The actin treadmill

1985 ◽  
Vol 63 (6) ◽  
pp. 414-421 ◽  
Author(s):  
Michael Wanger ◽  
Thomas Keiser ◽  
Jean-Marc Neuhaus ◽  
Albrecht Wegner
Keyword(s):  
Adenosine Triphosphate ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Actin Assembly ◽  
Tight Coupling ◽  
Pointed End

Actin filaments can assemble at the barbed end and disassemble simultaneously at the pointed end. Prerequisites for this treadmilling reaction are the structural polarity of actin filaments and tight coupling of the actin assembly reaction and the adenosine triphosphate hydrolysis occurring during actin polymerization. In this article, investigations on the actin treadmill are reviewed.

scholarly journals Mechanism of actin polymerization revealed by cryo-EM structures of actin filaments with three different bound nucleotides

2018 ◽  
Author(s):  
Steven Z. Chou ◽  
Thomas D. Pollard
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Atp Hydrolysis ◽  
Regulatory Proteins ◽  
Actin Assembly ◽  
Cryo Electron Microscopy ◽  
Pointed End ◽  
Binding Loop

AbstractWe used electron cryo-micrographs to reconstruct actin filaments with bound AMPPNP (β,γ-imidoadenosine 5’-triphosphate, an ATP analog), ADP-Pi (ADP with inorganic phosphate) or ADP to resolutions of 3.4 Å, 3.4 Å and 3.6 Å. Subunits in the three filaments have nearly identical backbone conformations, so assembly rather than ATP hydrolysis or phosphate dissociation is responsible for their flattened conformation in filaments. Polymerization increases the rate of ATP hydrolysis by changing the conformations of the three ATP phosphates and the side chains of Gln137 and His161 in the active site. Flattening also promotes interactions along both the long-pitch and short-pitch helices. In particular, conformational changes in subdomain 3 open up favorable interactions with the DNase-I binding loop in subdomain 2 of the adjacent subunit. Subunits at the barbed end of the filament are likely to be in this favorable conformation, while monomers are not. This difference explains why filaments grow faster at the barbed end than the pointed end. Loss of hydrogen bonds after phosphate dissociation may account for the greater flexibility of ADP-actin filaments.Significance StatementActin filaments comprise a major part of the cytoskeleton of eukaryotic cells and serve as tracks for myosin motor proteins. The filaments assemble from actin monomers with a bound ATP. After polymerization, actin rapidly hydrolyzes the bound ATP and slowly dissociates the γ-phosphate. ADP-actin filaments then disassemble to recycle the subunits. Understanding how actin filaments assemble, disassemble and interact with numerous regulatory proteins depends on knowing the structure of the filament. High quality structures of ADP-actin filaments were available, but not of filaments with bound ATP- or with ADP and phosphate. We determined structures of actin filaments with bound AMPPNP (a slowly hydrolyzed ATP analog), ADP and phosphate and ADP by cryo-electron microscopy. These structures show how conformational changes during actin assembly promote ATP hydrolysis and faster growth at one end of the filament than the other.

scholarly journals The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly.

1993 ◽  
Vol 120 (4) ◽  
pp. 909-922 ◽  
Author(s):  
C P Chia ◽  
A Shariff ◽  
S A Savage ◽  
E J Luna
Keyword(s):  
Membrane Protein ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Lipid Vesicles ◽  
Integral Membrane Protein ◽  
Actin Binding ◽  
Actin Assembly ◽  
Nucleation Activity

Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma membranes, was isolated by detergent extraction from cytoskeletons and purified to homogeneity. Ponticulin is an abundant membrane protein, averaging approximately 10(6) copies/cell, with an estimated surface density of approximately 300 per microns2. Ponticulin solubilized in octylglucoside exhibited hydrodynamic properties consistent with a ponticulin monomer in a spherical or slightly ellipsoidal detergent micelle with a total molecular mass of 56 +/- 6 kD. Purified ponticulin nucleated actin polymerization when reconstituted into Dictyostelium lipid vesicles, but not when a number of commercially available lipids and lipid mixtures were substituted for the endogenous lipid. The specific activity was consistent with that expected for a protein comprising 0.7 +/- 0.4%, by mass, of the plasma membrane protein. Ponticulin in octylglucoside micelles bound F-actin but did not nucleate actin assembly. Thus, ponticulin-mediated nucleation activity was sensitive to the lipid environment, a result frequently observed with transmembrane proteins. At most concentrations of Dictyostelium lipid, nucleation activity increased linearly with increasing amounts of ponticulin, suggesting that the nucleating species is a ponticulin monomer. Consistent with previous observations of lateral interactions between actin filaments and Dictyostelium plasma membranes, both ends of ponticulin-nucleated actin filaments appeared to be free for monomer assembly and disassembly. Our results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly. To date, ponticulin is the only integral membrane protein known to directly nucleate actin polymerization.

scholarly journals Interactions of tensin with actin and identification of its three distinct actin-binding domains.

1994 ◽  
Vol 125 (5) ◽  
pp. 1067-1075 ◽  
Author(s):  
S H Lo ◽  
P A Janmey ◽  
J H Hartwig ◽  
L B Chen
Keyword(s):  
Amino Acids ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Sh2 Domain ◽  
Actin Binding ◽  
Molar Ratio ◽  
Actin Assembly ◽  
Binding Domains ◽  
Focal Contacts ◽  
End Capping

Tensin, a 200-kD phosphoprotein of focal contacts, contains sequence homologies to Src (SH2 domain), and several actin-binding proteins. These features suggest that tensin may link the cell membrane to the cytoskeleton and respond directly to tyrosine kinase signalling pathways. Here we identify three distinct actin-binding domains within tensin. Recombinant tensin purified after overexpression by a baculovirus system binds to actin filaments with Kd = 0.1 microM, cross-links actin filaments at a molar ratio of 1:10 (tensin/actin), and retards actin assembly by barbed end capping with Kd = 20 nM. Tensin fragments were constructed and expressed as fusion proteins to map domains having these activities. Three regions from tensin interact with actin: two regions composed of amino acids 1 to 263 and 263 to 463, cosediment with F-actin but do not alter the kinetics of actin assembly; a region composed of amino acids 888-989, with sequence homology to insertin, retards actin polymerization. A claw-shaped tensin dimer would have six potential actin-binding sites and could embrace the ends of two actin filaments at focal contacts.

scholarly journals Role of Tropomyosin in Formin-mediated Contractile Ring Assembly in Fission Yeast

2009 ◽  
Vol 20 (8) ◽  
pp. 2160-2173 ◽  
Author(s):  
Colleen T. Skau ◽  
Erin M. Neidt ◽  
David R. Kovar
Keyword(s):  
Fission Yeast ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Actin Assembly ◽  
Animal Cells ◽  
Contractile Ring ◽  
Direct Role ◽  
Ring Assembly

Like animal cells, fission yeast divides by assembling actin filaments into a contractile ring. In addition to formin Cdc12p and profilin, the single tropomyosin isoform SpTm is required for contractile ring assembly. Cdc12p nucleates actin filaments and remains processively associated with the elongating barbed end while driving the addition of profilin-actin. SpTm is thought to stabilize mature filaments, but it is not known how SpTm localizes to the contractile ring and whether SpTm plays a direct role in Cdc12p-mediated actin polymerization. Using “bulk” and single actin filament assays, we discovered that Cdc12p can recruit SpTm to actin filaments and that SpTm has diverse effects on Cdc12p-mediated actin assembly. On its own, SpTm inhibits actin filament elongation and depolymerization. However, Cdc12p completely overcomes the combined inhibition of actin nucleation and barbed end elongation by profilin and SpTm. Furthermore, SpTm increases the length of Cdc12p-nucleated actin filaments by enhancing the elongation rate twofold and by allowing them to anneal end to end. In contrast, SpTm ultimately turns off Cdc12p-mediated elongation by “trapping” Cdc12p within annealed filaments or by dissociating Cdc12p from the barbed end. Therefore, SpTm makes multiple contributions to contractile ring assembly during and after actin polymerization.

scholarly journals A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Uri Manor ◽  
Sadie Bartholomew ◽  
Gonen Golani ◽  
Eric Christenson ◽  
Michael Kozlov ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Actin Assembly ◽  
Mitochondrial Division

Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division.

scholarly journals Yeast actin patches are networks of branched actin filaments

2004 ◽  
Vol 166 (5) ◽  
pp. 629-635 ◽  
Author(s):  
Michael E. Young ◽  
John A. Cooper ◽  
Paul C. Bridgman
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Branch Ratio ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Capping Protein ◽  
Negative Stain ◽  
Actin Structure ◽  
Negative Stain Electron Microscopy ◽  
Actin Patch

Cortical actin patches are the most prominent actin structure in budding and fission yeast. Patches assemble, move, and disassemble rapidly. We investigated the mechanisms underlying patch actin assembly and motility by studying actin filament ultrastructure within a patch. Actin patches were partially purified from Saccharomyces cerevisiae and examined by negative-stain electron microscopy (EM). To identify patches in the EM, we correlated fluorescence and EM images of GFP-labeled patches. Patches contained a network of actin filaments with branches characteristic of Arp2/3 complex. An average patch contained 85 filaments. The average filament was only 50-nm (20 actin subunits) long, and the filament to branch ratio was 3:1. Patches lacking Sac6/fimbrin were unstable, and patches lacking capping protein were relatively normal. Our results are consistent with Arp2/3 complex-mediated actin polymerization driving yeast actin patch assembly and motility, as described by a variation of the dendritic nucleation model.

scholarly journals Mechanism of actin polymerization revealed by cryo-EM structures of actin filaments with three different bound nucleotides

2019 ◽  
Vol 116 (10) ◽  
pp. 4265-4274 ◽  
Author(s):  
Steven Z. Chou ◽  
Thomas D. Pollard
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Atp Hydrolysis ◽  
Release Site ◽  
Cryo Electron Microscopy ◽  
C Terminus ◽  
Pointed End ◽  
Binding Loop

We used cryo-electron microscopy (cryo-EM) to reconstruct actin filaments with bound AMPPNP (β,γ-imidoadenosine 5′-triphosphate, an ATP analog, resolution 3.1 Å), ADP-Pi(ADP with inorganic phosphate, resolution 3.1 Å), or ADP (resolution 3.6 Å). Subunits in the three filaments have similar backbone conformations, so assembly rather than ATP hydrolysis or phosphate dissociation is responsible for their flattened conformation in filaments. Polymerization increases the rate of ATP hydrolysis by changing the positions of the side chains of Q137 and H161 in the active site. Flattening during assembly also promotes interactions along both the long-pitch and short-pitch helices. In particular, conformational changes in subdomain 3 open up multiple favorable interactions with the DNase-I binding loop in subdomain 2 of the adjacent subunit. Subunits at the barbed end of the filament are likely to be in this favorable conformation, while monomers are not. This difference explains why filaments grow faster at the barbed end than the pointed end. When phosphate dissociates from ADP-Pi-actin through a backdoor channel, the conformation of the C terminus changes so it distorts the DNase binding loop, which allows cofilin binding, and a network of interactions among S14, H73, G74, N111, R177, and G158 rearranges to open the phosphate release site.

scholarly journals Direct visualization of how Actin Depolymerizing Factor’s filament severing and depolymerization synergizes with Capping Protein's "monomer funneling" to promote rapid polarized growth of actin filaments

2017 ◽  
Author(s):  
Shashank Shekhar ◽  
Marie-France Carlier
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Molecular Mechanisms ◽  
Leading Edge ◽  
Bulk Solution ◽  
Direct Visualization ◽  
Actin Networks ◽  
Capping Protein ◽  
Visual Evidence ◽  
Pointed End

AbstractA living cell’s ability to assemble actin filaments in intracellular motile processes is directly dependent on the availability of polymerizable actin monomers which feed polarized filament growth. Continued generation of the monomer pool by filament disassembly is therefore crucial. Disassemblers like ADF/cofilin and filament cappers like Capping Protein (CP) are essential agonists of motility, but the exact molecular mechanisms by which they accelerate actin polymerization at the leading edge and filament turnover has been debated for over two decades. While filament fragmentation by ADF/cofilin has long been demonstrated by TIRF, filament depolymerization was only inferred from bulk solution assays. Using microfluidics-assisted TIRF microscopy, we provide the first direct visual evidence of ADF's simultaneous severing and rapid depolymerization of individual filaments. We have also built a conceptually novel assay to directly visualize ADF’s effect on a filament population. We demonstrate that ADF’s enhanced pointed-end depolymerization leads to an increase in polymerizable actin monomers co-existing with filaments, thus promoting faster barbed-end growth. We further reveal how ADF-enhanced filament depolymerization synergizes with CP’s long-predicted “monomer funneling” and leads to skyrocketing of filament growth rates, close to estimated rates in the lamellipodia. The “Funneling model” hypothesized, on thermodynamic grounds, that at high enough extent of capping, the few noncapped filaments transiently grow much faster, an effect proposed to be very important for motility. We provide the first direct microscopic evidence of monomer funneling by CP at the scale of individual filaments. We believe that these results enlighten our understanding of the turnover of cellular actin networks.

scholarly journals Chlamydomonas reinhardtii formin FOR1 and profilin PRF1 are optimized for acute rapid actin filament assembly

2016 ◽  
Author(s):  
Jenna R. Christensen ◽  
Evan W. Craig ◽  
Michael J. Glista ◽  
David M. Mueller ◽  
Yujie Li ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Apical Cell ◽  
Actin Assembly ◽  
Filamentous Actin ◽  
Tubule Formation ◽  
Actin Networks ◽  
Cellular Processes ◽  
The Right

ABSTRACTThe regulated assembly of multiple filamentous actin (F-actin) networks from an actin monomer pool is important for a variety of cellular processes. Chlamydomonas reinhardtii is a unicellular green alga expressing a conventional and divergent actin that is an emerging system for investigating the complex regulation of actin polymerization. One actin network that contains exclusively conventional F-actin in Chlamydomonas is the fertilization tubule, a mating structure at the apical cell surface in gametes. In addition to two actin genes, Chlamydomonas expresses a profilin (PRF1) and four formin genes (FOR1-4), one of which (FOR1) we have characterized for the first time. We found that unlike typical profilins, PRF1 prevents unwanted actin assembly by strongly inhibiting both F-actin nucleation and barbed end elongation at equimolar concentrations to actin. However, FOR1 stimulates the assembly of rapidly elongating actin filaments from PRF1-bound actin. PRF1 further favors FOR1-mediated actin assembly by potently inhibiting Arp2/3 complex-mediated actin assembly. Furthermore, for1 and prf1-1 mutants, as well as the small molecule formin inhibitor SMIFH2, prevent fertilization tubule formation in gametes, suggesting that polymerization of F-actin for fertilization tubule formation is a primary function of FOR1. Together, these findings indicate that FOR1 and PRF1 cooperate to selectively and rapidly assemble F-actin at the right time and place.SUMMARY STATEMENTThe Chlamydomonas reinhardtii formin FOR1 initiates rapid assembly of fertilization tubule actin filaments from monomers associated with the actin-assembly inhibitor profilin PRF1.

scholarly journals Different Localizations and Cellular Behaviors of Leiomodin and Tropomodulin in Mature Cardiomyocyte Sarcomeres

2010 ◽  
Vol 21 (19) ◽  
pp. 3352-3361 ◽  
Author(s):  
Aneta Skwarek-Maruszewska ◽  
Malgorzata Boczkowska ◽  
Allison L. Zajac ◽  
Elena Kremneva ◽  
Tatyana Svitkina ◽  
...  
Keyword(s):  
Muscle Contraction ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Capping Protein ◽  
Cellular Behaviors ◽  
Nucleation Activity ◽  
End Capping ◽  
Pointed End ◽  
Narrow Bands ◽  
Cultured Cardiomyocytes

Leiomodin (Lmod) is a muscle-specific F-actin–nucleating protein that is related to the F-actin pointed-end–capping protein tropomodulin (Tmod). However, Lmod contains a unique ∼150-residue C-terminal extension that is required for its strong nucleating activity. Overexpression or depletion of Lmod compromises sarcomere organization, but the mechanism by which Lmod contributes to myofibril assembly is not well understood. We show that Tmod and Lmod localize through fundamentally different mechanisms to the pointed ends of two distinct subsets of actin filaments in myofibrils. Tmod localizes to two narrow bands immediately adjacent to M-lines, whereas Lmod displays dynamic localization to two broader bands, which are generally more separated from M-lines. Lmod's localization and F-actin nucleation activity are enhanced by interaction with tropomyosin. Unlike Tmod, the myofibril localization of Lmod depends on sustained muscle contraction and actin polymerization. We further show that Lmod expression correlates with the maturation of myofibrils in cultured cardiomyocytes and that it associates with sarcomeres only in differentiated myofibrils. Collectively, the data suggest that Lmod contributes to the final organization and maintenance of sarcomere architecture by promoting tropomyosin-dependent actin filament nucleation.

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