Filament Barbed Ends Research Articles

A barbed end interference mechanism reveals how capping protein promotes nucleation in branched actin networks

Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP potentiates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we combine structural biology with in vitro reconstitution to demonstrate that CP not only terminates filament elongation, but indirectly stimulates the activity of Arp2/3 activating nucleation promoting factors (NPFs) by preventing their association to filament barbed ends. Key to this function is one of CP’s C-terminal “tentacle” extensions, which sterically masks the main interaction site of the terminal actin protomer. Deletion of the β tentacle only modestly impairs capping. However, in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes—capping and nucleation—in branched actin network assembly.

Structural and Functional Analyses of the Gelsolin Homology Domains of Flightless‐I in Actin Dynamics

Flightless-I is a unique member of the gelsolin superfamily alloying six gelsolin homology domains and leucine-rich repeats. Flightless-I is an established regulator of the actin cytoskeleton, however, its biochemical activities in actin dynamics are still largely elusive. To better understand the background of the biological functioning of Flightless-I we studied the actin activities of Drosophila Flightless-I by in vitro bulk fluorescence spectroscopy and single filament fluorescence microscopy. Our work identifies the first three gelsolin homology domains (1–3) of Flightless-I as the main actin-binding site; neither the other three gelsolin homology domains (4–6) nor the leucine-rich repeats bind actin. Flightless-I inhibits polymerization by high-affinity (∼nM) filament barbed end capping, moderately facilitates nucleation by low-affinity (∼ µM) monomer binding, and does not sever actin filaments. Our work reveals that in the presence of profilin Flightless-I is only able to cap actin filament barbed ends but fails to promote actin assembly. Flightless-I was found to interact with actin and affect actin dynamics in a calcium-independent fashion in vitro, suggesting the lack of calcium-mediated activation and conformational change of protein. For the comparative structural analysis of the six gelsolin homology domains (GH16) of gelsolin and Flightless-I, we used a combination of biophysical and biochemical approaches. The use of both internal (tryptophans) and external (8-anilinonaphthalene-1-sulfonic acid; ANS) fluorophores revealed that calcium-binding induces structural changes in gelsolin but the conformational behavior of Flightless-I GH16 was not significantly affected by the divalent cation. Different kinetics of limited proteolysis observed for gelsolin and Flightless-I GH16 further strengthened this conclusion. Our experimental findings are supported by bioinformatics analysis predicting that the sequence elements responsible for Ca2+-activation of GSN are not conserved in Flightless-I GH16.

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.

Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks.

Coordinated polymerization of actin filaments provides force for cell migration, morphogenesis and endocytosis. Capping protein (CP) is a central regulator of actin dynamics in all eukaryotes. It binds to actin filament (F-actin) barbed ends with high affinity and slow dissociation kinetics to prevent filament polymerization and depolymerization. However, in cells, CP displays remarkably rapid dynamics within F-actin networks, but the underlying mechanism remains unclear. Here, we report that the conserved cytoskeletal regulator twinfilin is responsible for CP's rapid dynamics and specific localization in cells. Depletion of twinfilin led to stable association between CP and cellular F-actin arrays, as well as to its retrograde movement throughout leading-edge lamellipodia. These were accompanied by diminished F-actin turnover rates. In vitro single-filament imaging approaches revealed that twinfilin directly promotes dissociation of CP from filament barbed ends, while enabling subsequent filament depolymerization. These results uncover a bipartite mechanism that controls how actin cytoskeleton-mediated forces are generated in cells.

AtFH14 crosslinks actin filaments and microtubules in different manners.

In many cellular processes including cell division, the synergistic dynamics of actin filaments and microtubules play vital roles. However, the regulatory mechanisms of these synergistic dynamics are not fully understood. Proteins such as formins are involved in actin filament-microtubule interactions and Arabidopsis thaliana formin 14 (AtFH14) may function as a crosslinker between actin filaments and microtubules in cell division, but the molecular mechanism underlying such crosslinking remains unclear. Without microtubules, formin homology (FH) 1/FH2 of AtFH14 nucleated actin polymerisation from actin monomers and capped the barbed end of actin filaments. However, in the presence of microtubules, quantitative analysis showed that the binding affinity of AtFH14 FH1FH2 to microtubules was higher than that to actin filaments. Moreover, microtubule-bound AtFH14 FH1FH2 neither nucleated actin polymerisation nor inhibited barbed end elongation. In contrast, tubulin did not affect AtFH14 FH1FH2 to nucleate actin polymerisation and inhibit barbed end elongation. Nevertheless, microtubule-bound AtFH14 FH1FH2 bound actin filaments and the bound actin filaments slid and elongated along the microtubules or elongated away from the microtubules, which induced bundling or crosslinking of actin filaments and microtubules. Pharmacological analyses indicated that AtFH14 FH1FH2 promoted crosslinking of actin filaments and microtubules in vivo. Additionally, co-sedimentation and fluorescent dye-labelling experiments of AtFH14 FH2-truncated proteins in vitro revealed the essential motifs of bundling actin filaments or microtubules, which were 63-92 aa and 42-62 aa in the AtFH14 FH2 N-terminal, respectively, and 42-62 aa was the essential motif to crosslink actin filaments and microtubules. Our results aid in explaining how AtFH14 functions as a crosslinker between actin filaments and microtubules to regulate their dynamics via different manners during cell division. They also facilitate further understanding of the molecular mechanisms of the interactions between actin filaments and microtubules.

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

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.

Capping protein is dispensable for polarized actin network growth and actin-based motility

Heterodimeric capping protein (CP) binds the rapidly growing barbed ends of actin filaments and prevents the addition (or loss) of subunits. Capping activity is generally considered to be essential for actin-based motility induced by Arp2/3 complex nucleation. By stopping barbed end growth, CP favors nucleation of daughter filaments at the functionalized surface where the Arp2/3 complex is activated, thus creating polarized network growth, which is necessary for movement. However, here using an in vitro assay where Arp2/3 complex-based actin polymerization is induced on bead surfaces in the absence of CP, we produce robust polarized actin growth and motility. This is achieved either by adding the actin polymerase Ena/VASP or by boosting Arp2/3 complex activity at the surface. Another actin polymerase, the formin FMNL2, cannot substitute for CP, showing that polymerase activity alone is not enough to override the need for CP. Interfering with the polymerase activity of Ena/VASP, its surface recruitment or its bundling activity all reduce Ena/VASP's ability to maintain polarized network growth in the absence of CP. Taken together, our findings show that CP is dispensable for polarized actin growth and motility in situations where surface-directed polymerization is favored by whatever means over the growth of barbed ends in the network.

A novel labeling strategy reveals that myosin Va and myosin Vb bind the same dendritically polarized vesicle population.

Neurons are specialized cells with a polarized geometry and several distinct subdomains that require specific complements of proteins. Delivery of transmembrane proteins requires vesicle transport, which is mediated by molecular motor proteins. The myosin V family of motor proteins mediates transport to the barbed end of actin filaments, and little is known about the vesicles bound by myosin V in neurons. We developed a novel strategy to visualize myosin V-labeled vesicles in cultured hippocampal neurons and systematically characterized the vesicle populations labeled by myosin Va and Vb. We find that both myosins bind vesicles that are polarized to the somatodendritic domain where they undergo bidirectional long-range transport. A series of two-color imaging experiments showed that myosin V specifically colocalized with two different vesicle populations: vesicles labeled with the transferrin receptor and vesicles labeled by low-density lipoprotein receptor. Finally, coexpression with Kinesin-3 family members found that myosin V binds vesicles concurrently with KIF13A or KIF13B, supporting the hypothesis that coregulation of kinesins and myosin V on vesicles is likely to play an important role in neuronal vesicle transport. We anticipate that this new assay will be applicable in a broad range of cell types to determine the function of myosin V motor proteins.

The Activities of the Gelsolin Homology Domains of Flightless-I in Actin Dynamics.

Flightless-I is a unique member of the gelsolin superfamily alloying six gelsolin homology domains and leucine-rich repeats. Flightless-I is an established regulator of the actin cytoskeleton, however, its biochemical activities in actin dynamics are still largely elusive. To better understand the biological functioning of Flightless-I we studied the actin activities of Drosophila Flightless-I by in vitro bulk fluorescence spectroscopy and single filament fluorescence microscopy, as well as in vivo genetic approaches. Flightless-I was found to interact with actin and affects actin dynamics in a calcium-independent fashion in vitro. Our work identifies the first three gelsolin homology domains (1–3) of Flightless-I as the main actin-binding site; neither the other three gelsolin homology domains (4–6) nor the leucine-rich repeats bind actin. Flightless-I inhibits polymerization by high-affinity (∼nM) filament barbed end capping, moderately facilitates nucleation by low-affinity (∼μM) monomer binding, and does not sever actin filaments. Our work reveals that in the presence of profilin Flightless-I is only able to cap actin filament barbed ends but fails to promote actin assembly. In line with the in vitro data, while gelsolin homology domains 4–6 have no effect on in vivo actin polymerization, overexpression of gelsolin homology domains 1–3 prevents the formation of various types of actin cables in the developing Drosophila egg chambers. We also show that the gelsolin homology domains 4–6 of Flightless-I interact with the C-terminus of Drosophila Disheveled-associated activator of morphogenesis formin and negatively regulates its actin assembly activity.

Z‐line‐like Structures Are Distinct from α‐actinin‐rich Dense Bodies in Nematode Obliquely Striated Muscle

Obliquely striated muscles are present widely in soft‐bodied invertebrates and differ from cross‐striated muscles in that thin and thick filaments are staggered and obliquely aligned. Distinct Z‐lines or Z‐discs are absent from obliquely striated muscles. In general, thin filaments in obliquely striated muscles are considered to be anchored at the dense bodies, although there is no direct evidence of a linkage between the ends of thin filaments and dense bodies. In this study, we used body wall muscle of the nematode Caenorhabditis elegans as a representative obliquely striated muscle and found that the barbed ends of sarcomeric actin filaments are not localized to the dense bodies, but instead aligned to linear Z‐line‐like structures. In C. elegans, α‐actinin (ATN‐1) is one of the major components of the dense bodies. However, sarcomeric actin, as probed by anti‐actin antibody, phalloidin, or GFP‐actin, was only marginally co‐localized with ATN‐1. To specifically localize the barbed ends of sarcomeric actin filaments, we expressed GFP‐tagged CAP‐1, an α subunit of heterodimeric actin capping protein. GFP‐CAP‐1 was localized in a striated pattern filling gaps between dense bodies but was not co‐localized with ATN‐1. These “Z‐line‐like” structures of GFP‐CAP‐1 assembled normally in atn‐1‐knockout worms, indicating that ATN‐1 is not required for linear alignment of the actin barbed ends. In atn‐1‐knockout worms, major cytoplasmic portions of the dense bodies are absent, and only moderate disorganization of sarcomeric actin filaments are observed (Mulder et al., 2010). However, RNA interference of cap‐1 caused larval lethality and strong disorganization of sarcomeric actin filaments in embryonic body wall muscle cells. These results indicate that the barbed ends of sarcomeric actin filaments are aligned to Z‐line‐like structures, which assemble independently of dense bodies, and that capping protein plays an essential role in early sarcomeric assembly.Support or Funding InformationSupported by NIH 5R01AR048615‐18.

Sliding Filament and Fixed Filament Mechanisms Contribute to Tension of the Fission Yeast Cytokinetic Ring

A fundamental challenge in cell biology is understanding how cells generate contractile force from actomyosin machineries. The contractile ring is a vital actomyosin machine that generates tension and constricts and divides the cell during cytokinesis. Following discovery of the contractile ring decades ago, a sliding filament mechanism for ring tension was proposed similar to that in muscle (Schroeder, 1972). However, contractile rings lack the highly ordered periodic sarcomeric architecture of muscle, and it remains unknown how tension emerges from the disordered ring organization. Here, we developed a highly coarse-grained continuum model of the fission yeast cytokinetic ring, severely constrained by experimentally measured component amounts. The model incorporates the two myosin-II isoforms, Myo2 in membrane-anchored protein complexes called nodes, and Myp2 clusters assumed unanchored. We found that the model does indeed predict a sliding filament mechanism, but somewhat different to that envisaged by the pioneering studies of Schroeder and others. The sliding filament mechanism is a spatially and temporally homogeneous version of that in muscle: pairs of oppositely oriented actin filaments are made tense as they are pulled toward one another by myosin-II clusters through which they slide. The mechanism relies on anchoring of actin filament barbed ends to the plasma membrane, which enables filaments to become tense when pulled. A second fixed filament component is generated by chains of like-oriented actin filaments encircling the ring, independent of lateral anchoring. Cellular actomyosin contractile machines are intrinsically vulnerable to contractile instabilities. We also studied how such instabilities are controlled. We find that tension loss due to runaway catastrophic aggregation of the contractile components Myo2 and Myp2 is prevented by fast turnover and sufficiently low myosin cluster mobilities, enforced by lateral anchoring and crowding.

Spire stimulates nucleation by Cappuccino and binds both ends of actin filaments.

The actin nucleators Spire and Cappuccino synergize to promote actin assembly, but the mechanism of their synergy is controversial. Together these proteins promote the formation of actin meshes, which are conserved structures that regulate the establishment of oocyte polarity. Direct interaction between Spire and Cappuccino is required for oogenesis and for in vitro synergistic actin assembly. This synergy is proposed to be driven by elongation and the formation of a ternary complex at filament barbed ends, or by nucleation and interaction at filament pointed ends. To mimic the geometry of Spire and Cappuccino in vivo, we immobilized Spire on beads and added Cappuccino and actin. Barbed ends, protected by Cappuccino, grow away from the beads while pointed ends are retained, as expected for nucleation-driven synergy. We found that Spire is sufficient to bind barbed ends and retain pointed ends of actin filaments near beads and we identified Spire’s barbed-end binding domain. Loss of barbed-end binding increases nucleation by Spire and synergy with Cappuccino in bulk pyrene assays and on beads. Importantly, genetic rescue by the loss-of-function mutant indicates that barbed-end binding is not necessary for oogenesis. Thus, increased nucleation is a critical element of synergy both in vitro and in vivo.

Geometrical Constraints Greatly Hinder Formin mDia1 Activity.

Formins are one of the central players in the assembly of most actin networks in cells. The sensitivity of these processive molecular machines to mechanical tension is now well established. However, how the activity of formins is affected by geometrical constraints related to network architecture, such as filament cross-linking and formin spatial confinement, remains largely unknown. Combining microfluidics and micropatterning, we reconstituted in vitro mDia1 formin-elongated filament bundles induced by fascin, with different geometrical constraints on the formins, and measured the impact of these constraints on formin elongation rate and processivity. When filaments are not bundled, the anchoring details of formins have only a mild impact on their processivity and do not affect their elongation rate. When formins are unanchored, we show that filament bundling by fascin reduces both their elongation rate and their processivity. Strikingly, when filaments elongated by surface-anchored formins are cross-linked together, formin elongation rate immediately decreases and processivity is reduced up to 24-fold depending on the cumulative impact of formin rotational and translational freedom. Our results reveal an unexpected crosstalk between the constraints at the filament and the formin levels. We anticipate that in cells the molecular details of formin anchoring to the plasma membrane strongly modulate formin activity at actin filament barbed ends.

The C-terminal domain of EFA6A interacts directly with F-actin and assembles F-actin bundles

The Arf6-specific exchange factor EFA6 is involved in the endocytic/recycling pathway for different cargos. In addition EFA6 acts as a powerful actin cytoskeleton organizer, a function required for its role in the establishment of the epithelial cell polarity and in neuronal morphogenesis. We previously showed that the C-terminus of EFA6 (EFA6-Ct) is the main domain which contributes to actin reorganization. Here, by in vitro and in vivo experiments, we sought to decipher, at the molecular level, how EFA6 controls the dynamic and structuring of actin filaments. We showed that EFA6-Ct interferes with actin polymerization by interacting with and capping actin filament barbed ends. Further, in the presence of actin mono-filaments, the addition of EFA6-Ct triggered the formation of actin bundles. In cells, when the EFA6-Ct was directed to the plasma membrane, as is the case for the full-length protein, its expression induced the formation of membrane protrusions enriched in actin cables. Collectively our data explain, at least in part, how EFA6 plays an essential role in actin organization by interacting with and bundling F-actin.

The Actin-Capping Protein Alpha-Adducin Is Required for T-Cell Costimulation.

Alpha-adducin (Add1) is a critical component of the actin-spectrin network in erythrocytes, acting to cap the fast-growing, barbed ends of actin filaments, and recruiting spectrin to these junctions. Add1 is highly expressed in T cells, but its role in T-cell activation has not been examined. Using a conditional knockout model, we show that Add1 is necessary for complete activation of CD4+ T cells in response to low levels of antigen but is dispensable for CD8+ T cell activation and response to infection. Surprisingly, costimulatory signals through CD28 were completely abrogated in the absence of Add1. This study is the first to examine the role of actin-capping in T cells, and it reveals a previously unappreciated role for the actin cytoskeleton in regulating costimulation.

Molecular dynamics study of interactions between polymorphic actin filaments and gelsolin segment-1.

The assembly of protein actin into double-helical filaments promotes many eukaryotic cellular processes that are regulated by actin-binding proteins (ABPs). Actin filaments can adopt multiple conformations, known as structural polymorphism, which possibly influences the interaction between filaments and ABPs. Gelsolin is a Ca2+ -regulated ABP that severs and caps actin filaments. Gelsolin binding modulates filament structure; however, it is not known how polymorphic actin filament structures influence an interaction of gelsolin S1 with the barbed-end of filament. Herein, we investigated how polymorphic structures of actin filaments affect the interactions near interfaces between the gelsolin segment 1 (S1) domain and the filament barbed-end. Using all-atom molecular dynamics simulations, we demonstrate that different tilted states of subunits modulate gelsolin S1 interactions with the barbed-end of polymorphic filaments. Hydrogen bonding and interaction energy at the filament-gelsolin S1 interface indicate distinct conformations of filament barbed ends, resulting in different interactions of gelsolin S1. This study demonstrates that filament's structural multiplicity plays important roles in the interactions of actin with ABPs.

Sliding filament and fixed filament mechanisms contribute to ring tension in the cytokinetic contractile ring.

A fundamental challenge in cell biology is to understand how cells generate actomyosin-based contractile force. Here we study the actomyosin contractile ring that divides cells during cytokinesis and generates tension by a mechanism that remains poorly understood. Long ago a muscle-like sliding filament mechanism was proposed, but evidence for sarcomeric organization in contractile rings is lacking. We develop a coarse-grained model of the fission yeast cytokinetic ring, incorporating the two myosin-II isoforms Myo2 and Myp2 and severely constrained by experimental data. The model predicts that ring tension is indeed generated by a sliding filament mechanism, but a spatially and temporally homogeneous version of that in muscle. In this mechanism all pairs of oppositely oriented actin filaments are rendered tense as they are pulled toward one another and slide through clusters of myosin-II. The mechanism relies on anchoring of actin filament barbed ends to the plasma membrane, which resists lateral motion and enables filaments to become tense when pulled by myosin-II. A second fixed filament component is independent of lateral anchoring, generated by chains of like-oriented actin filaments. Myo2 contributes to both components, while Myp2 contributes to the sliding filament component only. In the face of instabilities inherent to actomyosin contractility, organizational homeostasis is maintained by rapid turnover of Myo2 and Myp2, and by drag forces that resist lateral motion of actin, Myo2 and Myp2. Thus, sliding and fixed filament mechanisms contribute to tension in the disordered contractile ring without the need for the sarcomeric architecture of muscle.

Ena/VASP processive elongation is modulated by avidity on actin filaments bundled by the filopodia cross-linker fascin.

Ena/VASP tetramers are processive actin elongation factors that localize to diverse F-actin networks composed of filaments bundled by different cross-linking proteins, such as filopodia (fascin), lamellipodia (fimbrin), and stress fibers (α-actinin). Previously, we found that Ena takes approximately threefold longer processive runs on trailing barbed ends of fascin-bundled F-actin. Here, we used single-molecule TIRFM (total internal reflection fluorescence microscopy) and developed a kinetic model to further dissect Ena/VASP’s processive mechanism on bundled filaments. We discovered that Ena’s enhanced processivity on trailing barbed ends is specific to fascin bundles, with no enhancement on fimbrin or α-actinin bundles. Notably, Ena/VASP’s processive run length increases with the number of both fascin-bundled filaments and Ena “arms,” revealing avidity facilitates enhanced processivity. Consistently, Ena tetramers form more filopodia than mutant dimer and trimers in Drosophila culture cells. Moreover, enhanced processivity on trailing barbed ends of fascin-bundled filaments is an evolutionarily conserved property of Ena/VASP homologues, including human VASP and Caenorhabditis elegans UNC-34. These results demonstrate that Ena tetramers are tailored for enhanced processivity on fascin bundles and that avidity of multiple arms associating with multiple filaments is critical for this process. Furthermore, we discovered a novel regulatory process whereby bundle size and bundling protein specificity control activities of a processive assembly factor.

A novel mode of capping protein-regulation by twinfilin.

Cellular actin assembly is controlled at the barbed ends of actin filaments, where capping protein (CP) limits polymerization. Twinfilin is a conserved in vivo binding partner of CP, yet the significance of this interaction has remained a mystery. Here, we discover that the C-terminal tail of Twinfilin harbors a CP-interacting (CPI) motif, identifying it as a novel CPI-motif protein. Twinfilin and the CPI-motif protein CARMIL have overlapping binding sites on CP. Further, Twinfilin binds competitively with CARMIL to CP, protecting CP from barbed-end displacement by CARMIL. Twinfilin also accelerates dissociation of the CP inhibitor V-1, restoring CP to an active capping state. Knockdowns of Twinfilin and CP each cause similar defects in cell morphology, and elevated Twinfilin expression rescues defects caused by CARMIL hyperactivity. Together, these observations define Twinfilin as the first 'pro-capping' ligand of CP and lead us to propose important revisions to our understanding of the CP regulatory cycle.

Processive Nanostepping of Formin mDia1 Loosely Coupled with Actin Polymerization.

Formins are actin-binding proteins that construct nanoscale machinery with the growing barbed end of actin filaments and serve as key regulators of actin polymerization and depolymerization. To maintain the regulation of actin dynamics, formins have been proposed to processively move at every association or dissociation of a single actin molecule toward newly formed barbed ends. However, the current models for the motile mechanisms were established without direct observation of the elementary processes of this movement. Here, using optical tweezers, we demonstrate that formin mDia1 moves stepwise, observed at a nanometer spatial resolution. The movement was composed of forward and backward steps with unitary step sizes of 2.8 and -2.4 nm, respectively, which nearly equaled the actin subunit length (∼2.7 nm), consistent with the generally accepted models. However, in addition to steps equivalent to the length of a single actin subunit, those equivalent to the length of two or three subunits were frequently observed. Our findings suggest that the coupling between mDia1 stepping and actin polymerization is not tight but loose, which may be achieved by the multiple binding states of mDia1, providing insights into the synergistic functions of biomolecules for the efficient construction and regulation of nanofilaments.