Faculty Opinions recommendation of Actin turnover-dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing.

2007 ◽  
Author(s):  
Pekka Lappalainen
Keyword(s):  
Actin Network ◽  
Capping Protein ◽  
Actin Turnover

scholarly journals Actin turnover–dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing

2006 ◽  
Vol 175 (6) ◽  
pp. 947-955 ◽  
Author(s):  
Takushi Miyoshi ◽  
Takahiro Tsuji ◽  
Chiharu Higashida ◽  
Maud Hertzog ◽  
Akiko Fujita ◽  
...  
Keyword(s):  
Single Molecule ◽  
Actin Filaments ◽  
Dissociation Rate ◽  
Actin Network ◽  
Lim Kinase ◽  
Capping Protein ◽  
Filament Dynamics ◽  
Actin Turnover ◽  
Kinetics Of

Actin forms the dendritic nucleation network and undergoes rapid polymerization-depolymerization cycles in lamellipodia. To elucidate the mechanism of actin disassembly, we characterized molecular kinetics of the major filament end-binding proteins Arp2/3 complex and capping protein (CP) using single-molecule speckle microscopy. We have determined the dissociation rates of Arp2/3 and CP as 0.048 and 0.58 s−1, respectively, in lamellipodia of live XTC fibroblasts. This CP dissociation rate is three orders of magnitude faster than in vitro. CP dissociates slower from actin stress fibers than from the lamellipodial actin network, suggesting that CP dissociation correlates with actin filament dynamics. We found that jasplakinolide, an actin depolymerization inhibitor, rapidly blocked the fast CP dissociation in cells. Consistently, the coexpression of LIM kinase prolonged CP speckle lifetime in lamellipodia. These results suggest that cofilin-mediated actin disassembly triggers CP dissociation from actin filaments. We predict that filament severing and end-to-end annealing might take place fairly frequently in the dendritic nucleation actin arrays.

scholarly journals MICAL2 acts through Arp3B isoform-specific Arp2/3 complexes to destabilize branched actin networks

2020 ◽  
Author(s):  
Chiara Galloni ◽  
Davide Carra ◽  
Jasmine V. G. Abella ◽  
Svend Kjær ◽  
Pavithra Singaravelu ◽  
...  
Keyword(s):  
Functional Properties ◽  
Actin Filament ◽  
Actin Assembly ◽  
Actin Network ◽  
Network Stability ◽  
Actin Networks ◽  
Cellular Processes ◽  
Actin Turnover ◽  
Filament Networks

AbstractThe Arp2/3 complex (Arp2, Arp3 and ARPC1-5) is essential to generate branched actin filament networks for many cellular processes. Human Arp3, ARPC1 and ARPC5 exist as two isoforms but the functional properties of Arp2/3 iso-complexes is largely unexplored. Here we show that Arp3B, but not Arp3 is subject to regulation by the methionine monooxygenase MICAL2, which is recruited to branched actin networks by coronin-1C. Although Arp3 and Arp3B iso-complexes promote actin assembly equally efficiently in vitro, they have different cellular properties. Arp3B turns over significantly faster than Arp3 within the network and upon its depletion actin turnover decreases. Substitution of Arp3B Met293 by Thr, the corresponding residue in Arp3 increases actin network stability, and conversely, replacing Arp3 Thr293 with Gln to mimic Met oxidation promotes network disassembly. Thus, MICAL2 regulates a subset of Arp2/3 complexes to control branched actin network disassembly.

scholarly journals A barbed end interference mechanism reveals how capping protein promotes nucleation in branched actin networks

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Johanna Funk ◽  
Felipe Merino ◽  
Matthias Schaks ◽  
Klemens Rottner ◽  
Stefan Raunser ◽  
...  
Keyword(s):  
Structural Biology ◽  
Actin Filaments ◽  
Actin Network ◽  
Essential Factor ◽  
Actin Networks ◽  
Capping Protein ◽  
Cellular Processes ◽  
In Vitro Reconstitution ◽  
Interference Mechanism

AbstractHeterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP potentiates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we combine structural biology with in vitro reconstitution to demonstrate that CP not only terminates filament elongation, but indirectly stimulates the activity of Arp2/3 activating nucleation promoting factors (NPFs) by preventing their association to filament barbed ends. Key to this function is one of CP’s C-terminal “tentacle” extensions, which sterically masks the main interaction site of the terminal actin protomer. Deletion of the β tentacle only modestly impairs capping. However, in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes—capping and nucleation—in branched actin network assembly.

scholarly journals Capping protein regulatory cycle driven by CARMIL and V-1 may promote actin network assembly at protruding edges

2014 ◽  
Vol 111 (19) ◽  
pp. E1970-E1979 ◽  
Author(s):  
I. Fujiwara ◽  
K. Remmert ◽  
G. Piszczek ◽  
J. A. Hammer
Keyword(s):  
Actin Network ◽  
Capping Protein

scholarly journals Instantaneous inactivation of cofilin reveals its function of F-actin disassembly in lamellipodia

2013 ◽  
Vol 24 (14) ◽  
pp. 2238-2247 ◽  
Author(s):  
Eric A. Vitriol ◽  
Ariel L. Wise ◽  
Mathew E. Berginski ◽  
James R. Bamburg ◽  
James Q. Zheng
Keyword(s):  
Actin Filaments ◽  
Flow Speed ◽  
Retrograde Flow ◽  
Actin Network ◽  
Principal Role ◽  
Actin Turnover ◽  
And Control ◽  
In Cells

Cofilin is a key regulator of the actin cytoskeleton. It can sever actin filaments, accelerate filament disassembly, act as a nucleation factor, recruit or antagonize other actin regulators, and control the pool of polymerization-competent actin monomers. In cells these actions have complex functional outputs. The timing and localization of cofilin activity are carefully regulated, and thus global, long-term perturbations may not be sufficient to probe its precise function. To better understand cofilin's spatiotemporal action in cells, we implemented chromophore-assisted laser inactivation (CALI) to instantly and specifically inactivate it. In addition to globally inhibiting actin turnover, CALI of cofilin generated several profound effects on the lamellipodia, including an increase of F-actin, a rearward expansion of the actin network, and a reduction in retrograde flow speed. These results support the hypothesis that the principal role of cofilin in lamellipodia at steady state is to break down F-actin, control filament turnover, and regulate the rate of retrograde flow.

scholarly journals New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales

2014 ◽  
Vol 25 (7) ◽  
pp. 1010-1024 ◽  
Author(s):  
Sawako Yamashiro ◽  
Hiroaki Mizuno ◽  
Matthew B. Smith ◽  
Gillian L. Ryan ◽  
Tai Kiuchi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Protein ◽  
Focal Adhesions ◽  
Actin Network ◽  
Actin Turnover ◽  
Flow Velocity Measurement ◽  
Flow Speeds ◽  
Green Fluorescent ◽  
User Friendly

Speckle microscopy directly visualizes the retrograde actin flow, which is believed to promote cell-edge protrusion when linked to focal adhesions (FAs). However, it has been argued that, due to rapid actin turnover, the use of green fluorescent protein–actin, the lack of appropriate analysis algorithms, and technical difficulties, speckle microscopy does not necessarily report the flow velocities of entire actin populations. In this study, we developed a new, user-friendly single-molecule speckle (SiMS) microscopy using DyLight dye-labeled actin. Our new SiMS method enables in vivo nanometer-scale displacement analysis with a low localization error of ±8–8.5 nm, allowing accurate flow-velocity measurement for actin speckles with lifetime <5 s. In lamellipodia, both short- and long-lived F-actin molecules flow with the same speed, indicating they are part of a single actin network. These results do not support coexistence of F-actin populations with different flow speeds, which is referred to as the lamella hypothesis. Mature FAs, but not nascent adhesions, locally obstruct the retrograde flow. Interestingly, the actin flow in front of mature FAs is fast and biased toward FAs, suggesting that mature FAs attract the flow in front and actively remodel the local actin network.

scholarly journals The Role of Actin Turnover in Retrograde Actin Network Flow in Neuronal Growth Cones

PLoS ONE ◽  
2012 ◽  
Vol 7 (2) ◽  
pp. e30959 ◽  
Author(s):  
David Van Goor ◽  
Callen Hyland ◽  
Andrew W. Schaefer ◽  
Paul Forscher
Keyword(s):  
Network Flow ◽  
Growth Cones ◽  
Actin Network ◽  
Neuronal Growth ◽  
Actin Turnover ◽  
Neuronal Growth Cones

scholarly journals Myosin-dependent actin stabilization as revealed by single-molecule imaging of actin turnover

2018 ◽  
Vol 29 (16) ◽  
pp. 1941-1947 ◽  
Author(s):  
Sawako Yamashiro ◽  
Soichiro Tanaka ◽  
Laura M. McMillen ◽  
Daisuke Taniguchi ◽  
Dimitrios Vavylonis ◽  
...  
Keyword(s):  
Single Molecule ◽  
Driving Force ◽  
Early Stage ◽  
Actin Binding ◽  
Actin Network ◽  
Turnover Rates ◽  
Time Resolved ◽  
Molecular Behavior ◽  
Actin Turnover ◽  
Actomyosin Contraction

How mechanical stress applied to the actin network modifies actin turnover has attracted considerable attention. Actomyosin exerts the major force on the actin network, which has been implicated in actin stability regulation. However, direct monitoring of immediate changes in F-actin stability on alteration of actomyosin contraction has not been achieved. Here we reexamine myosin regulation of actin stability by using single-molecule speckle analysis of actin. To avoid possible errors attributable to actin-binding probes, we employed DyLight-labeled actin that distributes identical to F-actin in lamellipodia. We performed time-resolved analysis of the effect of blebbistatin on actin turnover. Blebbistatin enhanced actin disassembly in lamellipodia of fish keratocytes and lamellar of Xenopus XTC cells at an early stage of the inhibition, indicating that actomyosin contraction stabilizes cellular F-actin. In addition, our data show a previously unrecognized relationship between the actin network-driving force and the actin turnover rates in lamellipodia. These findings point to the power of direct viewing of molecular behavior in elucidating force regulation of actin filament turnover.

scholarly journals A structure-derived mechanism reveals how capping protein promotes nucleation in branched actin networks

2021 ◽  
Author(s):  
Johanna Funk ◽  
Felipe Merino ◽  
Matthias Schaks ◽  
Klemens Rottner ◽  
Stefan Raunser ◽  
...  
Keyword(s):  
Binding Site ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Network ◽  
Essential Factor ◽  
Interaction Site ◽  
Actin Networks ◽  
Capping Protein ◽  
Cellular Processes ◽  
Terminal Filament

Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP stimulates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we report the structure of capped actin filament barbed ends, which reveals how CP not only prevents filament elongation, but also controls access to both terminal filament subunits. In addition to its primary binding site that blocks the penultimate subunit, we find that the CP sterically occludes the central interaction site of the terminal actin protomer through one of its C-terminal tentacle extensions. Deletion of this β tentacle only modestly impairs capping. However in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors (NPFs) by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes -capping and nucleation- in branched actin network assembly.

scholarly journals Turnover of branched actin filament networks by stochastic fragmentation with ADF/cofilin

2011 ◽  
Vol 22 (14) ◽  
pp. 2541-2550 ◽  
Author(s):  
Anne-Cécile Reymann ◽  
Cristian Suarez ◽  
Christophe Guérin ◽  
Jean-Louis Martiel ◽  
Christopher J. Staiger ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Actin Network ◽  
Comet Tail ◽  
Actin Networks ◽  
Capping Protein ◽  
Nucleation Sites ◽  
Actin Regulatory Proteins ◽  
Filament Networks ◽  
Actin Comet Tails

Cell motility depends on the rapid assembly, aging, severing, and disassembly of actin filaments in spatially distinct zones. How a set of actin regulatory proteins that sustains actin-based force generation during motility work together in space and time remains poorly understood. We present our study of the distribution and dynamics of Arp2/3 complex, capping protein (CP), and actin-depolymerizing factor (ADF)/cofilin in actin “comet tails,” using a minimal reconstituted system with nucleation-promoting factor (NPF)-coated beads. The Arp2/3 complex concentrates at nucleation sites near the beads as well as in the first actin shell. CP colocalizes with actin and is homogeneously distributed throughout the comet tail; it serves to constrain the spatial distribution of ATP/ADP-Pi filament zones to areas near the bead. The association of ADF/cofilin with the actin network is therefore governed by kinetics of actin assembly, actin nucleotide state, and CP binding. A kinetic simulation accurately validates these observations. Following its binding to the actin networks, ADF/cofilin is able to break up the dense actin filament array of a comet tail. Stochastic severing by ADF/cofilin loosens the tight entanglement of actin filaments inside the comet tail and facilitates turnover through the macroscopic release of large portions of the aged actin network.

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