scholarly journals Twinfilin bypasses assembly conditions and actin filament aging to drive barbed end depolymerization

2020 ◽  
Vol 220 (1) ◽  
Author(s):  
Shashank Shekhar ◽  
Gregory J. Hoeprich ◽  
Jeff Gelles ◽  
Bruce L. Goode
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Atp Hydrolysis ◽  
Tirf Microscopy ◽  
Actin Networks ◽  
Rapid Elongation ◽  
In Cells

Cellular actin networks grow by ATP-actin addition at filament barbed ends and have long been presumed to depolymerize at their pointed ends, primarily after filaments undergo “aging” (ATP hydrolysis and Pi release). The cytosol contains high levels of actin monomers, which favors assembly over disassembly, and barbed ends are enriched in ADP-Pi actin. For these reasons, the potential for a barbed end depolymerization mechanism in cells has received little attention. Here, using microfluidics-assisted TIRF microscopy, we show that mouse twinfilin, a member of the ADF-homology family, induces depolymerization of ADP-Pi barbed ends even under assembly-promoting conditions. Indeed, we observe in single reactions containing micromolar concentrations of actin monomers the simultaneous rapid elongation of formin-bound barbed ends and twinfilin-induced depolymerization of free barbed ends. The data show that twinfilin catalyzes dissociation of subunits from ADP-Pi barbed ends and thereby bypasses filament aging prerequisites to disassemble newly polymerized actin filaments.

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.

scholarly journals Profilin and formin constitute a pacemaker system for robust actin filament growth

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Johanna Funk ◽  
Felipe Merino ◽  
Larisa Venkova ◽  
Lina Heydenreich ◽  
Jan Kierfeld ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filament ◽  
Growth Rates ◽  
Actin Filaments ◽  
Mammalian Cells ◽  
Biological Processes ◽  
Muscle Actin ◽  
Cell Morphogenesis ◽  
Actin Networks ◽  
In Cells

The actin cytoskeleton drives many essential biological processes, from cell morphogenesis to motility. Assembly of functional actin networks requires control over the speed at which actin filaments grow. How this can be achieved at the high and variable levels of soluble actin subunits found in cells is unclear. Here we reconstitute assembly of mammalian, non-muscle actin filaments from physiological concentrations of profilin-actin. We discover that under these conditions, filament growth is limited by profilin dissociating from the filament end and the speed of elongation becomes insensitive to the concentration of soluble subunits. Profilin release can be directly promoted by formin actin polymerases even at saturating profilin-actin concentrations. We demonstrate that mammalian cells indeed operate at the limit to actin filament growth imposed by profilin and formins. Our results reveal how synergy between profilin and formins generates robust filament growth rates that are resilient to changes in the soluble subunit concentration.

scholarly journals Profilin and formin constitute a pacemaker system for robust actin filament growth

2019 ◽  
Author(s):  
Johanna Funk ◽  
Felipe Merino ◽  
Larisa Venkova ◽  
Pablo Vargas ◽  
Stefan Raunser ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filament ◽  
Growth Rates ◽  
Actin Filaments ◽  
Mammalian Cells ◽  
Biological Processes ◽  
Muscle Actin ◽  
Cell Morphogenesis ◽  
Actin Networks ◽  
In Cells

AbstractThe actin cytoskeleton drives many essential biological processes, from cell morphogenesis to motility. Assembly of functional actin networks requires control over the speed at which actin filaments grow. How this can be achieved at the high and variable levels of soluble actin subunits found in cells is unclear. Here we reconstitute assembly of mammalian, non-muscle actin filaments from physiological concentrations of profilin-actin. We discover that under these conditions, filament growth is limited by profilin dissociating from the filament end and the speed of elongation becomes insensitive to the concentration of soluble subunits. Profilin release can be directly promoted by formin actin polymerases even at saturating profilin-actin concentrations. We demonstrate that mammalian cells indeed operate at the limit to actin filament growth imposed by profilin and formins. Our results reveal how synergy between profilin and formins generates robust filament growth rates that are resilient to changes in the soluble subunit concentration.

scholarly journals Actin-depolymerizing Factor and Cofilin-1 Play Overlapping Roles in Promoting Rapid F-Actin Depolymerization in Mammalian Nonmuscle Cells

2005 ◽  
Vol 16 (2) ◽  
pp. 649-664 ◽  
Author(s):  
Pirta Hotulainen ◽  
Eija Paunola ◽  
Maria K. Vartiainen ◽  
Pekka Lappalainen
Keyword(s):  
Cell Motility ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Depolymerizing Factor ◽  
Cytoskeletal Dynamics ◽  
Nonmuscle Cells ◽  
In Cells

Actin-depolymerizing factor (ADF)/cofilins are small actin-binding proteins found in all eukaryotes. In vitro, ADF/cofilins promote actin dynamics by depolymerizing and severing actin filaments. However, whether ADF/cofilins contribute to actin dynamics in cells by disassembling “old” actin filaments or by promoting actin filament assembly through their severing activity is a matter of controversy. Analysis of mammalian ADF/cofilins is further complicated by the presence of multiple isoforms, which may contribute to actin dynamics by different mechanisms. We show that two isoforms, ADF and cofilin-1, are expressed in mouse NIH 3T3, B16F1, and Neuro 2A cells. Depleting cofilin-1 and/or ADF by siRNA leads to an accumulation of F-actin and to an increase in cell size. Cofilin-1 and ADF seem to play overlapping roles in cells, because the knockdown phenotype of either protein could be rescued by overexpression of the other one. Cofilin-1 and ADF knockdown cells also had defects in cell motility and cytokinesis, and these defects were most pronounced when both ADF and cofilin-1 were depleted. Fluorescence recovery after photobleaching analysis and studies with an actin monomer-sequestering drug, latrunculin-A, demonstrated that these phenotypes arose from diminished actin filament depolymerization rates. These data suggest that mammalian ADF and cofilin-1 promote cytoskeletal dynamics by depolymerizing actin filaments and that this activity is critical for several processes such as cytokinesis and cell motility.

scholarly journals The dynamic instability of actin filament barbed ends

2021 ◽  
Vol 220 (4) ◽  
Author(s):  
Guillaume Romet-Lemonne ◽  
Antoine Jégou
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Molecular Mechanisms ◽  
Dynamic Instability ◽  
Filament Networks ◽  
In Cells

The turnover of actin filament networks in cells has long been considered to reflect the treadmilling behavior of pure actin filaments in vitro, where only the pointed ends depolymerize. Newly discovered molecular mechanisms challenge this notion, as they provide evidence of situations in which growing and depolymerizing barbed ends coexist.

scholarly journals The Arp1/11 minifilament of dynactin primes the endosomal Arp2/3 complex

2021 ◽  
Vol 7 (3) ◽  
pp. eabd5956 ◽  
Author(s):  
Artem I. Fokin ◽  
Violaine David ◽  
Ksenia Oguievetskaia ◽  
Emmanuel Derivery ◽  
Caroline E. Stone ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Networks ◽  
Capping Protein ◽  
Related Proteins ◽  
In Cells

Dendritic actin networks develop from a first actin filament through branching by the Arp2/3 complex. At the surface of endosomes, the WASH complex activates the Arp2/3 complex and interacts with the capping protein for unclear reasons. Here, we show that the WASH complex interacts with dynactin and uncaps it through its FAM21 subunit. In vitro, the uncapped Arp1/11 minifilament elongates an actin filament, which then primes the WASH-induced Arp2/3 branching reaction. In dynactin-depleted cells or in cells where the WASH complex is reconstituted with a FAM21 mutant that cannot uncap dynactin, formation of branched actin at the endosomal surface is impaired. Our results reveal the importance of the WASH complex in coordinating two complexes containing actin-related proteins.

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 Computing on actin bundles network

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Andrew Adamatzky ◽  
Florian Huber ◽  
Jörg Schnauß
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Ionic Currents ◽  
Computational Experiments ◽  
Two Dimensional ◽  
Actin Bundle ◽  
Actin Bundles ◽  
Actin Networks

Abstract Actin filaments are conductive to ionic currents, mechanical and voltage solitons. These travelling localisations can be utilised to generate computing circuits from actin networks. The propagation of localisations on a single actin filament is experimentally unfeasible to control. Therefore, we consider excitation waves propagating on bundles of actin filaments. In computational experiments with a two-dimensional slice of an actin bundle network we show that by using an arbitrary arrangement of electrodes, it is possible to implement two-inputs-one-output circuits.

scholarly journals Mechanisms of actin disassembly

2013 ◽  
Vol 24 (15) ◽  
pp. 2299-2302 ◽  
Author(s):  
William Brieher
Keyword(s):  
Actin Cytoskeleton ◽  
Cell Motility ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Actin Depolymerization ◽  
Filament Dynamics ◽  
Physiological Demands ◽  
In Cells

The actin cytoskeleton is constantly assembling and disassembling. Cells harness the energy of these turnover dynamics to drive cell motility and organize cytoplasm. Although much is known about how cells control actin polymerization, we do not understand how actin filaments depolymerize inside cells. I briefly describe how the combination of imaging actin filament dynamics in cells and using in vitro biochemistry progressively altered our views of actin depolymerization. I describe why I do not think that the prevailing model of actin filament turnover—cofilin-mediated actin filament severing—can account for actin filament disassembly detected in cells. Finally, I speculate that cells might be able to tune the mechanism of actin depolymerization to meet physiological demands and selectively control the stabilities of different actin arrays.

scholarly journals Activation of the Arp2/3 Complex by the Actin Filament Binding Protein Abp1p

2001 ◽  
Vol 153 (3) ◽  
pp. 627-634 ◽  
Author(s):  
Bruce L. Goode ◽  
Avital A. Rodal ◽  
Georjana Barnes ◽  
David G. Drubin
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Mammalian Cells ◽  
Actin Assembly ◽  
Related Protein ◽  
Genetic Studies ◽  
Actin Networks ◽  
Specific Step

The actin-related protein (Arp) 2/3 complex plays a central role in assembly of actin networks. Because distinct actin-based structures mediate diverse processes, many proteins are likely to make spatially and temporally regulated interactions with the Arp2/3 complex. We have isolated a new activator, Abp1p, which associates tightly with the yeast Arp2/3 complex. Abp1p contains two acidic sequences (DDW) similar to those found in SCAR/WASp proteins. We demonstrate that mutation of these sequences abolishes Arp2/3 complex activation in vitro. Genetic studies indicate that this activity is important for Abp1p functions in vivo. In contrast to SCAR/WASp proteins, Abp1p binds specifically to actin filaments, not monomers. Actin filament binding is mediated by the ADF/cofilin homology (ADF-H) domain of Abp1p and is required for Arp2/3 complex activation in vitro. We demonstrate that Abp1p recruits Arp2/3 complex to the sides of filaments, suggesting a novel mechanism of activation. Studies in yeast and mammalian cells indicate that Abp1p is involved functionally in endocytosis. Based on these results, we speculate that Abp1p may link Arp2/3-mediated actin assembly to a specific step in endocytosis.

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