Membrane identity and GTPase cascades regulated by toggle and cut-out switches

Perla Del Conte-Zerial^1,2,a, Lutz Brusch^1,a, Jochen C Rink^2,a, Claudio Collinet², Yannis Kalaidzidis^2,3, Marino Zerial² & Andreas Deutsch¹

^aThese authors contributed equally to this work

^aPresent address: University of Utah School of Medicine, 401 MREB, 20 North 1900 East, Salt Lake City, UT 84132-3401, USA

Synopsis

The transition from early to late endosomes is regulated by the loss of the small GTPase Rab5 and the concomitant acquisition of Rab7 in a mechanism termed Rab conversion (Rink et al, 2005). The behaviour of the two master GTPases creates a paradox: on the one hand, early endosomes are required to maintain Rab5 and increase Rab5's activity as they (1) receive incoming cargo from the plasma membrane, (2) grow in size through homotypic early endosome fusion and (3) package cargo destined for degradation while sorting recycling cargo to the cell surface. On the other hand, when cargo has sufficiently accumulated in fewer and larger endosomes and the surface density of Rab5 reaches its peak, Rab5 needs to be switched off and substituted by Rab7 to irreversibly commit cargo for degradation. To resolve this paradox, we considered these two master GTPases as modules and applied a combination of theoretical and experimental approaches to unravel the yet unknown design principles of the switch system.

Here, we identified two design principles that can explain the maintenance and dynamic transition between successive Rab domains or Rab modules, each requiring only a single inhibitory interaction (Figure 2):

Figure 2

Two competing models for Rab domain identity generation and endosome conversion. Green arrows denote reported interactions and red arrows denote proposed interactions that each may be essential (solid arrow) or just tolerated (dashed arrow). (A) Model 1 corresponds to a toggle switch that can switch off Rab5 as a result of weakened activation of Rab5 by its GEF (solid green arrow). Switching on of Rab7 follows if the repression (red arrow) by Rab5 becomes inferior to the activation (green dashed arrows). (B) Model 2 represents a cut-out switch that can switch off Rab5 as a result of enhanced (e.g. cargo-dependent, pH-dependent) activation of Rab5, as Rab5 fosters Rab7 activity (upper green arrow) to pass a threshold above which Rab7 self-sustains (right green arrow) its own activation. Rab7 activation at supra-threshold values strongly suppresses Rab5 (red arrow).

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(1) Toggle switch. Rab5 displays cooperative auto-activation and suppresses Rab7.

(2) Cut-out switch. Rab5 activates Rab7; Rab7 displays cooperative auto-activation and suppresses Rab5.

According to model 1, Rab5 auto-activates and controls the level of Rab7 by a negative feedback loop. To perform such a task, it is necessary for Rab5 to maintain its level above a threshold; therefore, by decreasing the level of Rab5, Rab5 will be replaced by Rab7.

According to model 2, Rab5 activates Rab7 until Rab7 reaches a threshold upon which it inactivates Rab5 through a negative feedback loop and hence Rab5 activity needs to increase for Rab7 activity to pass its threshold.

So far, model 2 is best supported by the experimental data (Figure 3 and Table I). Therefore, we propose that Rab conversion is operated by a cut-out switch analogous to an electrical safety-breaker (Morecoft and Hehre, 1933; Oliver, 1990) controlled by Rab7. To our knowledge, this is the first example of a cut-out switch used in a biological system.

Figure 3

Rab5 membrane density increases towards the conversion event. (A) Time-course data of Rab5 density on 23 individual endosomes tracked in 3 independent experiments (each value per time point shown by a dot) and a representative time course (black) are shown as a function of time. The average over 23 data points at each time point is given by the green curve together with the 70% confidence interval in grey. For each tracked endosome, the time point of conversion was determined by visual inspection and the recorded time courses were shifted by the conversion time point such that all 23 conversion events were synchronized at the chosen time point 0. Rab5 density has been computed by dividing the integral fluorescence by twice the cross-sectional area (Rink et al, 2005). Red curves denote model simulations for model 1 (dashed) and model 2 (solid curve), which were rescaled with respect to Figure 4C and D by (t-825)/4 t, (R₅-0.18)13 000+10 000 R₅ for model 1 and (t-850)/4-t, (R₅-0.6)20 000+10 000 R₅ for model 2. (B) Analogously obtained and analysed data for Rab7 density on 15 converting endosomes from an independent experiment.

Figure 4

Bifurcation diagrams showing quasi-steady-state solutions and simulation of models. (A) Bifurcation diagram for model 1 (toggle switch). When a parameter of the kinetic terms of Rab5 (density of Rab5:GTP, green line) varies as a function of time (here the hydrolysis rate k_h,5 increases and other parameters are fixed), then the membrane density of Rab5 decreases until it suddenly drops during the conversion event (green arrow). Opposite to Rab5 switching off, Rab7 (density of Rab7:GTP, blue line) switches on at the same critical parameter value (blue arrow). (B) Bifurcation diagram for model 2 (cut-out switch; line colours as in (A); the exchange rate k_e,5 increases and other parameters are fixed). Here the membrane density of Rab5 increases before dropping during the conversion event (green arrow). For graphical reasons, the parameter values chosen for both bifurcations differ from those of the simulations (see Supplementary information). (C) Numerical simulation of model 1 (solutions plotted in (A)). (D) Numerical simulation of model 2 (solutions plotted in (B)).

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Table 1: Experimental tests (numbers, see text for details), models and model predictions

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We propose that the design principle shown here is not limited to Rab conversion but may underlie other modules posing a similar paradox. This proposal is supported by a series of experimental evidences for the necessary components of predicted cut-out switches in other modules centred on Rab as well as other GTPases in intracellular transport.

In conclusion, the description of the Rab5–Rab7 system as small functional units, or modules, represented by individual Rab domains (Miaczynska and Zerial, 2002) gives the possibility to further develop a more comprehensive model of the entire endocytic pathway, taking into account the recycling branch as well as further molecular components of the endocytic machinery, for example, coats and SNARE proteins (Heinrich and Rapoport, 2005). It is conceivable that the cut-out switch described here could be a design principle shared by other regulatory cascades from a broad range of biological processes.

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