Honors & Awards
Novartis-Sponsored Fellow, Helen Hay Whitney Foundation (12/1/2011 - 11/30/2014)
Doctor of Philosophy, Harvard University (2009)
Rajat Rohatgi, Postdoctoral Faculty Sponsor
WAVE proteins link upstream signals to actin nucleation by activating the Arp2/3 complex and are at the core of regulatory pathways driving membrane protrusion. They are found in heteropentameric complexes whose role in regulating WAVE function is presently unclear. Here we demonstrate that purified native WAVE complexes are basally inactive; previous reports of constitutive activity are artifacts of in vitro manipulation. Further, the native complexes are not activated by Rac alone. Activation of the WAVE2 complex requires simultaneous interactions with prenylated Rac-GTP and acidic phospholipids, as well as a specific state of phosphorylation. Together these signals promote full activation in a highly cooperative process on the membrane surface, by inducing an allosteric change in the complex rather than by simple recruitment or by dissociation of the subunits. These results explain how the WAVE complex can integrate coincident signals to promote localized actin nucleation during cell motility.
View details for DOI 10.1016/j.molcel.2009.10.024
View details for Web of Science ID 000272506200017
View details for PubMedID 19917258
Identification of small-molecule targets remains an important challenge for chemical genetics. We report an approach for target identification and protein discovery based on functional suppression of chemical inhibition in vitro. We discovered pirl1, an inhibitor of actin assembly, in a screen conducted with cytoplasmic extracts. Pirl1 was used to partially inhibit actin assembly in the same assay, and concentrated biochemical fractions of cytoplasmic extracts were added to find activities that suppressed pirl1 inhibition. Two activities were detected, separately purified, and identified as Arp2/3 complex and Cdc42/RhoGDI complex, both known regulators of actin assembly. We show that pirl1 directly inhibits activation of Cdc42/RhoGDI, but that Arp2/3 complex represents a downstream suppressor. This work introduces a general method for using low-micromolar chemical inhibitors to identify both inhibitor targets and other components of a signaling pathway.
View details for DOI 10.1016/j.chembiol.2006.02.009
View details for Web of Science ID 000237376400013
View details for PubMedID 16632257
In the accompanying chapter, we describe an in vitro system that uses Xenopus egg extracts to study actin assembly induced by phosphatidylinositol (4,5)bisphosphate (PIP2) and Cdc42. Biochemical fractionation and candidate screening experiments conducted in the extract system have identified the Arp2/3 complex, the N-WASP-WIP (or N-WASP-CR16) complex, and the Cdc42-binding protein Toca-1 as important mediators of PIP2- and Cdc42-actin signaling. Toward our ultimate goal of reconstituting an in vitro system that recapitulates the signaling properties observed in vivo, we then developed a purified actin assembly assay system consisting of the regulatory components that we discovered from extracts. In these assays, the stereotypical sigmoidal kinetics of actin polymerization are monitored by pyrene-actin fluorescence in the presence of defined recombinant or purified proteins, enabling the detailed study of mechanism and protein function. In this chapter, we describe the preparation of the components used in these purified actin assembly reactions, as well as the assay conditions under which we monitor actin polymerization kinetics in vitro.
View details for DOI 10.1016/S0076-6879(06)06014-9
View details for Web of Science ID 000235750600014
View details for PubMedID 16472658
Xenopus egg cytoplasmic extracts have been used to study a variety of complex cellular processes. Given their amenability to biochemical manipulation and physiological balance of regulatory proteins, these extracts are an ideal system to dissect signal transduction pathways leading to actin assembly. We have developed methods to study Cdc42 and PI(4,5)P2-induced actin assembly in Xenopus egg extracts. In this chapter, we describe detailed procedures to prepare Xenopus egg extracts, Cdc42, and PI(4,5)P2 for use in actin assembly experiments. We also describe a fluorometric pyrene actin assay for quantitative kinetic analysis of actin polymerization and a microscopic rhodamine actin assay for quick measurement of actin rearrangements in extracts. Finally we provide a protocol for immunodepletion of proteins and discuss the use of immunodepletion and rescue experiments for functional analysis of components in the extracts.
View details for DOI 10.1016/S0076-6879(06)06013-7
View details for Web of Science ID 000235750600013
View details for PubMedID 16472657
An important signaling pathway to the actin cytoskeleton links the Rho family GTPase Cdc42 to the actin-nucleating Arp2/3 complex through N-WASP. Nevertheless, these previously identified components are not sufficient to mediate Cdc42-induced actin polymerization in a physiological context. In this paper, we describe the biochemical purification of Toca-1 (transducer of Cdc42-dependent actin assembly) as an essential component of the Cdc42 pathway. Toca-1 binds both N-WASP and Cdc42 and is a member of the evolutionarily conserved PCH protein family. Toca-1 promotes actin nucleation by activating the N-WASP-WIP/CR16 complex, the predominant form of N-WASP in cells. Thus, the cooperative actions of two distinct Cdc42 effectors, the N-WASP-WIP complex and Toca-1, are required for Cdc42-induced actin assembly. These findings represent a significantly revised view of Cdc42-signaling and shed light on the pathogenesis of Wiskott-Aldrich syndrome.
View details for Web of Science ID 000222966000009
View details for PubMedID 15260990