrometastases. *, P < 0.02; **, P < 0.05. F, restoring metastasis-forming ability by CD2–FAK expression. The effects of CD2–FAK expression on the number of lung macrometastases formed by the control, Rif-, and mDia2-knockdown D2A1 cells following tail vein injection were tested. ns, P > 0.1; *, P < 0.005. Scale bars, 10 μm (A and B), 2 mm (D and F), and 0.1 mm (E). Values = means ± SEM (n = 150; A) or means ± SD (n = 3; B, D, and E).
- Figure 7.
Rif/mDia2 signaling in primary tumor formation and spontaneous metastasis. A, cell biologic and associated signaling events critical to the initial proliferation of cancer cells within foreign tissue parenchyma. B and C, role of FLP-regulating proteins in primary tumor formation and spontaneous metastasis. The D2A1-GFP cells (B) and TS/A-GFP cells (C), each engineered as indicated, were implanted into mouse mammary fat pads. Subsequent formation of primary tumors and the incidences of spontaneous metastases in the lungs and liver were analyzed. B, Ki67 staining positivity was scored on the primary tumor sections (right). Values = means ± SD (n = 3). C, a macrometastasis and micrometastases are indicated by blue and pink arrowheads, respectively, on the representative lung images (right). *, P < 0.04; **, P = 0.03 (vs. control) by the Fisher exact test. ns, P > 0.2; ***, P < 0.05 by the Student t test. D, role of Rif in metastasis formation by the intracardially injected B16F10 cells. The control and Rif-knockdown B16F10 cells were intracardially injected into mice. Metastasis formation in the lungs, liver, and bone marrow was analyzed. *, P < 0.05 (vs. sh scrambled) by the Fisher exact test. **, P < 0.02 by the Student t test. Scale bars, 0.1 mm (B); 2 mm [C (low mag.), D], and 0.5 mm [C (high mag.)].
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Methods - PDF file - 216K
- Supplementary Figures 1-17 - PDF file - 1.8MB
- Supplementary Movie 1 - MOV file - 3.4MB, Kinetics of the assembly and disassembly of filopodia in cells cultured under monolayer conditions
- Supplementary Movie 2 - MOV file - 3.4MB, Kinetics of the assembly and disassembly of FLPs in cells cultured under MoT conditions
- Supplementary Movie 3 - MOV file - 3.4MB, Direct conversion of FLP-associated integrin clumps into the ones constituting adhesion plaques
Volume 2, Issue 8
