Cryo-EM structure of NPF-bound human Arp2/3 complex and activation mechanism

Actin-related protein (Arp) 2/3 complex nucleates branched actin networks that drive cell motility. It consists of seven proteins, including two actin-related subunits (Arp2 and Arp3). Two nucleation-promoting factors (NPFs) bind Arp2/3 complex during activation, but the order, specific interactions, and contribution of each NPF to activation are unresolved. Here, we report the cryo–electron microscopy structure of recombinantly expressed human Arp2/3 complex with two WASP family NPFs bound and address the mechanism of activation. A cross-linking assay that captures the transition of the Arps into the activated filament-like conformation shows that actin binding to NPFs favors this transition. Actin-NPF binding to Arp2 precedes binding to Arp3 and is sufficient to promote the filament-like conformation but not activation. Structure-guided mutagenesis of the NPF-binding sites reveals their distinct roles in activation and shows that, contrary to budding yeast Arp2/3 complex, NPF-mediated delivery of actin at the barbed end of both Arps is required for activation of human Arp2/3 complex.

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CRYO-Electron Microscopy of the Acto-Myosin-ATP Complex

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100179993 ◽

1990 ◽

Vol 48 (1) ◽

pp. 248-249

Author(s):

John Trinickt ◽

Howard White

Keyword(s):

Electron Microscopy ◽

Atp Hydrolysis ◽

Myosin Head ◽

Bound State ◽

Cross Linking ◽

Weakly Bound ◽

Cryo Electron Microscopy ◽

Myosin Subfragment 1 ◽

Attached State ◽

High Fraction

The primary force of muscle contraction is thought to involve a change in the myosin head whilst attached to actin, the energy coming from ATP hydrolysis. This change in attached state could either be a conformational change in the head or an alteration in the binding angle made with actin. A considerable amount is known about one bound state, the so-called strongly attached state, which occurs in the presence of ADP or in the absence of nucleotide. In this state, which probably corresponds to the last attached state of the force-producing cycle, the angle between the long axis myosin head and the actin filament is roughly 45°. Details of other attached states before and during power production have been difficult to obtain because, even at very high protein concentration, the complex is almost completely dissociated by ATP. Electron micrographs of the complex in the presence of ATP have therefore been obtained only after chemically cross-linking myosin subfragment-1 (S1) to actin filaments to prevent dissociation. But it is unclear then whether the variability in attachment angle observed is due merely to the cross-link acting as a hinge.We have recently found low ionic-strength conditions under which, without resorting to cross-linking, a high fraction of S1 is bound to actin during steady state ATP hydrolysis. The structure of this complex is being studied by cryo-electron microscopy of hydrated specimens. Most advantages of frozen specimens over ambient temperature methods such as negative staining have already been documented. These include improved preservation and fixation rates and the ability to observe protein directly rather than a surrounding stain envelope. In the present experiments, hydrated specimens have the additional benefit that it is feasible to use protein concentrations roughly two orders of magnitude higher than in conventional specimens, thereby reducing dissociation of weakly bound complexes.

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Control of microtubule dynamics using an optogenetic microtubule plus end–F-actin cross-linker

The Journal of Cell Biology ◽

10.1083/jcb.201705190 ◽

2017 ◽

Vol 217 (2) ◽

pp. 779-793 ◽

Cited By ~ 9

Author(s):

Rebecca C. Adikes ◽

Ryan A. Hallett ◽

Brian F. Saway ◽

Brian Kuhlman ◽

Kevin C. Slep

Keyword(s):

Blue Light ◽

Actin Binding ◽

Cross Linking ◽

Dependent Manner ◽

Exclusion Zone ◽

Actin Networks ◽

Drosophila Melanogaster S2 Cells ◽

Actin Binding Domain ◽

Specific Factors ◽

Insight Into

We developed a novel optogenetic tool, SxIP–improved light-inducible dimer (iLID), to facilitate the reversible recruitment of factors to microtubule (MT) plus ends in an end-binding protein–dependent manner using blue light. We show that SxIP-iLID can track MT plus ends and recruit tgRFP-SspB upon blue light activation. We used this system to investigate the effects of cross-linking MT plus ends and F-actin in Drosophila melanogaster S2 cells to gain insight into spectraplakin function and mechanism. We show that SxIP-iLID can be used to temporally recruit an F-actin binding domain to MT plus ends and cross-link the MT and F-actin networks. Cross-linking decreases MT growth velocities and generates a peripheral MT exclusion zone. SxIP-iLID facilitates the general recruitment of specific factors to MT plus ends with temporal control enabling researchers to systematically regulate MT plus end dynamics and probe MT plus end function in many biological processes.

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Leukosialin (CD43, sialophorin) redistribution in uropods of polarized neutrophils is induced by CD43 cross-linking by antibodies, by colchicine or by chemotactic peptides

Journal of Cell Science ◽

10.1242/jcs.110.13.1465 ◽

1997 ◽

Vol 110 (13) ◽

pp. 1465-1475

Author(s):

S. Seveau ◽

S. Lopez ◽

P. Lesavre ◽

J. Guichard ◽

E.M. Cramer ◽

...

Keyword(s):

Electron Microscopy ◽

Cell Motility ◽

Small Proportion ◽

Cytochalasin B ◽

Cell Polarization ◽

Cross Linking ◽

Chemotactic Peptides ◽

Butanedione Monoxime ◽

Major Surface ◽

Triton X

We investigated a possible association of leukosialin (CD43), the major surface sialoglycoprotein of leukocytes, with neutrophil cytoskeleton. We first analysed the solubility of CD43 in Triton X-100 and observed that CD43 of resting neutrophils was mostly soluble. The small proportion of CD43 molecules, which ‘spontaneously’ precipitated in Triton, appeared associated with F-actin, as demonstrated by the fact that this insolubility did not occur when cells were incubated with cytochalasin B or when F-actin was depolymerized with DNase I in the Triton precipitate. Cell stimulation with anti-CD43 mAb (MEM59) enhanced this CD43-cytoskeleton association. By immunofluorescence as well as by electron microscopy, we observed a redistribution of CD43 on the neutrophil membrane, initially in patches followed by caps, during anti-CD43 cross-linking at 37 degrees C. This capping did not occur at 4 degrees C and was inhibited by cytochalasin B and by a myosin disrupting drug butanedione monoxime, thus providing evidence that the actomyosin contracile sytem is involved in the capping and further suggesting an association of CD43 with the cytoskeleton. Some of the capped cells exhibited a front-tail polarization with CD43 caps located in the uropod at the rear of the cell. Surprisingly, colchicine and the chemotactic factor fNLPNTL which induce neutrophil polarization associated with cell motility, also resulted in a clustering of CD43 in the uropod, independently of a cross-linking of the molecule by mAbs. An intracellular redistribution of F-actin, mainly at the leading front and of myosin in the tail, was observed during CD43 clustering induced by colchicine and in cells polarized by anti-CD43 mAbs cross-linking. We conclude that neutrophil CD43 interacts with the cytoskeleton, either directly or indirectly, to redistribute in the cell uropod under antibodies stimulation or during cell polarization by colchicine, thus highly suggesting that CD43 may be involved in cell polarization.

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Formation of Hirano Bodies Induced by Expression of an Actin Cross-Linking Protein with a Gain-of-Function Mutation

Eukaryotic Cell ◽

10.1128/ec.2.4.778-787.2003 ◽

2003 ◽

Vol 2 (4) ◽

pp. 778-787 ◽

Cited By ~ 25

Author(s):

Andrew Maselli ◽

Ruth Furukawa ◽

Susanne A. M. Thomson ◽

Richard C. Davis ◽

Marcus Fechheimer

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Cellular Response ◽

Biological Function ◽

Point Mutations ◽

Actin Binding ◽

Cross Linking ◽

Hirano Bodies ◽

Transmission Electron ◽

Ef Hand

ABSTRACT Hirano bodies are paracrystalline actin filament-containing structures reported to be associated with a variety of neurodegenerative diseases. However, the biological function of Hirano bodies remains poorly understood, since nearly all prior studies of these structures were done with postmortem samples of tissue. In the present study, we generated a full-length form of a Dictyostelium 34-kDa actin cross-linking protein with point mutations in the first putative EF hand, termed 34-kDa ΔEF1. The 34-kDa ΔEF1 protein binds calcium normally but has activated actin binding that is unregulated by calcium. The expression of the 34-kDa ΔEF1 protein in Dictyostelium induces the formation of Hirano bodies, as assessed by both fluorescence microscopy and transmission electron microscopy. Dictyostelium cells bearing Hirano bodies grow normally, indicating that Hirano bodies are not associated with cell death and are not deleterious to cell growth. Moreover, the expression of the 34-kDa ΔEF1 protein rescues the phenotypes of cells lacking the 34-kDa protein and cells lacking both the 34-kDa protein and α-actinin. Finally, the expression of the 34-kDa ΔEF1 protein also initiates the formation of Hirano bodies in cultured mouse fibroblasts. These results show that the failure to regulate the activity and/or affinity of an actin cross-linking protein can provide a signal for the formation of Hirano bodies. More generally, the formation of Hirano bodies is a cellular response to or a consequence of aberrant function of the actin cytoskeleton.

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3D Reconstruction of Smooth Muscle A-Actinin by Cryo Electron Microscopy

Microscopy and Microanalysis ◽

10.1017/s1431927600033717 ◽

2000 ◽

Vol 6 (S2) ◽

pp. 244-245

Author(s):

Jun Liu ◽

Dianne Taylor ◽

Kenneth A. Taylor

Keyword(s):

Electron Microscopy ◽

Smooth Muscle ◽

Calcium Binding ◽

3D Structure ◽

Unit Cell Dimension ◽

Lipid Monolayer ◽

Actin Binding ◽

Cryo Electron Microscopy ◽

Ef Hand ◽

Polypeptide Chains

α-Actinin is an actin crosslinking protein identified in a wide variety of cells. Both muscle and nonmuscle isoforms of α-actinin have been characterized [1]. The molecule consists of two polypeptide chains that form a rod-shaped antiparallel dimer. Each polypeptide is composed of three distinct structural regions: actin-binding domain, four triple helical repeats that form a central rod, and a carboxyl terminal domain that contains two EF hand calcium-binding sites. Here we present the 3D structure of the smooth muscle α-actinin, as determined by cryo electron microscopy at 2.0 nm resolution.Two dimensional crystals of chicken gizzard α-actinin were formed on positively charged lipid monolayer [2] and preserved frozen hydrated for TEM. The crystal has a unit cell dimension of a = 26.31 nm, b = 20.37 nm, γ= 107.10°, based on internal calibration against TMV.

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Structure of the posttranslational Sec protein-translocation channel complex from yeast

Science ◽

10.1126/science.aav6740 ◽

2018 ◽

Vol 363 (6422) ◽

pp. 84-87 ◽

Cited By ~ 32

Author(s):

Samuel Itskanov ◽

Eunyong Park

Keyword(s):

Electron Microscopy ◽

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Binding Sites ◽

Protein Translocation ◽

Secretory Proteins ◽

Conducting Channel ◽

Cryo Electron Microscopy ◽

Lateral Gate

The Sec61 protein-conducting channel mediates transport of many proteins, such as secretory proteins, across the endoplasmic reticulum (ER) membrane during or after translation. Posttranslational transport is enabled by two additional membrane proteins associated with the channel, Sec63 and Sec62, but its mechanism is poorly understood. We determined a structure of the Sec complex (Sec61-Sec63-Sec71-Sec72) from Saccharomyces cerevisiae by cryo–electron microscopy (cryo-EM). The structure shows that Sec63 tightly associates with Sec61 through interactions in cytosolic, transmembrane, and ER-luminal domains, prying open Sec61’s lateral gate and translocation pore and thus activating the channel for substrate engagement. Furthermore, Sec63 optimally positions binding sites for cytosolic and luminal chaperones in the complex to enable efficient polypeptide translocation. Our study provides mechanistic insights into eukaryotic posttranslational protein translocation.

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Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites

eLife ◽

10.7554/elife.29795 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 54

Author(s):

Baoyu Chen ◽

Hui-Ting Chou ◽

Chad A Brautigam ◽

Wenmin Xing ◽

Sheng Yang ◽

...

Keyword(s):

Electron Microscopy ◽

Binding Sites ◽

Actin Polymerization ◽

Rho Gtpase ◽

Biochemical Data ◽

Actin Assembly ◽

Cellular Processes ◽

Cryo Electron Microscopy ◽

Regulatory Complex ◽

Rac1 Gtpase

The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly.

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Structure of the RSC complex bound to the nucleosome

Science ◽

10.1126/science.aay0033 ◽

2019 ◽

Vol 366 (6467) ◽

pp. 838-843 ◽

Cited By ~ 31

Author(s):

Youpi Ye ◽

Hao Wu ◽

Kangjing Chen ◽

Cedric R. Clapier ◽

Naveen Verma ◽

...

Keyword(s):

Electron Microscopy ◽

Gene Transcription ◽

Chromatin Structure ◽

Adenosine Triphosphatase ◽

Related Protein ◽

Dna Translocation ◽

Auxiliary Subunit ◽

Cryo Electron Microscopy ◽

Rsc Complex ◽

Shed Light

The RSC complex remodels chromatin structure and regulates gene transcription. We used cryo–electron microscopy to determine the structure of yeast RSC bound to the nucleosome. RSC is delineated into the adenosine triphosphatase motor, the actin-related protein module, and the substrate recruitment module (SRM). RSC binds the nucleosome mainly through the motor, with the auxiliary subunit Sfh1 engaging the H2A-H2B acidic patch to enable nucleosome ejection. SRM is organized into three substrate-binding lobes poised to bind their respective nucleosomal epitopes. The relative orientations of the SRM and the motor on the nucleosome explain the directionality of DNA translocation and promoter nucleosome repositioning by RSC. Our findings shed light on RSC assembly and functionality, and they provide a framework to understand the mammalian homologs BAF/PBAF and the Sfh1 ortholog INI1/BAF47, which are frequently mutated in cancers.

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Cryo-electron microscopy structures of pyrene-labeled ADP-Pi- and ADP-actin filaments

Nature Communications ◽

10.1038/s41467-020-19762-1 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Steven Z. Chou ◽

Thomas D. Pollard

Keyword(s):

Electron Microscopy ◽

Skeletal Muscle ◽

Active Site ◽

Actin Filaments ◽

Actin Polymerization ◽

Actin Binding ◽

Muscle Actin ◽

Hydrophobic Cavity ◽

Cryo Electron Microscopy ◽

Kinetics Of

AbstractSince the fluorescent reagent N-(1-pyrene)iodoacetamide was first used to label skeletal muscle actin in 1981, the pyrene-labeled actin has become the most widely employed tool to measure the kinetics of actin polymerization and the interaction between actin and actin-binding proteins. Here we report high-resolution cryo-electron microscopy structures of actin filaments with N-1-pyrene conjugated to cysteine 374 and either ADP (3.2 Å) or ADP-phosphate (3.0 Å) in the active site. Polymerization buries pyrene in a hydrophobic cavity between subunits along the long-pitch helix with only minor differences in conformation compared with native actin filaments. These structures explain how polymerization increases the fluorescence 20-fold, how myosin and cofilin binding to filaments reduces the fluorescence, and how profilin binding to actin monomers increases the fluorescence.

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Cortactin signalling and dynamic actin networks

Biochemical Journal ◽

10.1042/bj20040737 ◽

2004 ◽

Vol 382 (1) ◽

pp. 13-25 ◽

Cited By ~ 197

Author(s):

Roger J. DALY

Keyword(s):

Tyrosine Kinase ◽

Actin Cytoskeleton ◽

Current Knowledge ◽

Actin Binding ◽

Related Protein ◽

Protein Targets ◽

Actin Networks ◽

Multiple Protein ◽

Initial Characterization

Cortactin was first identified over a decade ago, and its initial characterization as both an F-actin binding protein and v-Src substrate suggested that it was likely to be a key regulator of actin rearrangements in response to tyrosine kinase signalling. The recent discovery that cortactin binds and activates the actin related protein (Arp)2/3 complex, and thus regulates the formation of branched actin networks, together with the identification of multiple protein targets of the cortactin SH3 domain, have revealed diverse cellular roles for this protein. This article reviews current knowledge regarding the role of cortactin in signalling to the actin cytoskeleton in the context of these developments.

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