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.

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 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 Synergy between Cyclase-associated protein and Cofilin accelerates actin filament depolymerization by two orders of magnitude

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Shashank Shekhar ◽  
Johnson Chung ◽  
Jane Kondev ◽  
Jeff Gelles ◽  
Bruce L. Goode
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
Binding Protein ◽  
Actin Dynamics ◽  
Actin Network ◽  
Cellular Mechanisms ◽  
Actin Networks ◽  
Binding Event

AbstractCellular actin networks can be rapidly disassembled and remodeled in a few seconds, yet in vitro actin filaments depolymerize slowly over minutes. The cellular mechanisms enabling actin to depolymerize this fast have so far remained obscure. Using microfluidics-assisted TIRF, we show that Cyclase-associated protein (CAP) and Cofilin synergize to processively depolymerize actin filament pointed ends at a rate 330-fold faster than spontaneous depolymerization. Single molecule imaging further reveals that hexameric CAP molecules interact with the pointed ends of Cofilin-decorated filaments for several seconds at a time, removing approximately 100 actin subunits per binding event. These findings establish a paradigm, in which a filament end-binding protein and a side-binding protein work in concert to control actin dynamics, and help explain how rapid actin network depolymerization is achieved in cells.

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 Modeling myosin Va liposome transport through actin filament networks reveals a percolation threshold that modulates transport properties

2021 ◽  
Author(s):  
Sam Walcott ◽  
David M Warshaw
Keyword(s):  
Phase Transition ◽  
Actin Filament ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Fold Increase ◽  
Coarse Grained ◽  
Actin Network ◽  
Cargo Delivery ◽  
Myosin Va ◽  
Filament Networks

Myosin Va (myoVa) motors transport membrane-bound cargo through three-dimensional, intracellular actin filament networks. We developed a coarse-grained, in silico model to predict how actin filament density (3-800 filaments) within a randomly oriented actin network affects fluid-like liposome (350nm vs. 1,750nm) transport by myoVa motors. 5,000 simulated liposomes transported within each network adopted one of three states: transport, tug of war, or diffusion. Diffusion due to liposome detachment from actin rarely occurred given at least 10 motors on the liposome surface. However, with increased actin density, liposomes transitioned from primarily directed transport on single actin filaments to an apparent random walk, resulting from a mixture of transport and tug of wars as the probability of encountering additional actin filaments increased. This phase transition arises from a percolation phase transition at a critical number of accessible actin filaments, Nc. Nc, is a geometric property of the actin network that depends only on the position and polarity of the actin filaments and the liposome diameter, as evidenced by a five-fold increase in liposome diameter resulting in a five-fold decrease in Nc. Thus, in cells, actin network density and cargo size may be regulated to match cargo delivery to the cell's physiological demands.

scholarly journals LC3 and STRAP regulate actin filament assembly by JMY during autophagosome formation

2018 ◽  
Author(s):  
Xiaohua Hu ◽  
R. Dyche Mullins
Keyword(s):  
Actin Filament ◽  
Network Formation ◽  
De Novo ◽  
Actin Binding ◽  
Actin Assembly ◽  
Autophagosome Formation ◽  
Actin Networks ◽  
Filament Assembly ◽  
Filament Networks ◽  
Actin Comet Tails

AbstractDuring autophagy actin filament networks move and remodel cellular membranes to form autophagosomes that enclose and metabolize cytoplasmic contents. Two actin regulators, WHAMM and JMY, participate in autophagosome formation, but the signals linking autophagy to actin assembly are poorly understood. We show that, in non-starved cells, cytoplasmic JMY co-localizes with STRAP, a regulator of JMY’s nuclear functions, on non-motile vesicles with no associated actin networks. Upon starvation, JMY shifts to motile, LC3-containing membranes that move on actin comet tails. LC3 enhances JMY’s de novo actin nucleation activity via a cryptic actin-binding sequence near JMY’s N-terminus, and STRAP inhibits JMY’s ability to nucleate actin and activate the Arp2/3 complex. Cytoplasmic STRAP negatively regulates autophagy. Finally, we use purified proteins to reconstitute LC3‐ and JMY-dependent actin network formation on membranes, and inhibition of network formation by STRAP. We conclude that LC3 and STRAP regulate JMY’s actin assembly activities in trans during autophagy.eTOC BlurbThe actin regulator JMY creates filament networks that move membranes during autophagy. We find that, in unstarved cells, JMY is inhibited by interaction with the STRAP protein, but upon starvation JMY is recruited away from STRAP and activated by LC3.

scholarly journals Synergy between Wsp1 and Dip1 may initiate assembly of endocytic actin networks

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Connor J Balzer ◽  
Michael L James ◽  
Heidy Y Narvaez-Ortiz ◽  
Luke A Helgeson ◽  
Vladimir Sirotkin ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Schizosaccharomyces Pombe ◽  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
Total Internal Reflection ◽  
Actin Assembly ◽  
Actin Network ◽  
Actin Networks ◽  
Co Activation

The actin filament nucleator Arp2/3 complex is activated at cortical sites in Schizosaccharomyces pombe to assemble branched actin networks that drive endocytosis. Arp2/3 complex activators Wsp1 and Dip1 are required for proper actin assembly at endocytic sites, but how they coordinately control Arp2/3-mediated actin assembly is unknown. Alone, Dip1 activates Arp2/3 complex without preexisting actin filaments to nucleate ‘seed’ filaments that activate Wsp1-bound Arp2/3 complex, thereby initiating branched actin network assembly. In contrast, because Wsp1 requires preexisting filaments to activate, it has been assumed to function exclusively in propagating actin networks by stimulating branching from preexisting filaments. Here we show that Wsp1 is important not only for propagation but also for initiation of endocytic actin networks. Using single molecule total internal reflection fluorescence microscopy we show that Wsp1 synergizes with Dip1 to co-activate Arp2/3 complex. Synergistic co-activation does not require preexisting actin filaments, explaining how Wsp1 contributes to actin network initiation in cells.

scholarly journals Cyclase-associated Protein (CAP) Acts Directly on F-actin to Accelerate Cofilin-mediated Actin Severing across the Range of Physiological pH

2012 ◽  
Vol 287 (42) ◽  
pp. 35722-35732 ◽  
Author(s):  
Kieran P. M. Normoyle ◽  
William M. Brieher
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Mammalian Cells ◽  
Actin Dynamics ◽  
Turnover Rates ◽  
Interacting Protein ◽  
Actin Depolymerization ◽  
Neutral Ph ◽  
Filament Networks ◽  
Actin Comet Tails

Fast actin depolymerization is necessary for cells to rapidly reorganize actin filament networks. Utilizing a Listeria fluorescent actin comet tail assay to monitor actin disassembly rates, we observed that although a mixture of actin disassembly factors (cofilin, coronin, and actin-interacting protein 1 is sufficient to disassemble actin comet tails in the presence of physiological G-actin concentrations this mixture was insufficient to disassemble actin comet tails in the presence of physiological F-actin concentrations. Using biochemical complementation, we purified cyclase-associated protein (CAP) from thymus extracts as a factor that protects against the inhibition of excess F-actin. CAP has been shown to participate in actin dynamics but has been thought to act by liberating cofilin from ADP·G-actin monomers to restore cofilin activity. However, we found that CAP augments cofilin-mediated disassembly by accelerating the rate of cofilin-mediated severing. We also demonstrated that CAP acts directly on F-actin and severs actin filaments at acidic, but not neutral, pH. At the neutral pH characteristic of cytosol in most mammalian cells, we demonstrated that neither CAP nor cofilin are capable of severing actin filaments. However, the combination of CAP and cofilin rapidly severed actin at all pH values across the physiological range. Therefore, our results reveal a new function for CAP in accelerating cofilin-mediated actin filament severing and provide a mechanism through which cells can maintain high actin turnover rates without having to alkalinize cytosol, which would affect many biochemical reactions beyond actin depolymerization.

scholarly journals Synergy between Wsp1 and Dip1 may initiate assembly of endocytic actin networks

2020 ◽  
Author(s):  
Connor J. Balzer ◽  
Michael L. James ◽  
Luke A. Helgeson ◽  
Vladimir Sirotkin ◽  
Brad J. Nolen
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
Actin Assembly ◽  
Actin Network ◽  
Tirf Microscopy ◽  
Actin Networks ◽  
In Cells

AbstractThe actin filament nucleator Arp2/3 complex is activated at cortical sites in S. pombe to assemble branched actin networks that drive endocytosis. Arp2/3 complex activators Wsp1 and Dip1 are required for proper actin assembly at endocytic sites, but how they coordinately control Arp2/3-mediated actin assembly is unknown. Alone, Dip1 activates Arp2/3 complex without preexisting actin filaments to nucleate “seed” filaments that activate Wsp1-bound Arp2/3 complex, thereby initiating branched actin network assembly. In contrast, because Wsp1 requires pre-existing filaments to activate, it has been assumed to function exclusively in propagating actin networks by stimulating branching from pre-existing filaments. Here we show that Wsp1 is important not only for propagation, but also for initiation of endocytic actin networks. Using single molecule TIRF microscopy we show that Wsp1 synergizes with Dip1 to co-activate Arp2/3 complex. Synergistic coactivation does not require pre-existing actin filaments, explaining how Wsp1 contributes to actin network initiation in cells.

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