Nebulin regulates actin filament lengths by a stabilization mechanism

Efficient muscle contraction requires regulation of actin filament lengths. In one highly cited model, the giant protein nebulin has been proposed to function as a molecular ruler specifying filament lengths. We directly challenged this hypothesis by constructing a unique, small version of nebulin (mini-nebulin). When endogenous nebulin was replaced with mini-nebulin in skeletal myocytes, thin filaments extended beyond the end of mini-nebulin, an observation which is inconsistent with a strict ruler function. However, under conditions that promote actin filament depolymerization, filaments associated with mini-nebulin were remarkably maintained at lengths either matching or longer than mini-nebulin. This indicates that mini-nebulin is able to stabilize portions of the filament it has no contact with. Knockdown of nebulin also resulted in more dynamic populations of thin filament components, whereas expression of mini-nebulin decreased the dynamics at both filament ends (i.e., recovered loss of endogenous nebulin). Thus, nebulin regulates thin filament architecture by a mechanism that includes stabilizing the filaments and preventing actin depolymerization.     

Uncoupling actin filament fragmentation by cofilin from increased subunit turnover

The actin depolymerizing factor (ADF)/cofilin family of proteins interact with actin monomers and filaments in a pH-sensitive manner. When ADF/cofilin binds F-actin it induces a change in the helical twist and fragmentation; it also accelerates the dissociation of subunits from the pointed ends of filaments, thereby increasing treadmilling or depolymerization. Using site-directed mutagenesis we characterized the two actin-binding sites on human cofilin. One target site was chosen because we previously showed that the villin head piece competes with ADF for binding to F-actin. Limited sequence homology between ADF/cofilin and the part of the villin headpiece essential for actin binding suggested an actin-binding site on cofilin involving a structural loop at the opposite end of the molecule to the alpha-helix already implicated in actin binding. Binding through the alpha-helix is primarily to monomeric actin, whereas the loop region is specifically involved in filament association. We have characterized the actin binding properties of each site independently of the other. Mutation of a single lysine residue in the loop region abolishes binding to filaments, but not to monomers. Using the mutation analogous to the phosphorylated form of cofilin (S3D), we show that filament binding is inhibited at physiological ionic strength but not under low salt conditions. At low ionic strength, this mutant induces both the twist change and fragmentation characteristic of wild-type cofilin, but does not activate subunit dissociation. The results suggest a two-site binding to filaments, initiated by association through the loop site, followed by interaction with the adjacent subunit through the "helix" site at the opposite end of the molecule. Together, these interactions induce twist and fragmentation of filaments, but the twist change itself is not responsible for the enhanced rate of actin subunit release from filaments.     

in vitro development of porcine enucleated oocytes reconstructed by the transfer of porcine fetal fibroblasts and cumulus cells

In the pig little information is available on cytoplasmic events during the reprogramming of oocytes reconstructed with somatic nuclei. The present study was conducted to determine the developmental potential of porcine cumulus cells (CC) and fetal fibroblasts (FF) after they were transferred into enucleated oocytes. Non-quiescent FF were fused to the enucleated oocytes using electrical pulse, whereas CC were directly injected into the oocytes. Transferred nuclei from both CC and FF underwent premature chromosome condensation (PCC), nuclear swelling and pronucleus formation. The remodeled oocytes developed to the mitotic and 2-cell stage at 18 to 24 h after nuclear transfer. The pattern of nuclear remodeling was similar regardless of the sources of karyoplasts or nuclear transfer methods. However, using FF, 24% of nuclear transferred embryos developed to the morula or blastocyst stage, whereas only 8% of those using CC developed to the morula or blastocyst stage. These results suggest that porcine oocyte cytoplasm can successfully reprogram somatic cell nuclei and support the development of nuclear transferred embryos to the blastocyst stage.     

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

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-P(i) 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.     

An actin filament matrix in hand-isolated nuclei of X. laevis oocytes

The nuclear gel of Xenopus oocytes contains a meshwork of randomly oriented microfilaments which have been identified as F-actin by decoration with rabbit skeletal muscle myosin subfragment-1 (S-1). Nuclear gel preparations treated with S-1 differ in several respects from control preparations incubated in either aqueous medium alone, or medium containing BSA. Actin filaments in control preparations appear less well preserved than those in S-1 treated preparations of the nuclear gel. The nucleoli of control preparations are extremely dense, while those of S-1 treated preparations have a more open, granular appearance. Large granular aggregates, which are a prominent feature of the controls, are seen much less frequently in S-1-treated preparations of the nuclear gel. These morphological differences appear to be correlated with the binding of protein to F-actin, since nuclear gel preparations incubated in tropomyosin, which also binds to actin filaments, appear similar to those treated with S-1. Approximately 63% of the total nuclear actin exists in a globular state, while 37% is filamentous.     

Role of intermonomer ionic bridges in the stabilization of the actin filament

Filament formation is required for most of the functions of actin. However, the intermonomer interactions that stabilize F-actin have not been elucidated because of a lack of an F-actin crystal structure. The Holmes muscle actin model suggests that an ionic interaction between Arg-39 of one monomer and Glu-167 of an adjacent monomer in the same strand contributes to this stabilization. Yeast actin has an Ala-167 instead. F-actin molecular dynamics modeling predicts another interaction between Arg-39 of one monomer and Asp-275 of an opposing strand monomer. In Toxoplasma gondii actin, which forms short stubby filaments, the Asp-275 equivalent is replaced by Arg leading to a potential filament-destabilizing charge-charge repulsion. Using yeast actin, we tested the effect of A167E as a potential stabilizer and A167R and D275R as potential filament disruptors. All mutations caused abnormal growth and mitochondrial malfunction. A167E and D275R actins polymerize normally and form relatively normal appearing filaments. A167R nucleates filaments more slowly and forms filament bundles. The R39D/A167R double mutant, which re-establishes an ionic bond in the opposite orientation, reverses this polymerization and bundling defect. Stoichiometric amounts of yeast cofilin have little effect on wild-type and A167E filaments. However, D275R and A167R actin depolymerization is profound with cofilin. Although our results suggest that disruption of an interaction between Arg-39 and Asp-275 is not sufficient to cause fragmentation, it suggests that it changes filament stability thereby disposing it for enhanced cofilin depolymerizing effects. Ala-167 results demonstrate the in vivo and in vitro importance of another potential Arg-39 ionic interaction.     

The enhanced ATPase activity of glutathione-substituted actin provides a quantitative approach to filament stabilization

[Cys374]glutathionyl-actin was prepared by isolation of the reaction product of G-actin with Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid], followed by reaction with glutathione. Filaments of this actin disulfide are susceptible to even weak shearing stress as exerted, for example, by heating to 37 degrees C. This treatment produces a 25-fold enhanced steady-state ATPase activity as compared to unsubstituted F-actin at room temperature. Monitoring the reduction of this enhanced ATPase activity is a reliable method for quantifying the effectiveness of filament-stabilizing agents and for determining their apparent dissociation constants. A detailed comparative study of filament-stabilizing agents was performed, and some hitherto unknown filament-protecting effects were revealed. Inorganic phosphate provides stabilization only to a maximum of 45% ATPase inhibition, but reaches this effect already at cytoplasmic Pi concentrations (approximately 4 mM). Arsenate seems to bind with similar affinity, but with distinctly less protective activity (maximum of 16%). High concentrations of alkali ions provide a more effective protection (maximum of 95%), Li+ being more efficient than Na+ and K+. Divalent cations (Ca2+, Mg2+) had a strong stabilizing effect on KCl-polymerized actin; we confirmed the presence of two distinct classes of binding sites for divalent metal ions with moderate and low affinities, apparent in a strong stabilizing effect on KCl-polymerized actin. The stabilizing effects of KCl and Pi are independent and additive. Correspondingly, at K2HPO4 concentrations greater than 4 mM, K+ ions contribute considerably to stabilization. In the presence of 100 mM KCl plus 4 mM Pi, conditions which mimic the physiological environment, filament protection is nearly as effective as with the mushroom toxin phalloidin. The strong stabilizing effect of phalloidin occurred at concentrations far below stoichiometric, suggesting a very high degree of cooperativity in its interaction with actin filaments.     

Injection of frozen-thawed porcine first polar bodies into enucleated oocytes results in fertilization and embryonic development

The objective was to determine whether enucleated oocytes injected with frozen porcine first polar bodies (pPB1s) could be fertilized and developed into viable embryos in vitro. Metaphase II (MII) oocytes with pPB1s were frozen (vitrified) and stored for 2 mo. The pPB1s were isolated from thawed MII oocytes and injected into enucleated recipient oocytes by micromanipulation. All recipients injected with thawed pPB1s were fertilized by intracytoplasmic sperm injection (ICSI), and the resulting recombinant zygotes were incubated to assess their developmental competence in vitro. Furthermore, double-antibody immunohistochemistry was used to verify that the nucleus of the pPB1 participated in fertilization and supported embryonic development. Porcine embryos (2- to 8-cell stage) were obtained from the recombinants. The average in vitro cleavage rate of 2-, 4-, and 8-cell stage recombinant embryos was 25.3, 17.7, and 9.3% (P < 0.05), respectively. Chromosomes in the labeled pPB1 participated in the formation of the two blastomere nuclei of 2-cell stage embryos derived from recombinant oocytes. In conclusion, nuclear materials of frozen-thawed pPB1 supported oocyte fertilization and subsequent embryonic development, thereby providing a new way to use frozen PB1s for preservation and reproduction of mammals.     

Mechanism of actin filament bundling by fascin

Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four β-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in β-trefoil domains 1 and 3. The site in β-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in β-trefoil-3 is related by pseudo-2-fold symmetry to that in β-trefoil-1. The two sites are ～5 nm apart, resulting in a distance between actin filaments in the bundle of ～8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.     

Stimulation of actin polymerization by filament severing

The extent and dynamics of actin polymerization in solution are calculated as functions of the filament severing rate, using a simple model of in vitro polymerization. The model is solved by both analytic theory and stochastic-growth simulation. The results show that severing essentially always enhances actin polymerization by freeing up barbed ends, if barbed-end cappers are present. Severing has much weaker effects if only pointed-end cappers are present. In the early stages of polymerization, the polymerized-actin concentration grows exponentially as a function of time. The exponential growth rate is given in terms of the severing rate, and the latter is given in terms of the maximum slope in a polymerization time course. Severing and branching are found to act synergistically.     

Replication of somatic micronuclei in bovine enucleated oocytes

Background

Microcell-mediated chromosome transfer (MMCT) was developed to introduce a low number of chromosomes into a host cell. We have designed a novel technique combining part of MMCT with somatic cell nuclear transfer, which consists of injecting a somatic micronucleus into an enucleated oocyte, and inducing its cellular machinery to replicate such micronucleus. It would allow the isolation and manipulation of a single or a low number of somatic chromosomes.

Methods

Micronuclei from adult bovine fibroblasts were produced by incubation in 0.05 μg/ml demecolcine for 46 h followed by 2 mg/ml mitomycin for 2 h. Cells were finally treated with 10 μg/ml cytochalasin B for 1 h. In vitro matured bovine oocytes were mechanically enucleated and intracytoplasmatically injected with one somatic micronucleus, which had been previously exposed [Micronucleus- injected (+)] or not [Micronucleus- injected (?)] to a transgene (50 ng/μl pCX-EGFP) during 5 min. Enucleated oocytes [Enucleated (+)] and parthenogenetic [Parthenogenetic (+)] controls were injected into the cytoplasm with less than 10 pl of PVP containing 50 ng/μl pCX-EGFP. A non-injected parthenogenetic control [Parthenogenetic (?)] was also included. Two hours after injection, oocytes and reconstituted embryos were activated by incubation in 5 μM ionomycin for 4 min + 1.9 mM 6-DMAP for 3 h. Cleavage stage and egfp expression were evaluated. DNA replication was confirmed by DAPI staining. On day 2, Micronucleus- injected (?), Parthenogenetic (?) and in vitro fertilized (IVF) embryos were karyotyped. Differences among treatments were determined by Fisher′s exact test (p≤0.05).

Results

All the experimental groups underwent the first cell divisions. Interestingly, a low number of Micronucleus-injected embryos showed egfp expression. DAPI staining confirmed replication of micronuclei in most of the evaluated embryos. Karyotype analysis revealed that all Micronucleus-injected embryos had fewer than 15 chromosomes per blastomere (from 1 to 13), while none of the IVF and Parthenogenetic controls showed less than 30 chromosomes per spread.

Conclusions

We have developed a new method to replicate somatic micronuclei, by using the replication machinery of the oocyte. This could be a useful tool for making chromosome transfer, which could be previously targeted for transgenesis.

     

Improvement in development of porcine embryos reconstituted with cells from blastocyst-derived cell lines and enucleated oocytes by optimization of reconstruction methods

The present study was conducted to establish the most suitable system for producing porcine reconstructed embryos by transferring cells from blastocyst-derived cell lines into enucleated oocytes. When the cells were fused to preactivated metaphase II oocytes, or the cells and arrested metaphase II oocytes were fused in medium without CaCl(2) and MgSO(4), the percentages (43-53%) of fused embryos were significantly lower than those (72-79%) produced by fusing the cells to arrested metaphase II oocytes in medium containing CaCl(2) and MgSO(4). High productive efficiency (7%) of blastocysts was obtained when reconstituted embryos produced by the last method were activated again at 3 hours after fusion (F/A --> Activation). Pronuclear formation was observed in 80-91% of the reconstructed embryos produced by F/A --> Activation, with no significant differences between different culture periods in the medium containing cytochalasin B. When cultured in the medium containing cytochalasin B for 0-1 h, almost all (83-85%) the embryos had one pronucleus and one polar body. However, the number of embryos with two pronuclei and no polar bodies was increased significantly by culturing in the medium containing cytochalasin B for 2-4 h. The cleavage rate (34-48%) of reconstructed embryos was not affected by the presence of cytochalasin B for 2 h after activation. However, the percentage of embryos that developed to the blastocyst stage was significantly higher in the presence (23%) than absence (5%) of cytochalasin B. The results indicate that F/A --> Activation and cytochalasin B treatment are effective for the production of porcine embryos reconstituted with cells from blastocyst-derived cell lines and enucleated oocytes.     

Nuclear actin in transcriptional reprogramming by oocytes

Comment on: Miyamoto K, et al. Genes Dev 2011; 25:946-58.     

Osmotic tolerance of in vitro produced porcine blastocysts assessed by their morphological integrity and cellular actin filament organization

This experiment investigated the osmotic tolerance limits of the morphology and the cellular actin filament organization of porcine blastocysts. In vitro produced Day 6 blastocysts were subjected to osmotic treatments with sucrose solutions of different osmolalities (75, 150, 210, 600, 1200, and 2400 mOsm) and one isotonic solution (NCSU-23, 285 mOsm). Blastocysts were then either fixed immediately, or cultured for 18 h and subsequently fixed with formalin. The morphology of the treated blastocysts was examined under a stereomicroscope and the integrity of the cellular actin filaments of the blastocysts was examined by confocal microscopy after staining with Alexa Fluor 488 phalloidin. The results indicated that there was a significant relationship between the osmotic levels and the probability of blastocysts exhibiting disrupted cellular actin filaments. In addition, blastocysts also collapsed in proportion to the levels of osmotic treatments. The osmotic tolerance limits which would maintain 70% of the blastocysts with their original morphology immediately after the treatment were 90 and 170%, respectively, of isotonicity. After 18 h of culture, the osmotic tolerance limits were 61 and 163%, respectively, of isotonicity. Similarly, the osmotic conditions relative to isotonicity which would maintain the integrity of cellular actin filaments in 70% of treated blastocysts had to be within the range of 87 and 147% immediately after the treatment and 87 and 169% after 18 h of culture. Collectively, these data indicate that in vitro produced porcine blastocysts are very sensitive to osmotic stress. This information can be used to optimize cryopreservation procedures for porcine embryos.     

Establishment of a porcine cell line from in vitro-produced blastocysts and transfer of the cells into enucleated oocytes

The present study was conducted to establish a porcine cell line from blastocysts produced in vitro and to examine the developmental ability of nuclear transfer embryos reconstituted with the cells and enucleated mature oocytes. When hatched blastocysts were cultured in Dulbecco's modified Eagle's medium with supplements, no colonies of embryo-derived cells were observed. In contrast, 56% of embryos that were attached to feeder layers of STO cells formed colonies in NCSU-23 with supplements. When the colonies were subcultured in the absence of feeder cells, a cell line with an epithelial-like cell morphology was obtained. This cell morphology was stable up to at least passage 30. Although no fused embryos were observed when a pulse of 100 V/mm was applied, the fusion rate increased significantly at 150 V/mm (28%) and 200 V/mm (64%). At 200 V/mm, 39% of fused embryos cleaved, but no embryos developed beyond the 3-cell stage. When cocultured with electro-activated oocytes, percentages of reconstructed embryos cleaved (65%) and developed to the 4-cell stage (23%) were significantly higher than percentages for those (cleavage: 38%; 4-cell stage: 3%) in the absence of activated oocytes. At 7 days after culture, one reconstructed embryo successfully developed to the blastocyst stage in the presence of activated oocytes. When green fluorescent protein-expressing cells and enucleated oocytes were fused and the fused embryos were cultured with electro-activated oocytes, 3 of 102 reconstructed embryos developed to the blastocyst stage. All of the blastocysts were positive for fluorescent green under ultraviolet light. The results of the present study indicate that a porcine cell line can be established from the hatched blastocyst and maintained in vitro for a long period, and that reconstructed embryos obtained by transferring the blastocyst-derived cells into enucleated oocytes have the ability to develop to the blastocyst stage in vitro.     

Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo

Porcine embryos produced in vitro have a small number of cells and low viability. The present study was conducted to examine the morphological characteristics and the relationship between actin filament organization and morphology of porcine embryos produced in vitro and in vivo. In vitro-derived embryos were produced by in vitro maturation, in vitro fertilization (IVF), and in vitro development. In vivo-derived embryos were collected from inseminated gilts on Days 2-6 after estrus. In experiment 1, in vitro-derived embryos (相似文献   

Development of androgenetic mouse embryos produced by in vitro fertilization of enucleated oocytes

Enucleated mouse oocytes were successfully fertilized in vitro, and the resultant androgenetic eggs developed to the blastocyst stage. The proportion of enucleated oocytes fertilized in vitro was high (87–99%) at sperm concentrations ranging from 10–100 × 10⁴/ml. At high sperm concentrations (100–1,000 × 10⁴), 35–45% of the fertilized eggs resulted in heterozygous bispermic androgenones. The proportion of hemizygous haploid and heterozygous diploid androgenones developing to blastocysts was 11% and 43%, respectively. Hemizygous diploidization, however, showed no positive effect on development. These results clearly show that the procedure reported here is efficient and reliable for the production of androgenetic eggs. © 1993 Wiley-Liss, Inc.     

The dynamic instability of actin filament barbed ends

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.

IntroductionIn cells, actin assembles into filament networks with diverse architectures and lifetimes, playing key roles in functions such as endocytosis, cell motility, and cell division. These filament networks are maintained and renewed by actin turnover, which implies that assembly and disassembly must take place simultaneously and in a controlled manner within the networks. Each actin filament end has the ability to either grow or shrink, depending on the concentration of actin and regulatory proteins, but pure actin treadmills at steady state: ATP-actin is added at the barbed end at a rate matching the departure of ADP-actin from the pointed end, and ATP hydrolysis takes place within the filament. This hallmark feature of actin dynamics has been known for decades (Wegner, 1976) and has been generalized to the cell context, in which it is commonly assumed that actin polymerization takes place at the barbed end, while depolymerization takes place only at the pointed end (whether it be the ends of filaments within the network or the ends of fragments that have detached from it). This notion is reinforced by the fact that the cytoplasm contains high concentrations of monomeric actin (G-actin) in complex with profilin (Funk et al., 2019), which is unable to bind to pointed ends and should drive the elongation of all noncapped barbed ends.Recently, however, in vitro studies have identified two seemingly independent mechanisms in which, in the presence of profilin-actin, filament barbed ends alternate between phases of growth and depolymerization. This behavior, referred to as “dynamic instability,” is widely observed for microtubules but was unexpected for actin filaments. It suggests that cells could use barbed ends for both elongation and disassembly.Driving the depolymerization of barbed ends with cofilin side-decorationProteins of the actin depolymerizing factor (ADF)/cofilin family (henceforth cofilin) are composed of a single ADF-homology (ADF-H) domain and are mostly known for their actin filament–severing activity (De La Cruz, 2009). Cofilin binds cooperatively to the sides of actin filaments, forming clusters where the conformation of the filament is locally altered, leading to its severing at cofilin cluster boundaries. In addition, the barbed ends of cofilin-decorated filaments steadily depolymerize, despite the presence of G-actin and profilin-actin (Fig. 1 A) and even capping protein (CP) in solution (Wioland et al., 2017, 2019). This unexpected result likely originates from the conformational change of actin subunits at the barbed end, induced by cofilin side-binding. As a consequence, filaments exposed to G-actin (with or without profilin), CP, and cofilin alternate between phases of barbed-end elongation and barbed-end depolymerization. In these conditions, actin filament barbed ends thus exhibit a form of dynamic instability.Open in a separate windowFigure 1.Two mechanisms that give rise to barbed-end depolymerization in elongation-promoting conditions. (A) When a cofilin side-decorated region reaches the barbed end, adding a new actin or profilin-actin becomes very difficult, and the barbed end depolymerizes. Not represented: Capping by CP can lead to depolymerization, as it allows the cofilin cluster to reach the barbed end, which then has a much weaker affinity for CP and steadily depolymerizes. Also, severing events occur at cofilin cluster boundaries, creating new barbed ends, either bare or cofilin-decorated. (B) Twinfilin binds to the barbed end, preventing its elongation and causing its depolymerization. Whether twinfilin remains processively attached to the depolymerizing barbed end or departs with the actin subunits is still unknown. Twinfilin has no impact on the elongation of mDia1-bearing barbed ends.Driving the depolymerization of barbed ends with twinfilin end-targetingTwinfilin has two ADF-H domains, but unlike cofilin, it binds poorly to the sides of actin filaments. Rather, twinfilin appears to mainly sequester ADP-actin monomers and target the barbed end to modulate its elongation and capping. Recent in vitro studies have shown that the interaction of twinfilin with actin filament barbed ends could drive their depolymerization, even in the presence of G-actin and profilin-actin (Johnston et al., 2015; Hakala et al., 2021; Shekhar et al., 2021). Very interestingly, the processive barbed-end elongator formin mDia1 is able to protect barbed ends from twinfilin, allowing them to sustain elongation (Shekhar et al., 2021). This leads to a situation in which, as filaments are exposed to profilin-actin and twinfilin, mDia1-bearing barbed ends elongate while bare barbed ends depolymerize (Fig. 1 B). It is safe to assume that, if filaments were continuously exposed to this protein mix including formin in solution, they would alternate between phases of growth and shrinkage over time, as formins come on and fall off the barbed end. This mix of proteins would therefore constitute another situation causing actin filament dynamic instability.From actin treadmilling to dynamic instability, in cells?This newly identified versatile behavior of actin filaments is reminiscent of microtubules. While dynamic instability is the hallmark behavior of microtubules, they can also be made to treadmill steadily by adding 4 microtubule-associated proteins (Arpağ et al., 2020). In cells, both microtubule dynamic instability and treadmilling have been clearly observed (Wittmann et al., 2003). In contrast, the disassembly of single actin filaments, either embedded in a network or severed from it, has not yet been directly observed in cells. Despite insights from techniques such as single-molecule speckle microscopy, it is still unclear from which end actin filaments depolymerize, even in networks that appear to globally treadmill, such as the lamellipodium. Pointed end depolymerization alone cannot account for what is observed in cells (Miyoshi et al., 2006) and alternative mechanisms have been proposed, including brutal filament-to-monomer transitions occurring in bursts, driven by cofilin, coronin, and Aip1 (Brieher, 2013; Tang et al., 2020).In cells, the high amounts of available G-actin (tens of micromolars; Funk et al., 2019) should limit barbed-end depolymerization. Based on the reported on-rate for ATP–G-actin at the barbed ends of cofilin-decorated filaments (Wioland et al., 2017, 2019), we can estimate that these barbed ends, under such conditions, would depolymerize for tens of seconds before being “rescued,” which is enough to remove tens of subunits from each filament. In contrast, twinfilin concentrations similar to those of G-actin appear necessary to drive barbed-end depolymerization (Hakala et al., 2021; Shekhar et al., 2021). As proteomics studies in HeLa cells report that twinfilin is 50-fold less abundant than actin, this may be difficult to achieve in cells (Bekker-Jensen et al., 2017). However, future studies may uncover proteins, or posttranslational modifications of actin, that enhance the ability of twinfilin to drive barbed-end depolymerization in the presence of high concentrations of profilin-actin.Molecular insights and possible synergiesWhile cofilin and twinfilin both interact with actin via ADF-H domains, they appear to drive barbed-end depolymerization through different mechanisms: twinfilin by directly targeting the barbed end, and cofilin by decorating the filament sides, thereby changing the conformation of the filament and putting its barbed end in a depolymerization-prone state.The two mechanisms, nonetheless, share clear similarities. For instance, cofilin side-binding and twinfilin end-targeting both slow down ADP-actin barbed-end depolymerization, compared with bare ADP-actin filaments (Wioland et al., 2017; Hakala et al., 2021; Shekhar et al., 2021). Strikingly, a crystal structure of the actin/twinfilin/CP complex indicates that the actin conformational change induced by twinfilin binding at the barbed end is similar to that induced by cofilin decorating the sides (Mwangangi et al., 2021). It is thus possible that the dynamic instability of actin filament barbed ends reflects the same conformation changes, triggered either by cofilin side-decoration or twinfilin end-targeting.In addition to decorating the filament sides, cofilin targets ADP-actin barbed ends. Unlike twinfilin, the direct interaction of cofilin with the barbed end cannot cause its depolymerization in the presence of ATP-actin monomers. Indeed, cofilin end-targeting accelerates the depolymerization of ADP-actin barbed ends in the absence of G-actin, but cofilin does not appear to interact with growing ATP-actin barbed ends (Wioland et al., 2017). This is in stark contrast with twinfilin end-targeting, which slows down ADP-actin depolymerization and accelerates ADP–Pi-actin depolymerization (Shekhar et al., 2021). These different behaviors regarding the nucleotide state of actin are intriguing and should be investigated further.Cofilin thus needs to decorate the filament sides in order to have an impact on barbed-end dynamics in elongation-promoting conditions. However, it is unknown whether cofilin side-decoration extends all the way to the terminal subunits and occupies sites that twinfilin would target. Thus, it is unclear whether cofilin and twinfilin would compete or synergize to drive barbed-end depolymerization.Synergies with other proteins are also worth further investigation, CP being an interesting candidate. Cofilin side-decoration drastically decreases the barbed-end affinity for CP, and capped filaments are thereby an efficient intermediate to turn growing barbed ends into depolymerizing barbed ends (Wioland et al., 2017). Twinfilin interacts with CP and the barbed end to enhance uncapping (Hakala et al., 2021; Mwangangi et al., 2021). Since CP can bind mDia1-bearing barbed ends and displace mDia1 (Bombardier et al., 2015; Shekhar et al., 2015), perhaps CP can also contribute to turn growing, mDia1-bearing barbed ends into depolymerizing barbed ends, by removing mDia1 from barbed ends and subsequently getting displaced from the barbed end by twinfilin.Finally, it is worth noting that profilin, which does not contain an ADF-H domain, also interacts with the barbed face of G-actin and with the barbed end of the filament. When profilin is in sufficient excess, it is able to promote barbed-end depolymerization in the presence of ATP–G-actin (Pernier et al., 2016). Unlike twinfilin, its depolymerization-promoting activity is not prevented by formin mDia1, and it thus does not lead to dynamic instability (bare and mDia1-bearing barbed ends all either grow or depolymerize). The coexistence of growing, mDia1-bearing barbed ends and depolymerizing, twinfilin-targeted barbed ends (Fig. 1 B) was observed in the presence of profilin (Shekhar et al., 2021), but profilin actually may not be required. Future studies should determine the exact role of profilin in this mechanism.ConclusionThe extent to which barbed-end dynamic instability contributes to actin turnover in cells is not known, but possible molecular mechanisms have now been identified. They should change the way we envision actin network dynamics, as we must now consider the possibility that cells also exploit the barbed end for disassembly. More work is needed to further document these mechanisms, but the idea of a “generalized treadmilling” has now been contradicted at its source: in vitro experiments.     

Mechanism of actin filament nucleation

We used computational methods to analyze the mechanism of actin filament nucleation. We assumed a pathway where monomers form dimers, trimers, and tetramers that then elongate to form filaments but also considered other pathways. We aimed to identify the rate constants for these reactions that best fit experimental measurements of polymerization time courses. The analysis showed that the formation of dimers and trimers is unfavorable because the association reactions are orders of magnitude slower than estimated in previous work rather than because of rapid dissociation of dimers and trimers. The 95% confidence intervals calculated for the four rate constants spanned no more than one order of magnitude. Slow nucleation reactions are consistent with published high-resolution structures of actin filaments and molecular dynamics simulations of filament ends. One explanation for slow dimer formation, which we support with computational analysis, is that actin monomers are in a conformational equilibrium with a dominant conformation that cannot participate in the nucleation steps.     

The interaction of cofilin with the actin filament

Cofilin is a key actin-binding protein that is critical for controlling the assembly of actin within the cell. Here, we present the results of molecular docking and dynamics studies using a muscle actin filament and human cofilin I. Guided by extensive mutagenesis results and other biophysical and structural studies, we arrive at a model for cofilin bound to the actin filament. This predicted structure agrees very well with electron microscopy results for cofilin-decorated filaments, provides molecular insight into how the known F- and G-actin sites on cofilin interact with the filament, and also suggests new interaction sites that may play a role in cofilin binding. The resulting atomic-scale model also helps us understand the molecular function and regulation of cofilin and provides testable data for future experimental and simulation work.