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Dissection of HEF1-dependent functions in motility and transcriptional regulation

Fox Chase Cancer Center, 7701 Burholme Ave, Philadelphia, PA 19111, USA

View larger version (51K): [in a new window]   Fig. 1. Generation of MCF7 stable cell lines in which HEF1 expression is regulated by tetracycline. (A) MCF7 cells were transfected with a plasmid encoding the tTA transactivator and a tetracycline-regulatable expression plasmid either without (CM1,2) or with a HEF1 cDNA insert (HEF1.M1-M5). Total cell lysates (35 µg) derived from uninduced (+ lanes) or induced/mock induced (- lanes) clones were processed by immunoblotting with HEF1-SB-R1 antisera to assess HEF1 production levels. The p105 and p115 proteins are differently phosphorylated forms of HEF1 (Law et al., 1998). (B) Lysates (30 µg) were isolated from HEF1.M1 cells induced for the indicated time intervals (in hours, labeled above each lane). Negative controls include lysates isolated after 24 hours from uninduced HEF1.M1 (lane 1) or mock induced CM1 cells (lane 9), probed with antibody HEF1-SB-R1, which is specific for HEF1. (C) Lysates from CM.1, HEF1.M1 and HEF1.M2 cells induced for the indicated time intervals (in hours) or maintained in tetracycline (+T) were immunoblotted with antibody to p130Cas (bottom). Corresponding levels of HEF1 at the same time points are also shown (top). (D) Cell lysates from CM1, HEF1.M1 or HEF1.M2 cells prepared at 0, 9 or 24 hours after induction, or in cells maintained in tetracycline (+T) were immunoprecipitated with antibody to p130Cas and then probed with antibody to phosphotyrosine (top), stripped and reprobed with antibody to p130Cas (bottom).

View larger version (159K): [in a new window]   Fig. 2. HEF1 induces a morphological conversion to crescent-shaped cells. HEF1.M1 (A,B), HEF1.M2 (C,D) and CM1 (E,F) cells were either uninduced (A,C,E) or induced/mock induced (B,D,F) for HEF1 production. Phase contrast CCD images were acquired with a 40x objective. Bar, 25 µm.

View larger version (134K): [in a new window]   Fig. 3. HEF1 localizes to prominent focal adhesion sites. Immunofluorescent staining of either uninduced (A-C) or induced (D-I) HEF1.M1 cells was performed using antisera specific for HEF1 (A,D,G) and antibodies specific for paxillin (B,E) or phalloidin-stained F-actin (H). Merged images (C,F,I) demonstrate the pronounced co-localization of HEF1 and paxillin to the prominent focal adhesion sites in the leading edge lamellipodia (F), with HEF1 present at the distal ends of F-actin-rich stress fibers (I, arrowheads). Images depict 1.4 µm sections, acquired using a Bio-Rad MRC 600 confocal microscope (60x objective). Bar, 25 µm.

View larger version (90K): [in a new window]   Fig. 4. HEF1 production increases cell spreading. HEF1.M1 cells were maintained for 18 hours in either non-inducing (A,C,E,G,I) or inducing (B,D,F,H,J) conditions and then replated on glass coverslips coated with 6 µg ml–1 human FN (1.25 µg cm–2). Cells were fixed at 30 minutes (A,B), 1 hour (C,D), 2 hours (E,F), 3 hours (G,H) and 6 hours (I,J) after plating. Phase contrast CCD images were acquired with a 40x objective. Cells were maintained in inducing or non-inducing conditions for the duration of the experiment. The graphs show the quantitation of increased cell spreading. HEF1.M1 or CM1 cells maintained for 18 hours in either non-inducing (–) or inducing (+) condition were replated for 6 hours on glass coverslips either uncoated in the presence of 10% FBS (gray) or coated with human FN in serum-free medium (black). CCD images were acquired with a 40x objective (8-10 fields per condition) and the area determined using Inovision ISEETM. Results shown are the means of three independent experiments±standard error.

View larger version (13K): [in a new window]   Fig. 5. HEF1 production enhances cell motility by increasing cell speed. The average speed of uninduced or induced HEF1.M1 (A) or CM1 (B) cells was determined by analyzing the movement of individual cells at 5-minute intervals over the course of 6 hours using Inovision ISEETM nanotracking software. (A) The average speed of uninduced (gray) or induced (black) HEF1.M1 cells, grouped into speed ranges. (B) The average speed of uninduced (gray) or mock induced (black) CM1 cells, grouped into speed ranges; the speed of CM1 cells was not altered by mock induction. These data represent the average speed of HEF1.M1 cells (uninduced, N=59; induced, N=55) and CM1 cells (uninduced, N=76; induced, N=60) derived from two independent experiments.

View larger version (24K): [in a new window]   Fig. 6. HEF1 production augments FN mediated haptotaxis. (A) Increase in the number of cells that traversed the membrane in a Boyden chamber assay in response to soluble FN, as opposed to the absence of stimulus. HEF1.M1 (black) or CM1 (gray) cells were seeded into the top well of Boyden chambers and assessed for their ability to migrate towards FN in either non-inducing or inducing (for 20 hours) conditions. The haptotactic response of populations maintained in either non-inducing (–) or inducing (+) conditions were grouped separately and normalized against the number of cells that traversed the membrane for each condition in the absence of stimulus. Results shown are the mean of multiple independent experiments for each cell line±standard error. (B) Induced lysates were probed with anti-phosphorylated-MAPK and anti-phosphorylated-p38 antibodies (top), and in parallel with anti-MAPK (lanes 1,2) and anti-p38 (lanes 3,4) (bottom) (C) Inhibition of HEF1.M1 cell haptotaxis toward FN in a Boyden chamber assay (as described above) following treatment with drug inhibitors for MAPK kinase (PD98059, 25 µM), p38 (SB202190, 25 µM) and control (DMSO).

View larger version (17K): [in a new window]   Fig. 7. Correlative analysis of cDNA expression array experiments. Scatter graphs showing pair-wise correlation in gene expression in (A) uninduced (x axis) and induced (y axis) HEF1.M1 cells, and (B) uninduced (x axis) and mock-induced (y axis) CM1 cells. A high degree of correlation (>90%) between the compared data sets is indicated by the R2 values (0.916 and 0.964, respectively).

View larger version (37K): [in a new window]   Fig. 8. HEF1 overproduction induces increased mRNA transcript and protein levels of downstream targets. (A) RT-PCR analysis of actin control and HEF1 targets predicted by the array analysis. (Top) Actin message was amplified from cDNA template derived from either uninduced (lanes 1-4) or HEF1 induced (lanes 5-8) HEF1.M1 cells for 23 (lanes 1 and 5), 28 (lanes 2 and 6), 33 (lanes 3 and 7) or 38 (lanes 4 and 8) cycles. M is a molecular weight marker. (Bottom) Comparative amplification of transcripts encoding MLCK, p160ROCK (ROCK), MDA7 and metalloprotease (MTLP) using normalized cDNA template derived from either uninduced (–) or HEF1 induced (+) HEF1.M1 cells. RT-PCR products shown were amplified for either 28 (MLCK, MDA7 and MTLP) or 33 (ROCK) cycles. (B) Whole-cell lysates of HEF1.M1 cells either uninduced (lanes 1, 3, 5; –) or induced (lanes 2, 4, 6; +) for 20 hours were probed with antibodies to MMP1, MMP14 or ErbB2.