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First published online 6 January 2009
doi: 10.1242/jcs.037564

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Spontaneous phosphoinositide 3-kinase signaling dynamics drive spreading and random migration of fibroblasts

Department of Chemical and Biomolecular Engineering, North Carolina State University, Box 7905, Raleigh, NC 27695, USA

View larger version (53K): [in this window] [in a new window]   Fig. 1. PI3K activation during fibroblast spreading on adhesive surfaces. Quiescent, EGFP-AktPH-expressing NIH3T3 mouse fibroblasts were imaged by TIRF microscopy as they attached and spread on adhesive surfaces. Subsequently, cells were uniformly stimulated with 5 nM PDGF (P) and later treated with 5 µM wortmannin (W) at the indicated times to fully activate and inhibit PI3K signaling, respectively (Schneider and Haugh, 2004). (A,B) Two representative cells spreading on fibronectin (A) (see also supplementary material Movie 1) or poly-D-lysine (B) (see also supplementary material Movie 2) show localized PI3K activation in conjunction with membrane protrusion events (arrowheads). Scale bars: 20 µm. (C) Comparison of cell-spreading rates, measured when the cell had spread to half of its maximum contact area, on poly-D-lysine and fibronectin. The black square and error bars report the mean and s.d., and the box reports the median and upper and lower quartiles. Cells spread faster on average on fibronectin (P<0.001, Student's t-test). (D) The extent of PI3K activation during spreading, relative to the level measured at the periphery of each cell after PDGF stimulation, is displayed in the same format as in panel C. (E) Representative control experiments showing sequential TIRF images of EGFP-AktPH-expressing cells that had been loaded with the cytoplasmic dye CellTracker Red. Scale bars: 20 µm.

View larger version (58K): [in this window] [in a new window]   Fig. 2. PI3K activation in the absence of classical integrin signaling. Quiescent, EGFP-AktPH-expressing NIH3T3 mouse fibroblasts were allowed to adhere and spread onto dishes coated with poly-D-lysine (PL) or fibronectin (FN). (A,B) Cell lysates were collected at the indicated times and probed for the indicated proteins by immunoblotting. Cells were held in suspension (Susp) for 30 minutes prior to seeding. Integrin signaling, assessed in terms of FAK and paxillin phosphorylation, was evident in cells spreading on fibronectin but not in cells spreading on poly-D-lysine; PI3K activation, assessed in terms of Akt phosphorylation, was seen in cells spreading on both surfaces. The blot shown in A is representative of three independent experiments. (B) Densitometry analysis, with β-actin used as a loading control (mean ± s.e.m., n=3). (C) Phase-contrast images of spreading cells, 90 minutes post-plating. Antibodies that block murine β1 integrin (AB; 20 µg/ml) strongly inhibit cell spreading on fibronectin but not poly-D-lysine, and this treatment does not grossly affect PI3K activation during spreading on poly-D-lysine (assessed as in Fig. 1). (D) Anti-β1-integrin antibodies block FAK and paxillin phosphorylation on fibronectin but do not block Akt phosphorylation on either fibronectin or poly-D-lysine. Cells were allowed to spread on poly-D-lysine or fibronectin for 90 minutes, either in the presence or absence of the blocking antibodies, and immunoblotting was performed and quantified as in B. A cell suspension control was included for comparison, and the results are normalized relative to the fibronectin, no integrin block condition and expressed as mean ± s.e.m. (n=2).

View larger version (39K): [in this window] [in a new window]   Fig. 3. PI3K is required for efficient spreading of fibroblasts. (A,B) Quiescent, EGFP-AktPH-expressing NIH3T3 mouse fibroblasts were treated with the PI3K inhibitor LY294002 prior to spreading on fibronectin, imaged by TIRF microscopy. (A) The normalized, whole-cell fluorescence level of PI3K signaling during spreading (f-rate) and the rate of spreading, assessed when each cell had reached half its maximum contact area plotted as a function of LY294002 concentration. The black square and error bars report the mean and s.d., and the box represents the median and upper and lower quartiles. The asterisks indicate significant inhibition of spreading relative to the DMSO control (P<0.05, Student's t-test). (B) Correlation of spreading rate versus PI3K signaling (f-rate). The vertical dashed line demarcates cells that showed minimal PI3K signaling (f-rate <0.1) from those with more significant PI3K activation levels. The horizontal dashed line demarcates cells that showed a low spreading rate (<60 µm2/minute, approximately one s.d. below the mean of the DMSO control) from those that spread at a higher rate. See the text for additional details. (C) NIH3T3 cells were co-transfected with EGFP-AktPH and dominant-negative PI3K regulatory subunit (DN p85); in parallel, control cells were transfected with EGFP-AktPH only. As indicated by the asterisks, spreading is significantly inhibited in those cells showing strong inhibition of PI3K, and a comparison of all DN p85-transfected cells with control cells was also statistically significant (P<0.01, Student's t-test). (D) EGFP-AktPH-expressing fibroblasts were treated with either 100 µM LY294002 (LY) or 100 nM wortmannin (W) while in the midst of active spreading. Two representative cells show significant inhibition of spreading. Scale bars: 20 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Disrupting actin polymerization halts spreading but not PI3K signaling. (A,B) Representative TIRF montages of EGFP-AktPH-expressing fibroblasts treated with either 1 µM cytochalasin D (CytoD, A) or 2 µM latrunculin B (LatB, B) while in the midst of active spreading (indicated by an asterisk). PDGF (P) and then wortmannin (W) were subsequently added to assess the extent of PI3K signaling as in Fig. 1D. Scale bars: 20 µm. (C) The extent of PI3K activation was similar in cells treated during spreading with a DMSO control (0.40±0.22), CytoD (0.43±0.29) or LatB (0.48±0.17). The black square and error bars report the mean and s.d., and the box reports the median and upper and lower quartiles.

View larger version (49K): [in this window] [in a new window]   Fig. 5. Dissociation of microtubules during spreading elicits dynamic, PI3K-dependent motility processes. (A) Representative TIRF montage of an EGFP-AktPH-expressing fibroblast treated with 10 µM nocodazole (indicated by an asterisk) during active spreading on fibronectin. Nocodazole induces uniform retraction of the cell, often followed by periods of dynamic protrusion and retraction events, as seen in the plot of contract area vs. time. PDGF (P) and then LY294002 (LY) were subsequently added to assess the extent of PI3K signaling, yielding uniform spreading and retraction of the contact area, respectively. Scale bars: 20 µm. See also supplementary material Movie 3. (B) Representative TIRF montages of cells treated first with nocodazole (*) and later with one of three PI3K inhibitors: 100 µM LY294002 (LY), 100 nM wortmannin (W) or 3 µM PI3K inhibitor IV. Scale bars: 20 µm.

View larger version (33K): [in this window] [in a new window]   Fig. 6. PI3K activation dynamics in randomly migrating fibroblasts. EGFP-AktPH-expressing fibroblasts were allowed to migrate in low serum conditions for 6 hours. Overall cell movement is tracked by calculating the coordinates of the cell contact area centroid as a function of time. (A) A representative fibroblast exhibiting persistent migration shows polarized PI3K signaling (arrowheads) that correlates with the direction of migration (inset arrows). See also supplementary material Movie 4. Scale bar: 50 µm. (B) A representative fibroblast with alternating PI3K activation events (arrowheads) in conjunction with protrusion of lamellipodial branches that together determine the overall direction of migration (inset arrows). See also supplementary material Movie 5. Scale bar: 50 µm. (C) Quantification of overall PI3K signaling directionality was performed as described under Materials and Methods. At an instant in time, a cell's signaling vector is defined as the sum of position vectors associated with regions of higher EGFP-AktPH fluorescence, normalized so that each position vector has a magnitude equal to the overall, background-subtracted fluorescence of the region. For each 12-minute interval, the angle from the signaling vector to the vector of cell centroid movement was recorded, and the polar plot shows the histogram of angles recorded for 18 migrating cells. (D) NIH3T3 cells were co-transfected with mCherry-AktPH and EGFP-Rac1 and monitored by TIRF microscopy during random migration. In the representative cell shown, regions with higher fluorescence are indicated by arrowheads. Scale bar: 20 µm.

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