FIGURE 9.

Individual contributions of the intermediate nanocluster configurations to the total immobile fraction and RBD recruitment. In our model, we assume that the number of Gal-1 proteins bound to a nanocluster affects its stability. The specific H-rasG12V mutant·Gal-1 complex formation tendency thus significantly determines the rate of nanocluster formation and the stability of nanoclusters. Both the fraction of H-ras in nanoclusters and the nanocluster stability (measured by the lifetime) affect the recruitment of the effector (here the Ras-binding domain of c-Raf, RBD). A, schematic representation of the five nanocluster configurations, where we define those with <4 Gal-1 as intermediate, as they do not yet possess the final number of four Gal-1 on four H-ras-GTP. Arrows pointing out of the nanoclusters correspond to rates at which a specific nanocluster configuration will cease to exist or actually change configuration. These arrows are therefore contributing to the lifetime of the particular configuration. Red arrows correspond to association rates with free Gal-1 that lead to nanoclusters with more Gal-1 incorporated. The sizes of the red arrows have been adjusted to show the increasingly higher association rates as the number of Gal-1 in a nanocluster increases. Arrows pointing into the formation of each nanocluster configuration have not been displayed, as they do not have any effect on the nanocluster lifetime. See Fig. 8 for a complete scheme of reactions. The same legend as in Fig. 8 applies. Detailed results of our nanocluster simulation for (B) depleted, (C) normal, and (D) overexpressed Gal-1 levels. In each panel we show the number of H-ras-GTP in the different nanocluster configurations (top, left), the total number of H-ras-GTP in nanoclusters (top, right), the lifetime of the different nanocluster configurations (bottom, left), and the resulting RBD recruitment (bottom, right). Total numbers of H-ras-GTP in nanoclusters is calculated by adding the number of H-ras-GTP in each nanocluster configuration. These values can be easily converted into the nanoclustered fraction as the total number of H-ras is 1000. RBD recruitment is calculated by adding the fractional contribution of each nanocluster configuration weighted by their individual lifetimes. Bars in blue correspond to data from mutant H-rasG12V-R169A/K170A, in gray to H-rasG12V, and green to H-rasG12V-R128A/R135A. Individual nanocluster configurations are shown below the bars, with description shown in A. Note that except for the lifetime-plot, the scales in B are only half that of those in C and D.