Purification of Arp2/3 Complex from Saccharomyces cerevisiae

Doolittle, Lynda K.; Rosen, Michael K.; Padrick, Shae B.

doi:10.1007/978-1-62703-538-5_15

Cited by 13 publications

(13 citation statements)

References 30 publications

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“…new filament from the side of an already existing actin filament at 70˚ angle. In addition to ARP2 and ARP3, it contains five more proteins (Robinson et al 2001;Doolittle et al 2013) and requires additional nucleation promoting factors for its activity. The structure of the Arp2/3 complex is described in (Nolen et al 2004).…”

Section: Discussionmentioning

confidence: 99%

Actin, actin-binding proteins, and actin-related proteins in the nucleus

Kristó

Bajusz

et al. 2016

Histochem Cell Biol

105

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Extensive research in the past decade has significantly broadened our view about the role actin plays in the life of the cell and added novel aspects to actin research. One of these new aspects is the discovery of the existence of nuclear actin which became evident only recently. Nuclear activities including transcriptional activation in the case of all three RNA polymerases, editing and nuclear export of mRNAs, and chromatin remodeling all depend on actin. It also became clear that there is a fine-tuned equilibrium between cytoplasmic and nuclear actin pools and that this balance is ensured by an export-import system dedicated to actin. After over half a century of research on conventional actin and its organizing partners in the cytoplasm, it was also an unexpected finding that the nucleus contains more than 30 actin-binding proteins and new classes of actin-related proteins which are not able to form filaments but had evolved nuclear-specific functions. The actin-binding and actin-related proteins in the nucleus have been linked to RNA transcription and processing, nuclear transport, and chromatin remodeling. In this paper, we attempt to provide an overview of the wide range of information that is now available about actin, actin-binding, and actin-related proteins in the nucleus.

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Section: Discussionmentioning

confidence: 99%

Actin, actin-binding proteins, and actin-related proteins in the nucleus

Kristó

Bajusz

et al. 2016

Histochem Cell Biol

105

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“…Actin and Arp2/3 complex were purified from rabbit muscle and bovine thymus, respectively, using established methods ( Doolittle et al, 2013a ; Doolittle et al, 2013b ). G-actin (1 µM, 10% rhodamine labeled) was added to p-Nephrin clusters containing 1 μM Nck and 2 μM N-WASP, with or without 10 nM Arp2/3 complex.…”

Section: Methodsmentioning

confidence: 99%

Phase transitions of multivalent proteins can promote clustering of membrane receptors

Banjade

Rosen

2014

eLife

522

576

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Clustering of proteins into micrometer-sized structures at membranes is observed in many signaling pathways. Most models of clustering are specific to particular systems, and relationships between physical properties of the clusters and their molecular components are not well understood. We report biochemical reconstitution on supported lipid bilayers of protein clusters containing the adhesion receptor Nephrin and its cytoplasmic partners, Nck and N-WASP. With Nephrin attached to the bilayer, multivalent interactions enable these proteins to polymerize on the membrane surface and undergo two-dimensional phase separation, producing micrometer-sized clusters. Dynamics and thermodynamics of the clusters are modulated by the valencies and affinities of the interacting species. In the presence of the Arp2/3 complex, the clusters assemble actin filaments, suggesting that clustering of regulatory factors could promote local actin assembly at membranes. Interactions between multivalent proteins could be a general mechanism for cytoplasmic adaptor proteins to organize membrane receptors into micrometer-scale signaling zones.DOI: http://dx.doi.org/10.7554/eLife.04123.001

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“…S . cerevisiae Arp2/3 complex was purified from commercially purchased baker’s yeast (Kastalia, Lesaffre, Marcq-en-Baroeul, France) based on a protocol modified from [61,62]. Droplets of liquid yeast culture were frozen in liquid nitrogen and ground in a steel blender (Waring, Winsted, CT, USA).…”

Section: Methodsmentioning

confidence: 99%

Sizes of actin networks sharing a common environment are determined by the relative rates of assembly

et al. 2019

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Within the cytoplasm of a single cell, several actin networks can coexist with distinct sizes, geometries, and protein compositions. These actin networks assemble in competition for a limited pool of proteins present in a common cellular environment. To predict how two distinct networks of actin filaments control this balance, the simultaneous assembly of actin-related protein 2/3 (Arp2/3)-branched networks and formin-linear networks of actin filaments around polystyrene microbeads was investigated with a range of actin accessory proteins (profilin, capping protein, actin-depolymerizing factor [ADF]/cofilin, and tropomyosin). Accessory proteins generally affected actin assembly rates for the distinct networks differently. These effects at the scale of individual actin networks were surprisingly not always correlated with corresponding loss-of-function phenotypes in cells. However, our observations agreed with a global interpretation, which compared relative actin assembly rates of individual actin networks. This work supports a general model in which the size of distinct actin networks is determined by their relative capacity to assemble in a common and competing environment.

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Purification of Arp2/3 Complex from Saccharomyces cerevisiae

Cited by 13 publications

References 30 publications

Actin, actin-binding proteins, and actin-related proteins in the nucleus

Actin, actin-binding proteins, and actin-related proteins in the nucleus

Phase transitions of multivalent proteins can promote clustering of membrane receptors

Sizes of actin networks sharing a common environment are determined by the relative rates of assembly

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