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Liquid-like VASP condensates drive actin polymerization and dynamic bundling

Nature Physics volume 19, pages 574–585 (2023)Cite this article

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Abstract

The organisation of actin filaments into bundles is required for cellular processes such as motility, morphogenesis and cell division. A network of actin-binding proteins, some of which can undergo liquid–liquid phase separation, controls filament bundling. However, it remains unclear how these liquid-like condensates contribute to filament bundling. Here we show that the processive actin polymerase and bundling protein VASP forms liquid-like droplets under physiological conditions. As actin polymerizes within VASP droplets, elongating filaments partition to the edges of the droplet to minimize filament curvature, forming an actin-rich ring within the droplet. The rigidity of this ring is balanced by the droplet’s surface tension. However, as the ring grows thicker, its rigidity increases and eventually overcomes the surface tension, deforming into a linear bundle. The fluid nature of the droplets is critical for bundling, as more solid droplets resist deformation and therefore prevent filaments from rearranging into bundles. This droplet-based bundling mechanism may be relevant to the assembly of cellular architectures rich in bundled actin filaments such as filopodia, stress fibres and focal adhesions.

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Fig. 1: VASP forms liquid-like droplets in vitro.
Fig. 2: Actin polymerization within VASP droplets drives droplet deformation through discrete intermediates.
Fig. 3: Peripheral actin accumulation drives assembly of actin shells, rings and discs.
Fig. 4: An elastic model predicts shape changes by balancing filament bending energy and droplet surface energy.
Fig. 5: Linear droplets formed at high actin-to-VASP ratios consist of parallel-bundled actin filaments.
Fig. 6: Liquid-like behaviour is required for robust actin bundling.

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Data availability

Source data are available for this paper. All other data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request.

Code availability

ImageJ (version 2.1.0) was used with the FRAP Profiler plugin, which is available online (https://worms.zoology.wisc.edu/ImageJ/FRAP_Profiler_v2.java). The code used to generate the simulation results is deposited in GitHub (https://github.com/achansek/VASPDroplet.git).

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Acknowledgements

This research was supported by grants from the National Institutes of Health to J.C.S. (R35GM139531) and P.R. (R01GM132106), by the National Science Foundation through a modulus grant BIO-1934411 to P.R. and J.C.S. and by the Welch Foundation through grant F-2047 to JCS. We thank S. Parekh and D. Dickinson for their feedback. A.C. thanks A. Ravichandran for thoughtful discussions on the model.

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, University of Texas at Austin, Austin, TX, USA

    Kristin Graham, Aly Ladak & Jeanne C. Stachowiak

  2. Department of Mechanical and Aerospace Engineering, University of California San Diego, San Diego, CA, USA

    Aravind Chandrasekaran & Padmini Rangamani

  3. Department of Biochemistry and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

    Liping Wang & Eileen M. Lafer

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Contributions

K.G., A.C., P.R. and J.C.S. designed experiments. K.G., A.C., P.R. and J.C.S. wrote and edited the manuscript. K.G., A.L., A.C., L.W., E.M.L., P.R. and J.C.S. performed experiments and analysed data. All authors consulted on manuscript preparation and editing.

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Correspondence to Padmini Rangamani or Jeanne C. Stachowiak.

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Extended data

Extended Data Fig. 1 Structure and disorder prediction of VASP by Jpred and IUPred.

Results from Jpred secondary structure prediction for the full length (residues 1-380), wild-type VASP sequence16. Prediction reveals the secondary structure prediction, with green arrows representing predicted sheets, and red ovals representing predicted helices. The confidence estimate of the prediction is noted on a scale from 0-9. Results from IUPred long disorder score (purple dashed line) predicts disordered domains of at least 30 consecutive residues, while short disorder score (green dashed line) predicts shorter disordered regions49,50. Results are depicted as a score from 0 to 1, with scores above 0.5 (red dotted line) indicate a higher probability of being disordered. Alignment of sequence predictions was achieved using Jalview51.

Extended Data Fig. 2 VASP droplet formation relies on weak electrostatic interactions between tetramers.

(a) Schematic of VASP domain organisation and designed mutants. (b) Droplet formation tests of various VASP mutants. VASP-IDR did not form droplets at a protein concentration of 100 μM and a PEG concentration of 10% (w/v). VASPΔtet was unable to form droplets at protein or PEG concentrations up to 56 μM VASP and up to 5% PEG. VASPΔEVH1 displays significantly impaired phase separation, as 100 μM VASPΔEVH1 and 5% PEG was required to generate droplets with a size comparable to 20 μM VASP. These data suggest that the EVH1 domain plays a role in LLPS of VASP, though the exact interaction remains unclear. Scale bar 10 μm. (c) Partitioning of 1 μM VASP and VASP mutants to 20 μM unlabeled VASP droplets. Scale bar 5 μm. (d) Quantification of partitioning of 1 μM of each of the mutants into droplets formed from 20 μM of full-length VASP. Partitioning is defined as the ratio of protein intensity inside the droplet to outside the droplet. Bars depict averages, and error bars represent standard error across n = 3 independent experiments with at least 3 images quantified per experiment. (e) Increasing salt concentration disrupts 15 μM VASP droplet formation, indicating that electrostatic interactions are important for VASP condensation. This finding is consistent with the high density of charged residues in the EVH1 and EVH2 domains of VASP, which may result in weak electrostatic interactions that help create a long-range network1,3,52. Scale bar 5 μm.

Source data

Extended Data Fig. 3 Controls for VASP droplet-catalyzed actin polymerization experiments.

a) 2 μM actin added to 15 μM VASP droplets formed with 3% PEG as described in the methods. (b) 15 μM VASP and 2 μM actin are premixed, incubated for 10 minutes, and PEG is added last to a final concentration of 3%. The resulting solution is then imaged after 15 minutes. Droplets still form and are deformed into elongated structures. Scale bar 5 μm. (c) Droplets formed by 15 μM VASP and associated VASP mutants upon addition of 2 μM actin. Scale bar 5 μm. (d) Phalloidin staining of 15 μM VASP-mutFAB droplets and 2 μM actin reveals that filamentous actin is localized to the exterior surfaces of the droplet. Scale bar 5 μm. Inset: magnified droplet from dashed square, scale bar 2 μm. (e) Quantification of actin partitioning into VASP-mutFAB and VASP-mutGAB-mutFAB droplets. Data are mean partitioning ± standard deviation (n = 3 replicates, with 3 images analyzed per trial). Insets: magnified droplets from dashed squares shown in A. Scale bar 1 μm.

Source data

Extended Data Fig. 4 Controls for actin shell, ring, and disc experiments.

(a) Schematic depicting the classification of the various droplet morphologies. Uniform, peripheral, and rod describe morphologies seen in 2D confocal images, while shell, ring and disc describe subdivisions of the peripheral structures, observed upon 3D reconstruction of confocal image stacks. (b) The presence or absence of EDTA/EGTA does not affect the distribution of droplets undergoing progressive bundling. The experiment performed in Fig. 3c-d was performed as discussed in the methods, with the modification that EDTA and EGTA were removed from the droplet buffer. Representative images of VASP droplets containing peripheral actin distribution in the absence of metal ion chelators. Note that the same previously observed 3D arrangements are still observed. Scale bar 1 μm. (c) The population of droplets containing shells, rings, and discs were quantified in the absence of EDTA/EGTA and compared to the original data set without significant variation in trend (compare to Fig. 3d in main text). Data indicates mean + standard deviation over n = 3 replicates, with at least 3 images analyzed per replicate. This data indicates that chelators do not affect the stability of actin filaments to the extent that droplet deformability is impacted. Thus, our model for droplet deformation by actin is likely not impacted by the presence of EDTA/EGTA. (d) Ring thickness is not determined by solution actin to VASP ratio. Quantification of ring thickness as a function of droplet diameter across the three actin to VASP ratios tested (n = 386 droplets). All droplets are for aspect ratios less than 1.1. (e) Histogram of the distributions of droplet sizes for 20 μM VASP droplets. Median droplet size is 2.86 μm. Maximum droplet size is 7.22 μm. n = 1656 droplets counted over at least 3 images from 3 independent replicates.

Source data

Extended Data Fig. 5 Energetic considerations to account for the shape change of a VASP droplet containing a bundled-actin ring.

a. A cartoon of a bundled-actin ring of thickness Tring is shown in a VASP-rich droplet. The interface between the VASP-rich and -depleted phases is shown in black. The total energy of the system is obtained by adding the following terms b. Filament energy – we represent the actin bundle as a linear polymer discretized into segments of size 100 nm. We consider the bundled-actin ring to be made of multiple actin filaments spaced 15 nm apart. The persistence length of the ring is assumed to scale linearly with the number of filaments. The total energy in the bundled-actin ring is given by the sum of stretching and bending energy. We assume that the bundled-actin ring thickness does not change during the structural transitions. c. Wetting energy – Interaction energy parameters (γAV R and γAV D) are chosen to ensure actin prefers to interact with the VASP-rich droplet. Interaction parameter at the interface is defined by a hyperbolic tangent function to ensure continuity and differentiability. d. Surface energy –While the total surface area of the droplet is held constant during the simulation, the energetic cost of changing droplet perimeter is given by γI.

Extended Data Fig. 6 Parameter sweep to determine the surface tension of the interface that bundled-actin ring shares with VASP-rich and VASP-dilute phases.

A) The fraction of actin bundle that is wetted by the VASP-rich droplet is shown. B) Heat map shows the energy of the system corresponding to the energy minimized configuration. As we are interested in the parameters that favor interaction of actin filaments with the VASP-rich phase, we only considered γAV R < γAV D.

Extended Data Fig. 7 Droplet shape depends on bundled-actin ring thickness and droplet shape change parameter, γI.

a-g. Each panel shows heat map of droplet aspect ratio at energy minimum as ring thickness of bundle is systematically varied (color bar shown to the right of each panel) at a given γI value (mentioned on top). We find that lower γI values of 1, and 2.5 pN allow for larger aspect ratios of droplets while increasing the surface energy parameter leads to less-deformable droplets.

Extended Data Fig. 8 High actin to VASP ratios result in increasingly high aspect ratio droplets.

(a) Independent replicates of data shown in Fig. 5b. Left: n = 1196 droplets counted over at least 3 images per condition. Right: n = 594 droplets counted over at least 3 images per condition. (b) Cartoon depicting hypothesized mechanism of droplet-driven actin bundling. Shaded region depicts droplets shown from 3D perspective, and unshaded regions depicts droplets shown from top-down perspective. (c) Addition of 2 μM actin added to 10 μM VASP droplets results in linear droplets that elongate over time. Scale bar 5 μm. (d) High actin to VASP ratios result in very long bundles of actin. Droplets formed with more physiologically relevant actin:VASP ratios result in long bundles. Actin to VASP ratios of 10:1 correspond to 5 μM VASP droplets with 50 μM actin, and an actin:VASP ratio of 28:1 corresponds to 2.5 μM VASP droplets with 70 μM actin. The resulting bundles span multiple microns, overlapping and zippering with other nearby bundles. These long bundles are consistent with our data and model predictions that higher actin:VASP ratios result in increasingly deformed structures. Scale bar 5 μm. (e) Simultaneous mixing of VASP, PEG, and actin results in high aspect ratio bundles. VASP, PEG, and actin diluted with droplet buffer to equal volumes, and were mixed simultaneously to yield a final concentration of 15 μM VASP, 3% PEG, and 2 μM actin. The resulting solution was imaged after 15 minutes. Scale bar 5 μm.

Source data

Extended Data Fig. 9 A liquid-like state is required for droplet bundling.

(a) 10 μM VASP droplets formed under increasing PEG. Scale bar 5 μm. (b) Droplets formed from 10% PEG are more solid than their 3% counterparts, and do not coalesce upon contact. Scale bar 5 μm. (c) Distribution of droplet size under increasing PEG concentrations. Data from n = 3 biologically independent experiments. (d) Droplets formed from increasing PEG do not bundle actin into elongated structures. Scale bar 5 μm. (e) Distribution of droplet aspect ratios of 10 μM VASP droplets containing 2 μM actin as a function of PEG concentration. Bar data are mean + s.d. and teal background lines represent data points. For 3% PEG n = 877 droplets, for 5% PEG n = 2888 droplets, for 10% PEG n = 2802 droplets, for 15% PEG n = 2527 droplets across 3 biologically independent experiments. Brackets indicate data that was tested for significance using an unpaired, two-tailed t-test. *** denotes p < 0.001.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. S1–S9 and methods.

Reporting Summary

Supplementary Video S1

VASP droplets fuse and re-round upon contact: 35 μM VASP droplets (labelled with Alexa Fluor-647, red) quickly fuse upon contact.

Supplementary Video S2

VASP droplets exhibit quick and complete fluorescence recovery after photobleaching. VASP droplets (labelled with Atto-488, red) were bleached at t = 9 s. Fluorescence recovery occurred in approximately 5 min post-bleach.

Supplementary Video S3

Actin localized to VASP droplets distributes peripherally into rings over time: 2 μM actin (labelled with Atto-488, shown in green) added to 13 μM VASP droplets is initially homogeneous but over time redistributes into a ring.

Supplementary Video S4

VASP droplets containing actin rings deform into rods: 10 μM VASP (labelled with Atto-594, red) droplets containing 2 μM actin (labelled with Atto-488, green). Actin is shown initially in a ring conformation and collapses into rods.

Supplementary Video S5

VASP droplets containing actin rings deform into rods that elongated into bundles: 10 μM VASP (labelled with Atto-594, red) droplets containing 2 μM actin (labelled with Atto-488, green). Actin is shown initially in a ring conformation and collapses into a rod that elongate bidirectionally into a bundle-like linear structure.

Supplementary Video S6

Elongation of VASP-actin bundles over time. Bundles formed by 10 μM VASP droplets (labelled with Alexa Fluor 647, red) and 2 μM actin (labelled with Atto-488, green) elongate into linear structures over time.

Supplementary Video S7

New actin monomers are added to the growing tip of the droplet bundles. Bundles formed by 10 μM VASP droplets (labelled with Atto-594, red) and 2 μM actin (labelled with Atto-488, green) were bleached at t = 9 s. Increased fluorescence at the tips indicates new actin monomers are being added to the tip, while the polymerized actin along the shaft remains dark.

Supplementary Video S8

Elongating droplet bundles zipper together upon contact. Bundles formed by 10 μM VASP droplets (labelled with Alexa Fluor 647, red) and 2 μM actin (labelled with Atto-488, green). Bundles elongate with time and, upon contact, zipper together.

Supplementary Video S9

New actin monomers enter the transforming droplet over time. An initially round droplet formed by 10 μM VASP (labelled with Alexa Fluor 647, red) in the presence of 2 μM actin (labelled with Atto-488, green) was bleached at t = 20 s. As the droplet transforms into an elongated bundle, both VASP and actin intensity increase over time, indicating that new proteins enter the droplet from the surrounding solution.

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Graham, K., Chandrasekaran, A., Wang, L. et al. Liquid-like VASP condensates drive actin polymerization and dynamic bundling. Nat. Phys. 19, 574–585 (2023). https://doi.org/10.1038/s41567-022-01924-1

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