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First published online 29 June 2004
doi: 10.1242/jcs.01205

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Disruption of Rho signal transduction upon cell detachment

¹ Department of Dermatology, SUNY at Stony Brook, Stony Brook, NY 11794, USA
² Department of Pharmacological Sciences, SUNY at Stony Brook, Stony Brook, NY 11794, USA
³ Department of Biomedical Engineering, SUNY at Stony Brook, Stony Brook, NY 11794, USA
⁴ Department of Cell Pharmacology, Nagoya University, 5 Tsurumai, Showa, Nagoya, Aichi 466-8550, Japan

View larger version (35K): [in a new window]   Fig. 1. ppMLCT18/S19 is dependent on serum, Rho and Rho kinase. (A) Human fibroblasts were serum starved for 0 minutes, 2 minutes, 5 minutes and 15 minutes, and collected for analysis of ppMLCT18/S19 by western blotting. (B) Cells were starved for 60 minutes, stimulated with 10% serum for 0 minutes, 1 minutes, 3 minutes and 10 minutes, and subjected to analysis of ppMLCT18/S19. (C) Human fibroblasts, either serum starved or in 10% FBS, were treated with 10 µM Y-27632 or 25 µM ML-7 for 30 minutes, and collected in TCA for the analyses of MLC phosphorylation. DMSO (1:1000) was included as a vehicle control for ML-7. Similar results were obtained in three independent experiments. (D) Cells were treated with C3 exoenzyme/Lipofectamine mixture and incubated under serum-free condition for 8 hours. PBS/Lipofectamine was used for mock transfection. Cells were then either not stimulated or stimulated with 10% FBS for 2 minutes and collected in TCA for the analyses of ppMLCT18/S19.

View larger version (56K): [in a new window]   Fig. 2. Effect of detachment and Y-27632 on ppMLCT18/S19 and pMLCS19. (A) Cells were detached with trypsin-EDTA for about 5 minutes at room temperature and the serum-containing medium was then added. The cell suspension was either directly transferred to 40 ml ice-cold PBS to stop the reaction (after 0.5 minutes) or incubated at 37°C for 3 minutes or 10 minutes before adding 40 ml ice-cold PBS. Cells were pelleted at 4°C and subjected to ppMLCT18/S19 analysis. Attached cells (Att) were included for comparison. (B) The pMLCS19 levels were analysed in human fibroblasts either detached and incubated for 3 minutes in the presence of serum or attached in the presence of serum or after serum starvation for 30 minutes. The ppMLCT18/S19 from the same samples is also shown. (C) Response of pMLCS19 and ppMLCT18/S19 to the treatment with 10 µM Y-27632 for the indicated times. The results from at least three independent experiments are summarized and presented (right) as mean±s.e.m. relative to the time 0. (D) Immunofluorescence staining of focal adhesions (vinculin) and actin stress fibers after Y-27632 treatment.

View larger version (27K): [in a new window]   Fig. 3. Analysis of RhoA activity, pMBST853 and in vitro Rho-kinase activity after detachment. (A) Cells were detached with trypsin-EDTA for about 5 minutes at room temperature and the serum-containing medium was then added. The cell suspension was either directly transferred to 40 ml ice-cold PBS to stop the reaction (after 0.5 minutes) or incubated at 37°C for 3 minutes or 10 minutes before adding 40 ml ice-cold PBS. Cells were then collected for RhoA activity assay. (B) Effect of Y-27632 on pMBST853 in attached and detached cells in the presence of serum. Levels of ppMLCT18/S19 in the same samples are also shown. (C) pMBST853 was analysed in human fibroblasts either detached and incubated for 3 minutes in the presence of serum (Det) or attached in the presence of serum or after serum starvation for 30 minutes. (D) In vitro Rho-kinase activity was assayed. Radiolabeling of MLC was absent without HA-Rho kinase (lane 1, mock immunoprecipitation using Protein-A beads only) or when 10 µM Y-27632 was included in the kinase reaction (lane 4), confirming that the kinase reaction is specific to Rho kinase.

View larger version (31K): [in a new window]   Fig. 4. pMBST696 and pCPI-17T38 levels are not significantly affected by detachment, serum starvation or Y-27632 treatment. (A) Cells were detached and incubated for 3 minutes in the presence of serum (Det), attached (Att) in the presence of serum, serum starved for 30 minutes or treated with 10 µM Y-27632 for 30 minutes in the presence of serum. Data from four independent experiments were summarized and presented as mean±s.e.m. relative to the serum-fed attached cells. (top right) pMBST696 decreased in COS-7 cells treated with 10 µM Y-27632 for 30 minutes in the presence of serum. (B) Cells were treated the same way as above for the detection of pCPI-17T38. (right) Fibroblasts were treated with 0.5 µM PMA or 1:2000 DMSO for 30 minutes.

View larger version (29K): [in a new window]   Fig. 5. Active Rho and Rho-kinase are unable to restore ppMLCT18/S19 in detached cells. (A) The HT-1080 cells were transfected with various cDNAs as indicated using the Effectene reagents for 20 hours in the presence of serum. Cells were either collected directly in TCA for the analysis of MLC and MBS phosphorylation or detached and incubated in the presence of serum for 3 minutes before being stopped with TCA. The results for ppMLCT18/S19 are summarized from three independent experiments and are shown as the mean±s.e.m. relative to adherent pEGFP-transfected cells. The insert shows one of the representative experiments with the same sample orders as for the chart. (B) The analysis of pMBST853 in the detached transfected cells in comparison with the adherent pEGFP-transfected cells. The ppMLCT18/S19 from the same samples is also shown.

View larger version (52K): [in a new window]   Fig. 6. Regulation of ppMLCT18/S19 by cell adhesion to FN. (A) Human fibroblasts were detached, resuspended in 10% FBS and immediately plated onto dishes coated with 20 µg ml–1 FN for the indicated times for the analysis of ppMLCT18/S19 and pMBST853. (B) Human fibroblasts were suspended in 1% FBS for 60 minutes and plated onto dishes coated with 20 µg ml–1 FN or PLL for indicated times. (bottom) NIH3T3 cells transiently expressing RhoAV14 or Rho-kinase CAT were suspended in serum-free medium for 30 minutes and plated onto dishes coated with 20 µg ml–1 FN or 100 µg ml–1 PLL for 30 minutes for the analysis of ppMLCT18/S19.