Three-color single molecule imaging shows WASP detachment from Arp2/3 complex triggers actin filament branch formation

Abstract

During cell locomotion and endocytosis, membrane-tethered WASP proteins stimulate actin filament nucleation by the Arp2/3 complex. This process generates highly branched arrays of filaments that grow toward the membrane to which they are tethered, a conflict that seemingly would restrict filament growth. Using three-color single-molecule imaging in vitro we revealed how the dynamic associations of Arp2/3 complex with mother filament and WASP are temporally coordinated with initiation of daughter filament growth. We found that WASP proteins dissociated from filament-bound Arp2/3 complex prior to new filament growth. Further, mutations that accelerated release of WASP from filament-bound Arp2/3 complex proportionally accelerated branch formation. These data suggest that while WASP promotes formation of pre-nucleation complexes, filament growth cannot occur until it is triggered by WASP release. This provides a mechanism by which membrane-bound WASP proteins can stimulate network growth without restraining it.

DOI: http://dx.doi.org/10.7554/eLife.01008.001

eLife digest

Most cells are neither perfect spheres nor amorphous blobs, but instead have characteristic shapes that enable them to carry out specific roles within tissues or organs. These shapes are established by a type of scaffolding, called the cytoskeleton, that gives structure to the cell, and also forms networks over which other proteins, and even organelles, can travel.

The filaments that make up the cytoskeleton are composed of various proteins, one of which is called actin. Cellular actin filaments can grow by adding new actin molecules, and actin filaments can also have ‘branches’ that fork out from the mother filament. Branches grow out of an assembly of seven proteins known as the Arp2/3 complex, which attaches to the side of the mother filament. Branch growth is triggered by binding to the Arp2/3 complex of an additional protein, WASP, but the sequence of events required to initiate a new branch is not well understood. In particular, WASP is bound to cell membranes; at some point it must detach from the Arp2/3 complex so that the nearness of the membrane does not interfere with the growth of branches. Now, Smith et al. uncover how branch formation is triggered, and define a new role played by WASP in this process.

It is known that a specific region of the WASP protein called the VCA domain binds to both the Arp2/3 complex and actin. Smith et al. studied how this domain could initiate branch formation, and showed that a pair of VCA domains linked to each other, along with an Arp2/3 complex, could interact jointly with an existing actin filament before a new branch formed. However, new branches did not form unless the VCA-domain pair detached from the actin filament, leaving the Arp2/3 complex behind. Additionally, Smith et al. found that mutant VCA-domain pairs detached from the actin filament at different rates, which then determined the chance that a new branch formed.

These findings—and those of Helgeson and Nolen published concurrently in eLife—suggest that, in cells, two WASP proteins first recruit the Arp2/3 complex to the membrane, and that together they interact with an existing actin filament. The WASP proteins then release the filament, and only then does the Arp2/3 complex initiate the formation of an actin branch. Since the Arp2/3 complex is no longer attached to WASP, subsequent growth of the branch is not physically limited by linkage to the membrane.

DOI: http://dx.doi.org/10.7554/eLife.01008.002

Contributors

Wesley Sundquist: Role: Reviewing editor

Journal

Journal ID (nlm-ta): eLife

Journal ID (iso-abbrev): Elife

Journal ID (hwp): eLife

Journal ID (publisher-id): eLife

Title: eLife

Publisher: eLife Sciences Publications, Ltd

ISSN (Electronic): 2050-084X

Publication date (Electronic): 03 September 2013

Publication date Collection: 2013

Volume: 2

Affiliations

[1 ]Department of Biochemistry, Brandeis University , Waltham, United States

[2 ]Department of Biophysics, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center , Dallas, United States

[3 ]Rosenstiel Basic Medical Sciences Research Center, Brandeis University , Waltham, United States

[4 ]Department of Biology, Brandeis University , Waltham, United States

[5 ]New England Biolabs , Ipswich, United States

University of Utah , United States

University of Utah , United States

Author notes

[* ]For correspondence: michael.rosen@ 123456utsouthwestern.edu (MKR);

[* ]For correspondence: gelles@ 123456brandeis.edu (JG)

[†]

These authors contributed equally to this work.

[‡]

Biogen Idec, Cambridge, United States.

Article

Publisher ID: 01008

DOI: 10.7554/eLife.01008

PMC ID: 3762362

PubMed ID: 24015360

Copyright statement: Copyright © 2013, Smith et al

License:

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Funding

Funded by: National Institutes of Health

Award ID: GM43369

Award Recipient : Jeff Gelles

Funded by: Howard Hughes Medical Institute

Award Recipient : Michael K Rosen

Funded by: Welch Foundation

Award ID: I-1544

Award Recipient : Michael K Rosen

Funded by: National Science Foundation

Award ID: MRSEC 0820492

Award Recipient : Karen Daugherty-Clarke Bruce L Goode

Funded by: National Institutes of Health

Award ID: GM56322

Award Recipient : Michael K Rosen

Funded by: National Institutes of Health

Award ID: GM63691

Award Recipient : Bruce L Goode

Funded by: National Institutes of Health

Award ID: GM098143

Award Recipient : Jeff Gelles

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Categories

Subject: Research Article

Subject: Biochemistry

Subject: Biophysics and Structural Biology

Custom metadata

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Author impact statement New insights into the mechanism responsible for the nucleation of branches on networks of actin filaments shed light on a process that is essential for eukaryotic cell motility and endocytosis.