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1.
Figure 6.

Figure 6. From: Migrating fibroblasts reorient directionality by a metastable, PI3K-dependent mechanism.

Conceptual model of fibroblast reorientation by branch-and-pivot dynamics. Cells with a defined front and rear at opposite ends are considered stable, and deviations from this morphology are unstable. Nascent branches are initiated stochastically and in a PI3K-independent manner. After the onset of protrusion, PI3K is localized there; PI3K signaling is required for lateral spreading and stabilization of the new lamellipodium. If PI3K signaling is not interrupted (for too long) in either of the two branches, the branched state is metastable and propagates. A turn of up to 90° is completed once one of the branches stops protruding and reverts to net retraction. The inset outlines a hypothetical feedback loop in which protrusion and PI3K signaling reinforce one another.

Erik S. Welf, et al. J Cell Biol. 2012 April 2;197(1):105-114.
2.
Figure 1.

Figure 1. From: Migrating fibroblasts reorient directionality by a metastable, PI3K-dependent mechanism.

Reorientation of fibroblast migration by branch and pivot of protrusions. NIH 3T3 fibroblasts expressing GFP-AktPH were monitored by TIRF microscopy during random migration on fibronectin (n = 28). (a) A pseudocolor montage showing the characteristic branching and pivoting of protrusions and localization of PI3K signaling (see also Video 1). The sketch at the right illustrates how protrusion velocity, mapped as a function of angular position and time, reveals branch-and-pivot behavior. Bar, 20 µm. (b) Spatiotemporal maps of protrusion (red)/retraction (blue) velocity, PI3K-signaling hotspots, and morphological extensions for the cell depicted in a. a.u., arbitrary unit. (c–e) Protrusion branching and switching between protrusion and retraction mediate sharp turns. A pseudocolor montage (c; see also Video 2), contact area centroid path (d), and spatiotemporal map of PI3K-signaling hotspots (e) show how abrupt changes in cell orientation correspond with changes in PI3K signaling (colored arrowheads). Bar, 20 µm.

Erik S. Welf, et al. J Cell Biol. 2012 April 2;197(1):105-114.
3.
Figure 4.

Figure 4. From: Migrating fibroblasts reorient directionality by a metastable, PI3K-dependent mechanism.

PI3K signaling is localized in response to protrusion induced by focally activated Rac. (a) Localization of mCherry-AktPH in fibroblasts coexpressing PA-Rac was monitored by TIRF microscopy, as shown in the pseudocolor montage. Photoactivation of PA-Rac was initiated at the 18-min mark in the region indicated by the red oval and was maintained there until after the 41-min image shown (see also Video 7). Bar, 20 µm. (b) For another cell, spatiotemporal maps of protrusion/retraction velocity and PI3K signaling localization show the typical patterns before, during (indicated by the dashed polygons), and after PA-Rac photoactivation. The results are representative of 42 cells. a.u., arbitrary unit. (c) PA-Rac still elicits protrusion in cells treated with 1 µM PI3K-α inhibitor IV (see also Video 8). The results are representative of six cells treated with 0.5, 1, or 3 µM inhibitor. Bar, 20 µm. (d) Blocking protrusion by treatment with 200 nM cytochalasin D before photoactivation of PA-Rac prevents dramatic relocalization of PI3K signaling (see also Video 9). The results are representative of 11 cells thus treated. Bar, 20 µm.

Erik S. Welf, et al. J Cell Biol. 2012 April 2;197(1):105-114.
4.
Figure 3.

Figure 3. From: Migrating fibroblasts reorient directionality by a metastable, PI3K-dependent mechanism.

PI3K signaling is localized after initiation of protrusion. (a) A pseudocolor montage illustrating local membrane protrusion followed by increases in GFP-AktPH localization in a randomly migrating fibroblast. White arrowheads indicate local protrusion events and ensuing increases in PI3K signaling. Bar, 5 µm. (b) Protrusion/retraction map for a representative cell migration experiment alongside a plot showing the time courses of positive protrusion velocity and PI3K signaling localization at the angular position marked by the black arrowhead. (c) Time-lagged correlations of PI3K signaling localization with positive protrusion velocity (left) and of their positive time derivatives (increase in signaling with increase in protrusion; middle) and of the negative time derivative of PI3K signaling localization with the negative derivative of retraction velocity (increase in retraction; right) for the cohort of randomly migrating fibroblasts. Correlation coefficients were calculated for each cell, and the aggregate values are reported as mean ± 95% confidence interval (n = 28). (d) Fibroblasts coexpressing mCherry-AktPH (top) and GFP-paxillin (bottom) were monitored by TIRF microscopy during random migration (see also Video 6). White arrowheads indicate local increases in PI3K signaling coinciding with transition of adhesions from nascent to mature. Bar, 5 µm.

Erik S. Welf, et al. J Cell Biol. 2012 April 2;197(1):105-114.
5.
Figure 5.

Figure 5. From: Migrating fibroblasts reorient directionality by a metastable, PI3K-dependent mechanism.

Fibroblast reorientation is biased by a PDGF gradient. (a–d) GFP-AktPH–expressing cells were monitored by TIRF microscopy during migration in the presence of alginate microspheres loaded with PDGF. (a) A pseudocolor montage indicating the calculated PDGF concentration field and outline of a chemotactic cell (see also Video 10). Bar, 50 µm. (b) Spatiotemporal maps of protrusion/retraction velocity and PI3K signaling localization for the cell depicted in a. The angular position of maximum PDGF concentration as a function of time is indicated by circle symbols to show the alignment of the cell with the external gradient. The results are representative of 25 cells that exhibited reorientation behavior. a.u., arbitrary unit. (c). A total of 30 branching events in the chemotaxing cells was scored according to whether they exhibited biases (markedly more in one branch than the other) in protrusion velocity and/or PI3K signaling localization and, if so, whether the dominant branch experienced the higher or lower PDGF concentration. (d) When the absolute angles of cell movement relative to the gradient (0–180°, with 0° corresponding to perfect alignment) before and after a successful branching event could be clearly determined, these angles were compared by scatter plot. Points below the y = x (dotted) line represent branching events that resulted in improved alignment. (e) Overlaid outlines of GFP-AktPH–expressing fibroblasts, each responding to a PDGF gradient introduced by a micropipette (position indicated by an asterisk) oriented roughly perpendicular to the cell’s long axis. The cell on the right shows the more characteristic behavior of cells coexpressing the dominant-negative PI3K regulatory subunit. Times after initiation of the gradient are indicated. Bars, 20 µm.

Erik S. Welf, et al. J Cell Biol. 2012 April 2;197(1):105-114.
6.
Figure 2.

Figure 2. From: Migrating fibroblasts reorient directionality by a metastable, PI3K-dependent mechanism.

PI3K signaling is required for propagation but not initiation of branched protrusions. (a) Protrusion/retraction map and pseudocolor TIRF images of a randomly migrating fibroblast expressing GFP-AktPH; PI3K-α inhibitor IV (500 nM) was added at approximately the midway point (see also Video 3). Red arrowheads in the image mark sites of protrusion/retraction. The results are representative of 12 cells. Bar, 20 µm. (b) Protrusion/retraction map and pseudocolor TIRF image of a randomly migrating fibroblast coexpressing the dominant-negative PI3K regulatory subunit and GFP-AktPH (see also Video 4). The results are representative of nine cells. Bar, 20 µm. (c) TIRF montage of a randomly migrating fibroblast expressing GFP-AktPH and tdTomato-Lifeact; PI3K-α inhibitor IV (500 nM) was added after ∼3 h (see also Video 5). Arrowheads mark sites of protrusion with F-actin at the leading edge. The results are representative of 10 cells. Bar, 10 µm. (d) A linescan showing temporal overlap of AktPH and Lifeact accumulation in a transient protrusion. The TIRF images at the right show the position of the linescan and correspond to the time indicated by arrowheads on the left. Bar, 10 µm. (e) Nascent protrusions (arrowheads) are thin and short lived in cells coexpressing the dominant-negative PI3K regulatory subunit and GFP-AktPH. Bar, 10 µm. (f) Quantification of branch initiation and successful branch propagation (total number of events divided by total time) with versus without PI3K inhibition (Inhib.). The control cells (n = 28) are the same as analyzed in Fig. 1, DN p85 refers to cells expressing the dominant-negative PI3K regulatory subunit (n = 9), and the other cohorts are cells before and after treatment with PI3K-α inhibitor IV (n = 12). (g) Cell motility metrics comparing the control and DN p85 cohorts as described in f. Mean cell centroid translocation speed, D/T ratio (direct distance from start to end divided by the total distance traveled), and mean protruded area generation were evaluated for 12-min intervals. The cell path axis ratio measures each cell’s degree of sideways movement relative to the major axis of migration.

Erik S. Welf, et al. J Cell Biol. 2012 April 2;197(1):105-114.

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