Structural effects and functional implications of phalloidin and jasplakinolide binding to actin filaments

Pospich, Sabrina; Merino, Felipe; Raunser, Stefan

doi:10.1101/794495

Cited by 6 publications

(8 citation statements)

References 79 publications

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“…1C). Interestingly and contrary to what we have 110 seen before in samples co-polymerized with phalloidin (Pospich et al, 2019), the D-loop is in 111 its closed conformation ( Fig. S3).…”

Section: Structure Of the Lifeact-f-actin Complex 89contrasting

confidence: 72%

“…Based on previous studies (Mentes et al, 2018;Merino et al, 2018b;Pospich et al, 2019), we 90 know that phalloidin stabilizes actin filaments. When it is added during polymerization, the 91 nucleotide binding pocket is occupied with an ADP and Pi and the D-loop is in the open 92 conformation (Pospich et al, 2019). We therefore polymerized actin in the presence of 93 phalloidin and added an excess of Lifeact to the formed filaments in order to fully decorate the 94 filaments with Lifeact.…”

Section: Structure Of the Lifeact-f-actin Complex 89mentioning

confidence: 70%

“…We could clearly identify densities corresponding to ADP, Mg 2+ and Pi in the 98 nucleotide-binding pocket of actin ( Fig. 1A) and a density corresponding to phalloidin at the 99 expected position (Mentes et al, 2018;Merino et al, 2018a;Pospich et al, 2019) in the center 100 of the filament (Fig. 1A).…”

Section: Structure Of the Lifeact-f-actin Complex 89mentioning

confidence: 90%

“…However, both molecules strongly stabilize F-actin and shift the cellular 40 actin equilibrium, largely limiting their use in live cell imaging (Bubb et al, 2000;Dancker et 41 al., 1975). Recent cryo-EM studies from our group and others have uncovered how phalloidin 42 and jasplakinolide affect the structure of F-actin and described the potential limitations of their 43 use (Mentes et al, 2018;Merino et al, 2018a;Pospich et al, 2019). 44…”

Section: Introduction 25mentioning

confidence: 99%

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Structure of the Lifeact–F-actin complex

Belyy

Merino

Sitsel

et al. 2020

Preprint

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9Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) 10 structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe 11 has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The 12 molecular basis for such artefacts is poorly understood. Here, we determined the high-13resolution structure of the Lifeact-F-actin complex using electron cryo-microscopy. The 14 structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and 15 stretches over two adjacent actin subunits, stabilizing the DNase I-binding loop of actin in the 16 closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding 17 proteins, such as cofilin and myosin and actin-binding toxins, such as TccC3HVR from 18Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays 19 and activity measurements demonstrate that Lifeact indeed competes with these proteins, 20providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, 21we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations 22 into the peptide, laying the foundation for designing improved actin probes for live cell 23 imaging. 24

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“…1C). Interestingly and contrary to what we have 110 seen before in samples co-polymerized with phalloidin (Pospich et al, 2019), the D-loop is in 111 its closed conformation ( Fig. S3).…”

Section: Structure Of the Lifeact-f-actin Complex 89contrasting

confidence: 72%

Section: Structure Of the Lifeact-f-actin Complex 89mentioning

confidence: 70%

Section: Structure Of the Lifeact-f-actin Complex 89mentioning

confidence: 90%

Section: Introduction 25mentioning

confidence: 99%

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Structure of the Lifeact–F-actin complex

Belyy

Merino

Sitsel

et al. 2020

Preprint

Self Cite

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“…Consistent with this notion, we showed that modification of filaments by the actinstabilizing drugs jasplakinolide and phalloidin have different effects on the binding dwell-time of utrophin ABD mutants. Recent evidence has suggested that jasplakinolide preferentially biases one state of f-actin, stabilizing the D-loop from subdomain 2 in a more open configuration (Pospich et al, 2019), which may partially explain our observations. CH1-CH2 domains have been shown to bind actin by making contacts both on and between actin subunits within the same protofilament (Iwamoto et al, 2018;Kumari et al, 2019) (n and n+2).…”

Section: Discussionsupporting

confidence: 67%

Biased localization of actin binding proteins by actin filament conformation

Harris¹,

Jreij²,

Belardi³

et al. 2020

Preprint

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The assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell movement, shape change, and mechanics, but how different sets of proteins localize to these structures within a shared cytoplasm remains unclear. Here, we show that the actin-binding domains of accessory proteins can be sensitive to filament conformational changes induced by perturbations that include other binding proteins, stabilizing drugs, and physical constraints on the filament. Using a combination of live cell imaging and in vitro single molecule binding measurements, we demonstrate that the affinity of tandem calponin homology domain (CH1-CH2) mutants varies as actin filament twist is altered, and we show differential localization of native and mutant CH1-CH2 domains to actin networks at the front and rear of motile cells. These findings suggest that conformational heterogeneity of actin filaments in cells could influence the biochemical composition of cytoskeletal structures through a biophysical feedback loop.

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The evolution of SPHIRE-crYOLO particle picking and its application in automated cryo-EM processing workflows

Wagner

Raunser

2020

Commun Biol

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Particle selection is a crucial step when processing electron cryo microscopy data. Several automated particle picking procedures were developed in the past but most struggle with non-ideal data sets. In our recent Communications Biology article, we presented crYOLO, a deep learning based particle picking program. It enables fast, automated particle picking at human levels of accuracy with low effort. A general model allows the use of crYOLO for selecting particles in previously unseen data sets without further training. Here we describe how crYOLO has evolved since its initial release. We have introduced filament picking, a new denoising technique, and a new graphical user interface. Moreover, we outline its usage in automated processing pipelines, which is an important advancement on the horizon of the field.The crYOLO particle picking procedure A major goal of electron cryo microscopy (cryo-EM) is to obtain high-resolution threedimensional (3D) reconstructions of proteins and protein complexes to gain novel biological insights. This process involves the selection of thousands to millions of noisy two-dimensional (2D) particle projections, a number that only keeps increasing with recent advances in hardware and software development.In our recent work in Communications Biology 1 we introduced the "crYOLO" particle picking procedure. It is based on a deep neural network and the You Only Look Once (YOLO) object detection framework 2 . This approach enables the automated picking of particles within cryo-EM micrographs with a low signal-to-noise ratio requiring minimal human supervision or intervention. CrYOLO is easy to configure and train on a specific data set. It is fast and can process up to six micrographs per second. As crYOLO sees the complete micrograph, it is able to learn the

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Structural effects and functional implications of phalloidin and jasplakinolide binding to actin filaments

Cited by 6 publications

References 79 publications

Structure of the Lifeact–F-actin complex

Structure of the Lifeact–F-actin complex

Biased localization of actin binding proteins by actin filament conformation

The evolution of SPHIRE-crYOLO particle picking and its application in automated cryo-EM processing workflows

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