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The effect of IQGAP1 on Xenopus embryonic ectoderm requires Cdc42
1Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Abstract:
IQGAP1 contains a number of protein recognition motifs through which it binds to targets. Several in vitro studies have documented that IQGAP1 interacts directly with calmodulin, actin, E-cadherin, beta-catenin, and the small GTPases Cdc42 and Rac. Nevertheless, direct demonstration of in vivo function of mammalian IQGAP1 is limited. Using a novel assay to evaluate in vivo function of IQGAP1, we document here that microinjection of IQGAP1 into early Xenopus embryos generates superficial ectoderm lesions at late blastula stages. This activity was retained by the mutated variants of IQGAP1 in which the calponin homology domain or the WW domain was deleted. By contrast, deletion of the IQ (IQGAP1-DeltaIQ), Ras-GAP-related (IQGAP1-DeltaGRD), or C-terminal (IQGAP1-DeltaC) domains abrogated the effect of IQGAP1 on the embryos. None of the latter mutants bound Cdc42, suggesting that the binding of Cdc42 by IQGAP1 is critical for its function. Moreover, overexpression of IQGAP1, but not IQGAP1-DeltaGRD, significantly increased the amount of active Cdc42 in embryonic cells. Co-injection of wild type IQGAP1 with dominant negative Cdc42, but not the dominant negative forms of Rac or Rho, blocked the effect of IQGAP1 on embryonic ectoderm. Together these data indicate that the activity of IQGAP1 in embryonic ectoderm requires Cdc42 function.
Insights
IQGAP1 protein causes ectoderm lesions in Xenopus embryos by interacting with Cdc42. This interaction is crucial for IQGAP1
Area of Science:
- Cell Biology
- Developmental Biology
- Molecular Biology
Background:
- IQGAP1 is known to interact with various proteins in vitro.
- The in vivo function of mammalian IQGAP1 remains largely uncharacterized.
Purpose of the Study:
- To investigate the in vivo function of IQGAP1 in early Xenopus embryos.
- To identify the specific domains and interactions of IQGAP1 critical for its embryonic function.
Main Methods:
- Microinjection of IQGAP1 and its mutants into early Xenopus embryos.
- Assessing ectoderm lesion formation at late blastula stages.
- Evaluating protein binding (Cdc42) and activity using mutated IQGAP1 variants and dominant-negative GTPases.
Main Results:
- Microinjected IQGAP1 induced superficial ectoderm lesions in Xenopus embryos.
- IQGAP1's activity required the IQ, Ras-GAP-related (GRD), and C-terminal domains.
- Deletion of these domains abolished Cdc42 binding and IQGAP1's embryonic effect.
- Overexpression of IQGAP1 increased active Cdc42 levels, and dominant-negative Cdc42 blocked IQGAP1's activity.
Conclusions:
- IQGAP1's function in embryonic ectoderm is dependent on its interaction with Cdc42.
- Specific domains of IQGAP1 are essential for mediating its effects via Cdc42 in vivo.
