Holmes WR et al. (2015), Modeling the roles of protein kinase C beta and...

XB-ART-51185

Mol Biol Cell 2015 Nov 05; doi: 10.1091/mbc.E15-06-0383.

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Modeling the roles of protein kinase C beta and eta in single cell wound repair.

Holmes WR , Liao L , Bement W , Edelstein-Keshet L .

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Wounded cells such as Xenopus oocytes respond to damage by assembly and closure of an array of actin filaments and myosin-2 controlled by Rho GTPases including Rho and Cdc42. Rho and Cdc42 are patterned around wounds in a characteristic manner, with active Rho concentrating in a ring-like zone inside a larger, ring-like zone of active Cdc42. How this patterning is achieved is unknown, but Rho and Cdc42 at wounds are subject to regulation by other proteins including the protein kinases C. Specifically, Cdc42 and Rho activity are enhanced by PKCβ and inhibited by PKCη. We adapt a pre-existing mathematical model of Simon et al. (2013) to probe the possible roles of these kinases. We show that PKCβ likely affects the magnitude of positive Rho - Abr feedback whereas PKCη acts on Cdc42 inactivation. The model explains both qualitative and some overall quantitative features of PKC-Rho GTPase regulation. It also accounts for the previous, peculiar observation that ∼20% of cells overexpressing PKCη display zone inversions-i.e. displacement of active Rho to the outside of the active Cdc42.

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Species referenced: Xenopus laevis
Genes referenced: abr ccdc42 cdc42 ebf2 ebf3 ednra rho rho.2 rhoa tbx2 uqcc6

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	FIGURE 1:. Schematic diagram showing the Rho-Abr-Cdc42 cross-talk as modeled in Simon et al. (2013). Here we consider the possible effects of PKCs (blue arrows) on either the basal activation rates of RhoA and Cdc42 or the magnitude of positive feedback from Cdc42 to itself and from Rho via Abr to itself.
	FIGURE 2:. Model geometry, control simulation, and failure of the simplest model. (a) Geometry of the wound, showing the Rho and Cdc42 zones (green and red, here and throughout all figures) and our one-dimensional model (x is distance from the wound edge). (b) A control simulation showing the distributions of Rho, Cdc42, and Abr for a “wild-type” model cell, with parameter values in Supplemental Table S1. Initial conditions are shown as dashed lines, final profiles 20 s later are shown as solid lines. (c–f) Model results conflict with experimental observations when we assume that PKCs affect only the basal activation or inactivation rates of Rho and Cdc42. (c) PKCβ DN (full suppression of basal activation rates bR, bC) fails to extinguish the Cdc42 zone. (d) PKCβ OE (bR, bC increased by a factor of 1.33) hardly changes the zone intensities, failing to make them significantly brighter. (e–f) PKCη is assumed to influence basal inactivation rates (dR and dC) by the indicated fold change. (e) PKCη DN (no major disagreement). (f) PKCη OE suppresses Rho but allows Cdc42 to invade the region next to the wound edge, contrary to what is observed experimentally. In all cases, simulations are run for 20 s to determine how zones localize before wound closure.
	FIGURE 3:. (a) Reduced Rho–Abr phase plane showing the Abr nullcline (black) and sample Rho nullclines (control as well as DN, OE2, and OE3) for multiple values of γAR (0.5, 1, 2, and threefold multiples of the value stated in Supplemental Table S1; all other parameters are as in that table). Intersections (shown in purple) are steady-state Rho intensity levels in the Rho zone, adjacent to the wound, and the black dot is background Rho activity level. (b) Bifurcation diagram showing steady-state Rho activity levels as a function of the Rho–Abr positive feedback strength γAR. The lower solid line represents the background Rho level; the upper branch represents the Rho zone intensity. Note that the two steady states can coexist over a range of the parameter values and that the intensity of the zone (but not of the background) increases sharply as the positive feedback strength γAR increases. (c) As in a, but for several values of the basal activation rate (bR). (d) As in b, but for the parameter bR. Note that both background and Rho-zone intensities increase with bR but that the zone intensity increases less strongly than in b. (e) Simulation of the model “control” (wild-type cells) using parameters in Supplemental Table S1. (Initial conditions, dashed lines; final profiles, solid lines.) (f) Similar to e, but with both feedback strengths γAR and γC multiplied by a factor of 2 to mimic PKCβ OE (equivalent to 2× in a). Note that zones get significantly “brighter.” (g) Similar to f, but with a threefold increase in the feedback strengths. (h) Similar to e–g, but with a factor of 0.5 reduction in feedback strengths to mimic PKCβ DN. Here all zones are abolished.
	FIGURE 4:. (a) Diagram showing the Cdc42 rates of activation (red curve) and three sample inactivation (black) rates vs. Cdc42 activity levels. The three black lines correspond to PKCη OE, WT, and DN (dηC = −0.15, 0, 0.1, respectively). Intersections of red and black curves are Cdc42 steady-state levels, with red dots representing the elevated Cdc42 intensities in the Cdc42 zone and the blue dots (practically all the same) indicating background Cdc42 activity levels. All other parameters are as in Supplemental Table S1. (b) Cdc42 bifurcation plot with respect to the strength of PKCη-mediated Cdc42 inactivation. (c) Control (WT) simulation for comparison purposes. (d) Spatial behavior. Formation of a single Cdc42 zone for spatially uniform PKCη OE (dηC and dηR = 0.1 with η = 1 uniformly on the domain.) (e) Simulation of reduced inactivation for PKCη DN: the parameters dηC and dηR are reduced, but zone intensity hardly changes.
	FIGURE 5:. Model predictions for graded PKCη activity and predicted zone inversion. (a) Bifurcation plot for the case where PKCη jointly influences Rho and Cdc42. For PKCη activity levels higher than the value indicated by green (respectively red) line, the Rho (respectively Cdc42) zone cannot form due to excessive inactivation (bistability is required for each zone formation and only exists below those PKCη activity levels). (b) The assumed PKCη spatial distribution (linearly graded, with value η = 3 at the wound edge and η = 0 at 20 μm away). Red and green lines have the same meaning as in a. (c) Control simulation for comparison. (d) Graded PKCη overexpression (dηR = dηC = 0.1) suppresses the Rho zone. The Cdc42 zone persists and stays in the same location. (e) Same as in d, but with an extended Rho initial condition that represents some fraction (20%) of PKCη OE cells. The initial condition for Rho is the same as in previous simulations but is extended to a distance of 20 μm from the wound edge (mimicking a possible extension of the zone of initial Rho activity). Zone inversion occurs.

References [+] :

Abreu-Blanco, Coordination of Rho family GTPase activities to orchestrate cytoskeleton responses during cell wound repair. 2014, Pubmed