IMPACTOS

individually, both EP2 and EP4 make transition from fast to slow states within a trajectory before and during activation (Fig. 4G,H). With respect to EP2, EP4 alone was found to make these transitions more often after stimulation, as indicated by an increase in slow fraction size and constant dwell times. Since EP4* showed a constant fraction size during signaling, thus differing from EP4 alone, we wondered whether the co-expression of EP2 would alter EP4* fast-to-slow transitions. Therefore, we performed the diffusion state

classification for the trajectories of EP4* before and during 1 μM PGE2 stimulation and

in the absence or presence of EP2 antagonist (Fig. 5D). We quantified the average dwell

time of EP4* in either the fast or the slow state before and upon activation by PGE2 and

observed no significant changes (Fig. 5D). This suggests that co-expression of EP2 limits the occurrences of EP4* fast-to-slow transitions. Furthermore, the radius of gyration analysis of EP4* trajectories upon EP2 block showed no alterations in the average dwell times. Therefore, the recovered shift from fast to slow component determined by CPD analysis for EP4* after EP2 block (Fig. 5A) can most likely be ascribed to a re-establishment of a higher number of occurrences of the fast-to-slow state transitions (Fig. 5D).

EP2 alone showed no changes in dwell times after activation by PGE2 (Fig. 4H). Similarly,

PGE2 stimulation had no effects on EP2* average dwell times, and this was not altered by

blocking EP4 (Fig. 5E), meaning that co-expression of EP4 has no impact on EP2* state transitions.

Altogether, these results demonstrate that co-expressed EP2 and EP4 influence each other’s lateral mobility during signaling. The effects of EP4 on EP2 mobility are modest

and restricted to a slight decrease in EP2 Ds at low PGE2 concentrations. Since blocking

of EP4 does not greatly affect EP2 mobility, but delays Gαs dynamics and results in lower

cAMP production, EP4 appears as the major regulator of PGE2 sensing at physiological

concentrations. On the other hand, the influence of EP2 co-expression on EP4 mobility

was significant, as the presence of EP2 prevents EP4 from slowing down upon PGE2

addition. Since blocking of EP2 re-establishes EP4 slowing down, does not affect Gαs

dynamics and at the same time favors cAMP production, we conclude that EP4 signaling competence is regulated by the fast-to-slow state transitions and that EP2 attenuates EP4 signaling capacity.

These results demonstrate the existence of crosstalk between EP2 and EP4, which initiates within the plasma membrane by influencing receptor mobility and therefore capacity to engage with downstream signaling molecules.

The microtubule network spatially modulates EP2 and EP4 signaling

GPCR mobility has been shown to be influenced by the cortical microtubule network [89, 301], and a dynamic interplay between Gα proteins and microtubules has also been documented [302, 303]. Therefore, we sought to investigate a possible role for the microtubule network in regulating EP2 and EP4 mobility and signaling.

First, we evaluated the effects of microtubule disruption by nocodazole on the lateral

mobility of the receptors. After treatment with nocodazole only a modest PGE2-induced

decrease in overall mobility was observed for EP4 alone while no nocodazole-induced effects were detected on EP4* (Fig. S6A,B). Similarly, EP2 and EP2* mobility were unaffected by the microtubule disruption (Fig. S6C,D). These data indicate that the cortical microtubule network does not have a significant impact on the receptor mobility parameters. However, the images generated using the state classification analysis showed that during the slow mobility state both EP4 and EP4* occupy areas with a

distinct elongated geometry that were observed both before and during PGE2 stimulation

and were no longer detectable after microtubule disruption (Fig. 6A,B). Overlay of the elongated area occupied by the slow states onto the trajectories obtained by CPD analysis showed that within the same trajectory, the tracked receptor regularly returns to the same elongated area when transitioning to the slow mobility state (Fig. 6A,B). Moreover, the same elongated areas appeared to be visited by multiple receptors, thus representing pre-defined areas where the receptors slow down (Fig. 6A,B). Remarkably, neither EP2 nor EP2* showed comparable geometries in their slow mobility states, both before and

during PGE2 addition, and no visible alteration was observed after microtubule disruption

i ii + Nocodazole iii iv

A

B

Figure 6. Microtubules are important for the dynamic distribution of EP4 fast and slow state The trajectories of EP4 (A) and EP4* (B) were analyzed using the state classification method to determine the mobility state at each position during the trajectories. (i) This analysis yields images displaying the fast (green dots) or slow (red dots) state of the receptors. (ii) Representative examples of the trajectories obtained using the CPD analysis within the same region of interest as used in (i). (iii) Zoom in on the state classification image where the elongated shape of slow states is marked (red dotted line) and overlaid (red area) on the same zoomed in region of the trajectories, showing recurrent positioning of the same molecule or positioning of another molecule within this area. (iv) Images obtained with the state classification method for cells stimulated for 10s with PGE2 upon treatment with nocodazole (20 min 5 μM).

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