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. 2006 Mar 28;103(13):4906-11.
doi: 10.1073/pnas.0508269103. Epub 2006 Mar 20.

Reconstitution of the transition from lamellipodium to filopodium in a membrane-free system

Lior Haviv  1 , Yifat Brill-Karniely, Rachel Mahaffy, Frederic Backouche, Avinoam Ben-Shaul, Thomas D Pollard, Anne Bernheim-Groswasser
Affiliations

Reconstitution of the transition from lamellipodium to filopodium in a membrane-free system

Lior Haviv et al. Proc Natl Acad Sci U S A. .

Abstract

The cellular cytoskeleton is a complex dynamical network that constantly remodels as cells divide and move. This reorganization process occurs not only at the cell membrane, but also in the cell interior (bulk). During locomotion, regulated actin assembly near the plasma membrane produces lamellipodia and filopodia. Therefore, most in vitro experiments explore phenomena taking place in the vicinity of a surface. To understand how the molecular machinery of a cell self-organizes in a more general way, we studied bulk polymerization of actin in the presence of actin-related protein 2/3 complex and a nucleation promoting factor as a model for actin assembly in the cell interior separate from membranes. Bulk polymerization of actin in the presence of the verprolin homology, cofilin homology, and acidic region, domain of Wiskott-Aldrich syndrome protein, and actin-related protein 2/3 complex results in spontaneous formation of diffuse aster-like structures. In the presence of fascin these asters transition into stars with bundles of actin filaments growing from the surface, similar to star-like structures recently observed in vivo. The transition from asters to stars depends on the ratio [fascin]/[G actin]. The polarity of the actin filaments during the transition is preserved, as in the transition from lamellipodia to filopodia. Capping protein inhibits star formation. Based on these experiments and kinetic Monte Carlo simulations, we propose a model for the spontaneous self-assembly of asters and their transition into stars. This mechanism may apply to the transition from lamellipodia to filopodia in vivo.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Confocal images of asters. Conditions were actin monomer (3.78 μM), GST-VCA (50 nM), and Arp2/3 complex (25 nM). (a and b) Individual asters and aster pairs (marked by white lines in a) are observed. (c) The intensity profile of actin fluorescence along an aster pair (right aster pair) is plotted. Typically, the two asters are almost identical in size and actin content. (d) The intensity profile of the fluorescence, I(r), is plotted along an aster (marked by a black line) versus the radial distance r from the aster center. I(r) decays symmetrically as a function of r from the aster center to the periphery.
Fig. 2.
Fig. 2.
Total internal reflection fluorescent images of an aster formation. Conditions were 1 μM actin monomer, 200 nM GST-VCA, and 10 nM Arp2/3 complex. (a) Images in the time-lapse series are spaced 30 s apart for a total time of 3.5 min. The experiments begin with buffer alone in the chamber. Actin, Arp2/3 complex, and activator are quickly mixed and pulled through the chamber. High concentrations of Arp2/3 complex lead to the formation of asters. Most likely, very small spontaneously nucleated seeds act as nucleation points from which Arp2/3 complex rapidly forms branches autocatalytically. The initial seeds are smaller than the optical resolution and thus appear as points at the center of the aster. Formation of a new branch (≈70° is characteristic to Arp2/3 branching) marked by a white arrow in the two last frames shows that the actin filaments barbed ends (+) point outward. (b) An image of the aster part that is close to the coverslip surface clearly shows branches pointing away from the aster center, three of them are marked in red. The growth direction is thus from the pointed (−) end to the barbed (+) end.
Fig. 3.
Fig. 3.
Kinetic Monte Carlo simulations. (a–c) Snapshots taken 10 (a), 13 (b), and 16 (c) s after nucleation. (d and e) Magnified regions from c and b, respectively. All filament tips are barbed ends and labeled in red. (f) (Lower) The volume fraction, ρ, of polymerized actin, and the orientational order parameter, η, as a function of the distance, r, from the nucleation center, after all actin monomer has been consumed and cluster growth completed. (Upper) The average number of polymerized subunits, d, between adjacent branching points on the same filament, as a function of r. The plots represent averages over eight simulation runs. The initial concentrations are similar to those used experimentally: 7.47 μM G actin and 100 nM Arp2/3 complex.
Fig. 4.
Fig. 4.
Star formation is observed when fascin is added to a solution of actin, VCA, and Arp2/3 complex. (a and b) Initial conditions were 7.28 μM actin monomer, 200 nM GST-VCA, 100 nM Arp2/3 complex, and 3 μM fascin; the [fascin]/[G actin] = 1/2. (a) After mixing for 2.5 min the star appears to be composed of a dense core surrounded by a low density cloud of actin. It is already possible to observe actin bundles in the cloudy regions. (b) After 7.5 min the cloudy corona turns into a fully developed star; the diffuse actin cloud disappears, turning into long actin bundles (10–30 μm) emanating from the dense core. (c) Addition of 40 nM CP results in inhibition of star formation. The number of stars is decreased and their bundles are shorter (≈2 μm). (d) Stars are not formed in the absence of Arp2/3 complex; instead, an entangled network of filament bundles is observed. Short white arrows mark branching points of actin bundles, and the long white arrow marks the bundling/splitting of several bundles. (e) The dynamic of star growth is followed in time steps of 20 s. Initial protein concentrations were 3.64 μM actin monomer, 200 nM GST-VCA, 100 nM Arp2/3 complex, and 1.5 μM fascin; [fascin]/[actin] = ½ as in a–d. The initial average growth rate of bundles is v̄ = 2.27 ± 0.80 μm/min. (f) Star pairs also form. Their bundles growth rate is the same as for individual stars.
Fig. 5.
Fig. 5.
The transition from asters to stars. (a) The first polymerization step begins by spontaneous nucleation of an actin seed. This step is followed by binding of Arp2/3 complexes to nascent actin filaments, initiating an autocatalytic process of branch formation and filament growth. With time growth of the cluster becomes isotropic, resulting in an aster of roughly spherical symmetry. The growth of the network advances with the barbed ends of the actin filaments (+) pointing outward. (b) The transition of an aster into a star is initiated by the reorganization of the network structure into bundles. The star is now composed of a dense network of actin filaments with short bundles emanating with their barbed ends pointing outward (+) from beyond this core. (c) In the final step of star formation, the bundles elongate by continuous actin polymerization in the outward direction while new bundles continue to emerge close to the star core. During the growth process the density of the star core continues to increase because of the continuous nucleation of new Arp2/3-actin branches.
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