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First published online 11 March 2003
doi: 10.1242/jcs.00386

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The formin-homology-domain-containing protein FHOD1 enhances cell migration

¹ Department of Oral Biology, College of Dentistry, University of Nebraska Medical Center, Lincoln, NE 68583, USA
² University of Minnesota Cancer Center, Minneapolis, MN 55455, USA
³ Department of Laboratory Medicine and Pathology, Minneapolis, MN 55455, USA
⁴ Department of Orthopaedic Surgery, Minneapolis, MN 55455, USA
⁵ Eppley Institute for Research in Cancer and Allied Diseases, Department of Biochemistry and Molecular Biology, and Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA

View larger version (31K): [in a new window]   Fig. 1. Generation of stable FHOD1-positive WM35 and NIH-3T3 cell lines. (A) Schematic representation of the HA-FHOD1 proteins used in these studies. (B) Immunoblot analysis of endogenous and ectopic FHOD1 proteins in NIH-3T3 and WM35 cells. Protein lysates from untransduced and MSCV-transduced cells were resolved by SDS-PAGE. Antibodies recognizing the FHOD1 N-terminus (FHOD1-N) were used to probe NIH-3T3 cells for endogenous and ectopic FHOD1 proteins. WM35 cell lysates were probed with anti-HA or FHOD1-C antibodies to detect ectopic and endogenous proteins, respectively. (C) HA-FHOD1 protein subcellular localization in WM35 cells. WM35 cells were grown on coverslips and FHOD1 proteins were detected by in situ immunofluorescence with anti-HA antisera.

View larger version (76K): [in a new window]   Fig. 2. FHOD1 induces cell elongation. (A) Fluorescent (left) and phase contrast (right) images of transduced and sorted NIH-3T3 cells showing the cell morphology of eGFP-expressing cells. Because the same vector encodes FHOD1 and eGFP, eGFP expression is used as a marker to detect FHOD1—expressing cells. (B) FHOD1-positive cell populations contain elongated cells. The length-to-width ratios of eGFP positive cells are plotted in the graph. The table indicates the mean and median values for the indicated population. *P<0.05 for FHOD1 versus MSCV and FHOD1 (1-421) cells.

View larger version (35K): [in a new window]   Fig. 3. FHOD1 interacts with F-actin and G-actin. (A) NIH-3T3 cells were transiently transfected with plasmids encoding full-length HA-FHOD1 (1-1165), HA-FHOD1 C (1-1010) or HA-FHOD1 (1-421). FHOD1 expression was detected by in situ staining with anti-HA followed by FITC-conjugated secondary antibodies. Cells were countered stained with phalloidin-rhodamine to detect F-actin. Images were collected by confocal microscopy. (B) HEK293T cells were transiently transfected with expression vectors for the indicated HA-FHOD1 proteins. Triton X-100 soluble cellular proteins were immunoprecipitated with anti-HA or anti-actin antibodies. Proteins were detected by immunoblotting with anti-HA or anti-actin antibodies and enhanced chemiluminescence.

View larger version (41K): [in a new window]   Fig. 4. Dominant-negative Rac1 and inhibitors of RhoA and ROCK block FHOD1 C-dependent stress fibers. (A) Rac1N17 blocks FHOD1-C-dependent stress fibers. NIH-3T3 cells were transiently transfected with pCMV5-FHOD1 C and pCMV-T7-Rac1N17. FHOD1- and RacN17-expressing cells were detected by in situ immunofluorescence. F-actin was detected with phalloidin-rhodamine. (B) RhoA activity is required for FHOD1-C-dependent stress fibers. FHOD1-C-transfected NIH-3T3 cells were incubated for 18 hours with 10 µM or 30 µM C3 transferase prior to in situ fluorescence analysis and confocal microscopy. (C) ROCK activity is required for FHOD1-C-dependent stress fiber formation. FHOD1-C-transfected NIH-3T3 cells were incubated for 10 minutes with 30 µM Y-27632. FHOD1 C and F-actin were detected with in situ fluorescence and confocal microscopy.

View larger version (21K): [in a new window]   Fig. 5. FHOD1 expression does not affect cell adhesion. (A) FHOD1 expression does not significantly affect WM35 cell adhesion to type-I collagen. WM35 cells were allowed to adhere to plates coated with the indicated concentrations of type-I collagen for 20 minutes. Adherent cells were quantified with a cell viability assay. The number of adherent cells was normalized to a standard curve generated by plating increasing numbers of cells. (B) FHOD1 expression does not significantly affect NIH-3T3 cell adhesion to fibronectin. NIH-3T3 cells were allowed to adhere to plates coated with the increasing concentrations of fibronectin for 20 minutes. The number of adherent cells was determined as described in A.

View larger version (22K): [in a new window]   Fig. 6. FHOD1 enhances cell migration. (A) Expression of full-length FHOD1 in WM35 cells enhanced migration to type-I collagen. The migration of control (MSCV) and FHOD1-expressing WM35 cells to type-I collagen (1 µg ml-1 on the left, indicated concentrations on the right) during a 4-5 hour period was measured in a Boyden chamber. (B) Expression of full-length FHOD1 in NIH-3T3 cells enhanced migration to fibronectin, laminin and BCS. Each protein was used at a final concentration of 1 µg ml-1. BCS was added at 10% (v/v). Cells were allowed to migrate for 4-5 hours in a modified Boyden chamber.

View larger version (23K): [in a new window]   Fig. 7. FHOD1 expression does not affect integrin expression or use. (A) FHOD1 does not significantly alter integrin expression on WM35 cells. Stable WM35 cells were incubated with PE-conjugated anti-integrin antibodies and analyzed by flow cytometry. The mean PE fluorescence intensity of eGFP-positive cells was determined with CellQuest software. (B) FHOD1 expression does not alter integrin usage during cell adhesion. Cells were preincubated for 20 minutes with 2.5 µg ml-1 of the isotype-matched control or the indicated -integrin mAb, or with 1 µg ml-1 of anti-ß1 mAb. The amount of cell adhesion to plates coated with 15 µg ml-1 type-I collagen within 20 minutes was determined with a cell viability assay. Values for each cell line were normalized to the control cells incubated with the isotype-matched mAb. (C) FHOD1 does not alter integrin usage during cell migration. WM35-MSCV control (left) or FHOD1-expressing (right) cells were preincubated for 20 minutes with 2.5 µg ml-1 of the isotype-matched control or the indicated -integrin mAb or with 1 µg ml-1 of anti-ß1 mAb. Cell migration during a 4.5 hour period was measured in a modified Boyden chamber assay.

View larger version (10K): [in a new window]   Fig. 8. FHOD1 expression does not affect ß1-integrin activation. MSCV and FHOD1-expressing WM35 cells were incubated with antibodies recognizing the ß1-integrin activation epitopes 15/7 and 9EG7, the pan-ß1-integrin epitope P5D2 or control IgG for 30 minutes on ice followed by a 30-minute incubation with a Cy5-conjugated secondary mAb. Mean fluorescent intensities of eGFP-positive cells were determined with CellQuest software and normalized to baseline levels of control antibodies.