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Files in this Data Supplement:
Fig. S1. Schematic representation of the two non-binding mutated version of the monomeric cAMP FRET sensor. The mutation R96K in the phosphate binding cassette (PBC) of the B-domain sensor was performed to mimic the mutation generated by Lopez De Jesus and collaborators (Lopez De Jesus et al., 2006) in Epac1 (a cAMP-binding protein). Such a mutation generates a cAMP-binding deficient Epac1 mutant (R279K) (Lopez De Jesus et al., 2006), as well as a B-domain non-binding mutant (as we show in the current paper). The R96E mutant was generated on the basis of the mutation performed by DiPilato and collaborators (DiPilato et al., 2004) in the Epac1-based cAMP FRET sensor. Such mutation generated cAMP-binding-deficient mutants in both the Epac1 sensor and the B-domain sensor.
Fig. S2. E87Q mutation in the B-domain creates a non-binding mutant. (A) The E87 in the PBC of the B-domain was mutated to Q in an attempt to decrease the affinity of the sensor for cAMP. Such a mutation was performed because the Epac1 protein (a cAMP-binding protein) has lower affinity for cAMP and its PBC contains a Q instead of an E. It has been reported that this mutation in the B domain of the human RIα gives rise to a 280-times reduction in cAMP affinity (Dao et al., 2006). (B) Time course of the FRET efficiency average in E87Q B domain/WT cells. The experiments were carried on as in Fig. 3. Mean ± s.e.m. values are presented.
Fig. S3. Chemoattractant-mediated adenylyl cyclase activity in WT and regA− cells. Graph depicting the adenylyl cyclase activity measured before and 30 seconds after the addition of 1 nM cAMP to whole cells. Adenylyl cyclase activity was measured as described in Comer and collaborators (Comer et al., 2006).
Movie 1. Change in FRET signal following chemoattractant stimulation in WT cell. Movie depicting the change in FRET signal observed as a function of time following a uniform saturating dose of chemoattractant (10 µM) in a representative B domain/WT cell. Frames were taken every 15 seconds and are presented at 8 frames/second. The movie runs for 10 minutes.
Movie 2. Reg− cells migrating to a point source of cAMP. Movie depicts WT (left) and regA− (right) cells migrating to a micropipette filled with 1 µM cAMP. Frames were taken every 10 seconds and presented at 10 frames/second (100× real time).
Movie 3. RegA− cells migrating in a shallow cAMP gradient. Movie depicts WT (left) and regA− (right) cells migrating in the EZ-Taxiscan chamber filled with 10 µM cAMP. Cells lacking RegA appear to migrate faster and form streams and cell aggregates earlier than WT cells. Frames were taken every 5 seconds and presented at 10 frames/second (50× real time).
Movie 4. Change in FRET signal in WT cells exposed to an external chemoattractant gradient. Movie depicts the change in FRET signal observed as a function of time in a representative B-domain or WT cell exposed to a chemoattractant gradient. The right movie shows the phase signal and the left movie depicts the FRET signal. Frames were taken every 15 seconds and are presented at 8 frames/second. The movie runs for 15 minutes.
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