# Understanding the roles of WASP in Arp2/3 complex activation and branched actin network assembly

> **NIH NIH F32** · UNIVERSITY OF OREGON · 2020 · $64,926

## Abstract

PROJECT SUMMARY
The actin-related-protein (Arp) 2/3 complex is a 225-kDa seven-subunit actin filament nucleator that nucleates
branched actin filaments. Polymerizing branched actin networks provide protrusive forces necessary to drive a
myriad of cellular processes, including motility, vesicle trafficking, and endocytosis. To orchestrate these
functions, cells utilize proteins that bind to and activate Arp2/3 complex known as nucleation promotion factors
(NPFs). The most ubiquitous and well-studied class of NPFs, Wiskott-Aldrich syndrome proteins (WASP), are
characterized by a conserved C-terminal “VCA” (verprolin homology, central, acidic) motif that binds to actin
monomers (V) and Arp2/3 complex (CA). In the absence of WASP, Arp2/3 complex is held in an inactive
conformation in which the two actin-related proteins Arp2 and Arp3 are arranged in an end-to-end orientation
referred to here as the splayed state. Activation depends on a large conformational change that moves Arp2 and
Arp3 into filament-like arrangement known as the short-pitch conformation. Previously, we demonstrated that
WASP binding stimulates formation of the short-pitch conformation and that this is the main activating function
of WASP. However, exactly how WASP binding shifts the splayed to short-pitch conformational equilibrium is
unclear, and is the focus of this proposal. We will address this from a structure-function perspective using high
resolution structures of the inactive state and hypothetical models of the active state to determine how the
complex is held inactive in the absence of WASP (Aim 1). In addition, we will address two fundamental aspects
of WASP-mediated regulation of the complex that are critical open questions in the field. First, while recent data
indicated that WASP binds to two distinct binding sites on the complex, how engagement at each site contributes
to activation of the complex and assembly of actin networks in vitro or in cells remains unknown. We will address
this question in Aim 2, taking advantage of a recently determined map of the WASP binding sites on Arp2/3
complex determined by crosslinking/mass-spectrometry. Second, recent data show that to activate Arp2/3
complex, WASP must first bind to stimulate the short pitch conformation, but then must be released to allow
nucleation to proceed. Because WASP binds membranes in cells, release of WASP is thought to play a critical
role in the assembly of force-producing actin networks; i.e., it provides a transient connection between the
network to the membrane that facilitates pushing, yet by releasing after nucleation ensures that polymerizing
networks are not so tightly bound they cause network compression. No studies have addressed how the
interactions of WASP with the complex are tuned to optimally balance its nucleation potency versus its ability to
serve as a tether between actin networks and membranes, despite the fact that both of these activities are critical
in assembling productive actin...

## Key facts

- **NIH application ID:** 10068587
- **Project number:** 1F32GM139362-01
- **Recipient organization:** UNIVERSITY OF OREGON
- **Principal Investigator:** Michael J Lynch
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $64,926
- **Award type:** 1
- **Project period:** 2020-07-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10068587

## Citation

> US National Institutes of Health, RePORTER application 10068587, Understanding the roles of WASP in Arp2/3 complex activation and branched actin network assembly (1F32GM139362-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10068587. Licensed CC0.

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