Figure S3.
Design of the Cdc42·GTP biosensor MCB-EGFP. (A) Schematic depicting the primary structure of full-length human MRCKβ and its functional domains. C1: Protein kinase C conserved region 1. PH: Pleckstrin homology. CH: Citron homology. CRIB: Cdc42/Rac interactive binding. (B) Schematic outlining the different constructs generated through stepwise truncation to obtain a Cdc42·GTP biosensor, using full-length MRCKβ-EGFP as a template. All constructs were expressed in MII oocytes via cRNA injection and examined for polarized localization in the cortex overlying maternal chromosomes. The total number of oocytes scored is indicated in parentheses for each construct, with an indication of cortical polarization (+) or absence of (−). While the CRIB domain alone was not sufficient for cortical localization, stepwise extensions toward the N-terminus allowed us to define a minimal Cdc42·GTP-binding fragment encompassing the PH, CH, and CRIB domains. This minimal fragment was used as a biosensor for Cdc42 activation, designated as MCB. Mutation of the two key histidine residues in the CRIB domain to alanine, corresponding to H1593/1596A in the full-length sequence, and indicated by double asterisks (**), abolished polarized localization. aa: Amino acids. (C) Confocal image of a live MII oocyte expressing the MCB-EGFP biosensor. (D) Confocal image of a live MII oocyte expressing the MCB-EGFP biosensor, and treated with ML-141 (5 µM, 1 h). (E) Confocal image of a live MII oocyte expressing the MCB-EGFP biosensor bearing the H1593/1596A double substitution. Chromosomes were stained with SiR-DNA (magenta). Scale bars are 10 µm.

Design of the Cdc42·GTP biosensor MCB-EGFP. (A) Schematic depicting the primary structure of full-length human MRCKβ and its functional domains. C1: Protein kinase C conserved region 1. PH: Pleckstrin homology. CH: Citron homology. CRIB: Cdc42/Rac interactive binding. (B) Schematic outlining the different constructs generated through stepwise truncation to obtain a Cdc42·GTP biosensor, using full-length MRCKβ-EGFP as a template. All constructs were expressed in MII oocytes via cRNA injection and examined for polarized localization in the cortex overlying maternal chromosomes. The total number of oocytes scored is indicated in parentheses for each construct, with an indication of cortical polarization (+) or absence of (−). While the CRIB domain alone was not sufficient for cortical localization, stepwise extensions toward the N-terminus allowed us to define a minimal Cdc42·GTP-binding fragment encompassing the PH, CH, and CRIB domains. This minimal fragment was used as a biosensor for Cdc42 activation, designated as MCB. Mutation of the two key histidine residues in the CRIB domain to alanine, corresponding to H1593/1596A in the full-length sequence, and indicated by double asterisks (**), abolished polarized localization. aa: Amino acids. (C) Confocal image of a live MII oocyte expressing the MCB-EGFP biosensor. (D) Confocal image of a live MII oocyte expressing the MCB-EGFP biosensor, and treated with ML-141 (5 µM, 1 h). (E) Confocal image of a live MII oocyte expressing the MCB-EGFP biosensor bearing the H1593/1596A double substitution. Chromosomes were stained with SiR-DNA (magenta). Scale bars are 10 µm.

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