Lamellar Phase of Stacked Two-Dimensional Rafts of Actin Filaments

We examined liquid crystalline phases of the cytoskeletal polyelectrolyte filamentous (F-)actin in the presence of multivalent counterions. As a function of increasing ion concentration, the F-actin rods in either an isotropic or a nematic phase will transform into a new and unexpected lamellar phase of cross-linked rafts ($L_{\mathrm{X}\mathrm{R}}$ phase), before condensing into a bundled phase of parallel, close-packed rods. This behavior is generic for alkali earth divalent ions ${\mathrm{M}\mathrm{g}}^{2+}$, ${\mathrm{C}\mathrm{a}}^{2+}$, ${\mathrm{S}\mathrm{r}}^{2+}$, and ${\mathrm{B}\mathrm{a}}^{2+}$, and the structural transitions are achieved without any architecture-specific actin-binding linker proteins.

Figure 1

A schematic representation of the $L_{\mathrm{X}\mathrm{R}}$ phase in which rafts of F-actin rods are cross-linked into a one-dimensional (1D) lamellar stack. Counterions (not shown for clarity), in the immediate vicinity of the rods due to Manning condensation, are localized to the crossing regions between actin rods. One of the cross-linked bilayer rafts (i.e., a 3D unit cell) has been highlighted in blue for clarity. The graph on the bottom depicts a side view of the structure shown on the top.

Figure 2

(a) Condensation of F-actin filaments ($2\mathrm{m}\mathrm{g}/\mathrm{m}\mathrm{l}$, average length $\sim 100\mathrm{n}\mathrm{m}$) with ${\mathrm{C}\mathrm{a}\mathrm{C}\mathrm{l}}_2$. The ${\mathrm{C}\mathrm{a}\mathrm{C}\mathrm{l}}_2$ concentrations are 2.5, 9.0, and 80 mM for the top, middle, and bottom capillaries, respectively. The 2D nematic LC network phase exists in the middle capillary, where a phase boundary can be observed. The distinct bundled phase can be seen in the bottom capillary for contrast. These samples are stable for months. (b),(c),(d) Polarized micrographs taken from the top, middle, and bottom capillaries, respectively. Note the strong birefringence in both the network phase and the bundled phase. The bottom panel shows laser scanning confocal microscope images of a dilute solution ($0.03\mathrm{m}\mathrm{g}/\mathrm{m}\mathrm{l}$) of $\sim 10\mu \mathrm{m}$ F-actin filaments at (e) 2.5, (f) 9, and (g) 80 mM ${\mathrm{C}\mathrm{a}\mathrm{C}\mathrm{l}}_2$. The field of view is $25\mu \mathrm{m}\times 25\mu \mathrm{m}$. Although the global concentration of F-actin is constant, the ion-induced condensation transitions have progressively increased the local concentrations in (e)–(g). In (f), the system is dominated by nonparallel filament cross-links, while in (g), the filaments have condensed into a parallel arrangement and have formed an ensemble of thick bundles. Note the dramatically increased fluorescence intensity from the multifilament bundles.

Figure 3

(a) Two-dimensional (2D) SAXS data of a partially aligned condensed $L_{\mathrm{X}\mathrm{R}}$ phase composed of 100 nm F-actin condensed by 3.0 mM ${\mathrm{C}\mathrm{a}\mathrm{C}\mathrm{l}}_2$. (b) Top: a cut of the diffraction pattern in (a) along axis 1, showing three strong harmonics of the layering peaks. Bottom: a cut of the diffraction pattern along axis 2, showing a distinct set of broad correlation peaks. The weak liquidlike, short-ranged correlation peak marked by the double-headed arrow corresponds to a nematic arrangement of F-actin at a direction orthogonal to that of the layering direction. Weak intensity from the lamellar peaks can still be detected (single arrows), due to the large mosaic distribution. (c) SAXS data from the same sample in (a) but on a different spot. A number of domains with different orientation can be seen as a $300\mu \mathrm{m}\times 300\mu \mathrm{m}$ synchrotron x-ray beam is scanned across the sample. This indicates that the $L_{\mathrm{X}\mathrm{R}}$ phase has a multiaxial structure, and does not have a single preferred orientation. (d) Inset: 2D SAXS pattern from a partially aligned sample in the bundled phase, with 100 nm F-actin condensed by 80 mM ${\mathrm{C}\mathrm{a}\mathrm{C}\mathrm{l}}_2$. The plot shows radially averaged SAXS data showing the close-packed inter-rod correlation characteristic of the bundled phase.

Figure 4

A phase diagram for four different divalent ions from the alkali earth column (${\mathrm{M}\mathrm{g}}^{2+}$, ${\mathrm{C}\mathrm{a}}^{2+}$, ${\mathrm{S}\mathrm{r}}^{2+}$, ${\mathrm{B}\mathrm{a}}^{2+}$, represented by different symbols), in which the phase behavior as a function of divalent ion concentrations and rod length is summarized. For example, the onset of ion-induced condensation of actin into the $L_{\mathrm{X}\mathrm{R}}$ lamellar raft phase with ${\mathrm{M}\mathrm{g}}^{2+}$ for $3000\AA$ actin filaments occurs at ${\mathrm{M}\mathrm{g}}^{2+}$ concentrations as low as 3 mM, and increases to 5 mM for $1\mu \mathrm{m}$ actin and 11 mM for $10\mu \mathrm{m}$ actin filaments. Furthermore, although the phase boundaries shift slightly for different ions, it can be seen that the basic phase behavior is general for all four ion species: As the divalent ion concentration is increased, the system evolves from an uncondensed isotropic phase to the condensed $L_{\mathrm{X}\mathrm{R}}$ raft phase, and finally to the condensed bundled phase.