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

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.

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

10.1101/736272 ◽

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.

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Mechanisms of actin disassembly

Molecular Biology of the Cell ◽

10.1091/mbc.e12-09-0694 ◽

2013 ◽

Vol 24 (15) ◽

pp. 2299-2302 ◽

Cited By ~ 36

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.153.3.627 ◽

2001 ◽

Vol 153 (3) ◽

pp. 627-634 ◽

Cited By ~ 139

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

10.1101/2020.07.03.187120 ◽

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

The Journal of Cell Biology ◽

10.1083/jcb.202006022 ◽

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.

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Insights into the evolution of regulated actin dynamics via characterization of primitive gelsolin/cofilin proteins from Asgard archaea

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2009167117 ◽

2020 ◽

Vol 117 (33) ◽

pp. 19904-19913 ◽

Cited By ~ 2

Author(s):

Caner Akıl ◽

Linh T. Tran ◽

Magali Orhant-Prioux ◽

Yohendran Baskaran ◽

Edward Manser ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Actin Filament ◽

Calcium Binding ◽

Mammalian Cells ◽

Actin Polymerization ◽

Calcium Release ◽

Duplication Event ◽

Cellular Level ◽

Actin Dynamics

Asgard archaea genomes contain potential eukaryotic-like genes that provide intriguing insight for the evolution of eukaryotes. The eukaryotic actin polymerization/depolymerization cycle is critical for providing force and structure in many processes, including membrane remodeling. In general, Asgard genomes encode two classes of actin-regulating proteins from sequence analysis, profilins and gelsolins. Asgard profilins were demonstrated to regulate actin filament nucleation. Here, we identify actin filament severing, capping, annealing and bundling, and monomer sequestration activities by gelsolin proteins from Thorarchaeota (Thor), which complete a eukaryotic-like actin depolymerization cycle, and indicate complex actin cytoskeleton regulation in Asgard organisms. Thor gelsolins have homologs in other Asgard archaea and comprise one or two copies of the prototypical gelsolin domain. This appears to be a record of an initial preeukaryotic gene duplication event, since eukaryotic gelsolins are generally comprise three to six domains. X-ray structures of these proteins in complex with mammalian actin revealed similar interactions to the first domain of human gelsolin or cofilin with actin. Asgard two-domain, but not one-domain, gelsolins contain calcium-binding sites, which is manifested in calcium-controlled activities. Expression of two-domain gelsolins in mammalian cells enhanced actin filament disassembly on ionomycin-triggered calcium release. This functional demonstration, at the cellular level, provides evidence for a calcium-controlled Asgard actin cytoskeleton, indicating that the calcium-regulated actin cytoskeleton predates eukaryotes. In eukaryotes, dynamic bundled actin filaments are responsible for shaping filopodia and microvilli. By correlation, we hypothesize that the formation of the protrusions observed from Lokiarchaeota cell bodies may involve the gelsolin-regulated actin structures.

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Evolutionarily diverse LIM domain-containing proteins bind stressed actin filaments through a conserved mechanism

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2004656117 ◽

2020 ◽

Vol 117 (41) ◽

pp. 25532-25542 ◽

Cited By ~ 3

Author(s):

Jonathan D. Winkelman ◽

Caitlin A. Anderson ◽

Cristian Suarez ◽

David R. Kovar ◽

Margaret L. Gardel

Keyword(s):

Actin Cytoskeleton ◽

Fission Yeast ◽

Actin Filaments ◽

Mammalian Cells ◽

Strain Sensing ◽

Lim Domain ◽

Lim Domains ◽

Mechanical Homeostasis ◽

Functionally Diverse

The actin cytoskeleton assembles into diverse load-bearing networks, including stress fibers (SFs), muscle sarcomeres, and the cytokinetic ring to both generate and sense mechanical forces. The LIM (Lin11, Isl- 1, and Mec-3) domain family is functionally diverse, but most members can associate with the actin cytoskeleton with apparent force sensitivity. Zyxin rapidly localizes via its LIM domains to failing SFs in cells, known as strain sites, to initiate SF repair and maintain mechanical homeostasis. The mechanism by which these LIM domains associate with stress fiber strain sites (SFSS) is not known. Additionally, it is unknown how widespread strain sensing is within the LIM protein family. We identify that the LIM domain-containing region of 18 proteins from the Zyxin, Paxillin, Tes, and Enigma proteins accumulate to SFSS. Moreover, the LIM domain region from the fission yeast protein paxillin like 1 (Pxl1) also localizes to SFSS in mammalian cells, suggesting that the strain sensing mechanism is ancient and highly conserved. We then used sequence and domain analysis to demonstrate that tandem LIM domains contribute additively, for SFSS localization. Employing in vitro reconstitution, we show that the LIM domain-containing region from mammalian zyxin and fission yeast Pxl1 binds to mechanically stressed F-actin networks but does not associate with relaxed actin filaments. We propose that tandem LIM domains recognize an F-actin conformation that is rare in the relaxed state but is enriched in the presence of mechanical stress.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e04-07-0555 ◽

2005 ◽

Vol 16 (2) ◽

pp. 649-664 ◽

Cited By ~ 240

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.

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Actin disassembly by cofilin, coronin, and Aip1 occurs in bursts and is inhibited by barbed-end cappers

The Journal of Cell Biology ◽

10.1083/jcb.200801027 ◽

2008 ◽

Vol 182 (2) ◽

pp. 341-353 ◽

Cited By ~ 120

Author(s):

Hao Yuan Kueh ◽

Guillaume T. Charras ◽

Timothy J. Mitchison ◽

William M. Brieher

Keyword(s):

Actin Filaments ◽

Mammalian Cells ◽

Cytochalasin D ◽

Reaction Intermediates ◽

Latrunculin B ◽

Interacting Protein ◽

Physiological Relevance ◽

High Concentrations ◽

End Capping ◽

In Cells

Turnover of actin filaments in cells requires rapid actin disassembly in a cytoplasmic environment that thermodynamically favors assembly because of high concentrations of polymerizable monomers. We here image the disassembly of single actin filaments by cofilin, coronin, and actin-interacting protein 1, a purified protein system that reconstitutes rapid, monomer-insensitive disassembly (Brieher, W.M., H.Y. Kueh, B.A. Ballif, and T.J. Mitchison. 2006. J. Cell Biol. 175:315–324). In this three-component system, filaments disassemble in abrupt bursts that initiate preferentially, but not exclusively, from both filament ends. Bursting disassembly generates unstable reaction intermediates with lowered affinity for CapZ at barbed ends. CapZ and cytochalasin D (CytoD), a barbed-end capping drug, strongly inhibit bursting disassembly. CytoD also inhibits actin disassembly in mammalian cells, whereas latrunculin B, a monomer sequestering drug, does not. We propose that bursts of disassembly arise from cooperative separation of the two filament strands near an end. The differential effects of drugs in cells argue for physiological relevance of this new disassembly pathway and potentially explain discordant results previously found with these drugs.

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Actin filament destruction by osmium tetroxide

The Journal of Cell Biology ◽

10.1083/jcb.77.3.837 ◽

1978 ◽

Vol 77 (3) ◽

pp. 837-852 ◽

Cited By ~ 223

Author(s):

P Maupin-Szamier ◽

TD Pollard

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Sodium Phosphate ◽

Osmium Tetroxide ◽

Time Span ◽

Cross Linking ◽

Muscle Actin ◽

Viscosity Change ◽

Sodium Phosphate Buffer ◽

Sulfur Containing

We have studied the destruction of purified muscle actin filaments by osmium tetroxide (OsO4) to develop methods to preserve actin filaments during preparation for electron microscopy. Actin filaments are fragmented during exposure to OsO4. This causes the viscosity of solutions of actin filaments to decrease, ultimately to zero, and provides a convenient quantitative assay to analyze the reaction. The rate of filament destruction is determined by the OsO4 concentration, temperature, buffer type and concentration, and pH. Filament destruction is minimized by treatment with a low concentration of OsO4 in sodium phosphate buffer, pH 6.0, at 0 degrees C. Under these conditions, the viscosity of actin filament solutions is stable and actin filaments retain their straight, unbranched structure, even after dehydration and embedding. Under more severe conditions, the straight actin filaments are converted into what look like the microfilament networks commonly observed in cells fixed with OsO4. Destruction of actin filaments can be inhibited by binding tropomyosin to the actin. Cross-linking the actin molecules within a filament with glutaraldehyde does not prevent their destruction by OsO4. The viscosity decrease requires the continued presence of free OsO4. During the time of the viscosity change, OsO4 is reduced and the sulfur-containing amino acids of actin are oxidized, but little of the osmium is bound to the actin. Over a much longer time span, the actin molecules are split into discrete peptides.

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